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Ablation of SNX6 leads to defects in synaptic function of CA1 pyramidal neurons and spatial memory

  1. Yang Niu
  2. Zhonghua Dai
  3. Wenxue Liu
  4. Cheng Zhang
  5. Yanrui Yang
  6. Zhenzhen Guo
  7. Xiaoyu Li
  8. Chenchang Xu
  9. Xiahe Huang
  10. Yingchun Wang
  11. Yun S Shi  Is a corresponding author
  12. Jia-Jia Liu  Is a corresponding author
  1. Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. Jinling Hospital, School of Medicine, Nanjing University, China
  4. Nanjing University, China
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Cite this article as: eLife 2017;6:e20991 doi: 10.7554/eLife.20991

Abstract

SNX6 is a ubiquitously expressed PX-BAR protein that plays important roles in retromer-mediated retrograde vesicular transport from endosomes. Here we report that CNS-specific Snx6 knockout mice exhibit deficits in spatial learning and memory, accompanied with loss of spines from distal dendrites of hippocampal CA1 pyramidal cells. SNX6 interacts with Homer1b/c, a postsynaptic scaffold protein crucial for the synaptic distribution of other postsynaptic density (PSD) proteins and structural integrity of dendritic spines. We show that SNX6 functions independently of retromer to regulate distribution of Homer1b/c in the dendritic shaft. We also find that Homer1b/c translocates from shaft to spines by protein diffusion, which does not require SNX6. Ablation of SNX6 causes reduced distribution of Homer1b/c in distal dendrites, decrease in surface levels of AMPAR and impaired AMPAR-mediated synaptic transmission. These findings reveal a physiological role of SNX6 in CNS excitatory neurons.

https://doi.org/10.7554/eLife.20991.001

eLife digest

Neurons are the building blocks of the nervous system. These cells generally consist of a round portion called the cell body and a long cable-like axon. The cell body bears numerous branches called dendrites, which are in turn covered in spines. Neurons communicate with one another at junctions – or synapses – that typically form between the end of the axon of one cell and a dendritic spine on another.

Specialized proteins stabilize the dendritic spines and enable the cells to exchange messages across the synapse. However, it is the cell body – rather than the dendrites – that produces most of these proteins. Structures called molecular motors transport proteins to their destinations within the cell along fixed tracks, similar to how a freight train carries cargo over the rail network. One of the key molecular motors within neurons is called dynein‒dynactin. This in turn interacts with other proteins called adaptors, enabling it to transport specific types of cargo.

Niu, Dai, Liu et al. have now examined the role of SNX6, an adaptor protein for the dynein‒dynactin motor. Mice that have been genetically modified to lack SNX6 in their brains have fewer spines on their dendrites compared with normal mice. This was particularly true for dendrites that contain AMPAR, a protein that receives signals sent across synapses. Niu, Dai, Liu et al. showed that SNX6 interacts with another protein called Homer1b/c and is responsible for distributing this protein in dendrites far from the cell body. The Homer1b/c protein helps to stabilize dendritic spines and to regulate the number of AMPAR proteins within them. Mice that lack SNX6 therefore have less Homer1b/c in the dendrites furthest from the cell body, and fewer spines on these dendrites too. These mice also have fewer AMPAR proteins at their synapses than control mice.

Mice that lack SNX6 show impaired learning and memory compared to control mice. This is consistent with the fact that changes in the strength of synapses that possess AMPAR proteins are thought to underlie learning and memory. Additional experiments are required to explore these relationships further, and to determine whether SNX6 helps to localize any other proteins that also contribute to changes in the strength of synapses.

https://doi.org/10.7554/eLife.20991.002

Introduction

SNX6 is a member of the sorting nexin (SNX) family that plays important roles in retromer-mediated, dynein−dynactin-driven retrograde vesicular transport from endosomes to the trans-Golgi network (TGN). The retromer complex functions in endosomal protein sorting and trafficking. It is composed of the VPS26-VPS29-VPS35 core complex and a SNX subunit or subcomplex (Gallon and Cullen, 2015). In mammalian epithelial cells, SNX6 serves as dynein adaptor in retromer-mediated vesicular transport to regulate both cargo recognition and release via its interaction with the motor and the target membrane. SNX6 contains an amino-terminal Phox Homology (PX) domain that is evolutionarily conserved among SNXs and a carboxyl-terminal Bin/Amphiphysin/Rvs (BAR) domain that allows for dimerization with BAR domains of other proteins. It dimerizes with the SNX1 subunit of retromer through its BAR domain and binds to dynactin p150Glued through its PX domain, linking the dynein−dynactin motor complex to retromer-associated vesicular cargoes (Hong et al., 2009; Wassmer et al., 2009). Its PX domain also interacts with the TGN-enriched phospholipid PtdIns(4)P, which inhibits the interaction between SNX6 and p150Glued to facilitate dissociation of the retrograde motor from the retromer-associated cargo at the TGN (Niu et al., 2013). Although retromer is involved in endosomal sorting and trafficking of amyloid precursor protein (APP) (Fjorback et al., 2012; Sullivan et al., 2011), and transport, surface expression and endocytic recycling of AMPAR (Choy et al., 2014; Munsie et al., 2015; Zhang et al., 2012) in neurons, the biological function of SNX6 in the CNS, whether retromer-dependent or not, remains to be explored.

In most of the principal neurons in the central nervous system (CNS), dendritic spines, the micron-sized membrane protrusions covering dendritic shaft, provide major sites of excitatory inputs. They are highly specialized postsynaptic structures containing transmembrane neurotransmitter receptors and proteins with signaling and scaffolding functions. Among them, scaffold proteins of the postsynaptic density (PSD) play crucial roles in glutamatergic neurotransmission by organizing glutamate receptors and signaling molecules at the postsynaptic terminal. One group of PSD scaffold proteins is the PSD95 membrane-associated guanylyl kinase (MAGUK) family proteins that anchor glutamate receptors to the PSD (Elias and Nicoll, 2007). Another group is the Homer and Shank family proteins. They interact with each other and form a high-order complex with a mesh-like network structure, which is believed to serve as a structural platform of the PSD essential for the structural integrity of dendritic spines (Hayashi et al., 2009). The Homer family proteins also regulate trafficking and signaling of the group one metabotropic glutamate receptors (mGluR1/5) and synaptic plasticity (Ango et al., 2001, 2002; Gerstein et al., 2012; Mao et al., 2005; Roche et al., 1999; Ronesi and Huber, 2008). Moreover, Homer1b and 1c, the long isoforms encoded by the Homer1 gene that are differentiated by an insertion of 12 amino acid (aa) residues at aa 177 in Homer1c (Xiao et al., 1998), regulate surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) at synaptic sites through endocytic recycling (Lu et al., 2007). Although mechanisms underlying glutamate receptor trafficking to dendrites as well as their local trafficking into and out of synaptic sites have been intensively studied (Anggono and Huganir, 2012; Hoerndli et al., 2013; Horak et al., 2014; Huganir and Nicoll, 2013; Ladépêche et al., 2014; Setou et al., 2000, 2002), the molecular basis for dendritic distribution and spine localization of most PSD scaffolding proteins including Homer remains largely unexplored.

In this study, we investigated the physiological function(s) of SNX6 in mouse CNS neurons using multiple approaches including mouse genetics, behavior assays and electrophysiology, biochemistry and fluorescence imaging. Ablation of SNX6 in the CNS causes deficits in spatial learning and memory, decrease in spine density of the distal dendrites of hippocampal CA1 neurons and impairment of their AMPAR-mediated synaptic transmission, suggesting a role for SNX6 in synaptic structure and function. SNX6 interacts with Homer1b/c and loss of SNX6 leads to a reduction in its distribution in distal dendrites. Intriguingly, although SNX6 is required for the motility of a subpopulation of Homer1c on vesicles in dendritic shaft, live imaging and FRAP analyses indicate that Homer1c enters dendritic spines via protein diffusion but not SNX6-dependent active transport. Overexpression of SNX6 or Homer1c restores the spine density and AMPAR surface levels of Snx6-/- neurons. These findings uncover a physiological function for SNX6 in hippocampal CA1 excitatory neurons.

Results

Ablation of SNX6 in the CNS causes deficits in hippocampal-dependent spatial learning and memory

Immunoblotting analysis of tissue lysates indicated that SNX6 was ubiquitously expressed in mouse (Figure 1—figure supplement 1A). In mouse brain, SNX6 was expressed in both the somatodendritic area and processes of neurons in the cortex and the hippocampal formation (Figure 1—figure supplement 1B–E). Co-immunostaining with antibodies to SNX6 and axonal or dendritic markers revealed its distribution in a punctate pattern in both axon and dendrites of hippocampal neurons (Figure 1—figure supplement 1F). Quantitative analysis of fluorescence signal intensity revealed that SNX6 was primarily located in dendrites (Figure 1—figure supplement 1G). Moreover, SNX6 partially colocalized with PSD95, a postsynaptic marker in dendritic spines but not synaptophysin, a presynaptic marker (Figure 1—figure supplement 1H).

To investigate physiological function(s) of SNX6 in the CNS, we generated a conditional allele by floxing exon 5 of the Snx6 gene (Figure 1A–C), and obtained CNS-specific knockout mice (CNS-Snx6 KO) by mating Snx6 conditional KO mice (Snx6fl/fl) to Nestin promoter-driven Cre recombinase transgenic mice (Nestin-Cre). Lack of SNX6 protein expression in the CNS of Nestin-Cre; Snx6fl/fl mice was confirmed by immunoblotting analysis of mouse brain homogenates (Figure 1D). The CNS neurons of Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice were hence referred to as Snx6+/+ and Snx6-/- neurons, respectively. CNS-Snx6 KO mice were born with the expected Mendelian ratio and appeared indistinguishable from wild-type littermates. Their brain size was comparable with that of wild-type (Figure 1E), and no gross abnormalities in the structure of the cortex, hippocampus and cerebellum were observed by histological examination (Figure 1F).

Figure 1 with 1 supplement see all
Generation and characterization of Snx6 CNS-specific knockout mice.

(A) Domain structure of SNX6. (B) Schematic diagram of the Snx6 gene locus, the targeting vector, and the mutant alleles after homologous recombination. FRTtF/FRTtR and loxtF/loxtR: primer pairs used for genotyping. The XhoI and HpaI probes used for Southern blotting analysis are shown. Neo: the neomycin resistance cassette. (C) Southern blotting analysis of wild-type (WT) and two independent clones of targeted ES cells (9 hr and 5D). (D) Immunoblots of tissue lysates from mouse littermates, probed with antibodies to SNX6. (E) Comparison of brain weight of Snx6fl/fl (15) and Nestin-Cre; Snx6fl/fl mice (12). Data represent mean ± SEM for each group. (F) Nissl staining of sagittal sections of whole brain from Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice. Also shown are magnification of the cerebellum (middle panel) and the hippocampus/cortex area (right panel). Scale bar: 1 mm.

https://doi.org/10.7554/eLife.20991.003

Next we conducted behavioral analyses on Nestin-Cre; Snx6fl/fl mice and their wild-type littermates. No change in locomotor activity was detected by rotarod and open field assays (Figure 2A,B), and the mood levels of CNS-Snx6 KO were also similar to that of wild-type mice in elevated plus maze, tail suspension and forced swimming tests (Figure 2C–E). In the Three-Chamber test, the CNS-Snx6 KO mice showed no abnormality in sociability and social novelty (Figure 2F), nor did they display repetitive behaviors (Figure 2G). We then focused on their performance in learning and memory. Although Nestin-Cre; Snx6fl/fl mice performed as well as their littermates in Y maze and shuttle box (Figure 2H,I), in the Morris water maze test, they were significantly retarded in spatial learning using latency traveled to reach the hidden platform as measures (Figure 2J). A probe trial showed that they were also severely impaired in spatial memory (Figure 2K). Moreover, these mice exhibited deficits in memory recall (Figure 2L,M). As the hippocampal region participates in the processes of the encoding, storage, consolidation and retrieval of spatial memory (Riedel et al., 1999), the behavioral phenotypes suggest that ablation of SNX6 affects synaptic function of hippocampal neurons.

Impaired spatial learning and memory in Nestin-Cre; Snx6fl/fl mice.

(A–I) No effects of SNX6 ablation on the performance in assays of rotarod (A) (13 Snx6fl/fl and 16 Nestin-Cre; Snx6fl/fl mice), open field (B) (23 Snx6fl/fl and 23 Nestin-Cre; Snx6fl/fl mice), elevated plus maze (C) (14 Snx6fl/fl and 13 Nestin-Cre; Snx6fl/fl mice), tail suspension (D) (14 Snx6fl/fl and 24 Nestin-Cre; Snx6fl/fl mice), forced swimming (E) (15 Snx6fl/fl and 25 Nestin-Cre; Snx6fl/fl mice), Three-Chamber test (F) (10 Snx6fl/fl and 9 Nestin-Cre; Snx6fl/fl mice), repetitive behaviors (G) (12 Snx6fl/fl and 10 Nestin-Cre; Snx6fl/fl mice), Y maze (H) (11 Snx6fl/fl and 15 Nestin-Cre; Snx6fl/fl mice) and shuttle box (I) (20 Snx6fl/fl and 13 Nestin-Cre; Snx6fl/fl mice). The data represent mean ± SEM for each group. (J–K) Increased escape latency at acquisition learning (J) (data represent mean ± SEM of four trials per day), decreased number of crossing and increased latency to first enter the 1.5x area at probe test (K) (the data represent mean ± SEM for each group) in Nestin-Cre; Snx6fl/fl mice in the Morris water maze. Subject numbers were 18 Snx6fl/fl and 22 Nestin-Cre; Snx6fl/fl mice. (L) After a 20-day rest, both Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice exhibited memory extinguishment. (M) Decreased number of crossing and increased latency to first enter the 1.5x area at probe test in Nestin-Cre; Snx6fl/fl mice after one recall training. The data represent mean ± SEM. N = 3 independent experiments.

https://doi.org/10.7554/eLife.20991.005

Ablation of SNX6 causes a decrease in spine density in distal apical dendrites of hippocampal CA1 pyramidal neurons

To investigate changes in synaptic function caused by SNX6 ablation at the cellular level, we examined neuronal morphology in the hippocampal region by crossing Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice with Thy1-EGFP transgenic mice and analyzing brain sections by confocal microscopy (Figure 3A). We focused on the morphology of CA1 and CA3 pyramidal cells for two reasons: first, neurons in the CA1 and CA3 region were sparsely labeled by EGFP and hence easily distinguishable from neighboring ones for the purpose of morphological assessment; second, changes in the morphology and density of dendritic spines have been linked to synaptic function and plasticity. For quantification of spine number and morphology, we imaged segments of dendrites that are easily distinguishable from those of neighboring neurons, i.e., the oriens/distal branches of the basal and radiatum/thin branches of the apical dendrites of CA1 neurons, and secondary/tertiary branches of the basal and apical dendrites of CA3 neurons in stratum oriens and stratum radiatum, respectively (Figure 3B). Quantitative analysis showed that, although spine morphology did not change in either CA1 or CA3 pyramidal cells (Figure 3C–F), there was a decrease in the spine density of the distal portion of apical dendrites of Snx6-/- CA1 neurons (Figure 3C,D). In contrast, no change in spine density was detected in Snx6-/- CA3 neurons (Figure 3E,F). Consistently, ultrastructural analysis revealed a decrease in the number of asymmetric/excitatory synapses in the CA1, but not in the CA3 region of Nestin-Cre; Snx6fl/fl mouse brain (Figure 3G,H). Together, these data indicate that SNX6 is required for spine morphogenesis and/or maintenance of distal apical dendrites of CA1 pyramidal neurons.

Decreases in spine density of hippocampal CA1 apical dendrites and number of excitatory synapses in the CA1 region in Nestin-Cre; Snx6fl/fl Mice.

(A) Confocal images of coronal sections of hippocampi from Snx6fl/fl; Thy1-GFP and Nestin-Cre; Snx6fl/fl; Thy1-GFP. (B) Schematic of the location of the dendritic segments selected for morphological analysis. (C) Representative 3D-reconstructed confocal images of dendrites of CA1 pyramidal cells. The z-dimension position is color-coded according to the color scale bar. (D) Quantification of spine density (n = 5 pairs of mice, apical/basal: 43/40 cells, 95/82 dendritic segments and 4979/3721 spines for Snx6fl/fl; Thy1-GFP; 40/38 cells, 96/78 dendritic segments and 3662/3341 spines for Nestin-Cre; Snx6fl/fl; Thy1-GFP) and morphology (n = 2 pairs, apical/basal: 16/14 cells, 1053/764 spines for Snx6fl/fl; Thy1-GFP; 18/17 cells, 1099/894 spines for Nestin-Cre; Snx6fl/fl; Thy1-GFP) of CA1 dendrites. (E) Representative 3D-reconstructed confocal images of CA3 dendrites. (F) Quantification of spine density (n = 5 pairs, apical/basal: 34/34 cells, 80/75 dendritic segments and 3155/2261 spines for Snx6fl/fl; Thy1-GFP; 34/34 cells, 77/73 dendritic segments and 3053/2216 spines for Nestin-Cre; Snx6fl/fl; Thy1-GFP) and morphology (n = 2 pairs, apical/basal: 13/12 cells, 822/595 spines for Snx6fl/fl; Thy1-GFP; 11/14 cells, 739/568 spines for Nestin-Cre; Snx6fl/fl; Thy1-GFP) of CA3 dendrites. (G) Representative TEM images of hippocampal CA1 regions of adult animals. Yellow solid arrowheads indicate asymmetric (excitatory) synapses. Insets are representative higher magnification images of synapses in the boxed areas. Yellow empty arrowheads indicate mitochondria. Yellow arrows indicate lysosomes. (H) Quantification of synapse density (n = 3 pairs, CA1: 1553 synapses for Snx6fl/fl and 1038 synapses for Nestin-Cre; Snx6fl/fl. CA3: 1102 synapses for Snx6fl/fl and 1069 synapses for Nestin-Cre; Snx6fl/fl ). Data represent mean ± SEM. Bars: 200 μm in (A), 2 μm in (C) and 500 nm in (G).

https://doi.org/10.7554/eLife.20991.006

SNX6 directly interacts with Homer1b/c

That ablation of SNX6 causes a decrease in spine density of distal dendrites suggests that it functions in the formation/stabilization of dendritic spines, probably via regulating dendritic distribution of postsynaptic proteins such as PSD components and/or neurotransmitter receptors. As the first step to investigate its molecular function, we determined the subcellular distribution of SNX6 in dendrites by co-immunostaining of SNX6 and vesicular markers in cultured mature hippocampal neurons. Confocal microscopy revealed that the majority of SNX6 signals colocalized with EEA1 and Rab5B, the early endosome markers (Figure 4A,B). SNX6 also partially colocalized with the late endosome marker Rab7 and Rab4, marker for the fast recycling pathway, though to a lesser extent, but not Rab11 recycling endosomes (Figure 4A,B). Intriguingly, although SNX6 signals did not colocalize with Golgin97, a TGN resident protein, they overlapped partially with TGN46 (Figure 4A,B), a protein involved in membrane traffic to and from the TGN (Ponnambalam et al., 1996), suggesting that SNX6 associates with endosomes and transport carriers in the dendrite.

Figure 4 with 1 supplement see all
SNX6 interacts with Homer1b/c and colocalizes with Homer1b/c on endosomes.

(A) Hippocampal neurons were transfected with pLL3.7.1 on DIV14 to express DsRed as volume marker, fixed on DIV17 and immunostained with antibodies to SNX6 and vesicular markers. DsRed is pseudocolored for presentation. White arrowheads indicate overlapped signals. (B) Quantification of colocalization in (A) from 45 dendritic segments of 15 neurons (mean ± SEM, N = 3. Total length of dendrites: 1568 μm for EEA1; 1447 μm for Rab5; 1637 μm for Rab4; 1489 μm for Rab7; 1319 μm for Rab11; 1207 μm for Golgi97 and 1462 μm for TGN46). (C) Mouse brain lysates were incubated with His-SNX1-N or His-SNX6-N immobilized on Ni-NTA agarose. Bound proteins were subjected to SDS-PAGE and mass spectrometry analysis. The table shows the number of Homer1b/c unique peptides identified by mass spec analysis and their sequence coverage. (D) Schematic representation of the domain structure of Homer1 isoforms and Homer1c fragments used in this study. (E) Upper panels: immunoblotting of bound proteins in (C). Lower panel: coomassie brilliant blue (CBB) stained SDS-PAGE gel shows purified recombinant proteins. (F) Mapping of SNX6-Homer1b/c interaction sites by in vitro binding assay. (G) In vitro binding assay of SNX6 and Homer family members. (H) Lysates from HEK293 cells overexpressing Flag-SNX6 and mEmerald-Homer1c were subjected to co-IP with Flag M2 beads, followed by immunoblotting with antibodies to Flag and Homer1b/c. (I) Total lysates and membrane fractions from mouse brain lysates were subjected to IP and immunoisolation with antibodies to Homer1b/c or SNX6, and antibodies to SNX6 coupled to Dynabeads Protein G, respectively. Shown are immunoblots probed with antibodies to SNX6, p150Glued, DIC, GluN1, GluN2A, GluN2B, Homer1b/c and Homer1a. (J) DIV18 neurons were immunostained with antibodies to Homer1b/c and SNX6. (K) Quantification of colocalization in (J) from 45 dendritic segments of 15 neurons (mean ± SEM, N = 3 independent experiments. Total length 1677 μm). (L) DIV18 neurons were immunostained with antibodies to Homer1b/c and EEA1. (M) Quantification of colocalization in (L) from 45 dendritic segments of 15 neurons (mean ± SEM, N = 3. Total length 1459 μm). (N) DIV18 neurons were immunostained with antibodies to EEA1, Homer1b/c, and SNX6. Superresolution images were captured by structured illumination microscopy (SIM). White arrowheads indicate overlaps of signals from different channels. Bars: 2 μm.

https://doi.org/10.7554/eLife.20991.007

Next we attempted to identify SNX6-interacting protein(s) in dendrite. We performed pull down experiment from mouse brain lysates using a His-tagged SNX6 N-terminus (aa 1–181, encompassing the PX domain) immobilized on Ni-NTA agarose. Mass spectrometry analysis revealed six matching peptides with 24% sequence coverage corresponding to Homer1b/c, a postsynaptic scaffold protein (Figure 4C). Immunoblotting analysis verified that Homer1b/c, not Homer1a, the shorter isoform encoded by the Homer1 gene, was pulled down by the N-terminus of SNX6 but not SNX1 (Figure 4D,E). Moreover, in vitro binding assays showed that SNX6-N interacted directly with the coiled-coil domain of Homer1b/c (Figure 4F), which is not present in Homer1a. In contrast, neither Homer2b nor Homer3, the longer isoforms of other Homer family members, interacted with SNX6-N (Figure 4G). Further, mEmerald-Homer1c co-immunoprecipitated with Flag-tagged SNX6 in transiently transfected HEK293T cells (Figure 4H). Consistently, reciprocal co-immunoprecipitations of endogenous proteins from mouse brain lysates verified that SNX6 and Homer1b/c interact with each other (Figure 4I, left and center panels). Moreover, immunoisolation of SNX6-positive vesicles from membrane fractions of mouse brain lysates detected Homer1b/c together with p150Glued and dynein intermediate chain (DIC), subunits of the dynein−dynactin complex (Figure 4I, right panel). In contrast, neither Homer1a nor subunits of the N-methyl-D-aspartate receptor (NMDAR) were detected on SNX6-positive vesicles (Figure 4I, right panel). In dendrites, Homer1b/c not only colocalized with SNX6 on vesicular structures (Figure 4J,K), but also colocalized with EEA1 (Figure 4L,M). Both wide-field microscopy with deconvolution and superresolution fluorescence microscopy revealed colocalization of EEA1, Homer1b/c and SNX6 in dendrites (Figure 4N, Table 1, Figure 4—figure supplement 1 and Video 1), indicating that SNX6 associates with Homer1b/c on endosomes.

Table 1

Quantitative analysis of colocalization of signals in superresolution images and statistical significance of colocalization (related to Figures 4N and 6D, and Figure 4—figure supplement 1).

https://doi.org/10.7554/eLife.20991.009

voxel colocolization values (%) / p value
EEA1-SNX6-Homer1b/c                
EEA1 with SNX6 and Homer1b/c15.68/ 0.0007912.05/ 0.01117.48/ 0.008615.18/ 0.010216.92/ 0.0086416.48/ 0.012715.33/ 0.009218.86/ 0.006915.76/ 0.008911.96/ 0.014121.87/ 0.0005315.98/ 0.0006514.58/ 0.009616.76/ 0.00119.04/ 0.0005
SNX6 with EEA1 and Homer1b/c18.56/ 0.0007913.12/ 0.01117.28/ 0.008613.96/ 0.010217.44/ 0.0086412.36/ 0.012715.92/ 0.009222.64/ 0.006915.08/ 0.008914.32/ 0.014116.34/ 0.0005319.92/ 0.0006515.12/ 0.009615.44/ 0.00117.56/ 0.0005
Homer1b/c with EEA1 and SNX614.24/ 0.0007913.16/ 0.01113.64/ 0.008610.22/ 0.010216.28/ 0.0086416.84/ 0.012713.24/ 0.009213.92/ 0.006914.84/ 0.008914.52/ 0.014118.56/ 0.0005315.36/ 0.0006513.92/ 0.009613.44/ 0.00117.63/ 0.0005
















p150Glued-SNX6-Homer1b/c                
p150Glued with SNX6 and Homer1b/c15.63/ 0.0048.13/ 0.0110.06/ 0.01510.06/ 0.010210.06/ 0.005310.06/ 0.004710.06/ 0.00310.06/ 0.006910.06/ 0.01877.62/ 0.020< p <0.046016.38/ 0.00319.35/ 0.00518.85/ 0.008< p <0.03409.97/ 0.001879.21/ 0.0071
SNX6 with Homer1b/c and p150Glued14.57/ 0.00412.89/ 0.0110.41/ 0.01513.02/ 0.010210.18/ 0.005312.67/ 0.004718.49/ 0.00312.33/ 0.00699.51/ 0.01879.54/ 0.010< p <0.025023.55/ 0.003111.24/ 0.00519.39/ 0.008< p <0.03209.18/ 0.0018710.7/ 0.0071
Homer1b/c with SNX6 and p150Glued10.32/ 0.0048.27/ 0.018.33/ 0.0157.15/ 0.01029.66/ 0.00539.8/ 0.004713.16/ 0.0038.49/ 0.00698.73/ 0.01878.69/ 0.0120< p <0.036615.07/ 0.00319.17/ 0.00518.24/ 0.0100< p <0.03708.43/ 0.001879.03/ 0.0071
                
DIC-SNX6-Homer1b/c                
DIC with SNX6 and Homer1b/c9.21/ 0.00679.65/ 0.006120.05/ 0.00497.9/ 0.00713.41/ 0.00615.25/ 0.00589.91/ 0.0018710.88/ 0.00729.41/ 0.00798.53/ 0.006110.61/ 0.00719.64/ 0.00839.78/ 0.008210.98/ 0.008616.12/ 0.0042
SNX6 with DIC and Homer1b/c12.00/ 0.006713.19/ 0.006114.91/ 0.004912.02/ 0.00713.07/ 0.00615.49/ 0.005811.32/ 0.001879.81/ 0.007210.1/ 0.007916.07/ 0.006110.79/ 0.007112.48/ 0.008311.77/ 0.00829.86/ 0.008623.11/ 0.0042
Homer1b/c with DIC and SNX611.43/ 0.00679.81/ 0.006113.78/ 0.00499.82/ 0.00710.71/ 0.00610.86/ 0.00589.77/ 0.001879.82/ 0.00729.22/ 0.007910.36/ 0.00619.75/ 0.00717.87/ 0.00839.73/ 0.008210.21/ 0.008617.17/ 0.0042
Video 1
3D-SIM movie of an enlarged region of interest from a hippocampal neuron dendrite shows the association of EEA1 (red), SNX6 (green) and Homer1b/c (blue).
https://doi.org/10.7554/eLife.20991.010

Ablation of SNX6 causes decrease in distribution of Homer1b/c in distal dendrites

Dendritic distribution of Homer1b/c is essential for its scaffolding and signaling functions at the PSD. To determine whether SNX6 regulates Homer1b/c distribution in dendrites, we examined Homer1b/c expression and subcellular distribution in Snx6-/- hippocampal neurons by immunofluorescence staining. Indeed, although no change in the protein levels of Homer1b/c was detected in the hippocampi of Nestin-Cre; Snx6fl/fl mice (Figure 5—figure supplement 1), quantitative analysis revealed not only a decrease in the number of Homer1b/c puncta in dendritic segments (30–120 μm from the cell body) of Snx6-/- neurons as compared with that of Snx6+/+, but also a significant reduction in the fluorescence intensity of Homer1b/c puncta in spines (Figure 5A,B). Overexpression of EGFP-SNX6 rescued both puncta number and spine distribution of Homer1b/c in Snx6-/- neurons (Figure 5A,B). In contrast, neither the number nor the fluorescence intensity of PSD95 puncta was significantly affected in Snx6-/- neurons (Figure 5C,D). Further, quantification of the Homer1b/c signal intensity over distance from the cell body revealed a decrease in both shaft and spines of the distal dendrites and concurrent accumulation in the soma of Snx6-/- neurons, which was rescued by mCherry-SNX6 (Figure 5E,F). Together these data indicate that SNX6 is required for Homer1b/c distribution in distal dendrites.

Figure 5 with 2 supplements see all
Partial loss of Homer1b/c from distal dendrites of Snx6-/- neurons.

(A) Neurons were co-transfected with pLL3.7.1 and EGFP or EGFP-SNX6 construct on DIV13, fixed on DIV18 and immunostained with antibodies to Homer1b/c. Shown are representative confocal images of dendritic segments. (B) Quantification of puncta number per 100 μm dendrite length and mean intensity in spines for Homer1b/c (mean ± SEM, n = 30, N = 3). (C) Neurons were transfected with pLL3.7.1 on DIV13, fixed on DIV18 and immunostained with antibodies to PSD95. (D) Quantification of PSD95 distribution in dendrites (mean ± SEM, n = 30, N = 3). (E) DIV14 neurons were co-transfected with constructs overexpressing EGFP and mCherry or mCherry-SNX6, fixed on DIV16 and immunostained with antibodies to Homer1b/c. shown are representative confocal images of transfected neurons. Dashed lines outline the cell bodies. (F) Quantification of Homer1b/c distribution in the cell body and dendrites, and its mean intensity in spines in (E) (mean ± SEM, n = 30, N = 3). (G) DIV13 neurons were co-transfected with constructs expressing DsRed and EGFP, EGFP-SNX6, mEmerald-Homer1c-FL or EGFP-Homer1c-C and fixed on DIV18. (H) Quantification of spine density in (G) (mean ± SEM, n = 30, N = 3). Bars: 20 μm in (E), 2 μm in other panels.

https://doi.org/10.7554/eLife.20991.011

Homer and Shank are among the most abundant postsynaptic scaffolding proteins that contribute to the structural and functional integrity of dendritic spines. Consistent with the in vivo data, the spine density of Snx6-/- neurons was lower than that of Snx6+/+ in dissociated cultures (Figure 5G,H, and Figure 5—figure supplement 2). Overexpression of EGFP-SNX6 or mEmerald-Homer1c but not a Homer1c fragment that is truncated of its mGluR1/5-binding EVH1 domain (Homer1c-C) (Shiraishi-Yamaguchi and Furuichi, 2007) restored the spine density of Snx6-/- neurons (Figure 5G,H), indicating that SNX6-dependent dendritic distribution of Homer1b/c contributes to spine formation/stabilization, and that the cellular function of Homer1b/c as postsynaptic scaffold protein is required to restore the number of spines.

Active transport of a fraction of Homer1b/c molecules in the dendritic shaft requires SNX6

Given that SNX6 is a cargo adaptor for the microtubule-based dynein−dynactin motor, we reasoned that SNX6 might mediate transport of Homer1b/c in dendrites. To this end, we transfected hippocampal neurons with constructs expressing mEmerald-Homer1c and mCherry-SNX6 (Figure 6—figure supplement 1) and performed live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM) to monitor their movement in dendrites. Indeed, we observed movement of SNX6-, Homer1c-double positive puncta in the shaft of both proximal and distal dendrites (Figure 6A–C, Figure 6—figure supplement 2A,B, Videos 2 and 3). A retrospective staining of MAP2 right after live imaging verified dendrite identity of the distal branch (Figure 6C). Quantitative analysis revealed that, similar to mobility characteristics of PSD95 clusters in dendrites (Gerrow et al., 2006), the majority (~90%) of Homer1c puncta (1022 out of 1128 puncta from 31 neurons) were stationary. Of note, the majority of motile SNX6-, Homer1c-positive puncta were smaller than 0.3 μm2 in size (<600 nm in apparent diameter), whereas most of the immotile ones were larger (Figure 6—figure supplement 2C), suggesting that the moving structures were vesicles rather than large protein aggregates. The SNX6-, Homer1c-positive puncta moved bidirectionally in the dendritic shaft, with the mean velocity of 0.416 ± 0.037 μm/s, over distances ranging from 2.026 to 18.324 μm (Figure 6—figure supplement 2D–F). Movement of SNX6-, Homer1b-double positive puncta in the dendritic shaft was also observed by live imaging (Video 4). In contrast, in neurons overexpressing EGFP fusion of the GluN1 subunit of NMDAR, no comovement of SNX6- and GluN1-positive vesicles in dendrite was observed (Video 5). Moreover, we performed live imaging of mEmerald-Homer1c in Snx6-/- neurons and found that compared with wild-type, there was a dramatic decrease in the fraction of motile Homer1c fluorescent puncta (29 motile puncta out of 311 from 10 Snx6+/+ neurons vs. 10 out of 1217 from 40 Snx6-/- neurons).

Figure 6 with 2 supplements see all
SNX6 is required for motility of Homer1b/c vesicles in dendritic shaft and their association with dynein−dynactin.

(A–C) Dynamic behavior of mCherry-SNX6 and mEmerald-Homer1c in distal dendrite. The last frame of motile SNX6-, Homer1c-positive puncta (arrowhead) in dendrite (A) with the respective kymograph of boxed area (B) is shown. A retrospective staining of MAP2 after live imaging (C) illuminates dendrite identity. (D) Superresolution images of DIV18 neurons immunostained with antibodies to SNX6, Homer1b/c, and p150Glued or DIC. Shown are representative images of dendrites outlined with dashed lines. White arrowheads indicate overlapped signals. (E) DIV14 neurons were transfected with construct expressing EGFP, treated with DMSO or Ciliobrevin D on DIV16 for 2 hr and immunostained with antibodies to Homer1b/c. (F) Quantification of puncta number per 100 μm dendrite length and mean intensity in spines for Homer1b/c in (E) (mean ± SEM, n = 30, N = 3). (G) Same as (E), shown are representative confocal images of EGFP-expressing neurons. Dashed lines outline the cell bodies. (H) Quantification of Homer1b/c distribution in the cell body and dendrites in (G) (mean ± SEM, n = 30, N = 3). (I) DIV14 neurons were transfected with construct overexpressing EGFP or p150Glued-N-EGFP, fixed on DIV16 and immunostained with antibodies to Homer1b/c. Dashed lines outline the cell bodies. (J) Quantification of Homer1b/c distribution in the cell body and dendrites in (I) (mean ± SEM, n = 30, N = 3). (K) Membrane fractions from mouse brain lysates were subjected to immunoisolation with antibodies to p150Glued or DIC coupled to Dynabeads Protein G, respectively. Shown are immunoblots probed with antibodies to SNX6, p150Glued, DIC, and Homer1b/c. (L) Hippocampal neurons cultured from Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice were transfected with pLL3.7.1 on DIV14, fixed on DIV17 and immunostained with antibodies to Homer1b/c and p150Glued, DIC or EEA1. Shown are representative confocal images of dendritic segments. (M) Quantification of colocalization in (L) from 45 dendritic segments of 15 neurons (mean ± SEM, N = 2. Total length of dendrites: Snx6fl/fl/Nestin-Cre; Snx6fl/fl: 1247 μm/1058 μm for p150Glued; 1264 μm/1301 μm for DIC; 1244 μm/1291 μm for EEA1). Bars, 2 μm in (A), (D), (E) and (L), 20 μm in (G) and (I).

https://doi.org/10.7554/eLife.20991.014
Video 2
Time-lapse live imaging showing movement of Homer1c-labeled and SNX6-labeled puncta in proximal dendrites.

Hippocampal neurons co-transfected with Emerald-Homer1c and mCherry-SNX6 expressing constructs were imaged live by TIR-FM. The trajectories of two mobile Homer1c-, SNX6-positive puncta are indicated by white arrowheads. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 2 μm.

https://doi.org/10.7554/eLife.20991.017
Video 3
Time-lapse live imaging showing movement of Homer1c-labeled and SNX6-labeled puncta in distal dendrites.

Hippocampal neurons co-transfected with Emerald-Homer1c and mCherry-SNX6 expressing constructs were imaged live by TIR-FM. The trajectories of two mobile Homer1c-, SNX6-positive puncta are indicated by white and yellow arrowheads respectively. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 2 μm.

https://doi.org/10.7554/eLife.20991.018
Video 4
Time-lapse live imaging showing movement of Homer1b-labeled and SNX6-labeled structures in a distal dendrite.

Hippocampal neurons co-transfected with mEmerald-Homer1b and mCherry-SNX6 expressing constructs were imaged live by TIR-FM. The trajectory of a mobile Homer1b-, SNX6-positive structure is indicated by white arrowheads. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 2 μm.

https://doi.org/10.7554/eLife.20991.019
Video 5
Time-lapse live imaging showing movement of GluN1-labeled vesicles in dendrite.

Hippocampal neurons co-transfected with mCherry-SNX6 and GluN1-EGFP expressing construct were imaged live by TIR-FM. The trajectory of the mobile GluN1-positive, SNX6-negative vesicle is indicated by white arrowheads. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.020

To verify that Homer1b/c-associated vesicles are of early endosome origin, we performed live imaging on neurons expressing fluorescently tagged EEA1. First, we imaged neurons coexpressing EEA1-YFP and mCherry-SNX6 and observed movement of EEA1-, SNX6-double positive vesicles in the dendritic shaft (Video 6). In neurons coexpressing EEA1-YFP and mCherry-Homer1c, more than 90% of EEA1 vesicles also contained Homer1c (461/482 = 95.6%from 20 Snx6+/+ and 796/872 = 91.3 % from 26 Snx6-/- neurons), whereas the majority of Homer1c puncta were also EEA1-positive (461/690 = 66.8 % from 20 Snx6+/+ and 796/1249 = 63.7% from 26 Snx6-/- neurons). Live imaging detected not only movement of EEA1-, Homer1c-double positive vesicles in the dendritic shaft (Video 7), but also a significant decrease in the fraction of motile EEA1-positive vesicles in Snx6-/- neurons (total EEA1-positive vesicles: 58/482 motile = 12% from 20 Snx6+/+ neurons vs. 37/872 motile = 4.2% from 26 Snx6-/- neurons; EEA1-, Homer1c-double positive vesicles: 53/461 motile = 11.5% from 20 Snx6+/+ neurons vs. 25/796 motile = 3.1% from 26 Snx6-/- neurons). In contrast, in dendrites of neurons coexpressing Homer1c and the late endosome marker Rab7, neither comovement of Rab7- and Homer1c-positive structures was observed (Videos 8 and 9), nor the fraction of motile Rab7-labeled structures changed significantly when SNX6 was ablated (331/601 motile = 55.1% from 27 Snx6+/+ neurons vs. 341/647 motile = 52.7% from 22 Snx6-/- neurons). Together, these data indicate that SNX6 is required for the motility of EEA1-positive early endosomes or early endosome-derived vesicles carrying Homer1b/c.

Video 6
Time-lapse live imaging showing movement of EEA1- and SNX6-labeled vesicles in dendrite.

Hippocampal neurons co-transfected with EEA1-YFP and mCherry-SNX6 expressing constructs were imaged live by TIR-FM. The trajectories of two mobile EEA1-, SNX6-positive vesicles are indicated by white arrowheads. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.021
Video 7
Time-lapse live imaging showing movement of EEA1- and Homer1c-labeled vesicles in dendrite.

Hippocampal neurons co-transfected with EEA1-YFP and mCherry-Homer1c expressing constructs were imaged live by TIR-FM. Yellow arrowheads indicate the trajectory of an EEA1-, Homer1c-positive vesicle detaching and moving away from a large structure, suggesting fission and formation of transport carriers from early endosomes. White arrowheads indicate the trajectory of another vesicle moving in the dendritic shaft. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.022
Video 8
Time-lapse live imaging showing movement of Rab7- and Homer1c-labeled structures in the dendrite of a wild-type neuron.

Snx6+/+ hippocampal neurons co-transfected with Rab7-RFP and mEmerald-Homer1c expressing constructs were imaged live by TIR-FM. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.023
Video 9
Time-lapse live imaging showing movement of Rab7- and Homer1c-labeled structures in the dendrite of a Snx6 KO neuron.

Snx6-/- hippocampal neurons co-transfected with Rab7-RFP and mEmerald-Homer1c expressing constructs were imaged live by TIR-FM. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.024

Next we determined whether dynein−dynactin is involved in SNX6-regulated dendritic distribution of Homer1b/c. Consistent with the immunoisolation data (Figure 4I), superresolution microscopy analysis revealed that a significant fraction of both p150Glued and DIC colocalized with SNX6 and Homer1b/c on vesicular structures (Figure 6D, Table 1, Figure 4—figure supplement 1 and Videos 10 and 11). Because dynein is essential for cell viability, we transiently treated neurons with Ciliobrevin D, an inhibitor of dynein activity (Firestone et al., 2012), and detected a decrease in both shaft and synaptic distribution of Homer1b/c in distal dendrites (Figure 6E–H). Further, overexpression of p150Glued-N, a dominant negative mutant that disrupts the interaction between SNX6 and dynactin (Hong et al., 2009), also caused a decrease in Homer1b/c signals in distal dendrites and concurrent increase in the soma (Figure 6I,J), indicating a role for dynein−dynactin in the dendritic distribution of Homer1b/c.

Video 10
3D-SIM movie of an enlarged region of interest from a hippocampal neuron dendrite shows association of p150Glued (red), SNX6 (green) and Homer1b/c (blue).
https://doi.org/10.7554/eLife.20991.025
Video 11
3D-SIM movie of an enlarged region of interest from a hippocampal neuron dendrite shows the association of DIC (red), SNX6 (green) and Homer1b/c (blue).
https://doi.org/10.7554/eLife.20991.026

Next we asked whether SNX6 serves as a linker between the vesicles carrying Homer1b/c and the dynein−dynactin motor complex. To determine whether association of Homer1b/c and the motor complex on vesicles requires SNX6, we attempted immunoisolation of Homer1b/c and dynein−dynactin-associated vesicles from mouse brain. Although antibodies to Homer1b/c failed to isolate Homer1b/c-positive vesicles from membrane fractions of mouse brain lysates, western blotting of vesicles immunoisolated with antibodies to both p150Glued and DIC detected Homer1b/c signals in wild-type but not CNS-SNX6 KO mice (Figure 6K). Moreover, we performed immunostaining and colocalization analysis of Homer1b/c on Snx6+/+ and Snx6-/- neurons. Quantitative analysis of confocal images showed that there was a decrease in colocalization of Homer1b/c with p150Glued and DIC, but not with EEA1, in Snx6-/- neurons (Figure 6L,M). Together, these data suggest that SNX6 mediates dynein−dynactin-driven transport of a fraction of Homer1b/c molecules in the dendritic shaft.

The Homer1b/c trafficking pathway is distinct from the PSD95 and secretory trafficking pathways in dendrite

Although SNX6 mediates association of Homer1b/c vesicles with dynein‒dynactin, the majority of Homer1b/c puncta were immotile in steady-state neurons, suggesting that SNX6 might regulate Homer1b/c levels in distal dendrites via mechanisms other than vesicular transport. Moreover, ablation of SNX6 did not cause complete loss of Homer1/c from distal dendrites, suggesting that SNX6-independent mechanism(s) is required for Homer1b/c distribution in dendritic regions far from the cell body. Previous studies have found that the postsynaptic scaffolding proteins PSD95, guanylate kinase domain-associated protein (GKAP) and Shank are transported in a preformed protein complex in dendrite (Gerrow et al., 2006). To determine whether Homer1b/c shares the same trafficking pathway with PSD95, we performed live imaging of neurons expressing PSD95-RFP and determined association of SNX6 and Homer1b/c with motile PSD95 clusters by retrospective immunofluorescence staining. No SNX6/Homer1b/c signals were detected on motile PSD95 clusters (seven motile PSD95 puncta from five cells, Figure 7A and Video 12), indicating that Homer1b/c is not cotransported with PSD95.

The dendritic trafficking pathway of Homer1b/c is distinct from the PSD95 and secretory trafficking pathways.

(A) TIR-FM of hippocampal neurons transfected with PSD95-RFP expressing construct. Left panel: still image of the last frame of time lapse imaging. Arrowheads mark the final positions of two motile puncta. Center panels: confocal images of retrospective staining of endogenous SNX6 and Homer1b/c after live imaging. Right panels: enlargement of arrowhead-indicated, numbered puncta in the center panels. 1 and 2: motile PSD95 puncta lacking both SNX6 and Homer1b/c; 3: vesicle containing endogenous SNX6 and Homer1b/c, but not PSD95; 4: an immobile PSD95 punctum that contacts with SNX6 signal. 5: an immobile PSD95 punctum that colocalizes with Homer1b/c. 6: an immobile PSD95 punctum that colocalizes with both Homer1b/c and SNX6. (B) DIV14 hippocampal neurons were transfected with construct overexpressing PKD-KD and immunostained with antibodies to Homer1b/c on DIV16. Shown are representative confocal images. (C) Quantification of the mean intensity of Homer1b/c signals in the cell body and dendrites in (B) (mean ± SEM, n = 30, N = 3). (D) The effect of PKD-KD overexpression on the distribution of Homer1b/c in dendrites and spines. (E) Quantification of Homer1b/c distribution in (D) (mean ± SEM, n = 30, N = 3). Bars: 1 μm in (A), 20 μm in (B), 2 μm in (D).

https://doi.org/10.7554/eLife.20991.027
Video 12
Time-lapse live imaging showing movement of PSD95-RFP-labeled puncta in dendrites.

Hippocampal neurons transfected with PSD95-RFP expressing construct were imaged live by TIR-FM. White arrowheads indicate two mobile puncta followed by fixation. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.028

Next we asked whether the secretory trafficking pathway in dendrite contributes to Homer1b/c distribution in dendritic shaft and spines. To address this question, we transfected neurons with plasmid overexpressing a kinase-dead version of protein kinase D1 (PKD1-K618N, or PKD-KD), which blocks secretory trafficking by preventing fission of transport carriers from the TGN (Liljedahl et al., 2001). Expression of PKD-KD did not affect either dendritic distribution or spine localization of Homer1b/c (Figure 7B–E), indicating that its dendritic trafficking and synaptic delivery does not rely on the secretory pathway.

Translocation of Homer1b/c from dendritic shaft to spines is SNX6- and vesicular transport-independent

As localization of Homer1b/c to synaptic sites in dendritic spines is crucial for its function, next we asked the question whether its translocation from shaft to spines requires SNX6 and vesicular transport. First, we determined whether or not shaft-localized Homer1b/c puncta enter spines. In our live imaging experiments using mEmerald-Homer1c, we did not detect entry of Homer1c puncta into spines in either wild-type or Snx6 KO neurons. We also imaged dendritic segments of neurons expressing mCherry-Homer1c (Snx6+/+: 30 cells, 50 dendritic segments, 791 spines; Snx6-/-: 30 cells, 50 dendritic segments, 547 spines). Under the experimental condition we used, most motile fluorescent puncta moved in shaft (Figure 8A and Video 13), only one event of Homer1c particle entry into spine was observed (Video 14). Since the plus ends of microtubules transiently invade dendritic spines (Jaworski et al., 2009) and dynein is a minus end-directed motor, it is conceivable that dynein-driven transport is not involved in transfer of Homer1b/c from shaft to spines. Moreover, we reasoned that if delivery of Homer1b/c from shaft to spines requires SNX6, there would be a decrease in spine distribution of Homer1b/c signals in dendrites of Snx6 KO neurons. Quantitative analysis showed that, although both total and spine Homer1b/c signals decreased in distal dendrites (Figure 5F), its spine:shaft ratio remained constant throughout the dendrite and did not change in Snx6-/- neurons (Figure 8B), indicating that in steady-state neurons, SNX6 is not involved in local trafficking of Homer1b/c from shaft to spines.

Homer1b/c enters spines by SNX6-independent protein diffusion.

(A) Representative images from a time-lapse video (Video 13) of a wild-type neuron co-expressing EGFP and mCherry-Homer1c. White solid lines indicate outline of the dendritic shaft and spines. White arrowheads indicate positions of a Homer1c-labeled structure moving in the shaft. Bar: 2 μm. (B) Spine:shaft ratios of Homer1b/c fluorescence intensity over distance from the cell body. DIV14 neurons from Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice were transfected with construct overexpressing EGFP as volume marker, fixed on DIV16 and immunostained with antibodies to Homer1b/c. Shown are the ratios of the mean intensity of Homer1b/c in spines to that in the corresponding shaft (mean ± SEM, 50 spines from 15 neurons/group, N = 2 independent experiments). (C) FRAP analysis of mEmerald-Homer1c in dendritic spines. Hippocampal neurons were co-transfected with constructs expressing mEmerald-Homer1c and DsRed on DIV13. FRAP analysis was performed on DIV16. Shown are examples of the fluorescence intensity of mEmerald-Homer1c before, immediately after, 10 s and 600 s after photobleaching of the spines indicated with white circles. Bar: 1 μm. (D) Averaged fluorescence recovery curves after photobleaching for mEmerald-Homer1c in spines of Snx6+/+ (31 spines, 10 cells) and Snx6-/- (32 spines, 10 cells) neurons. Data represent mean ± SEM.

https://doi.org/10.7554/eLife.20991.029
Video 13
A motile Homer1c-labeled structure in dendritic shaft did not enter spines.

Snx6+/+ hippocampal neurons co-transfected with EGFP and mCherry-Homer1c expressing constructs were imaged live by TIR-FM. Images were acquired at 2 frames/s. Video plays at 10 frames/s. White solid lines indicate outline of the shaft and spines. White arrowheads indicate the trajectory of a Homer1c-labeled structure moving in the dendritic shaft. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.030
Video 14
A motile Homer1c-labeled structure in dendritic shaft entered a spine.

Snx6+/+ hippocampal neurons co-transfected with EGFP and mCherry-Homer1c expressing constructs were imaged live by TIR-FM. Images were acquired at 2 frames/s. Video plays at 10 frames/s. White solid lines indicate outline of the shaft and spines. White arrowheads indicate the trajectory of a Homer1c-labeled structure entering a spine. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.031

As a fraction of overexpressed fluorescently tagged Homer1c is cytosolic, next we tested the possibility that Homer1c enters spines via protein diffusion by fluorescence recovery after photobleaching (FRAP) assay on neurons expressing mEmerald-Homer1c. Consistent with previous findings on Homer1c turnover rates measured by FRAP using EGFP-Homer1c (Kuriu et al., 2006), following full synapse photobleaching of fluorescent signals, 7 ~ 9 % of recovery after 10 s was observed in both wild-type and Snx6 KO neurons (8.09 ± 0.91% in Snx6+/+ and 8.14 ± 0.73% in Snx6-/- neurons, Figure 8C,D), indicating that although the fraction of fast fluorescence recovery in spines attributable to entry of soluble cytosolic proteins (Blanpied et al., 2008; Kerr and Blanpied, 2012; Kuriu et al., 2006) is minor, diffusion of Homer1c protein molecules into spines does not require SNX6. After 10 min, about half of Homer1c fluorescence was recovered in both wild-type and Snx6 KO neurons with similar recovery half-time (Recovery level Rfinal = 54.55 ± 4.03%, τ1/2 = 108.4 ± 9.4 s in Snx6+/+ and Rfinal = 52.63 ± 3.53%, τ1/2 = 107.2 ± 13.1 s in Snx6-/-, Figure 8C,D). Further, similar results were obtained by fitting a single-exponential recovery curve to the average recovery time trace (τ1/2 = 117.6 ± 11.3 s, Rfinal = 51.63 ± 0.86% in Snx6+/+ and τ1/2 = 108.9 ± 9.1 s, Rfinal = 48.57 ± 0.73% in Snx6-/-). These results are in good agreement with previous findings that there are immobile and mobile fractions of Homer1c in spines (Kuriu et al., 2006) and indicate that the dynamic turnover of Homer1c in spines is not affected by ablation of SNX6. Taken together, these data indicate that in steady-state neurons, neither SNX6 nor vesicular transport is required for recruitment of Homer1b/c from shaft to spines, and that synaptic Homer1b/c exchanges with the soluble protein pool that enters the spine by diffusion.

Ablation of SNX6 causes impairment of AMPAR-mediated synaptic transmission and decrease in AMPAR surface expression

As ablation of SNX6 causes decrease in Homer1b/c distribution in distal dendrites, given the role of Homer1b/c in synaptic structure and function, next we sought to determine whether synaptic transmission is impaired in Snx6-/- neurons by electrophysiological analysis. We eliminated the Snx6 gene in a small subset of hippocampal neurons by injection of organotypic hippocampal slice culture from Snx6fl/fl mouse with recombinant adeno-associated virus (AAV) coexpressing EGFP and the Cre recombinase (Figure 9A and Figure 9—figure supplement 1). By simultaneous recording the evoked EPSCs (eEPSCs) on infected and adjacent uninfected CA1 pyramidal neurons, we found that AMPAR-mediated eEPSCs were significantly impaired by about 50% with ablation of SNX6 (Figure 9B), whereas NMDAR-mediated eEPSCs and the pair-pulse ratio of AMPAR eEPSCs were unaffected (Figure 9C,D), indicating that the impairment of AMPAR eEPSCs is due to decrease in the number of AMPARs in the postsynaptic membrane but not reduction of presynaptic glutamate release. Indeed, no change in surface expression of the GluN2B subunit of NMDAR was detected in Snx6-/- neurons by immunostaining and quantitative analysis (Figure 9E,F). In contrast, there was a decrease in the surface expression of GluA1 and GluA2, components of AMPAR, which was fully rescued by overexpressing SNX6 or Homer1c (Figure 9G–J).

Figure 9 with 4 supplements see all
Impaired synaptic transmission and decreased surface AMPAR levels of Snx6-/- neurons.

(A) Hippocampal slice culture from Snx6fl/fl mouse was partially infected with AAV-EGFP-2A-Cre. Lower panel shows a CA1 neuron infected with AAV-EGFP-2A-Cre and an adjacent control neuron (indicated with red arrows) that were chosen to be recorded simultaneously. (B) Dual recording analysis of AMPAR-mediated synaptic responses (n = 18 pairs). Scatterplots show amplitudes of AMPAR eEPSCs (absolute values) for single pairs of neurons (open circles) and mean ± SEM (filled circle) across all neuron pairs collected. The current amplitudes of infected neurons were plotted on the ordinate and the current amplitudes of the control neurons were plotted on the abscissa. Inset shows sample current traces from an infected (green) and a control (black) neurons. Bar graph shows mean ± SEM of AMPAR amplitudes represented in the scatterplots. (C) NMDAR-mediated eEPSCs (n = 12 pairs). (D) Paired-pulse recording of AMPAR eEPSCs (n = 11 pairs). Two identical stimulus pulses were delivered in an interval of 50 ms and AMPAR eEPSCs were recorded at −70 mV. Left are sample traces of eEPSCs from a pair of infected (green) and control neurons. The paired-pulse ratio (PPR) was the enhancement of the second eEPSC relative to the first eEPSC. Bar graph shows mean ± SEM of PPRs. (E) Hippocampal neurons transfected with pLL3.7.1 were fixed on DIV18 and immunostained with antibodies to surface GluN2B. (F) Quantification of puncta number per 100 μm dendrite length and fluorescence mean intensity in spines for surface GluN2B (mean ± SEM, n = 30, N = 3). (G) DIV13 neurons were co-transfected with constructs expressing DsRed and EGFP, EGFP-SNX6, mEmerald-Homer1c-FL or EGFP-Homer1c-C, fixed on DIV18 and immunostained with antibodies to surface GluA1. (H) Quantification of signal intensity and spine distribution of surface GluA1 (mean ± SEM, n = 32, N = 3). (I) Same as (G), except that neurons were immunostained with antibodies to surface GluA2. (J) Quantification of surface GluA2 (mean ± SEM, n = 33, N = 3). (K) Hippocampal neurons from Snx6fl/fl (WT) and Nestin-Cre; Snx6fl/fl (KO) littermates were cultured till DIV16. Surface levels of mGluR5, GluA1 and GluA2 were then measured by cleavable surface biotinylation followed by immunoblotting with antibodies to mGluR5, GluA1 and GluA2. SNX6 serves as a negative control for surface proteins. Shown are immunoblots from two pairs of Snx6fl/fl and Nestin-Cre; Snx6fl/fl littermates. (L) Antibody uptake assay was performed on neurons transfected with pLL3.7.1. Shown are representative images of dendrites immunostained for internalized and surface AMPAR signals. (M) Quantification of AMPAR endocytosis rate (mean ± SEM, 45 dendritic segments, n = 15, N = 3). (N) DIV14 neurons were treated with inverse agonists for mGluRs for 48 hr, fixed and immunostained with antibodies to surface GluA1. (O) Quantification of surface GluA1 (mean ± SEM, n = 30, N = 3). (P) Same as (N), except that neurons were immunostained with antibodies to surface GluA2. (Q) Quantification of surface GluA2 (mean ± SEM, n = 30, N = 3). Bar: 2 μm.

https://doi.org/10.7554/eLife.20991.032

Next we investigated mechanism(s) underlying reduced AMPAR surface expression in Snx6-/- neurons. The longer isoforms of Homer inhibit not only trafficking to synaptic membrane but also constitutive activities of mGluR1/5 (Ango et al., 2001, 2002 ; Roche et al., 1999). mGluR activation down-regulates surface expression of AMPAR by increasing its endocytosis rate (Snyder et al., 2001). Indeed, there was an increase in surface levels of mGluR5 in Snx6-/- neurons (Figure 9K, Figure 9—figure supplements 2 and 3), which was restored by overexpression of SNX6 or Homer1c but not Homer1c-C (Figure 9—figure supplement 2). Moreover, ratiometric analysis of internalized and surface AMPAR detected an increase in the rate of AMPAR endocytosis in Snx6-/- neurons ( Figure 9L,M ). To test the possibility that loss of Homer1b/c leads to decrease in surface AMPAR via increased surface levels and/or constitutive activation of mGluRs, we treated neurons with inverse agonists for mGluR1/5 and determined surface levels of GluA1 and GluA2 by immunostaining. Quantitative analysis indicated that inhibition of mGluRs did rescue surface expression of AMPAR in Snx6-/- neurons (Figure 9N–Q). Together, these data indicate that dendritic loss of Homer1b/c caused by ablation of SNX6 leads to increase in mGluR surface expression and mGluR-regulated AMPAR endocytic trafficking.

Another role of Homer1b/c in maintaining AMPAR surface levels is via the positioning of the endocytic zone (EZ) near the PSD (Lu et al., 2007). In the dendritic spine, an EZ adjacent to the PSD captures and retrieves AMPAR diffusing out of the synapse through local endocytic trafficking (Blanpied et al., 2002; Lu et al., 2007), which is crucial for the supply of a mobile pool of receptor molecules required for synaptic transmission and potentiation (Lu et al., 2007; Petrini et al., 2009). To determine whether ablation of SNX6 caused uncoupling of the EZ from the PSD, we performed co-immunostaining of PSD95 and clathrin heavy chain, a marker for the EZ. Indeed, ablation of SNX6 caused ~2 fold increase in the fraction of EZ (clathrin)-negative synapses (Figure 9—figure supplement 4), indicating that the coupling of the EZ to the PSD is impaired in Snx6-/- neurons, which might also contribute to lower surface levels of AMPAR in dendritic spines.

Activity of the retromer core complex is not required for SNX6-regulated dendritic distribution of Homer1b/c

Previous studies have established a role for SNX6 as cargo adaptor in retromer-mediated vesicular transport (Hong et al., 2009; Wassmer et al., 2009). To test whether retromer is involved in SNX6-regulated dendritic distribution of Homer1b/c, we depleted VPS35, a core component of the retromer complex, by siRNA-mediated RNA interference in hippocampal neurons (Figure 10A,B and Figure 10—figure supplement 1). Immunofluorescence staining and quantitative analysis of confocal images indicated that depletion of VPS35 in mature neurons caused a decrease not only in surface GluA1 but also in the number of Homer1b/c puncta in dendrites (Figure 10C–E), possibly resulted from reduced spine density as VPS35 has been found to promote spine formation and maturation (Tian et al., 2015; Wang et al., 2012). Nevertheless, compared with the phenotypes of Snx6-/- neurons, there was decrease in Homer1b/c signals throughout the cell body and dendrites, but no change in spine distribution of Homer1b/c or surface levels of mGluR5 when VPS35 was depleted (Figure 10C and E–H), indicating that it serves distinct function(s) from SNX6 in dendritic distribution of postsynaptic proteins. Moreover, live imaging of hippocampal neurons coexpressing mEmerald-Homer1c and VPS35-mCherry did not detect comovement of Homer1c- and VPS35-labeled vesicles in dendrite (Figure 10I,J and Videos 15 and 16). Further, retrospective staining of MAP2 and VPS35 right after live imaging confirmed the dendrite identity and absence of endogenous VPS35 at the base of the spine where a Homer1c-positive structure stopped (Figure 10J, rightmost panels). To further confirm that SNX6 does not cooperate with the retromer to regulate motility of Homer1b/c vesicles, we performed coimmunostaining of Homer1b/c and VPS35 on Snx6+/+ and Snx6-/- neurons. Quantitative analysis showed that colocalization between Homer1b/c and VPS35 was very little compared with that between Homer1b/c and SNX6 or EEA1. Moreover, it was not affected by ablation of SNX6 (Figure 10—figure supplement 2A,B). Conversely, no change in colocalization between Homer1b/c and SNX6 was detected in VPS35-depleted neurons either (Figure 10—figure supplement 2C,D). Taken together, these data indicate that SNX6 functions independent of retromer to regulate dendritic distribution of Homer1b/c.

Figure 10 with 2 supplements see all
The retromer core complex is not required for SNX6-regulated dendritic distribution of Homer1b/c.

(A) Hippocampal neurons were transfected with lentiviral vector expressing siRNA along with GFP at DIV12, fixed on DIV18 and immunostained with antibodies to VPS35. (B) Quantification of VPS35 signal intensity and puncta number in neurons in (A) (mean ± SEM, n = 10, N = 3). (C) Neurons transfected with siRNA constructs were immunostained with antibodies to surface GluA1 and Homer1b/c. (D–E) Quantification of surface GluA1 (D) (mean ± SEM, scrambled: 33 neurons; VPS35 RNAi #1: 30 neurons; VPS35 RNAi #2: 30 neurons, N = 3.) or Homer1b/c distribution in spines (E) (mean ± SEM, scrambled: 35 neurons; VPS35 RNAi #1: 32 neurons; VPS35 RNAi #2: 30 neurons. N = 3 ). (F) Quantification of Homer1b/c distribution in the cell body and dendrites of hippocampal neurons expressing scrambled or VPS35-targeting siRNA (mean ± SEM, n = 15, N = 3). The results show a decrease in signal intensity of Homer1b/c throughout the cell when VPS35 was depleted. (G) Same as (C), except that neurons were immunostained with antibodies to surface mGluR5. (H) Quantification of surface mGluR5 in (G) (mean ± SEM, scrambled: 35 neurons; VPS35 RNAi #1: 44 neurons; VPS35 RNAi #2: 52 neurons. N = 3 ). (I–J) TIR-FM of hippocampal neurons transfected with Homer1c and VPS35-expressing constructs. Shown in (I) is a VPS35-positive vesicle (white arrow) moving away from the cell body and bypassing a static Homer1c-positive structure (yellow arrow) with their respective kymographs to the right. Shown in (J) are still images of representative time points: a Homer1c-positive structure reached the base of spine and part of which entered the spine after a brief lag. White arrowheads indicate the mobile Homer1c structure. A retrospective staining of MAP2 and VPS35 after live imaging (right panels) illuminates the dendrite identity and the absence of endogenous VPS35 at the base of the spine. Yellow arrowhead indicates the position of the Homer1c-positive structure right before fixation. White circles indicate VPS35 puncta appearing in both retrospective staining and live imaging. Bars: 5 μm in (A) and (C), 20 μm in (G) and 1 μm in (I) and (J).

https://doi.org/10.7554/eLife.20991.037
Video 15
Time-lapse live imaging showing movement of a VPS35 vesicle in dendrite.

Hippocampal neurons co-transfected with mEmerald-Homer1c and VPS35-6G-mCherry expressing constructs were imaged live by TIR-FM. White arrowheads indicate the trajectory of a VPS35-positive vesicle moving away from the cell body and bypassing a static Homer1c-positive structure (indicated with yellow arrowhead). Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.040
Video 16
Time-lapse live imaging showing movement of Homer1c and VPS35 puncta in dendrites.

Hippocampal neurons co-transfected with mEmerald-Homer1c and VPS35-6G-mCherry expressing construct were imaged live by TIR-FM. White arrowheads indicate the initial and pausal sites of a Homer1c-positive, VPS35-negative structure. Yellow arrowhead indicates the spine position. Images were acquired at 2 frames/s. Video plays at 50 frames/s. Bar: 1 μm.

https://doi.org/10.7554/eLife.20991.041

Discussion

In this study, we demonstrate that ablation of SNX6 in the CNS causes deficits in spatial learning and memory, a hippocampal-dependent brain function. At the cellular level, loss of SNX6 causes a decrease in spine density in the distal apical dendrites of CA1 hippocampal cells and impairment of their AMPAR-mediated synaptic transmission, indicating that SNX6 is required for synaptic structure and function of these excitatory neurons. We also show that SNX6 directly interacts with Homer1b/c, a PSD scaffolding protein crucial for the structural and functional integrity of dendritic spines, and that there is decrease in Homer1b/c distribution in distal dendrites in Snx6-/- neurons. Moreover, the spine density and surface AMPAR level phenotypes of Snx6-/- neurons could be rescued by overexpressing Homer1b/c or SNX6. These findings reveal an important physiological function of SNX6 in the CNS excitatory neurons.

The long isoforms of Homer1 are implicated in learning and memory. Homer1 knockout mice exhibit deficits in spatial learning and a decrease in LTP in the CA1 region (Gerstein et al., 2012; Jaubert et al., 2007), which could be rescued by AAV-mediated expression of Homer1c in the hippocampus (Gerstein et al., 2012). Although Homer1b/c is widely expressed in the brain, in the hippocampus it is predominantly distributed to the CA1 region (Shiraishi et al., 2004), which partially explains why ablation of SNX6 function causes a decrease in spine density of CA1 distal apical dendrites and impairs hippocampal-dependent spatial learning and memory that mainly involves the Schaffer collateral-CA1 synapses. Whether there are other SNX6-interacting proteins that are also required for CA1 neuron function awaits further investigation. As spatial learning and memory involve not only the hippocampus but also other cortical areas such as the entorhinal cortex and the medial prefrontal cortex (Jo et al., 2007; Nagahara et al., 1995; Nakazawa et al., 2004; Steffenach et al., 2005; Zhou et al., 1998), it also remains to be determined whether and how ablation of SNX6 affects the synaptic structure and function of neurons in other parts of the cortex.

PSD scaffolding proteins interact, anchor and stabilize glutamate receptors. Change in their protein content in dendritic shaft and spines influences synaptic transmission through receptor localization and distribution at synaptic sites. Among the PSD scaffolding proteins, PSD95 has been intensively studied. Dendritic trafficking and synaptic targeting of PSD95 requires a C-terminal tyrosine-based signal, palmitoylation of the N-terminus and its interaction with both Myosin V and dynein through binding of GKAP to these molecular motors (Craven and Bredt, 2000; El-Husseini et al., 2000; Hruska et al., 2015; Naisbitt et al., 2000). Synaptic localization of Shank also requires GKAP (Naisbitt et al., 1999; Sala et al., 2001), whilst recruitment of Homer1b to dendritic spines requires synaptically targeted Shank (Sala et al., 2001). We found that the motility of Homer1c-associated vesicles in dendritic shaft requires SNX6, and that ablation of SNX6 or inhibition of dynein-dynactin activity causes reduction in the amount of Homer1b/c in distal dendrites. Previously imaging assays and quantitative modeling have established that dynein-driven bidirectional transport contributes to the polarized targeting of dendrite-specific cargo (Kapitein et al., 2010). Therefore, lack of dynein‒dynactin-driven transport in the dendritic shaft provides a possible mechanism for the Homer1b/c distribution phenotype in Snx6-/- neurons. However, since the majority of Homer1c puncta are immobile in dendrites of steady-state neurons, and little is known about the cellular functions of SNX6 apart from its role as dynein cargo adaptor, it is also possible that SNX6 regulates the distribution of Homer1b/c in dendrites via mechanism(s) distinct from dynein‒dynactin-driven transport. Moreover, ablation of SNX6 does not cause complete loss of Homer1b/c from distal dendrites, indicating that mechanism(s) other than SNX6-mediated transport contributes to its localization to dendritic shaft far from the cell body. Since disruption of the secretory pathway does not affect Homer1b/c localization to dendritic shaft and spines, alternative mechanisms for its distribution in dendrites include diffusion of free protein molecules, cotransport with protein(s) other than the PSD95-GKAP-Shank complex or vesicular transport mediated by different motor(s) and/or adaptor(s).

Notably, in Snx6-/- neurons, although there was a decrease in the amount of Homer1b/c in both shaft and spines of distal dendrites (Figure 5F), the spine:shaft ratio of its signals remained constant throughout the dendrite (Figure 8B), indicating that once in dendrite, Homer1b/c could enter the spines via SNX6-independent mechanism(s). In dendrites, several mechanisms exist for the transfer of postsynaptic components from shaft to synaptic sites in spines, including cytosolic diffusion, exocytosis of transmembrane proteins at the plasma membrane and their lateral diffusion to synaptic sites, and active transport by molecular motors. The AMPARs enter dendritic spines via both lateral diffusion and actin-based, Myosin V-driven transport of recycling endosomes (Adesnik et al., 2005; Correia et al., 2008; Makino and Malinow, 2009; Wang et al., 2008; Yudowski et al., 2007). Since Homer1b/c is a scaffolding protein, its entry into spines might rely on diffusion of free molecules, possibly released from endosomal carriers or from the cytosolic pool in the shaft, or transport of the vesicular cargo directly from the shaft by a different motor. Our results from live imaging, FRAP and quantitative analyses show that direct spine entry of Homer1c puncta is an extremely rare event, and that the dynamic turnover of Homer1c in spines is not affected by ablation of SNX6. Collectively, these data indicate that in steady-state neurons, Homer1b/c enters spines by cytosolic diffusion, and SNX6 is not required for its spine localization.

In retromer-mediated vesicular transport of transmembrane proteins, although the VPS26−VPS29−VPS35 core complex of retromer has been shown to interact with cargo proteins in the endosomal membrane (Arighi et al., 2004; Fjorback et al., 2012; Nothwehr et al., 2000) and was hence termed the cargo selection complex (CSC), the SNX subunits of the retromer also contribute to cargo recognition (Harterink et al., 2011; Strochlic et al., 2007; Temkin et al., 2011; Voos and Stevens, 1998; Zhang et al., 2011). Our study not only shows that SNX6 directly interacts with Homer1b/c, but also provides evidence that SNX6-regulated distribution of Homer1b/c in dendritic shaft is retromer-independent. The CSC is involved in retrieval of APP from endosomes to the TGN and plasma membrane delivery and endocytic recycling of the AMPAR in dendritic spines (Choy et al., 2014; Fjorback et al., 2012; Munsie et al., 2015). SNX27, another member of the SNX family and a component of retromer (Temkin et al., 2011), interacts with both NMDAR and AMPAR and regulates their recycling to the cell surface (Cai et al., 2011; Hussain et al., 2014; Loo et al., 2014). It remains to be determined whether or not SNX6 functions in retromer-mediated dendritic transport. Notably, SNX6 shares the highest sequence similarity (85%) with SNX32, another SNX with unknown function. The mild neurodevelopmental phenotype exhibited by Nestin-Cre; Snx6fl/fl mice suggests functional redundancy among the SNX family members. Further studies are needed to characterize the roles of evolutionarily conserved SNXs, including SNX6 and SNX32, in sorting and trafficking of neuronal proteins and their functions in synaptic development and activity.

Materials and methods

Animals

All experiments were performed in compliance with the guidelines of the Animal Care and Use Committee of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The Nestin-Cre transgenic C57BL/6 J mice were obtained from the Nanjing Biomedical Institution of Nanjing University (Tronche et al., 1999). The Thy1-EGFP transgenic mice were obtained from the Jackson Laboratory (Feng et al., 2000). The Snx6fl/fl mice were generated at the Nanjing Biomedical Institution of Nanjing University. SNX6 CNS-specific knockouts were generated by crossing Snx6fl/fl mice with Nestin-Cre mice.

Generation of Snx6 CNS-specific knockout mice

A 7.1 kb genomic fragment of Snx6 containing exon 5, 6 and 7 was cloned from a bacterial artificial chromosome (BAC) clone (Clone name: RP23-422H22, BACPAC Resource Center, Oakland, CA, USA) to construct targeting vector. The first loxP site was inserted 248 bp upstream of exon 5, and a 2.1 kb FRT-neo-loxP-FRT cassette with the second loxP site was inserted 424 bp downstream of exon 5. The targeting vector was linearized with NotI and electroporated into C57BL/6NTac embryonic stem (ES) cells. ES cells were selected in G418- and ganciclovir-containing medium as described previously (Liu et al., 2003). Genomic DNA of 96 drug-resistant colonies were isolated, digested with XhoI or HpaI and analyzed by southern blotting. Two independent ES cell clones (9 hr and 5D) were chosen to inject into C57BL/6 J blastocysts to obtain chimeric mouse. CNS-specific Snx6 knockout mice were obtained by crossing Snx6fl/fl with Nestin-Cre mice. The Snx6 frameshift mutation was generated by deleting exon five via Cre-loxp mediated site-specific recombination. Deletion of exon five resulted in premature stop in mRNA translation at nt 270. Genotyping of mouse lines was performed by genomic PCR. PCR genotyping of tail prep DNA from offspring was performed with the following primer pairs:

FRTtF/loxtR:

5'-TTTGGCATTATCAAAACGTTGTTGTA-3'

5'-GAGATGCTCAGCACACTTTCTCTAC-3'

(PCR primer locations are shown in Figure 1B resulting in a PCR product of 295 base pairs in Nestin-Cre; Snx6fl/fl mice and none in Snx6fl/fl mice).

loxtF/loxtR:

5'-AGAGTCACTATCAGAGCCTCTTCAG-3'

5'-GAGATGCTCAGCACACTTTCTAC-3’

(PCR primer locations are shown in Figure 1B resulting in a PCR product of 366 base pairs in Snx6fl/flmice and none in Nestin-Cre; Snx6fl/fl mice).

The Nestin-Cre transgene was detected using the following primer pairs:

5'-TGCCACGACCAAGTGACAGCAATG-3'

5'-ACCAGAGAGACGGAAATCCATCGCTC-3'

Mice with a sparsely distributed population of GFP-expressing neurons for analysis of single-cell morphology were generated by crossing Nestin-Cre; Snx6fl/flmice to Thy1-EGFP transgenic mice. Thy1-EGFP transgene was detected using the following primer pairs:

5'-TCTGAGTGGCAAAGGACCTTAGG-3'

5'-CGCTGAACTTGTGGCCGTTTACG-3'

Constructs and viruses

The mEmerald-Homer1c construct was a generous gift from Michael Davidson (Addgene plasmid # 54120, Addgene, Cambridge, MA). The PSD-95-pTagRFP was a generous gift from Johannes Hell (Addgene plasmid # 52671). The FUmGW construct expressing membrane bound GFP (mGFP) was a generous gift from Connie Cepko (Addgene plasmid # 22479). The mCherry-Homer1C construct was generated by cloning full-length Homer1c amplified from mEmerald-Homer1c into pmCherry-C2 (Clontech Laboratories, Inc., Mountain View, CA). The mEmerald-Homer1b construct was derived from mEmerald-Homer1c by deletion of its aa 177–188. EGFP-Homer1C-N/C constructs were generated by cloning Homer1c N-terminus (aa1-187)/C-terminus (aa188-366) amplified from mEmerald-Homer1c into pEGFP-C2 (Clontech Laboratories, Inc.). His-sumo fusion of full-length Homer1c, Homer1c-N (aa1-111), Homer1c-M1 (aa111-177), Homer1c-M2 (aa111-187) and Homer1c-C (aa188-366) were generated by cloning fragments amplified from mEmerald-Homer1C into pET28a-sumo. His-sumo-Homer2b (NCBI Accession: AF093260.1) and Homer3 (NCBI Accession: NM_001146153.1) constructs were generated by PCR amplification of full-length Homer2b and Homer3 from mouse brain cDNA and cloning into pET28a-sumo. The GluN1-EGFP (NR1-EGFP) construct was a generous gift from A. Kimberley McAllister (Department of Neurology, University of California at Davis, USA). The EEA1-YFP construct was a generous gift from Li Yu (Tsinghua University, China). The Rab7-RFP construct was a generous gift from Hong Tang (Institute of Biophysics, Chinese Academy of Sciences, China). The pEGFP-PKD-K618N construct was a generous gift from Vivek Malhotra (Center for Genomic Regulation, Spain). pEGFP-N3-p150Glued-N (aa 1–1060) and constructs expressing full-length SNX1, SNX2, SNX5, and SNX6 fused with EGFP, mCherry or FLAG tag were described previously (Hong et al., 2009). To obtain expression constructs for His-SNX1-N, His-SNX6-N, GST-SNX1-N and GST-SNX6-N, N termini of SNX1 (aa 1–271) and SNX6 (aa 1–181) were PCR-amplified from the full-length constructs and subcloned into pET28a and pGEX-4T-1, respectively. To avoid disrupting the subunit interactions of VPS35-VPS29-VPS26 (Munsie et al., 2015), VPS35-6G-EGFP was constructed by PCR amplifying human VPS35 coding sequence from HeLa cDNA with a pair of primers in which nucleotides encoding six glycines were added to the reverse primer and inserting into pCMV-EGFPN3.To construct VPS35-6G-mCherry, EGFP sequence of VPS35-6G-EGFP was replaced by mCherry coding sequence from pCMV-mCherryC1. Viral particles of adeno-associated virus (AAV) carrying pAOV-CaMKIIα-EGFP-2A-Cre and the control construct pAOV-CaMKIIα-EGFP-2A-3FLAG were purchased from Obio Technology (Shanghai) Corp. Ltd., (Shanghai, China).

Antibodies

Antibodies used in this study are: mouse anti-GluA1 (MAB2263), mouse anti-GluA2 (MAB397), mouse anti-Tau1 (MAB3420), and mouse anti-MAP2 (MAB3418) from Millipore (Billerica, MA); mouse anti-SYP (D-4) (sc-17750), mouse anti-PSD95 (6G6) (sc-32291), mouse anti-DIC (74-1) (sc-13524), goat anti-Homer1a (M-13) (sc-8922), rabbit anti-Homer1b/c (H-174) (sc-20807), mouse anti-SNX6 (D-5) (sc-365965), goat anti-SNX6 (N-19) (sc-8679), and rabbit anti-Rab5B (A-20) (sc-598) from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-p150Glued (610474), mouse anti-EEA1 (610457) from BD Biosciences (San Diego, CA); rabbit anti-clathrin (ab21679), rabbit anti-TGN46 (ab50595), rat anti-CTIP2 (ab18465), rabbit anti-SNX1 (ab134126) and rabbit anti-Rab4 (ab109009) from Abcam; rabbit anti-GluN1 (D65B7) from Cell Signaling Technology (Mississauga, ON, Canada); rabbit anti-GluN2B (AGC-003) and rabbit anti-mGluR5 (AGC-007) from Alomone labs (Jerusalem, Israel); rabbit and mouse anti-GFP (MBL598, D153-3), rabbit and mouse anti-RFP (PM005, M155-3) from Medical and Biological Laboratories (Naka-kuNagoya, Japan); mouse anti-β-actin (A5441) (Sigma-Aldrich, St. Louis, MO); mouse anti-Golgi97 (A21270) from Invitrogen (Carlsbad, CA); goat anti-VPS35 (PAB7499) from Abnova (Taipei, Taiwan) and rabbit anti-GluN2A (612–401-D89) from Rockland Immunochemicals (Limerick, PA); rabbit anti-Rab7 (#9367) from Cell Signaling Technology (Mississauga, ON, Canada); rabbit anti-Rab11 (3H18L, 700184) from Life Technologies (Carlsbad, CA); Rabbit anti-SNX6 was described previously (Hong et al., 2009).

Histology and immunohistochemistry

For SNX6 immunohistochemistry, 10 weeks-old mice were anesthetized and pre-fixed by perfusion with 4% paraformaldehyde (pH 7.4, Sigma-Aldrich) transcardially. Brains were removed, post-fixed overnight at 4°C followed by dehydration with gradient sucrose (30%, 40%, 50%), and sectioned at 25 μm on a LEIGA CM 1950 vibratome (Leica Biosystems, Germany). Braine slices were pasted on glass slides coated with gelatin/chromium potassium sulfate solution (gelatin, 3 g, and chromium potassium sulfate, 0.05 g from Sigma-Aldrich were dissolved in 200 ml sterile water) and antigen retrieval was performed in water bath with Tri-Sodium citrate (10 mM, pH 6.0, dihydrate, Sigma-Aldrich) at above 90°C for 20 min. Immunohistochemistry was performed following instructions from the manufacturer of polinker-2 plus polymer HRP detection system (GBI, Bothell, WA). Briefly, After permeabilization with 1% TritonX-100 for 15 min followed by an endogenous peroxidase activity quenching in 3% hydrogen peroxide, sections were rinsed with PBS, blocked in 1% BSA plus 5% normal goat serum, and incubated with anti-SNX6 primary antibody (Santa Cruz Biotechnology, diluted in PBS containing 1% BSA, 1% normal goat serum) overnight at room temperature (RT). Sections were then rinsed and incubated with polymer helper for 15 min at RT, poly-HRP anti-goat IgG for 20 min at 37°C followed by DAB color development, hematoxylin dyeing for the nucleus, and xylene clearing. Samples were analyzed using a Nikon ECLIPSE TE2000-U microscope.

Behavioral analyses

All mice used for behavior analysis were 10 weeks old male with normal eating and movement in cages by eye observation. One day before test, mice were transferred into the room installed with test platform.

Open field test

The mouse was fed in a separate cage one day before the test and was gently placed in an open-field test chamber and allowed to freely explore for 10 min. The locomotor activity (total distance traveled in the whole chamber) and the emotionality (the percentage of distance and time spent in the center area) were monitored and analyzed by an automated system (the Anilab System, AniLab Software and Instruments Co., Ltd, Ningbo, China).

Rotarod

On the first training day, the mouse was placed on the rotating rod with straight line acceleration of 9.9 rpm/s from minimal (10 rpm) to maximal speed (30 rpm) (the acceleration process takes about 10 min) followed by fixed speed at 30 rpm for another 5 min. On the second day, the mouse was placed on the rotating rod with fixed speed (30 rpm), the motor function and coordination were determined by the latency to fall off the rod.

Elevated plus maze

The mouse was placed on a platform consisting of four perpendicularly intersected arms (two open arms without walls and two arms enclosed by walls) 50 cm high from the ground and allowed to freely explore for 6 min after a 4 min pre-adaptation by allowing the mouse to move freely in an open chamber with high walls placed on the ground after leaving the feeding cage. The ration of retention time staying at and numbers entering the open arms to closed arms were monitored and analyzed by an automated system (the Anilab System, AniLab Software and Instruments Co., Ltd).

Tail suspension test

The mouse was suspended by its tail above 50 cm high from the ground for 5 min. Due to innate aversion to this tail-up situation, the mouse will struggle until immobile after multiple failures. A camera was installed as closely as possible in order to obtain the highest possible resolution of the animals. The immobile time was quantified and used to evaluate the depression condition.

Forced swimming test

On the first day, the mouse was gently and slowly placed into a round tank (height: 27 cm, diameter: 18.5 cm) of which two-thirds was filled with water to, typically, avoid the animal's head from being submerged under the water for 90 s for pre-adaptation. On the second day, the mouse was placed in the same round tank for 5 min. The immobile time was quantified and used to evaluate the depression condition.

Three-Chamber test

Subject mouse was placed into the middle chamber and habituated for 5 min. Then, the wall between the chambers was removed to allow the mouse access freely to explore the three chambers with two empty wire cup-like cup housing in both left and right chambers for the first 10 min. The duration of subject mouse stretching into a 5 cm circular area around the cup is monitored as an active contact within 10 min. Then ‘stranger 1’ mouse was placed into cup housing in the left chamber for a second 10 min. For a third 10 min, ‘stranger 2’ mouse was placed into cup housing in the right chamber.

Repetitive behavior test

The mouse was observed for a 10 min period in 20 cm × 30 cm quadrate cage with bedding. The duration of each mouse spending in the following behaviors was measured: cage-lid flipping/jumping, rearing, grooming and digging.

Y maze spontaneous alternation

Testing was performed in a Y-shaped maze consisting of three radial arms at a 120° angle. The mouse was put in the center of the maze and allowed to explore freely its three arms for 6 min. Typically, the mouse prefers to explore a new arm rather than the one that was just visited. The percentage of alternation (the number of trials/the number of arm entries) is calculated for evaluating the spatial working memory.

Shuttle box

The mouse was placed in a two-compartment shuttle box for about 5 ~ 10 min to make it quiet. A trial constituted of 5 s tone on followed by 5 s footshock (0.39 ~ 0.4 mA) was given to make mouse build the association between tone and footshock. The mouse can avoid to receive the shock by escaping to the opposite compartment during tone on (active escape), or can receive a foot shock shorter than 5 s during shock on after tone off (passive escape). Each day 25 trials were performed with 15 s interval for five days. The time latency of active or passive escape was monitored and analyzed by an automated system (the Anilab System, AniLab Software and Instruments Co., Ltd). The average time latency of 25 trials each day was used to evaluate conditioned memory.

Morris water maze

The Morris water maze procedure consists of hidden platform acquisition training, probe trial testing and recall training. The water tank is a 120 cm diameter circular pool with a circular goal platform submerged 0.5 cm below the water surface. Water temperature was about 22°C to 24°C. Cues with different shapes and colors were pasted on the wall of the tank above the water surface in four different directions. A circular black curtain around the tank eliminated competing environmental cues. The mouse trajectory in the pool was monitored and analyzed by an automated system (Smart 3.0, Panlab SMART video tracking system, Barcelona, Spain). The day before the experiment, the mouse was gently placed into the pool without a platform to freely swim for 90 s for pre-adaptation. Acquisition training was then performed for eight days and four trials per day with different water-entering the site (at north, south, east, and west positions adjacent to the pool wall). During each trial, mouse must learn to use cues to navigate a path to the hidden platform within 90 s. If failed to find the platform, mouse will be led to the platform with a stick, and kept on it for 10 s. The escape latency (the average value of the time duration from entering the water to finding the platform of four trials per day) was calculated for each mouse. After acquisition training, the hidden platform was removed and probe testing was performed for five days and one trial each day at the distal water-entering site away from the platform. A 1.5x platform circle area where the platform was placed was monitored. The latency to first enter 1.5x area (time duration from entering the water to first enter the 1.5x area) and numbers of crossing 1.5x platform circle area of each mouse within 90 s were analyzed. For recall training, after a 20 day-rest, mouse was placed in the same pool without a platform to examine memory extinguishment. Similarly, the latency to first enter 1.5x area and numbers of crossing 1.5x area of each mouse within 90 s were analyzed. Afterward, the platform was placed back to the pool and recall training was performed for one day with four trials with different water-entering sites. The second day, the platform was removed again, and the mouse was placed in the pool at the farthest water-entering site away from the platform. The latency to first enter 1.5x area and numbers of crossing 1.5x area of each mouse was analyzed.

Spine imaging, three-dimensional reconstructions and measurement of spine density in brain slices

The 100 μm-thick brain slices from Snx6fl/fl; Thy1-GFP andNestin-Cre; Snx6fl/fl; Thy1-GFP mice were prepared as described in Histology and immunohistochemistry and mounted onto slides. For quantitative analysis of the morphology and density of dendrite spines, only those dendrite segments located in similar branches of the dendritic tree (oriens/distal or radiatum/thin branches for CA1 basal/apical dendrites (Megías et al., 2001), and secondary and tertiary branches in stratum oriens or stratum radiatum for CA3 basal/apical dendrites (Baker et al., 2011) from GFP-expressing and relatively isolated dorsal hippocampal CA1 or CA3 pyramidal neurons were selected for imaging by z-stack sectioning with a 0.40 μm interval using a Nikon confocal microscopy (EZ-C1, 100x oil, 4x optical zoom) with the same acquisition parameters. Three-dimensional reconstructions were performed and spine density was quantified using the Filament module of IMARIS software as described previously (Shen et al., 2008). The parameters were: the minimums pine head diameter (thinnest diameter) was ≥ 0.1 μm, the ratio of branch length to trunk radius was ≥ 1.5 μm, and the branch length ≥ 0.5 μm. The spine numbers each segment was further verified by manual counting. Spine density was defined as spine numbers/3D-segment length.

Transmission electron microscopy (TEM)

Tissue preparation and electron microscopy were conducted as described (Chen et al., 2008) with slight modification. Briefly, eight-week-old mice (Snx6fl/fl and Nestin-Cre;Snx6fl/fl) were anesthetized with 2% pentobarbital sodium and perfusion-fixed with cold phosphate buffer (PB, 0.1 M, pH 7.4) containing 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) and 1% PFA (Electron Microscopy Sciences). Following removal of mouse brain, the hippocampal CA1 and CA3 regions were sliced into 1 mm-thickness sections transverse to its longitudinal axis. Sections were fixed in the same fixative overnight at 4°C, rinsed with PB and postfixed with 1% osmium tetroxide (OsO4) for 1 hr at 4°C in dark. Sections were then rinsed with distilled water and dehydrated in an ascending series of acetone (50%, 70%, 80%, 90% and 100%, 15 min per dilution). Samples were embedded in Embed 812 (Electron Microscopy Sciences) and polymerized at 60°C for 48 hr. Ultrathin sections (60 nm) were mounted on carbon-coated copper grids, stained with 2% uranyl acetate for 15 min, rinsed with distilled water and stained with lead citrate for 5 min, rinsed with distilled water and air dried. Sections were imaged on a JEM-1400 electron microscope (JEOL) at 80 kV. Electron micrographs were captured with a Gatan 832-CCD (4 k x 3.7 k pixels, Gatan Inc., Pleasanton, CA) at 30,000× magnification. All image analysis was conducted blind to the genotype.

Primary culture of mouse hippocampal neurons

Hippocampi from C57BL/6 J mouse embryos (E17.5) were removed and trypsinized (0.125% trypsin, 15 min at 37°C). Dissociated cells were suspended in Dulbecco’s modified Eagle medium (DMEM, Hyclone, Logan, UT) supplemented with 10% horse serum and 10% F12, then plated on coverslips pre-coated with poly-D-lysine (100 μg/ml, Sigma-Aldrich) in 24-well plates at a density of 2000 ~ 5000 cells/well. Four hours later, the medium was replaced with neuronal culture medium (Neurobasal medium, 2% B27, 1% Glutamax). Half of the media were changed every three days until use.

Primary neuron transfection and siRNA

Neurons were transfected at DIV12 ~14 using Lipofectamine 2000 (Invitrogen) or Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. For VPS35 knockdown, Lentiviral vectors coexpressing VPS35-targeting siRNA and EGFP were purchased from Applied Biological Materials Inc., USA. Target sequences for VPS35: siRNA #1: 5’-GGTGTAAATGTGGAACGTTACAAACAGAT-3'; siRNA #2: 5’-AGCTGTTATGTGCTTAGTAATGTTCTGGA-3'. Scrambled (non-targeting) siRNA: 5’-GGGTGAACTCACGTCAGAA-3'. Neurons were fixed for immunostaining analysis six days after siRNA transfection.

Immunofluorescence staining, confocal image acquisition and analysis

The procedures for immunofluorescence staining and confocal microscopy were performed essentially as previously described (Hong et al., 2009). Briefly, hippocampal neurons were fixed at DIV 16 ~ 18 with 4% paraformaldehyde supplied with 4% sucrose in phosphate-buffered saline (PBS) for 10 min at RT, then permeabilized and blocked in PBS containing 1% BSA, 0.4% Triton X-100 for 15 min at RT. Then primary and secondary antibodies conjugated with Alexa Fluor 488/555/647 were used for detection. For goat anti-SNX6 and rabbit anti-Rab5B (Santa Cruz Biotechnology), antigen retrieval was performed as described in Histology and Immunohistochemistry for optimal staining. Surface staining of GluA1 and mGluR5 was performed as previously described by Peebles et al. (2010). Surface staining of GluN2B and GluA2 was performed as previously described by Swanger et al. (2013). For digitonin extraction of cytosolic proteins before immunostaining, neurons were rinsed with KHM buffer (20 mM HEPES (pH 7.4), 110 mM CH3CO2K, and 2 mM Mg (CH3COO)2) and then treated with KHM buffer containing 25 μg/ml digitonin for 5 min on ice. Neurons were then rinsed once with KHM buffer and fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT, blocked with PBS containing 5 % BSA and 0.2% Triton X-100 for 15 min followed by overnight incubation with primary antibody. Images were acquired by confocal microscopy (EZ-C1, 100x oil, 4x optical zoom) and analyzed with the NIS-Elements AR3.1 software. Some confocal images (Figure 5A,C,E,G; Figure 6E,G and I; Figure 7B,D; Figure 9E,G,I,N,P; Figure 5—figure supplement 2A; Figure 9—figure supplement 2; Figure 9—figure supplement 3A; Figure 9—figure supplement 4) were collected using z-stack with a 0.40 μm interval and analyzed with ImageJ. Confocal imaging after applying 31 × 31 median followed by Costes' auto-threshold subtraction was done to quantify colocalization (Mander’s colocalization coefficient (MCC), values are %; as previously described [Dunn et al., 2011]). Control and experimental group neurons which were to be directly compared were imaged with the same acquisition parameters. To reduce variability, only segments of the secondary and tertiary dendrites (distance from the cell body: 30–120 μm; length: 30–40 μm /segment) were imaged. Ten to fifteen neurons each group in each independent experiment, and 90–120 μm dendrites per neuron were analyzed. Two-dimensional, background-subtracted, maximum projection reconstructions of Z-stack images were used for morphologic analysis and quantification. To examine the size, number, and fluorescence intensity of signal puncta in shaft and spines, the EGFP- or DsRed-labeled dendrites or spines were outlined manually. Numbers of puncta were measured manually and the size of Homer1 puncta as well as the mean intensity of fluorescent signals in individual spines was measured using the ImageJ function ‘Analyze > measure’. Quantification of spine:shaft ratios of Homer1b/c was conducted as described (Smith et al., 2014). Briefly, the dendritic shaft values of Homer1b/c signals was calculated as the mean fluorescence intensity of three regions of shaft along the dendritic region within 1–2 μm of analyzed spines, and used with corresponding spine values to produce spine:shaft ratios. All image analyses were conducted blind to the genotype. For quantitative analysis of VPS35 knockdown in neurons, the fluorescence intensity or VPS35 puncta from EGFP-expressing neurons in each group was normalized by that of untransfected neurons in the same group. The normalized values in each VPS35 siRNA group were further presented as relative values to the scrambled siRNA group.

Protein identification with mass spectrometry

For identification of SNX6-interacting proteins, mouse brain was homogenized with lysis buffer (20 mM Tris.HCl, 10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 20 mM imidazole, 1% Triton) supplemented with protease inhibitors. Lysates were centrifuged at 12,000 × g for 20 min. The supernatants were incubated with recombinant His-tagged SNX1-N (aa 1–271) or SNX6-N (aa 1–181) immobilized on Ni-NTA agarose overnight at 4°C. Beads were rinsed with wash buffer (150 mM NaCl, 20 mM Tris.HCl, 10 mM Hepes, 1 mM EDTA, 100 mM imidazole, 1% Triton) for five times and bound proteins were resolved by SDS-PAGE. The SDS-PAGE gel containing the protein sample was cut into pieces and destained with 25 mM ammonium bicarbonate/50% acetonitrile. Proteins in the sliced gels were reduced with 10 mM DTT at 37°C for 1 hr, and then alkylated with 25 mM iodoacetamide at RT for 1 hr in the dark before digested with trypsin (Sigma T1426; enzyme-to-substrate ratio 1:50) in 25 mM ammonium bicarbonate at 37°C overnight. Tryptic peptides were extracted from gel by sonication with a buffer containing 5% trifluoroacetic acid and 50% acetonitirile. The liquid was dried by SpeedVac, and peptides were resolubilized in 0.1% formic acid and filtered with 0.45 μm centrifugal filters before analysis with a TripleTOF 5600 mass spectrometer (AB SCIEX, Canada) coupled to an Eksigent nanoLC. Proteins were identified by searching the MS/MS spectra against the Mus musculus SwissProt database using the ProteinPilot 4.2 software. Carbamidomethylation of cysteine was set as the fixed modification. Trypsin was specified as the proteolytic enzyme with a maximum of 2 miss cleavages. Mass tolerance was set to 0.05 Da and the false discovery rates for both proteins and peptides were set at 1%.

Co-immunoprecipitation (IP) and GST-pull down

For immunoprecipitation, HEK293T cells transfected with constructs expressing Flag-SNX6 and mEmerad-Homer1c were washed with ice-cold PBS and lysed with lysis buffer 1 (0.5% [vol/vol] NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with protease inhibitors. The following steps were performed as previously described (Hong et al., 2009). HEK293T (ATCC, Manassas, VA) used for protein overexpression and immunoprecipitation in this study were negative for mycoplasma. Cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin and streptomycin (HyClone). For endogenous IP, whole brains of 10 week-old C57 mice were homogenized with 10 times volume of lysis buffer 2 (150 mM NaCl, 20 mM Tris-HCl, 10 mM Hepes, 1 mM EDTA, 1% TritonX-100, pH 7.4) supplemented with protease inhibitors and rotated for 30 min at 4°C. Lysates were centrifuged at 12,000 × g for 20 min. The supernatants were collected and incubated with 5 μg rabbit IgG (control) or mouse anti-SNX6 antibody bound to 20 μL of pre-washed Protein A/G Sepharose beads overnight at 4°C. Beads were washed five times with lysis buffer and bound proteins were eluted with 2× loading buffer and subjected to SDS-PAGE and immunoblotting. For GST-pull down assays, 2 μg of recombinant His-sumo-Homer1c-FL, His-sumo-Homer2b, His-sumo-Homer3, His-sumo-Homer1c-N, His-sumo-Homer1c-M1, His-sumo-Homer1c-M2 or His-sumo-Homer1c-C was incubated with GST, GST-SNX1-N or GST-SNX6-N immobilized on glutathione-Sepharose beads overnight at 4°C and proceeded as described for endogenous IP. For membrane IP/immunoisolation assays, mouse brain was homogenized with lysis buffer (20 mM Tris-HCl, 10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25 M sucrose) supplemented with protease inhibitors and centrifuged at 800 × g for 15 min. The supernatants were collected and subjected to high-speed centrifugation at 100,000 × g for 1 hr (TLS-55 rotor, Optima MAX Ultracentrifuge, Beckman Coulter, USA). The pellets (p100, the membrane fraction) were resuspended in lysis buffer and subjected to immunoisolation with Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA) coupled with 3–5 μg of mouse anti-SNX6 antibody. Then the beads were eluted by boiling in 2× SDS gel loading buffer and bound proteins were subjected to SDS-PAGE and immunoblotting.

3D-SIM superresolution microscopy and image analysis

3D-SIM images of immunostained neurons were acquired as previously described (Niu et al., 2013) on the DeltaVision OMX V4 imaging system (Applied Precision Inc, USA) with a 100 × 1.4 oil objective (Olympus UPlanSApo), solid state multimode lasers (488, 593 and 642 nm) and EMCCD cameras (Evolve 512 × 512, Photometrics, USA). For quantitative analysis of 3D-SIM images with three colors/triple channels, first raw images were committed to costes auto-threshold subtraction for unbiased background subtraction as previously described (Dunn et al., 2011). Then Biobprob ImageJ plugin was used to measure the colocalilzation of triple channels (Fletcher et al., 2010). The statistical significance of triple colocalization was determined by comparing the values of voxel colocalization of the original images with those of images generated by randomizing spatial locations of signals in the original ones (i.e., colocalization occurred by chance), which was illustrated with p values produced by Biobprob ImageJ plugin (parameters: voxel size: 40, 40, 125 nm; confidence interval: 95; p<0.05 means significantly more colocalization than chance).

Live imaging acquisition and analysis

Hippocampal neurons isolated from embryonic day 17.5 (E17.5) or newborn (P0) mice were transfected at DIV 10 ~ 13 with Lipofectamine LTX (Invitrogen) and imaged live by TIR-FM (Nikon TE2000-E equipped with 488 and 561 nm solid laser, 473/543 filter, 60 × 1.49 oil objective (Nikon CFI Apochromat TIRF) and EMCCD camera (iXon Ultra 897, ANDOR, UK)) at DIV 11 ~ 16 at 37°C with 5% CO2based on the optimal fluorescent protein expression. Image acquisition (512 × 512 pixels, two frames/sec for 5–10 min) was controlled by μManager software. Kymographs were prepared using NIH ImageJ functions: ‘reslice’ with one pixel Z-spacing (pixels) and ‘Z projection’ with ‘Standard deviation’ type. The distance between start position of each track in Kymographs and cell body was recorded and used for aligning these tracks of the motile SNX6-, Homer1c- positive vesicles along dendrites. Average velocities (run length / [ total time- pause time]) were acquired with ImageJ plugins ‘Macros > read velocities from tsp’. For retrospective staining, cells were fixed with 4% PFA immediately after live imaging, and immunostained with antibodies to MAP2 or endogenous SNX6, Homer1b/c and VPS35 as previously described (Hong et al., 2009). For size measurement of fluorescent puncta, the shape of puncta was distinguished by the naked eye from the diffuse cytosolic signal and the area value was obtained by the ImageJ function ‘Analyze>measure’.

Fluorescence recovery after photobleaching

FRAP was performed as described by Kerr and Blanpied (2012). Briefly, hippocampal neurons were transfected with constructs expressing mEmerald-Homer1c and pLL3.7.1-DsRed as spine volume marker on DIV13 and FRAP was performed on DIV15 ~ 16. Photobleaching of entire spines with synaptic Homer1c signals was achieved using 50 ~ 60% 488 nm laser power, while the following acquisition immediately after bleaching was achieved using 5 ~ 6% 488 nm laser power. To analyze the recovery of fluorescence, raw images were background subtracted frame-by-frame. The bleached spine and an additional ‘control’ spine were targeted as ROI. The recovery rate was calculated as R = (I(t)-I(0))/(I(before bleaching)-I(0)), with I(0) being the intensity immediately after bleaching. After normalization using the ‘control’ spine, recovery trace of each bleached spine over time was drawn. The fluorescence recovery at the end of recording time was determined as the Rfinal. After averaging the intensity of five time points, the first time point at which the intensity recovered to the half of Rfinal was determined as the half-time. By averaging all individual recovery traces and fitting a single exponential recovery curve using ‘Graphpad Prism 5>analysis>fit>exponential>one-phase association>least squares fit’, the value of Rfinal and half-time obtained through the exponential fitting curve are similar to the average values obtained from each individual recovery trace.

Brain slice culture and electrophysiology

Cultured hippocampal slices were prepared from P6-P8 Snx6fl/fl male mice as previously described (Herring et al., 2013). After one day in culture (DIV1), the slices were injected with adeno-associated virus (AAV) carrying EGFP-2A-Cre driven by the CaMKIIα promoter. Infected slices were cultured for an additional two weeks before recording. For recording, cultured slices were perfused with artificial cerebrospinal fluid (ACSF), containing (in mM) NaCl 119, KCl 2.5, NaHCO3 26, NaH2PO4 1, glucose 11, CaCl2 4, MgCl2 4, 2-chloroadenosine 0.01 (to dampen epileptiform activity) and saturated with 95% O2/5% CO2. Isolation of currents from glutamatergic (AMPA and NMDA) receptors was achieved by adding picrotoxin (0.1 mM) to the ACSF to block GABAA receptors. CA1 pyramidal cells were visualized by infrared differential interference contrast microscopy. The intracellular solution contained (in mM) CsMeSO4 135, NaCl 8, HEPES 10, Na3GTP 0.3, MgATP 4, EGTA 0.3, QX-314 5, and spermine 0.1. Cells were recorded with 3–5 MΩ borosilicate glass pipettes, following stimulation of Schaffer collaterals with bipolar metal electrodes placed in the stratum radiatum of the CA1 region. Series resistance was monitored and not compensated, and cells in which series resistance varied by 25% during a recording session were discarded. Synaptic responses were collected with a Multiclamp 700B-amplifier (Axon Instruments, Foster City, CA), filtered at 2 kHz and digitized at 10 kHz. GFP-positive neurons were identified by epifluorescence microscopy. All paired recordings involved simultaneous whole-cell recordings from one GFP-positive neuron and a neighboring GFP-negative neuron. The stimulus was adjusted to evoke a measurable, monosynaptic eEPSC in both cells. AMPAR eEPSCs were measured at a holding potential of −70 mV, and NMDAR eEPSCs were measured at +40 mV 150 ms after the stimulus, at which point the AMPAR eEPSC has completely decayed. All paired recording data were analyzed statistically with a Wilcoxon Sign Rank Test for paired data. A p value of <0.05 was considered statistically significant. Error bars represent standard error measurement.

CiliobrevinD and mGluR inverse agonist treatment

At DIV 16, cultured hippocampal neurons were treated with 20 μM Ciliobrevin D (EMD Chemicals, Gibbstown, NJ) for 2 hr and fixed for immunostaining analysis. For mGluR inverse agonist treatment, experiments were performed as previously (Hu et al., 2010). Briefly, DIV14 neurons were treated with Bay 36–7620 (Bay, 10 μM, Sigma-Aldrich) and MPEP (5 μM, Sigma-Aldrich) dissolved in DMSO for 48 hr and fixed for the subsequent assay.

GluA1 endocytosis assay

Endocytosis assay of GluA1 in steady state neurons was performed essentially as previously described (Lu et al., 2007). Briefly, DIV16 hippocampal neurons were pre-chilled on ice for 5 min and incubated with GluA1 N-terminal antibody (1:200, Millipore, Billerica, MA) for 30 min. After antibody washout, neurons were transferred to 37°C for 30 min before fixation in PFA-sucrose. Surface-bound GluA1 was immunostained by using Alexa Fluor 647-conjugated goat anti-mouse secondary antibodies (1:1000, Invitrogen) followed by blocking surface-remaining GluA1 with unconjugated goat anti-mouse secondary antibodies (undiluted stock, Jackson ImmunoResearch, West Grove, PA). Then neurons were post-fixed with PFA and permeabilized with blocking buffer containing 0.4%Triton-100. The internalized GluA1 was immunostained with Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1000, Invitrogen). Confocal images were captured and used for calculating the ratio of internalized GluR1 as follows after applying median and costes auto-threshold subtraction: mean intensity of internalized GluA1/(mean intensity of surface GluA1 + mean intensity of internalized GluA1).

Biotinylation assay of surface proteins

Biotinylation assay was performed as previously described (Fu et al., 2011). Briefly, DIV16 hippocampal neurons cultured from newborn (P1) mice were washed twice with ice-cold PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 (PBS-Ca-Mg), then incubated with PBS-Ca-Mg supplemented with 0.25 mg/ml EZ-link Sulfo-NHS-LC-biotin (Pierce, Thermo Fisher Scientific, Rockford, IL) for 1 hr at RT with mild shaking. The biotinylation reaction was quenched and unbound biotin was removed by washing the cells twice with PBS-Ca-Mg containing 100 mM glycine for 15 min at 4°C. Neurons were then lysed in lysis buffer (50 mM Tris-Cl, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1% Triton) supplemented with protease inhibitors. After centrifugation at 8000 × g at 4°C for 10 min, the supernatants were collected and incubated with streptavidin beads (Thermo Fisher Scientific, Rockford, IL) overnight at 4°C. Beads were washed five times with lysis buffer. Bound proteins were eluted with 2× loading buffer and subjected to SDS-PAGE and immunoblotting.

Statistical analysis

All data are presented as mean ± SEM. GraphPad Prism 5 (GraphPad Software) was used for statistical analysis. The two-tailed unpaired t-test was used for statistical analysis of immunoblotting data and to evaluate statistical significance of two groups of samples. One-way analysis of variance with a Tukey post hoc test was used to evaluate statistical significance of three or more groups of samples. For TEM data, non-parametric two-sided statistical testing Mann–Whitney was used to avoid the restriction of sample sizes without a normal distribution (Morris, 2000). A p value of less than 0.05 was considered statistically significant.

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Decision letter

  1. Anna Akhmanova
    Reviewing Editor; Utrecht University, Netherlands

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers. Further to an appeal, the editors stood by their decision, however offering the option of a resubmission.]

Thank you for submitting your work entitled "Synaptic Delivery of Homer1b/c by SNX6-mediated Dendritic Vesicular Transport" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

All the reviewers found that the paper contains a significant amount of novel data, that the description of the SNX6 knockout mice is interesting and solid, and that the biochemical link between SNX6 and Homer1b/c is well described and relevant. However, two of the three reviewers found that the major conclusion of the paper on SNX6 mediating a novel dynein-dependent dendritic transport pathway responsible for the synaptic delivery of Homer1b/c is not sufficiently supported by data. In particular, the reviewers had major concerns about the quality of the live imaging data and the rather weak biochemical and functional evidence supporting a specific connection to dynein. Furthermore, the data proving that the proposed novel transport pathway is retromer-independent were found not be sufficiently strong. The third reviewer has pointed out that the behavioral data were not complete and the data that support the increased level of surface mGlu5 were not convincing. Based on these major criticisms concerning the most important conclusions of the paper, we cannot proceed with this manuscript in eLife. However, we hope that you will find the comments of the reviewers useful for further developing your study.

Reviewer #1:

This paper describes the neuronal function of the sorting nexin family member SNX6. The authors show that SNX6 knockout mice exhibit defects in learning and memory, a phenotype that they connect to the reduction in the number of dendritic spines in a subpopulation of hippocampal pyramidal cells. Further, they uncover a biochemical connection between SNX6 and the postsynaptic scaffolding protein Homer1b/c, and show that SNX6 knockout leads to a reduction in the intensity of Homer1b/c in dendrites and affects surface levels of AMPA receptors and synaptic transmission. They also demonstrate co-motility of SNX6 and Homer1b/c, and propose that SNX6 mediates dynein-dynactin mediated transport and synaptic delivery of Homer1/b. Finally, the authors provide some data suggesting that this function of SNX6 is independent of other components of the retromer complex, a part of which SNX6 forms.

This paper is very rich in data, and provides solid new information on the function of SNX6 in neurons. However, the data supporting the major mechanistic conclusion of the authors on SNX6 mediating the dendritic transport and synaptic delivery of Homer1b/c to dendritic spines is not very strong.

Major comments:

1) For the proposed model to work, SNX6 should be able to bind to Homer1b/c and dynactin at the same time. However, the PX domain of SNX seems to be necessary for binding to both Homer1b/c and to dynactin, and it should be clarified whether both interactions can occur simultaneously.

2) Figure 5D demonstrates a dramatic reduction of Homer1b/c intensity throughout the dendrite in a SNX6 knockout neuron. It should be investigated whether the expression level of Homer1b/c is affected by SNX6 knockout, and if not, in which part of the neuron Homer1b/c accumulates. Is it possible that SNX6 regulates not the transport but stability of Homer1b/c?

3) The authors do not show that the motility of Homer1b/c is affected by the loss of SNX6. If the authors would like to make conclusion about SNX6 as a factor involved in Homer1b/c transport, such data should be included.

4) Triple colocalizations of EEA1/SNX6/Homer1b/c (Figure 4M), and p150 or DIC/ SNX6/Homer1b/c (Figure 5H) show very little actual colocalization between the three markers, and could just as well reflect spurious overlap between abundant punctate labeling patterns. Quantifications should be provided illustrating the significance of colocalizations by using shifted images as controls.

5) When the authors discuss Homer1b/c transport, do they mean transport from the cell body, or redistribution along the dendrite? If they mean transport from the cell body, why would an early endosome serve as a carrier? If relocalization of Homer1b/c along the dendrite is affected, why is the Homer1b/c intensity reduced throughout the dendrite? More clear data and a better discussion are needed here, including alternative models of the functional connection between SNX6 and Homer1b/c.

6) The strong conclusion on retromer-independent function of SNX6 in Homer1b/c transport seems to be mostly based on the observation that while the loss of both SNX6 and VPS35 reduces the number of Homer1b/c puncta, only the knockout of SNX6 affect Homer1b/c intensity is spines. However, this comparison is not fair, because a SNX6 knockout and VPS35 knockdown are used, and the data are thus not directly comparable. The observation that VPS35 and Homer1b/c do not colocalize may be due to the fact that the two proteins do not bind directly but only through SNX6, which becomes limiting when both VPS35 and Homer1b/c are overexpressed. Is colocolalization of VPS35 and Homer1b/c observed when SNX6 is overexpressed as well? Altogether, the authors should either provide additional data showing that SNX6-dependent distribution of Homer1b/c does not require retromer, or remove this conclusion.

Reviewer #2:

In this paper Niu et al. demonstrate that the CNS-specific Snx6 knockout mice exhibit deficits in spatial learning and memory, accompanied with loss of spines from apical dendrites of hippocampal CA1 pyramidal cells.

Interestingly they found that SNX6 functions independently of retromer to mediate vesicular transport of Homer1b/c to PSD. Indeed with a number of experiments they showed that the ablation of SNX6 causes loss of Homer1b/c from spines as well as decreases in surface levels of AMPAR and AMPAR-mediated synaptic transmission. Thus this paper potentially identifies a novel dendritic transport pathway that contributes to synaptic structure and function. This is a nice and well presented paper, however the following experiments and controls are absolutely required to really complete the paper.

Major points:

– Alteration of mGlu5 signaling and altered Homer1 localization to synapses has been associated to impaired social behavior in mice. Thus it will be interesting to characterize the social and repetitive behavior in the SNX6 KO mice.

– Dendrite spine morphology and synapse structure should be also analyzed in cortex.

– The quality of EM images is poor and should be improved substantially.

– The surface staining of mGlu5 is not very convincing. The higher expression of surface mGlu5 in the SNX6 KO mice should be proved also with biochemical experiments.

Reviewer #3:

Niu et al., investigates the physiological role of SNX6, which is a known component of the retrograde complex. Loss of SNX6 caused deficits in a/ spine density in hippocampal CA1 neurons, b/ hippocampal-dependent spatial learning and memory, c/ synaptic homer and AMPA receptor levels, d/ AMPA receptor-mediated synaptic transmission and e/ coupling of the endocytic zone to PSD. These data suggest an important role for SNX6 in synaptic structure and function. The biochemical, electrophysiology and behavioral experiments in this paper are well executed and are high quality but the cell biological part is poorly developed. The cell biological / localization analysis / live imaging data presented here (Figure 4, Figure 5 and some panels in Figures 6 and 7) are insufficient to support the major conclusions of the paper. It is possible that SNX6 directly transport homer1 but this is not shown by the data presented in this manuscript. Moreover, the paper lacks evidence for the claim that SNX6 mediates dynein-driven long range dendritic transport of Homer1b/c,

Major concerns:

1) From the current data it cannot be concluded that there is a "SNX6-mediated Homer1b/c transport pathway (Videos 24)". Overexpression of the fluorescently tagged SNX6 and homer1 look completely different than the endogenous staining patterns and do agree with previous reported subcellular distributions. Most of the SNX6 and homer1 clusters are static and do not move and are not present at synaptic sites.

2) There is no evidence in this paper that "SNX6 mediates vesicular transport of Homer1b/c to synaptic sites in dendrites". Other than previous data on an interaction between dynein and SNX6 and the pulldown experiments shown in Figure 4 there is no functional evidence for the role dynein in this transport pathway. The authors should provide functional data on the link between dynein and SNX6 and homer in dendritic transport.

3) There is no evidence in this paper that there is "a novel dendritic transport pathway that contributes to synaptic structure and function" (last sentence in the Abstract). Authors should first perform high-quality imaging experiments to exclude that other 'dendritic' trafficking pathways are not involved. Such as secretory pathways (work from Mike Ehlers) and other routes transporting mobile scaffolding proteins (Gerrow et al., Neuron, 2016). Moreover, the data do not convincingly show that the phenotype in SNX6 knockout mice is retromer independent.

[Editors’ note: what now follows is the decision letter after the appeal].

Thank you for choosing to send your work entitled "Synaptic Delivery of Homer1b/c by SNX6-mediated Dendritic Vesicular Transport" for consideration at eLife. Your article and your letter of appeal have been considered by a Senior Editor and a Reviewing Editor, and we regret to inform you that we are upholding our original decision. The concerns raised by the reviewers about the strength of the evidence supporting your major conclusions were very serious, and therefore, the paper in its current form is not suitable for publication in eLife. However, should you be able to thoroughly address the issues raised by the three reviewers with additional experiments, we will be prepared to consider a new submission for a formal review by the same reviewers.

If you choose to resubmit your paper, please give particular consideration to the following points:

1) It would be important to improve the quality of live imaging data by showing that the fluorescent proteins used for these experiments faithfully recapitulate the distribution of endogenous proteins.

2) It would also be important to provide clear proof for the idea that SNX6 indeed serves as a linker between Homer1b/c and cytoplasmic dynein. Please note that various strategies of inhibiting dynein are by themselves not sufficient here, as the inhibition of dynein is well known to affect most microtubule-based transport pathways independent of their identity. One would like to see proof that SNX6 really connects Homer1b/c to dynein, for example, by showing a reduced colocalization or a reduced biochemical interaction between Homer1b/c and the dynein complex in SNX6 knockout. Alternatively, you might consider a possibility that SNX6 participates in transporting Homer1b/c by simply recruiting it to endosomes, which then bind to microtubule motors independently of SNX6. It would also be useful to consider alternative ways of how SNX6 could regulate the abundance of Homer1b/c at the synapses.

3) On the technical side, please note that the use of SIM microscopy is an asset, but since the improvement in resolution provided by this technique is modest, its use does not overcome the need for proper unbiased quantifications, especially when the analyzed samples are highly crowded.

4) For proving that the pathway you are analyzing is indeed retromer-independent, it would be important to demonstrate better the efficiency of the knockdown of VPS35 in neurons, because if the knockdown is only partial and not complete, this might explain the differences between VPS35 knockdown and the SNX6 knockout phenotype, especially as the loss VPS35 does affect the number of Homer1b/c puncta in dendrites. Please also refer to the comments of reviewer 1 on this issue.

[Editors’ note: Further to the previous decision, the authors submitted a new version of the manuscript. The decision letter after another round of full peer review follows.]

Thank you for resubmitting your work entitled "Synaptic Delivery of Homer1b/c by SNX6-mediated Dendritic Vesicular Transport" for further consideration at eLife. Your revised article has been evaluated by Anna Akhmanova (Senior editor), a Reviewing editor, and two reviewers.

The reviewers agree that the part of the paper describing the mouse phenotype is well done. However, although the revisions have alleviated some technical concerns on the cell biological part, the evidence about SNX6 and Homer1 participating in a novel dendritic transport pathway remains insufficiently convincing.

First, if Homer1b/c localizes to early endosomes and is transported with them, then it is not a novel pathway. Moreover, as the authors show in Figure 6J, SNX6 knockout does not affect the 40% colocalisation between Homer1b/c and an early endosome marker. But then, surprisingly, SNX6 knockout does affect Homer1 motility and colocalization with dynein. Do the authors actually mean that the complex of SNX6 and Homer1b/c is required to transport early endosomes into dendrites? The authors should be much more explicit on this issue, as other molecules have been implicated in endosome transport. If Homer1b/c and SNX6 are transported on early endosomes, then SNX6 knockout should affect endosome motility. This should be easy to measure, and such data should be included. Alternatively, if SNX6 in complex with dynein transports the non-endosomal Homer1 population, what kind of vesicles are these, and what is then the relevance of colocalization of Homer1 with endosomes?

Furthermore, the new data provided by the authors show that fluorescently tagged Homer1b/c is present in large structures, many of which do not colocalize with a PSD marker. In contrast, the endogenous Homer1b/c is well known to show a synaptic localization. It is thus possible that the non-synaptic fluorescently tagged Homer 1b/c is present in aggregates, and that loss of SNX6 affects the very infrequent motility of these aggregates. The motility of fluorescent Homer1b/c particles might then be irrelevant to the distribution of synaptic Homer1. It is possible that the synaptic localization of Homer1b/c actually depends on protein diffusion and not on microtubule-based transport, an option that is not even properly discussed. The authors should use FRAP to investigate the turnover of the synaptic population of Homer1b/c in order to find out whether synaptic Homer1 exchanges with the soluble cytosolic pool of the protein or traffics into synapses as particles, and whether any of these processes are affected by the loss of SNX6.

If the authors cannot satisfactory uncover the nature of the "new dendritic transport pathway" that they propose or if it turns out that Homer1b/c accumulates in the synapses by exchanging with the soluble pool of the protein, the title, Abstract, the text and the conclusions of the paper will need to be very thoroughly revised accordingly.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Dendritic Delivery of Homer1b/c by SNX6-mediated Long-range Vesicular Transport" for further consideration at eLife. Your revised article has been favorably evaluated by Anna Akhmanova (Senior editor), a Reviewing editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

In the revised version of the paper, the authors performed the experiments suggested by the reviewers. The results most relevant to the overall message of the paper are that the labeled Homer1c structures only very rarely enter spines and that photobleached Homer1c in spines shows SNX6-independent recovery by 50% within 10 minutes through exchange with the soluble pool. These are very significant results, because together, they provide strong support for the view that Homer1c is not delivered into spines on endosomes but simply exchanges with the soluble pool in a SNX6-independent manner, which is not unexpected. Since most of dendritic Homer1c is present in spines, but vesicular transport is not responsible for delivering Homer1c into spines, the role of SNX6-dependent vesicle transport in "dendritic delivery of Homer1b/c" becomes confusing. Is it needed to redistribute soluble Homer1c through dendrites, so that it can then diffuse into spines? Or does Homer1c on endosomes represent part of a Homer1c pathway distinct from Homer1c function in dendritic spines? Based on the data presented in the paper, these questions are impossible to address. However, the data as they stand now certainly bring the importance of the observed SNX6-dependent transport of Homer1c on endosomes into question.

Given the large amount of work the authors have invested in revising the paper, the good quality of much of the data and the complexity of the problem, the reviewers felt that the paper can still be published. However, it would be essential to very thoroughly revise the text of the paper in a way that would do justice to the data shown, and not just to the model that the authors were trying to prove. To achieve this, it would be essential to very strongly downplay the "SNX6-mediated transport and delivery" angle, particularly by removing it from the title, re-writing the Abstract and Introduction, adding the data on FRAP and the failure to detect vesicle-based delivery of Homer1c into spines into the main figures, and writing a very balanced discussion. The revised version of the paper should include all the data but should not attempt to create an impression that the authors have proven that the phenotypes of SNX6 knockout mice are due to altered transport and delivery of Homer1c on SNX6-positive endosomes into dendritic spines, thus explaining the spine phenotypes observed. The reviewers would like to emphasize that "cosmetic" changes to the writing will not be sufficient in this case.

It is possible that the authors might disagree with the opinion of the reviewers, and in this case they are advised to seek publication in another journal.

Should you decide to resubmit, please address this additional point: the data of panel K of Figure 8 on the surface staining of mGlu5 do not look convincing. Please move them to the Supplement and move the supplementary biochemical data (Figure 8—figure supplement 1) to the main figure.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for choosing to send your work entitled "A Neuronal Role for SNX6 in Dendritic Trafficking of the Postsynaptic Scaffold Protein Homer1b/c" for consideration at eLife. The new version of your manuscript was reviewed by the Editor and one of the previous reviewers and, once again, they are not satisfied with the limited revisions you have made in response to their criticisms. Their initial reaction was to reject your paper with no chance for reconsideration but I requested they give you one last chance and here is their firmly written opinion that you simply must take seriously if you wish to have this work published in eLife:

Specifically, in the previous decision letters we have very clearly indicated that we cannot offer to publish your paper in the current form, because the reviewers found that your major claim that " SNX6 mediates long-range vesicular transport of Homer1b/c to distal dendrites" is not sufficiently supported by data. With other words, it is possible that SNX6 indeed mediates long-range transport of Homer1b/c, but it is also possible that Homer1b/c arrives to the dendritic spines by diffusion alone, and the interactions that you described serve another function (if any). Given the complexity of the subject and the large amount of work that you have put into revising this manuscript, we have offered you an opportunity to completely and thoroughly revise the whole paper in a way that would make it clear to the reader from the start to the end that your transport-related conclusions are not more than one of the possible explanations of the mouse phenotypes that you nicely describe. Such re-writing would entail not a minor but a major revision of your paper. Given your new suggestion for the title and the minor edits you have done, it is very clear to us that you are not willing to perform such a major revision. We of course fully respect your decision, because this is your paper, and you should write it in the way you find most appropriate. We would like to emphasize that your English writing is perfectly fine, and that our negative decision is only due to the fact the reviewers were not convinced that your major conclusion is sufficiently supported by data. We would also like to emphasize that since the reviewers are not co-authors of your paper, it is not their task to search for "sentences or words in the manuscript still misleading or overstated" – it is up to authors to make such changes.

We are willing to give you one more chance to re-write the paper. However, you would need to really thoroughly rethink and revise the whole manuscript to provide a more balanced view of different ways in which their data can be explained. You are very much in favor of pushing through the model that SNX6 mediates transport of Homer1b/c on vesicles, but the experimental support for this model is insufficient and the FRAP data directly contradict it. Therefore, the paper needs to be re-written in a way that would make it clear to the reader that transport of Homer1b/c on endosomes is not more than one of the possible explanations of the observed mouse phenotypes. Trying to solve the problem by adding a few disclaimers here and there is not enough, and also not what we have asked for. For example, in the latest version, you proposed to revert to the title with transport being the main message, while we asked for this change one revision before.

https://doi.org/10.7554/eLife.20991.044

Author response

[Editors’ note: this article was originally rejected after discussions between the reviewers. The authors’ rebuttal follows.]

Reviewer #1:

This paper describes the neuronal function of the sorting nexin family member SNX6. The authors show that SNX6 knockout mice exhibit defects in learning and memory, a phenotype that they connect to the reduction in the number of dendritic spines in a subpopulation of hippocampal pyramidal cells. Further, they uncover a biochemical connection between SNX6 and the postsynaptic scaffolding protein Homer1b/c, and show that SNX6 knockout leads to a reduction in the intensity of Homer1b/c in dendrites and affects surface levels of AMPA receptors and synaptic transmission. They also demonstrate co-motility of SNX6 and Homer1b/c, and propose that SNX6 mediates dynein-dynactin mediated transport and synaptic delivery of Homer1/b. Finally, the authors provide some data suggesting that this function of SNX6 is independent of other components of the retromer complex, a part of which SNX6 forms.

This paper is very rich in data, and provides solid new information on the function of SNX6 in neurons. However, the data supporting the major mechanistic conclusion of the authors on SNX6 mediating the dendritic transport and synaptic delivery of Homer1b/c to dendritic spines is not very strong.

Major comments:

1) For the proposed model to work, SNX6 should be able to bind to Homer1b/c and dynactin at the same time. However, the PX domain of SNX seems to be necessary for binding to both Homer1b/c and to dynactin, and it should be clarified whether both interactions can occur simultaneously.

We thank the reviewer for the very insightful comment. Two proteins binding to the same domain of another protein does not necessarily mean they bind to the same amino acid residues. By immunoisolation, we detected SNX6, Homer1b/c and dynein−dynactin on SNX6-positive vesicles (Figure 4H in the original manuscript). Although the interaction between SNX6 and Homer1b/c is pretty strong in both in vitro binding assay (Figure 4D–F in the original manuscript) and coIP with overexpressing proteins (Figure 4G in the original manuscript), given the fact that the interaction between dynein−dynactin and SNX6 is weak and transient, and can only be detected when both proteins (endogenous) are associated with membranes, which is well documented in our previous studies (Hong et al., 2009; Niu et al., 2013), it is not practical to coIP all three proteins with buffer containing detergent. Therefore, we decided to take an alternative approach to determine whether SNX6 is required for association of Homer1b/c cargo with dynein−dynactin: we performed immunoisolation experiment of membrane fractions from mouse brain with antibodies to Homer1b/c, p150Glued or DIC and examined whether these proteins still associate with the same vesicles in the absence of SNX6. Although the anti-Homer1b/c antibody failed to immunoisolate/enrich Homer1b/c-positive vesicles, we are very pleased that Homer1b/c was detected on p150Glued- or DIC-positive vesicles isolated from wild-type but not SNX6 knockout mouse brain (Figure 6H). This piece of data indicates that association of Homer1b/c and the motor on the same vesicles requires SNX6.

Moreover, we also performed co-immunostaining of neurons and found that the colocalization between Homer1b/c and p150Glued/DIC decreased in SNX6 knockout neurons, whereas its colocalization with the endosome marker EEA1 did not change (Figure 6I–J). Together these biochemical and cell biological data indicate that SNX6 serves as linker between Homer1b/c and the dynein−dynactin motor complex. These results are now incorporated in the text and the data is shown in new Figure 6.

2) Figure 5D demonstrates a dramatic reduction of Homer1b/c intensity throughout the dendrite in a SNX6 knockout neuron. It should be investigated whether the expression level of Homer1b/c is affected by SNX6 knockout, and if not, in which part of the neuron Homer1b/c accumulates. Is it possible that SNX6 regulates not the transport but stability of Homer1b/c?

We thank the reviewer for the very thoughtful comments. First we determined whether the expression level of Homer1b/c is affected by SNX6 knockout by immunoblotting of hippocampal lysates. Quantitative analysis of results from 3 pairs of wild-type and knockout littermates indicate that there was no significant difference in Homer1b/c levels, which means that ablation of SNX6 did not affect its protein stability. Second, in the original manuscript we only quantified the mean intensity of Homer1b/c signals in dendritic segments 30-120 μm from the cell body of Snx6+/+ and Snx6-/- neurons. In light of the reviewer’s comments, we quantified the dendritic distribution of Homer1b/c signals relative to the cell body. We are very pleased to find out that consistent with the phenotype caused by inhibition of dynein−dynactin activity (new Figure 6D–G), there is a decrease in Homer1b/c signals in distal dendrite with concurrent increase in the cell body, indicating that indeed loss of SNX6 impairs long-range transport of Homer1b/c from the cell body to dendrite. These results are incorporated as Figure 5—figure supplement 3 and Figure 5H–I in the manuscript.

3) The authors do not show that the motility of Homer1b/c is affected by the loss of SNX6. If the authors would like to make conclusion about SNX6 as a factor involved in Homer1b/c transport, such data should be included.

Thanks a lot for the very insightful comments. To address this issue, we performed live imaging of mEmerald-Homer1c in dendrites of SNX6 knockout neurons. Quantification of motile Homer1c puncta indicates that compared with wild-type neurons, in which 29 out of 311 puncta from 10 neurons are motile, only 10 out of 1217 puncta from 40 knockout neurons are motile, indicating that indeed dendritic transport of Homer1b/c requires SNX6 activity. These results are incorporated in the text under the subtitle “Dendritic vesicular transport and spine localization of Homer1b/c require SNX6”.

4) Triple colocalizations of EEA1/SNX6/Homer1b/c (Figure 4M), and p150 or DIC/ SNX6/Homer1b/c (Figure 5H) show very little actual colocalization between the three markers, and could just as well reflect spurious overlap between abundant punctate labeling patterns. Quantifications should be provided illustrating the significance of colocalizations by using shifted images as controls.

Thanks a lot for the constructive comment on colocalization analysis of superresolution images. In the original manuscript we used the conventional method to quantify the overlap between three fluorescent labels using Mander’s coefficient as a measure of colocalization. We redid the colocalization analysis on the images (15 neurons/group) and assessed the statistical significance of the data with the methodology developed by Costes et al. (Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells. Biophys. J. 86, 3993-4003.) (Costes et al., 2004) and Fletcher et al. (Multi-Image Colocalization and Its Statistical Significance. Biophys. J. 99, 1996-2005) (Fletcher et al., 2010). Basically we evaluated the statistical significance of the values of voxel (corresponds to pixel in 2D images) colocalization by comparing them with those of images generated by randomizing spatial locations of signals in original images (i.e., colocalization occurred by chance). The results show that the voxel colocalization of EEA1-SNX6-Homer1b/c, p150Glued-SNX6-Homer1b/c and DIC-SNX6-Homer1b/c in the 3D-SIM images are significantly more than chance. The quantification data are added to the manuscript as Table 1 and Figure 4—figure supplement 1.

5) When the authors discuss Homer1b/c transport, do they mean transport from the cell body, or redistribution along the dendrite? If they mean transport from the cell body, why would an early endosome serve as a carrier? If relocalization of Homer1b/c along the dendrite is affected, why is the Homer1b/c intensity reduced throughout the dendrite? More clear data and a better discussion are needed here, including alternative models of the functional connection between SNX6 and Homer1b/c.

We thank the reviewer for the thoughtful comments. The question as to whether or not Homer1b/c is transported from the cell body is similar to that raised in Comment #2. First, it has been well established that early endosomes serve as carrier that is transported from the cell body to dendrites to provide membrane supply and other materials for dendrite development (Satoh et al., 2008; Zheng et al., 2008). As we have discussed in the original manuscript, microtubules in the dendrite are of mixed polarity and dynein serves as the motor to transport cargoes from the cell body to dendrite, our data that Homer1b/c transport is driven by dynein is in full agreement with previous studies (Satoh et al., 2008; Zheng et al., 2008; Kapitein et al., 2010). Second, in our initial analysis of Homer1b/c distribution in dendrite, we quantified mean intensity of Homer1b/c in dendritic segments ranging from 30 to 120 μm from the cell body, and focused on spine localization of Homer1. In light of reviewer’s comments we have performed quantitative analysis of confocal images to determine whether its dendritic distribution decreases towards the distal end of dendrites. We are very pleased to find that indeed, similar to phenotypes caused by inhibition of dynein−dynactin activity (new Figure 6D–G), the signal intensity of Homer1b/c decreases in distal dendrites in SNX6 knockout neurons, accompanied with its accumulation in the cell body, which was rescued by overexpression of mCherry-SNX6 (new Figure 5H–I, please refer to Response to Comment #2). These data together indicate that transport of Homer1b/c from the cell body to dendrite requires dynein−dynactin, which support and greatly strengthen our original conclusions and are added to the manuscript as Figure 5H–I.

6) The strong conclusion on retromer-independent function of SNX6 in Homer1b/c transport seems to be mostly based on the observation that while the loss of both SNX6 and VPS35 reduces the number of Homer1b/c puncta, only the knockout of SNX6 affect Homer1b/c intensity is spines. However, this comparison is not fair, because a SNX6 knockout and VPS35 knockdown are used, and the data are thus not directly comparable. The observation that VPS35 and Homer1b/c do not colocalize may be due to the fact that the two proteins do not bind directly but only through SNX6, which becomes limiting when both VPS35 and Homer1b/c are overexpressed. Is colocolalization of VPS35 and Homer1b/c observed when SNX6 is overexpressed as well? Altogether, the authors should either provide additional data showing that SNX6-dependent distribution of Homer1b/c does not require retromer, or remove this conclusion.

Thanks for the very insightful and constructive comments on VPS35. Because VPS35 knockout is lethal (Wen et al., 2011), in previous studies researchers studied its cellular and molecular functions by either using heterozygous mice (Vps35+/m, expression of which is ~50% of wild-type, (Wen et al., 2011)), suppressing its expression by RNA interference (Wang et al., 2012) or overexpressing the Parkinson’s Disease-related D620N point mutant (Munsie et al., 2015; Tang et al., 2015). Since we do not have the Vps35+/m mice, based on the previous findings on VPS35 haploinsufficiency and the role of the retromer core complex in neuronal survival and degeneration (Wen et al., 2011; Liu et al., 2014; Wang et al., 2014; Tang et al., 2015), i.e., even ~50% reduction in VPS35 protein levels causes severe phenotypes in neuronal function and survival, we chose to transiently knockdown its expression in hippocampal neurons to assess its role in dendritic transport of Homer1b/c. Quantitative analysis shows that the VPS35 knockdown efficiency is 70-80% (Author response image 1). In fact, hippocampal neurons transfected with VPS35 siRNA-expressing construct were not as healthy as those expressing the scrambled siRNA, which is in good agreement with previous findings that VPS35 function is essential for cell survival.

Author response image 1
Quantitative analysis of VPS35 knockdown efficiency.

(A) HEK 293 cells were transfected with lentiviral vector expressing siRNA along with EGFP followed by immunoblotting with antibodies to VPS35 and β-actin after 72 h. Right panel shows quantification of immunoblots. Relative amount of VPS35 was determined with NIH ImageJ (N = 3 experiments). (B) Hippocampal neurons were transfected with lentiviral vector expressing siRNA along with GFP at DIV12, fixed on DIV18 and immunostained with antibodies to VPS35. Right panels are quantification of VPS35 signals in the outlined area of neurons (mean ± SEM, n = 10 neurons from three independent experiments). Scale bar, 5 μm.

https://doi.org/10.7554/eLife.20991.042

To determine whether VPS35 and Homer1b/c bind through SNX6, we performed co-immunostaining of endogenous proteins (to avoid potential artifacts caused by overexpression of fluorescent fusion proteins) and found that, first of all, colocalization between Homer1b/c and VPS35 in dendrite is low compared with colocalization of Homer1b/c with SNX6 and EEA1; second of all, it was not affected by ablation of SNX6 (Figure 9—figure supplement 2A–B). Consistently, no change was detected in Homer1b/c-SNX6 colocalization in VPS35-depleted neurons (Figure 9—figure supplement 2C–D). Together these data indicate that VPS35 is not involved in SNX6-mediated Homer1b/c transport.

Moreover, to determine whether VPS35 depletion causes defect in Homer1b/c transport from the cell body to dendrites, we also performed quantitative analysis of Homer1b/c distribution in VPS35-depleted neurons and found that, in contrast to accumulation of Homer1b/c signals in the cell body and decrease in distal dendrite in SNX6 knockout neurons, the signal intensity of Homer1b/c decreases throughout the cell when VPS35 is suppressed. The decrease in Homer1b/c distribution in dendrites of VPS35-depleted neurons might be due to impaired cell viability/function and lower spine density as VPS35 has been found to promote spine formation and maturation (Wang et al., 2012; Tian et al., 2015). Together these data indicate that SNX6 and VPS35 function in synaptic localization of postsynaptic proteins via different mechanisms. These results are now incorporated in the manuscript as Figure 9B,F and Figure 9—figure supplements 1 and 2.

Reviewer #2:

In this paper Niu et al. demonstrate that the CNS-specific Snx6 knockout mice exhibit deficits in spatial learning and memory, accompanied with loss of spines from apical dendrites of hippocampal CA1 pyramidal cells.

Interestingly they found that SNX6 functions independently of retromer to mediate vesicular transport of Homer1b/c to PSD. Indeed with a number of experiments they showed that the ablation of SNX6 causes loss of Homer1b/c from spines as well as decreases in surface levels of AMPAR and AMPAR-mediated synaptic transmission. Thus this paper potentially identifies a novel dendritic transport pathway that contributes to synaptic structure and function. This is a nice and well presented paper, however the following experiments and controls are absolutely required to really complete the paper.

Major points:

– Alteration of mGlu5 signaling and altered Homer1 localization to synapses has been associated to impaired social behavior in mice. Thus it will be interesting to characterize the social and repetitive behavior in the SNX6 KO mice.

Thanks for the very insightful comments and suggestions. Since Homer and Shank are binding partners in the PSD, and genes encoding Shank family members are causative genes for autism spectrum disorders (ASD) (Hulbert and Jiang, 2016), it is natural to reason that changes in Homer1 functions/levels/distribution might also cause autism-like phenotypes in mice. We searched literature and found that intriguingly, although mutations or deletion of Shank family members causes abnormal social and repetitive behaviors in mouse models (Jiang and Ehlers, 2013), no link between Homer and human ASD has been reported (Hulbert and Jiang, 2016). Interestingly, previous studies report that Homer1 knockout mice that lack both long and short isoforms in the brain spent more time in social interaction with a naïve WT stranger in the social dyad test than wild-type and no difference in repetitive behaviors (Yuan et al., 2003; Jaubert et al., 2007). Though counterintuitive, this might be explained by functional redundancy among the Homer family members and/or distinct molecular and cellular functions of Homer and Shank besides their common role in PSD scaffolding. Nevertheless we performed social interaction and repetitive behavior assays on the SNX6 KO mice. The results show that the KO mice did not behave significantly different from the wild-type in these assays (Figure 2F and G), indicating that ablation of SNX6 did not affect exactly the same higher brain functions involving Homer1 and its interaction partner Shank in brain regions other than the hippocampus. As we have pointed out in the Discussion, among the Homer family members, Homer1b/c is predominantly expressed in the hippocampal CA1 region (Shiraishi et al., 2004). Homer1 knockout mouse exhibited similar phenotypes in spatial learning and memory as SNX6 knockout, which could be rescued by AAV-mediated Homer1c expression in the hippocampus (Gerstein et al., 2012). Furthermore, AAV-mediated delivery of Homer1c also improved the spatial learning deficits in a rat model of cognitive learning (Gerstein et al., 2013). Based on the previous findings on Homer1 involvement in hippocampal-dependent spatial learning and memory and our data, we conclude that ablation of SNX6 specifically impairs Homer1 functions in the hippocampus.

– Dendrite spine morphology and synapse structure should be also analyzed in cortex.

Thanks for the very insightful comments. Our behavior screen detected deficits in hippocampal-dependent spatial learning and memory, but not in motor coordination, mood levels and social or repetitive behaviors, indicating that ablation of SNX6 did not cause major structural/functional changes in other circuits or regions of the brain. Since our behavior tests identified impairment in hippocampal –dependent function but not functions involving other brain regions (please refer to behavior screen results shown in Figure 2 and Response to major point 1), in the current study we focused on analyses of changes in the hippocampal region at cellular and subcellular levels. We agree with the reviewer that to thoroughly investigate physiological changes in the CNS caused by SNX6 knockout, we should analyze spine morphology and synapse structure in other regions of the brain including the cerebral cortex. However, technically it is very difficult because currently we have no clue other than deficits in spatial learning and memory to help us locate the exact regions or neural circuits in the cortex for analyses at cellular and subcellular levels. Like identifying other vesicular cargoes mediated by SNX6 or other neural circuits affected by SNX6 knockout, it is our long-term goal to analyze brain regions of the KO mice other than the hippocampus and is beyond the scope of this manuscript.

– The quality of EM images is poor and should be improved substantially.

We apologize for the quality of the EM images. We have now replaced them with better ones, which clearly show not only excitatory synapses, but also membranous structures such as mitochondria and lysosomes.

– The surface staining of mGlu5 is not very convincing. The higher expression of surface mGlu5 in the SNX6 KO mice should be proved also with biochemical experiments.

Thanks for the constructive comments. To verify that surface levels of mGluR5 are increased in SNX6 KO neurons, we cultured hippocampal neurons from two pairs of WT and KO mice and performed biotinylation assay of surface proteins. We are very pleased that the results are fully consistent with the IF staining data, i.e., there is increase in surface mGluR5 and decrease in surface GluA1 and GluA2. These data are incorporated in the manuscript as Figure 8—figure supplement 1.

Reviewer #3:

Niu et al., investigates the physiological role of SNX6, which is a known component of the retrograde complex. Loss of SNX6 caused deficits in a/ spine density in hippocampal CA1 neurons, b/ hippocampal-dependent spatial learning and memory, c/ synaptic homer and AMPA receptor levels, d/ AMPA receptor-mediated synaptic transmission and e/ coupling of the endocytic zone to PSD. These data suggest an important role for SNX6 in synaptic structure and function. The biochemical, electrophysiology and behavioral experiments in this paper are well executed and are high quality but the cell biological part is poorly developed. The cell biological / localization analysis / live imaging data presented here (Figure 4, Figure 5 and some panels in Figures 6 and 7) are insufficient to support the major conclusions of the paper. It is possible that SNX6 directly transport homer1 but this is not shown by the data presented in this manuscript. Moreover, the paper lacks evidence for the claim that SNX6 mediates dynein-driven long range dendritic transport of Homer1b/c,

Major concerns:

1) From the current data it cannot be concluded that there is a "SNX6-mediated Homer1b/c transport pathway (Videos 24)". Overexpression of the fluorescently tagged SNX6 and homer1 look completely different than the endogenous staining patterns and do agree with previous reported subcellular distributions. Most of the SNX6 and homer1 clusters are static and do not move and are not present at synaptic sites.

We thank the reviewer for the thoughtful comments. Please note that since Homer1b/c is a scaffold protein, the cystosolic distribution of overexpressed protein generates diffuse signals in the cell in addition to puncta, which could be extracted with the mild detergent digitonin before IF staining, while transmembrane proteins or proteins associated with membranous structures remain in the cytoplasm. First of all, to determine whether the fluorescent proteins overexpressed in cultured neurons faithfully recapitulate the distribution of endogenous proteins, we transfected neurons with corresponding constructs and performed immunofluorescence staining. To remove overexpressed proteins in cytosol we conducted digitonin extraction of live neurons before fixation and staining (please see Immunofluorescence staining, confocal image acquisition and analysis in Materials and methods for details). Confocal images indicate that mEmerald-Homer1c indeed partially colocalizes with both the endosomal marker EEA1 and endogenous SNX6 in dendrites; conversely, mCherry-SNX6 colocalizes with EEA1 and endogenous Homer1b/c. Moreover, both mEmerald-Homer1c and mCherry-SNX6 also partially overlapped with PSD95, indicating that like the endogenous proteins, the overexpressed fluorescent fusion proteins could localize to dendritic spines. These data are added to the revised manuscript as Figure 5—figure supplement 1.

Second of all, we compared the motility of mEmerald-Homer1c to that of PSD95-GFP (Gerrow et al., 2006) and found that similar to PSD95, the majority of Homer1c are immotile in dendrite. Unlike mitochondria (~30% are motile) or endosomes (Yap et al., 2008), which are actively transported in neuronal cells to fulfill their cellular functions, both Homer1c and PSD95 are scaffold proteins that stay anchored to the postsynaptic cytomatrix once they have been delivered to the postsynaptic site. Since we performed live imaging of neurons in which the fusion protein has been overexpressed to levels high enough to detect the fluorescent signals, by the time of imaging most of the protein molecules might have already been delivered to the spines. Nevertheless, in light of reviewer’s comments, we performed live imaging of mEmerald-Homer1c in Snx6+/+ and Snx6-/- neurons. We are very pleased to find out that compared with wild-type neurons, there was approximately 10-fold decrease in the fraction of motile Homer1c puncta in Snx6-/- neurons (29 out of 311 mEmerald-Homer1c puncta from 10 Snx6+/+ neurons are motile, whereas only 10 out of 1217 puncta from 40 Snx6-/- neurons are motile). These data indicate that the dendritic vesicular transport of Homer1c is indeed SNX6-dependent, which further strengthened our original conclusion. This piece of data is incorporated in the text under the subtitle “Dendritic vesicular transport and spine localization of Homer1b/c require SNX6”.

2) There is no evidence in this paper that "SNX6 mediates vesicular transport of Homer1b/c to synaptic sites in dendrites". Other than previous data on an interaction between dynein and SNX6 and the pulldown experiments shown in Figure 4 there is no functional evidence for the role dynein in this transport pathway. The authors should provide functional data on the link between dynein and SNX6 and homer in dendritic transport.

We thank the reviewer for the very insightful comments. However, we would like to respectfully point out that we have addressed the issue already in the original manuscript with significant amount of experimentation. We determined whether SNX6-mediated dendritic transport of Homer1b/c is dynein-dependent with two approaches: 1) we treated hippocampal neurons with ciliobrevin D, an inhibitor of dynein activity, and analyzed dendritic distribution of Homer1b/c by IF staining and confocal microscopy; 2) we overexpressed the N-terminal fragment of p150Glued, a dominant negative mutant that disrupts SNX6-p150Glued interaction, in hippocampal neurons and analyzed distribution of Homer1b/c in dendrites. Quantification results indicate that indeed, inhibition of dynein activity caused decrease in synaptic distribution of Homer1b/c (Figure 5J–K), and disruption of motor-adaptor interaction caused accumulation of Homer1b/c in the cell body and its decrease in distal dendrites (Figure 5L–M). Moreover, in the revised manuscript, to further confirm that dynein−dynactin drives Homer1b/c transport from the cell body to dendrites, we repeated the ciliobrevin D treatment experiment and compared the subcellular distribution of Homer1b/c in the cell body and dendritic segments. The results show that, consistent with data in the original manuscript, inhibition of dynein activity causes accumulation of Homer1b/c in the cell body and its decrease in the distal region of dendrites. Further, we performed two new experiments to determine whether SNX6 serves as cargo adaptor to link the dynein-dynactin motor complex to Homer1b/c vesicular cargo: 1) colocalization analysis of Homer1b/c and dynein−dynactin in SNX6 knockout neurons; 2) immunoisolation of Homer1b/c- and dynein−dynactin-double positive vesicles from SNX6 knockout mouse brain. The results show that 1) the colocalization between Homer1b/c with DIC and p150Glued is decreased when SNX6 is ablated, whereas the colocalization between Homer1b/c and EEA1 is not affected; 2) although the antibodies against Homer1b/c did not work for immunoisolation, antibodies to both p150Glued and DIC worked and western blotting detected Homer1b/c on DIC- or p150Glued-positive vesicles from wild-type but not SNX6-/- mouse brain. All together, these cell biological and biochemical data indicate that SNX6-mediated dendritic transport of Homer1b/c requires dynein−dynactin activity. To make it clear, we have reorganized the data, combined Figure 5H–M with the new data and made them into the new Figure 6.

3) There is no evidence in this paper that there is "a novel dendritic transport pathway that contributes to synaptic structure and function" (last sentence in the Abstract). Authors should first perform high-quality imaging experiments to exclude that other 'dendritic' trafficking pathways are not involved. Such as secretory pathways (work from Mike Ehlers) and other routes transporting mobile scaffolding proteins (Gerrow et al., Neuron, 2016). Moreover, the data do not convincingly show that the phenotype in SNX6 knockout mice is retromer independent.

We thank the reviewer for the very insightful comments and information on the previous findings. However, we would like to respectfully point out that we have already addressed the first issue in the original manuscript with experimental data. We have shown that 1) neither SNX6 nor Homer1b/c signals were detected on motile PSD95 clusters in dendrites; 2) block of the secretory pathway from the TGN in neurons by overexpressing the PKD-KD dominant mutant (Liljedahl et al., 2001) did not affect dendritic/synaptic distribution of Homer1b/c. We dedicated a section (subtitled “The SNX6-mediated Homer1b/c transport pathway is distinct from other trafficking pathways in dendrite”) and a whole figure to this question (Figure 6) in the original manuscript. It is now presented as Figure 7 in the revised version.

For the second question, we have also performed significant amount of experiments to verify that the phenotype in SNX6 knockout is retromer independent (please refer to our response to comment 6 by reviewer #1). Because VPS35 knockout is lethal (Wen et al., 2011), and we do not have the Vps35+/m mice in which the expression level of VPS35 is ~50% of wild-type, we chose to transiently knockdown its expression in hippocampal neurons (knockdown efficiency is 70-80%, Figure 9A–B and Figure 9—figure supplement 1) to assess its role in dendritic transport of Homer1b/c.

In the revised manuscript, taking into account that SNX6 mediates transport of Homer1b/c from the cell body to dendrites, we also performed quantitative analysis of Homer1b/c distribution in VPS35-depleted neurons and found that, in contrast to accumulation of Homer1b/c signals in the cell body and decrease in distal dendrites in SNX6 knockout neurons, the signal intensity of Homer1b/c decreases throughout the cell when VPS35 is suppressed (Figure 9F). Please note that this phenotype is distinct from that caused by not only ablation of SNX6 (Figure 5), but also inhibition of dynein activity or disruption of motor-cargo interaction (Figure 6).

Further, to address reviewer #1’s concern whether VPS35 and Homer1b/c bind through SNX6, we performed co-immunostaining and confocal microscopy of endogenous proteins and found that, the colocalization between VPS35 and Homer1b/c (~10%) was not affected at all by ablation of SNX6 (Figure 9—figure supplements 2A–B). Colocalization between Homer1b/c and SNX6 (~30%) was not affected by VPS35 knockdown either (Figure 9—figure supplement 2C–D). Together these data indicate that VPS35 is not involved in SNX6-mediated dendritic transport of Homer1b/c that contributes to its synaptic localization. These results are now incorporated in the manuscript as Figure 9B,F and Figure 9—figure supplements 1 and 2.

[Editors’ note: what now follows is the authors’ response after rejection of the appeal.]

If you choose to resubmit your paper, please give particular consideration to the following points:

1) It would be important to improve the quality of live imaging data by showing that the fluorescent proteins used for these experiments faithfully recapitulate the distribution of endogenous proteins.

Thanks for the great suggestion. Since Homer1b/c is a scaffold protein, the cystosolic distribution of overexpressed protein generates diffuse signals in the cell in addition to puncta, which could be extracted with the mild detergent digitonin before IF staining, while transmembrane proteins or proteins associated with membranous structures remain in the cytoplasm. To determine whether the fluorescent proteins overexpressed in cultured neurons faithfully recapitulate the distribution of endogenous proteins, we performed immunofluorescence staining of digitonin-extracted neurons transfected with corresponding constructs. Confocal images indicate that mEmerald-Homer1c indeed partially colocalizes with both the endosomal marker EEA1 and endogenous SNX6 in dendrites; conversely, mCherry-SNX6 colocalizes with both EEA1 and endogenous Homer1b/c (Please see below). Moreover, since digitonin extraction removes cytosolic proteins, we could not use overexpressed cytosolic GFP or RFP as volume marker to determine whether or not mEmerald-Homer1c and mCherry-SNX6 also distribute in dendritic spines. Instead, we performed immunostaining of the postsynaptic marker PSD95 on transfected neurons. We are very pleased to report that both mEmerald-Homer1c and mCherry-SNX6 signals partially colocalize with PSD95 (Please see below), indicating that like the endogenous proteins, the overexpressed fluorescent proteins localize in both dendritic shaft and spines. These data are added to the revised manuscript as Figure 5—figure supplement 1.

2) It would also be important to provide clear proof for the idea that SNX6 indeed serves as a linker between Homer1b/c and cytoplasmic dynein. Please note that various strategies of inhibiting dynein are by themselves not sufficient here, as the inhibition of dynein is well known to affect most microtubule-based transport pathways independent of their identity. One would like to see proof that SNX6 really connects Homer1b/c to dynein, for example, by showing a reduced colocalization or a reduced biochemical interaction between Homer1b/c and the dynein complex in SNX6 knockout. Alternatively, you might consider a possibility that SNX6 participates in transporting Homer1b/c by simply recruiting it to endosomes, which then bind to microtubule motors independently of SNX6. It would also be useful to consider alternative ways of how SNX6 could regulate the abundance of Homer1b/c at the synapses.

Thanks a lot for the excellent suggestions on the linker role of SNX6 in dynein-driven transport of Homer1b/c. We followed the suggestions for cell biological and biochemical assays to determine whether the association between Homer1b/c and dynein−dynactin is impaired in SNX6 knockout: 1) co-IF staining followed by colocalization analysis of confocal images shows that there is indeed a decrease in the colocalization of Homer1b/c and p150Glued/DIC in SNX6 knockout neurons, whereas the colocalization of Homer1b/c and EEA1 was not affected; 2) immunoisolation of membranous structures from mouse brain extracts with antibodies to p150Glued or DIC detects Homer1b/csignals in wild-type but not in SNX6 knockout mice, indicating that Homer1b/c and dynein-dynactin are not on the same vesicles when SNX6 is ablated. Please note that we attempted immunoisolation with antibodies to Homer1b/c but unfortunately they did not work for this type of experiment. Moreover, we performed live imaging of mEmerald-Homer1c in dendrites of Snx6-/- neurons and found that there was approximately 10-fold decrease in the fraction of motile Homer1b/c puncta compared with that in wild-type neurons (29 out of 311 puncta from 10 Snx6+/+ neurons are motile, whereas only 10 out of 1217 puncta from 40 Snx6-/- neurons are motile). These data together indicate that SNX6 serves as cargo adaptor to link Homer1b/c vesicular cargo and the motor complex, which strongly support and strengthen our original conclusion that SNX6 mediates dynein-driven vesicular transport of Homer1b/c. The results are now incorporated in the text and figures as new Figure 6H–J.

3) On the technical side, please note that the use of SIM microscopy is an asset, but since the improvement in resolution provided by this technique is modest, its use does not overcome the need for proper unbiased quantifications, especially when the analyzed samples are highly crowded.

To confirm our quantification results of the 3D-SIM images (EEA1-SNX6-Homer1b/c in Figure 4M and N, and p150Glued/DIC-SNX6-Hoemr1b/c in Figure 5H and I), in which we quantified the overlap among three fluorescent labels using Mander’s coefficient as a measure of colocalization, we redid the colocalization analysis and assessed the statistical significance of the data with the methodology developed by Costes et al. (Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells. Biophys. J. 86, 3993-4003.) (Costes et al., 2004) and Fletcher et al. (Multi-Image Colocalization and Its Statistical Significance. Biophys. J. 99, 1996-2005) (Fletcher et al., 2010). Basically we evaluated the statistical significance of the values of voxel (corresponds to pixel in 2D images) colocalization by comparing them with those of images generated by randomizing spatial locations of signals in original images (i.e., colocalization occurred by chance). The results show that the voxel colocalization of EEA1-SNX6-Homer1b/c and that of p150Glued/DIC-SNX6-Homer1b/c are significantly more than chance. The quantification data are added to the manuscript as Table 1 and Figure 4—figure supplement 1 and shown below.

4) For proving that the pathway you are analyzing is indeed retromer-independent, it would be important to demonstrate better the efficiency of the knockdown of VPS35 in neurons, because if the knockdown is only partial and not complete, this might explain the differences between VPS35 knockdown and the SNX6 knockout phenotype, especially as the loss VPS35 does affect the number of Homer1b/c puncta in dendrites. Please also refer to the comments of reviewer 1 on this issue.

Because VPS35 knockout is lethal (Wen et al., 2011), in previous studies researchers studied its cellular and molecular functions by either using heterozygous mice (Vps35+/m, expression of which is ~ 50% of wild-type, (Wen et al., 2011)), suppressing its expression by RNA interference (Wang et al., 2012) or overexpressing the Parkinson’s Disease-related D620N point mutant (Munsie et al., 2015; Tang et al., 2015). Based on the previous findings on VPS35 haploinsufficiency and the role of the retromer core complex in neuronal survival and degeneration (Wen et al., 2011; Liu et al., 2014; Wang et al., 2014; Tang et al., 2015), we chose to transiently knockdown its expression in hippocampal neurons to assess its role in dendritic transport of Homer1b/c. We performed quantitative analysis of both western blots and confocal images, the results show that VPS35 knockdown efficiency is 70-80% in hippocampal neurons, which are incorporated in the manuscript as Figure 9A,B and Figure 9—figure supplement. Please note that when we were analyzing the fluorescent signals of VPS35, we not only quantified the mean intensity of VPS35 fluorescent signals but also the number of VPS35 puncta in the somatodendritic area of each neuron (Choy et al., 2014).

Further, we also quantified the subcellular distribution of Homer1b/c in VPS35 knockdown neurons to determine whether VPS35 acts in SNX6-mediated transport pathway. Quantification results show that VPS35 knockdown causes decrease in Homer1b/c signals throughout the cell (Figure 9F), whereas the signals accumulate in the soma and decrease in dendritic segments distal to the cell body of SNX6 knockout neurons. Please note that this phenotype is distinct from that caused by not only ablation of SNX6, but also inhibition of dynein activity or disruption of motor-cargo interaction shown in Figure 6.

Moreover, we performed confocal microscopy and colocalization analysis of Homer1b/c and VPS35 in wild-type and SNX6 knockout neurons. The results show that the colocalization of Homer1b/c with VPS35 in dendrite, which is much lower than its colocalization with SNX6 and EEA1, is not affected by SNX6 knockout (Figure 9—figure supplement 2A,B). Conversely, colocalization of Homer1b/c with SNX6 is not affected by VPS35 knockdown either (Figure 9—figure supplement 2C,D). Collectively these results indicate that VPS35 is not involved in dynein-driven, SNX6-mediated vesicular transport of Homer1b/c from the hippocampal cell body to dendrites.

Taken together, these results are supportive of the findings reported in the original manuscript. Based on the extended amount of experimental data we re-arranged the figures to better support the flow of the story. In total, this improved manuscript contains one new figure, one table, six new supplementary figures, and numerous modifications/improvements to the existing figures, all of which support and greatly strengthen our original conclusions. Since we could not find enough room for new data in the original figures, we have reorganized the figures to accommodate the new results in the revised manuscript as the following: 1) we split Figure 5 into two, the new Figures 5 and 6, so Figures 58 are now Figures 59, respectively; 2) we combined Figure 4, Figure 4—figure supplements 1 and 2 to make it the new Figure 4; 3) we added immunostaining and confocal data for mEmerald-Homer1c and mCherry-SNX6 as Figure 5—figure supplement 1; 4) we added data from quantitative analysis of 3D-SIM images as Table 1 and new Figure 4—figure supplements 1;,2) we added data for subcellular distribution of mEmerald-Homer1c and mCherry-SNX6 as Figure 5—supplement 1; 6) we added data for biotinylation assay of surface receptors in wild-type and KO neurons as Figure 8—figure supplements 1;,2) we added data for VPS35 knockdown efficiency as Figure 9—figure supplements 1;,5) we added data for Homer1b/c colocalization with SNX6 (in VPS35 KD) and VPS35 (in Snx6-/-) as Figure 9—figure supplement 2.

[Editors’ note: the authors’ response after a second round of full peer review follows].

First, if Homer1b/c localizes to early endosomes and is transported with them, then it is not a novel pathway. Moreover, as the authors show in Figure 6J, SNX6 knockout does not affect the 40% colocalisation betweev n Homer1b/c and an early endosome marker. But then, surprisingly, SNX6 knockout does affect Homer1 motility and colocalization with dynein. Do the authors actually mean that the complex of SNX6 and Homer1b/c is required to transport early endosomes into dendrites? The authors should be much more explicit on this issue, as other molecules have been implicated in endosome transport. If Homer1b/c and SNX6 are transported on early endosomes, then SNX6 knockout should affect endosome motility. This should be easy to measure, and such data should be included. Alternatively, if SNX6 in complex with dynein transports the non-endosomal Homer1 population, what kind of vesicles are these, and what is then the relevance of colocalization of Homer1 with endosomes?

We appreciate the comments of the reviewers. It looks like the reviewers get the impression that SNX6 transports all early endosomes into dendrites. We apologize for the confusion/misunderstanding that might have been caused by our failure to make this aspect of the paper clearer. Based on the data that Homer1b/c and SNX6 colocalize on early endosomes and dendritic transport of Homer1b/c requires SNX6 and dynein‒dynactin, we propose that SNX6 serves as dynein cargo adaptor to mediate vesicular transport of Homer1b/c. In the absence of SNX6, the link between the motor and Homer1b/c-associated vesicles is missing, so that there is a decrease in colocalization between Homer1 and dynein. Please note that we did not claim that SNX6 links Homer1 to endosomes. Instead, current data support that SNX6 recognizes the endosomal carrier for Homer1b/c via its direct interaction with the cargo molecule. Indeed, it is an excellent question how active transport of endosomes carrying different cargoes is regulated by their cargo adaptors. In fact, during the study on SNX6’s role in dendrite we searched the literature on dendritic trafficking. We found that 1) most studies focus on recycling endosomes and receptor trafficking to the postsynaptic site, mechanisms for long-range transport of endosomal cargoes and their adaptors for molecular motors remain to be identified; 2) the motility of endosomes labeled by different cargoes is not the same. Research conducted by Dr. Bettina Winckler and her colleagues at University of Virginia has identified Neep21 as a regulator for early endosomal sorting and trafficking in dendrites (Yap et al., 2008). Intriguingly, they found by live imaging that only 7% of EEA1-positive endosomes are motile, compared with 63% of Neep21-positive endosomes (Lasiecka et al., 2014). Moreover, the run length and velocity of these two types of vesicles are also quite different from each other (Lasiecka et al., 2014). These findings suggest that there are multiple mechanisms underlying endosomal transport. To make sure we have not missed any most recent progress on this topic, we consulted an expert about endosome motility in dendrites, who was very nice to provide us the following information based on their published and unpublished imaging data:

1) Motility of vesicles labeled with various endosomal markers vary a lot.

2) There are many types of endosomes, so the motor adaptor for one specific cargo might not control the behavior of all endosomes equally and it might be difficult to see an effect of gene knockout with just any endosomal marker.

3) If less than 10% of vesicles are motile, then a decrease in motility will be hard to observe.

In retrospect, given the role of early endosomes as sorting station for a wide variety of cargoes, and the ~ 15% colocalization among EEA1, Homer1 and SNX6 (Figure 4—figure supplement 1, measured with more stringent method for quantification of images obtained by 3D-SIM, rather than conventional confocal microscopy), it is highly possible that the motile SNX6-, Homer1-positive vesicles are transport carriers derived from EEA1-positive endosomes and they might have lost the marker(s) upon sorting/fission from the endosomal membrane or en route to their final destination. That is to say, they do not represent all early endosomes or endosome-derived vesicles carrying different cargoes. Given the heterogeneity of early endosomes in composition and motility and the various transport pathways they might adopt (recycling to the plasma membrane, retrograde transport to the TGN, and trafficking to, conversion to late endosome and fusion with lysosomes, etc.), even if we carry out the live imaging experiment with a well-known endosomal marker protein, e.g., EEA1 or Rab5, questions still remain as to whether EEA1- or Rab5-labeled vesicles represent a proportion or all types of endosomal carriers derived from early endosomes, and whether SNX6 mediates transport of all endosomal carriers derived from EEA1- or Rab5-positive endosomes. Therefore, we would appreciate your opinion on this issue. To avoid confusion, we will tone down the statement about novel transport pathway (How about “previously unknown” instead of “novel”?), rewrite the sentences in the Results and Discussion sections to make it clearer and modify the Abstract accordingly.

Furthermore, the new data provided by the authors show that fluorescently tagged Homer1b/c is present in large structures, many of which do not colocalize with a PSD marker. In contrast, the endogenous Homer1b/c is well known to show a synaptic localization. It is thus possible that the non-synaptic fluorescently tagged Homer 1b/c is present in aggregates, and that loss of SNX6 affects the very infrequent motility of these aggregates. The motility of fluorescent Homer1b/c particles might then be irrelevant to the distribution of synaptic Homer1. It is possible that the synaptic localization of Homer1b/c actually depends on protein diffusion and not on microtubule-based transport, an option that is not even properly discussed. The authors should use FRAP to investigate the turnover of the synaptic population of Homer1b/c in order to find out whether synaptic Homer1 exchanges with the soluble cytosolic pool of the protein or traffics into synapses as particles, and whether any of these processes are affected by the loss of SNX6.

If the authors cannot satisfactory uncover the nature of the "new dendritic transport pathway" that they propose or if it turns out that Homer1b/c accumulates in the synapses by exchanging with the soluble pool of the protein, the title, Abstract, the text and the conclusions of the paper will need to be very thoroughly revised accordingly.

First, we would respectfully disagree with reviewer’s comment on large structures of Homer1c-EGFP as “aggregates”. As those fluorescent puncta are digitonin resistant, they are either membrane- or PSD-bound (shown by confocal images of colocalization between Homer1c-EGFP and PSD95, Figure 5—figure supplement 1A), making them no longer cytosolic and easily removed by detergent extraction. Second, we very much appreciate the wish to fully understand the mechanism of Homer1b/c delivery from dendritic shaft to spines and the role of SNX6 in this process. We apologize for not being able to explain explicitly that our current findings do not suggest that SNX6 is directly responsible for transport of Homer1b/c, whether it associates with vesicles or not, from dendritic shaft to spines. Besides the live imaging data showing that the motility of Homer1b/c vesicles is greatly impaired in SNX6 knockout neurons, IF staining and microscopy analysis show that Homer1b/c distribution in distal dendrites is reduced in the absence of SNX6, indicating that long-range vesicular transport of Homer1b/c from the cell body to dendrite is SNX6-dependent. To verify that failure of Homer1b/c transport to distal dendrites leads to its loss from dendritic spines and decrease in spine density of distal dendrites, we went back to the original confocal images and quantified spine localization of Homer1b/c over distance from the soma. The results are as expected and are in good agreement with a decrease in Homer1b/c signals in distal dendrite (Author response image 2), which we would incorporate in the manuscript. Although we did detect SNX6 signals in spines by IF staining, we have not obtained any direct evidence showing that transport of Homer1b/c into spines, whether microtubule- and dynein-dependent or not, is mediated by SNX6. Therefore, our main conclusion in this study is that SNX6 mediates long-range transport of Homer1b/c in dendritic shaft, which is required for its synaptic localization.

Author response image 2
DIV14 neurons were cotransfected with constructs overexpressing EGFP and mCherry or mCherry-SNX6, fixed on DIV16 and immunostained with antibodies to Homer1b/c.

Shown is quantification of Homer1b/c signal intensity in dendritic spines over distance from the cell body (mean ± SEM, n = 30, N = 3).

https://doi.org/10.7554/eLife.20991.043

To determine whether SNX6 is also required for delivery of Homer1b/c from shaft to spines, which is short-distance transport compared with long-range movement on microtubule tracks, we need to test the role of microtubules and actin filaments as well as dynein and the actin-based motor myosin, which is beyond the scope of this manuscript. We appreciate the editor/reviewer suggesting the FRAP experiment to determine whether Homer1b/c enters dendritic spine by active transport or diffusion. However, since overexpressed Homer1c-EGFP is in excess, in addition to its presence on vesicles, lots of the molecules are cytosolic, and those that have been delivered to spine incorporate into the PSD, forming higher-order polymerized complex with Shank that is necessary for the structural and functional integrity of spines (Bosch et al., 2014; Hayashi et al., 2009; Meyer et al., 2014), As a consequence of overexpression, there would be three pools of Homer1c-EGFP, namely vesicular, cytosolic and PSD-associated. Given that Homer1 in spines complexes with other PSD scaffold proteins to form a matrix structure, for neurons at steady state, it is highly unlikely the PSD-associated Homer1 exchanges with the free molecules in the cytosolic pool. Instead, we would rather expect addition of Homer1 to the expanding PSD during synaptic plasticity when the spine head expands. However, testing this idea is beyond the scope of this manuscript. Besides, if there are plenty of proteins in the cytosol, most likely those free molecules enter and leave spines by diffusion, which might just be an overexpression artifact. So we would expect that FRAP measures the diffusion rate of cytosolic Homer1 in spine and shaft area adjacent to the spine, rather than exchange between the synaptically incorporated and the cytosolic pools. As for vesicular Homer1, since we have already found that only ~ 10% of Homer1 puncta are motile (similar to PSD95 puncta in the 2006 Gerrow paper), considering the limited number of spines in the viewfield of high magnification lens under microscope, it is technically very difficult to catch those vesicles entering spines if there are any, let alone monitor the exchange of the motile puncta with synaptic Homer1 by FRAP. Moreover, previous studies using FRAP to measure mobility of PSD95 bound in the PSD do not provide strong support for the presumption that there is rapid exchange between synaptic and cytosolic PSD scaffold protein molecules (Blanpied et al., 2008). Again, we appreciate the wonderful question and suggestion from reviewers, and we would be more than happy to try our best to do the FRAP experiment. However, with the caveats and potential problems discussed above, FRAP results might not provide useful information to answer the question whether SNX6 is required for delivery of Homer1 from shaft to spine but rather means for investigation of Homer1 turnover in spine.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

In the revised version of the paper, the authors performed the experiments suggested by the reviewers. The results most relevant to the overall message of the paper are that the labeled Homer1c structures only very rarely enter spines and that photobleached Homer1c in spines shows SNX6-independent recovery by 50% within 10 minutes through exchange with the soluble pool. These are very significant results, because together, they provide strong support for the view that Homer1c is not delivered into spines on endosomes but simply exchanges with the soluble pool in a SNX6-independent manner, which is not unexpected. Since most of dendritic Homer1c is present in spines, but vesicular transport is not responsible for delivering Homer1c into spines, the role of SNX6-dependent vesicle transport in "dendritic delivery of Homer1b/c" becomes confusing. Is it needed to redistribute soluble Homer1c through dendrites, so that it can then diffuse into spines? Or does Homer1c on endosomes represent part of a Homer1c pathway distinct from Homer1c function in dendritic spines? Based on the data presented in the paper, these questions are impossible to address. However, the data as they stand now certainly bring the importance of the observed SNX6-dependent transport of Homer1c on endosomes into question.

Given the large amount of work the authors have invested in revising the paper, the good quality of much of the data and the complexity of the problem, the reviewers felt that the paper can still be published. However, it would be essential to very thoroughly revise the text of the paper in a way that would do justice to the data shown, and not just to the model that the authors were trying to prove. To achieve this, it would be essential to very strongly downplay the "SNX6-mediated transport and delivery" angle, particularly by removing it from the title, re-writing the Abstract and Introduction, adding the data on FRAP and the failure to detect vesicle-based delivery of Homer1c into spines into the main figures, and writing a very balanced discussion. The revised version of the paper should include all the data but should not attempt to create an impression that the authors have proven that the phenotypes of SNX6 knockout mice are due to altered transport and delivery of Homer1c on SNX6-positive endosomes into dendritic spines, thus explaining the spine phenotypes observed. The reviewers would like to emphasize that "cosmetic" changes to the writing will not be sufficient in this case.

It is possible that the authors might disagree with the opinion of the reviewers, and in this case they are advised to seek publication in another journal.

We would like to thank the editors and the reviewers for their thoughtful comments and advice. We have revised the text accordingly.

1) As requested by editors/reviewers, we have changed the title from “Dendritic Delivery of Homer1b/c by SNX6-mediated Long-range Vesicular Transport” to “A Neuronal Role for SNX6 in Dendritic Trafficking of the Postsynaptic Scaffold Protein Homer1b/c”.

2) We revised the Abstract as the following:

“SNX6 is a ubiquitously expressed PX-BAR protein that plays important roles in dynein‒dynactin-driven, retromer-mediated retrograde vesicular transport from endosomes. Here we show that CNS-specific Snx6 knockout mice exhibit deficits in spatial learning and memory, accompanied with loss of spines from distal dendrites of hippocampal CA1 pyramidal cells. We find that SNX6 functions independently of retromer to mediate dynein‒dynactin-driven dendritic vesicular transport of Homer1b/c, a postsynaptic scaffold protein crucial for synaptic distribution of other postsynaptic proteins and structural integrity of dendritic spines. Ablation of SNX6 causes loss of Homer1b/c from distal dendrites as well as decreases in surface levels of AMPAR and AMPAR-mediated synaptic transmission. As trafficking of Homer1b/c from its site of biosynthesis to dendrites is vital for synapse formation and functioning, these findings reveal an important physiological role of SNX6-mediated long-range vesicular transport in CNS neurons.”

We also revised the Introduction as requested, adding a paragraph to distinguish trafficking of postsynaptic proteins from the cell body to dendrites from their trafficking from dendritic shaft to spines (please see highlighted text in the Introduction). Accordingly, in the Results section we present data to show that SNX6 is required for transport of Homer1b/c from the cell body to dendrites, but not translocation of Homer1b/c from shaft to spines. To do this, we added the data on FRAP, live imaging and quantitative results from confocal microscopy to the main figures as new Figure 8, under a new subtitle “Translocation of Homer1b/c from dendritic shaft to spines is SNX6-independent”, after the data showing that SNX6 mediates dynein-driven transport of Homer1b/c to distal dendrites.

3) We agree with editor/reviewers’ view that Homer1c is not delivered into spines on endosomes but rather in the form of free cytosolic proteins. We have modified the Discussion to make it more balanced. We modified the opening paragraph to tone down our conclusions, discussed possible mechanisms for translocation of Homer1b/c from shaft to spines, and discussed our new data indicating that entry of Homer1b/c into spines is SNX6-independent as the following:

“Once in dendrites, several mechanisms exist for transfer of postsynaptic components from shaft to synaptic sites in spines, including cytosolic diffusion, exocytosis of transmembrane proteins at the plasma membrane and lateral diffusion to synaptic sites, and active transport by molecular motors. The AMPARs enter dendritic spines via both lateral diffusion and actin-based, Myosin V-driven transport of recycling endosomes (Adesnik et al.et al., 2005; Correia et al.et al., 2008; Makino and Malinow, 2009; Wang et al.et al., 2008; Yudowski et al.et al., 2007). Since Homer1b/c is a scaffolding protein, its entry into spines might rely on diffusion of free molecules, possibly released from endosomal carriers, or transport of the vesicular cargo directly from the shaft by a different motor. Our results indicate that direct spine entry of Homer1c vesicles is an extremely rare event, and that neither spine:shaft ratio nor dynamic turnover of Homer1b/c in spines is affected by ablation of SNX6, suggesting that most likely Homer1b/c enters spines in the form of free cytosolic molecules.”

4) Following the editors’ and reviewers’ advice, to interpret the data precisely, we also changed one of the subtitles from “Dendritic vesicular transport and spine localization of Homer1b/c require SNX6” to “Dendritic vesicular transport of Homer1b/c requires SNX6”.

Should you decide to resubmit, please address this additional point: the data of panel K of Figure 8 on the surface staining of mGlu5 do not look convincing. Please move them to the Supplement and move the supplementary biochemical data (Figure 8—figure supplement 1) to the main figure.

Changes made as requested.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Specifically, in the previous decision letters we have very clearly indicated that we cannot offer to publish your paper in the current form, because the reviewers found that your major claim that " SNX6 mediates long-range vesicular transport of Homer1b/c to distal dendrites" is not sufficiently supported by data. With other words, it is possible that SNX6 indeed mediates long-range transport of Homer1b/c, but it is also possible that Homer1b/c arrives to the dendritic spines by diffusion alone, and the interactions that you described serve another function (if any). Given the complexity of the subject and the large amount of work that you have put into revising this manuscript, we have offered you an opportunity to completely and thoroughly revise the whole paper in a way that would make it clear to the reader from the start to the end that your transport-related conclusions are not more than one of the possible explanations of the mouse phenotypes that you nicely describe. Such re-writing would entail not a minor but a major revision of your paper. Given your new suggestion for the title and the minor edits you have done, it is very clear to us that you are not willing to perform such a major revision. We of course fully respect your decision, because this is your paper, and you should write it in the way you find most appropriate. We would like to emphasize that your English writing is perfectly fine, and that our negative decision is only due to the fact the reviewers were not convinced that your major conclusion is sufficiently supported by data. We would also like to emphasize that since the reviewers are not co-authors of your paper, it is not their task to search for "sentences or words in the manuscript still misleading or overstated" – it is up to authors to make such changes.

We are willing to give you one more chance to re-write the paper. However, you would need to really thoroughly rethink and revise the whole manuscript to provide a more balanced view of different ways in which their data can be explained. You are very much in favor of pushing through the model that SNX6 mediates transport of Homer1b/c on vesicles, but the experimental support for this model is insufficient and the FRAP data directly contradict it. Therefore, the paper needs to be re-written in a way that would make it clear to the reader that transport of Homer1b/c on endosomes is not more than one of the possible explanations of the observed mouse phenotypes. Trying to solve the problem by adding a few disclaimers here and there is not enough, and also not what we have asked for. For example, in the latest version, you proposed to revert to the title with transport being the main message, while we asked for this change one revision before.

We thank the editors and reviewers for their thoughtful comments and advice. After careful scrutiny of our data and conclusions, we realized that indeed, first, SNX6-mediated transport serves as just one of the possible mechanisms for Homer1 distribution in distal dendrites, and there are other possibilities that have not been explored in our current study. Second, defects in Homer1 distribution might also provide just one of the explanations for the mouse phenotypes we observed. Besides Homer1, SXN6 might interact with proteins that also play important roles in synaptic structure and function, a possibility we did not discuss in the previous versions of the manuscript. We also thank the reviewer for the great suggestion on FRAP, which shows SNX6-independent diffusion of free Homer1c molecules into the spine and dynamic exchange between the synaptic and the soluble pools. The FRAP data, in combination with the results from live imaging and quantitative analysis of the spine:shaft ratio of endogenous Homer1b/c, indicates that SNX6 and vesicular transport are not involved in its translocation from shaft to spines, and that Homer1 can enter spines by diffusion.

Therefore, we have revised the text throughout the manuscript to make the points above clear to the reader. Since it is very difficult to reorganize the huge amount of data in this study, we did not change the data organization of the manuscript significantly. Nevertheless, we have changed the title, rewritten the Abstract and revised the concluding paragraph of the Introduction; we have also revised the results, reorganized Figures 5 and 6 to downplay the contribution of SNX6-mediated vesicular transport to the phenotypes, changed the conclusions for some of the subsections, and changed some of the subtitles for figures and results accordingly; we have also rewritten the Discussion section to provide a more critical and balanced review of our data. Specifically, we have made it clear that ablation of SNX6 only impairs the motility of a fraction of Homer1c molecules on vesicles in the dendritic shaft; we have also pointed out that mechanisms other than vesicular transport could contribute to the distribution of Homer1b/c in distal dendrites in both Results and Discussion; in addition to data presented in Results, discussion about the possible mechanisms for shaft to spine translocation of Homer1 and our conclusions in Discussion, we have added the conclusion on SNX6-independent Homer1 diffusion into spine in both Abstract and Introduction. The major changes in the revised manuscript are listed below.

1) To summarize our findings on the physiological role of SNX6 in CA1 neurons, we have changed the Title to “Ablation of SNX6 leads to defects in synaptic function of CA1 pyramidal neurons and spatial memory”. We think this descriptive title is the most objective.

2) We revised the Abstract as follows:

“SNX6 is a ubiquitously expressed PX-BAR protein that plays important roles in retromer-mediated retrograde vesicular transport from endosomes. Here we report that CNS-specific Snx6 knockout mice exhibit deficits in spatial learning and memory, accompanied with loss of spines from distal dendrites of hippocampal CA1 pyramidal cells. SNX6 interacts with Homer1b/c, a postsynaptic scaffold protein crucial for synaptic distribution of other postsynaptic density (PSD) proteins and structural integrity of dendritic spines. We show that SNX6 functions independently of retromer to regulate distribution of Homer1b/c in the dendritic shaft. We also find that Homer1b/c translocates from shaft to spines by protein diffusion, which does not require SNX6. Ablation of SNX6 causes reduced distribution of Homer1b/c in distal dendrites, decrease in surface levels of AMPAR and impaired AMPAR-mediated synaptic transmission. These findings reveal a physiological role of SNX6 in CNS excitatory neurons.”

3) To revise the Introduction, in the opening paragraph we described the known functions of SNX6 and the rationale for this study. In the second paragraph we introduced functions of the PSD scaffold proteins in dendritic spines and raised the question about mechanisms for their proper distribution in dendrites. In the last paragraph we described our main findings as follows:

“In this study, we investigated the physiological function(s) of SNX6 in mouse CNS neurons using multiple approaches including mouse genetics, behavior assays and electrophysiology, biochemistry and fluorescence imaging. Ablation of SNX6 in the CNS causes deficits in spatial learning and memory, decrease in spine density of the distal dendrites of hippocampal CA1 neurons and impairment of their AMPAR-mediated synaptic transmission, suggesting a role for SNX6 in synaptic structure and function. SNX6 interacts with Homer1b/c and loss of SNX6 leads to reduction in its distribution in distal dendrites. Intriguingly, although SNX6 is required for the motility of a subpopulation of Homer1c on vesicles in dendritic shaft, live imaging and FRAP analyses indicate that Homer1c enters dendritic spines via protein diffusion but not SNX6-dependent active transport. Overexpression of SNX6 or Homer1c restores the spine density and AMPAR surface levels of Snx6-/- neurons. These findings uncover a physiological function for SNX6 in hippocampal CA1 excitatory neurons.”

4) In the Results section, to downplay the role of SNX6-mediated transport in dendritic distribution of Homer1b/c, we moved the data on live imaging of Homer1c from Figure 5 to Figure 6. We hope that this change would help to prevent creating the impression that reduced Homer1b/c distribution in distal dendrite is solely caused by defect in SNX6-mediated vesicular transport. In the new Figure 5, we present the data that ablation of SNX6 causes reduced distribution of Homer1 in distal dendrites. In Figures 68, we present a series of data obtained in an effort to understand mechanism(s) underlying the Homer1b/c distribution phenotype. First we present the data about the effect of SNX6 knockout on the motility of Homer1 vesicles in the dendritic shaft, and the involvement of dynein-dynactin in Homer1 distribution in dendrite (new Figure 6). Then we explore the possibility whether two known trafficking pathways are involved in dendritic trafficking of Homer1 (Figure 7). In Figure 8, we present data on SNX6-independent translocation of Homer1b/c from shaft to spines via protein diffusion, indicating that SNX6 only functions in distribution of Homer1b/c in the dendritic shaft but not its spine localization.

In addition, we have also modified the text, replacing “SNX6-mediated transport” with “SNX6-regulated dendritic distribution” wherever applicable to avoid confusion or overstatement about the role of vesicular transport in regulating dendritic distribution of Homer1b/c.

5) We have rewritten the Discussion to make it more balanced. To this end, we changed the focus of discussion from SNX6’s role in trafficking to mechanisms underlying dendritic distribution and synaptic localization of Homer1c. We not only discussed alternative mechanisms for dendritic distribution of Homer1b/c, but also removed the part discussing the potential role of SNX6-mediated vesicular transport of Homer1 in synaptic plasticity.

i) In the opening paragraph, we described our findings on the mouse phenotypes caused by SNX6 knockout:

“In this study, we demonstrate that ablation of SNX6 in the CNS causes deficits in spatial learning and memory, a hippocampal-dependent brain function. At the cellular level, loss of SNX6 causes decrease in spine density in the distal apical dendrites of CA1 hippocampal cells and impairment of their AMPAR-mediated synaptic transmission, indicating that SNX6 is required for synaptic structure and function of these excitatory neurons. We also show that SNX6 directly interacts with Homer1b/c, a PSD scaffolding protein crucial for the structural and functional integrity of dendritic spines, and that there is decrease in Homer1b/c distribution in distal dendrites in Snx6-/- neurons. Moreover, the spine density and surface AMPAR level phenotypes of Snx6-/- neurons could be rescued by overexpressing Homer1b/c or SNX6. These findings reveal an important physiological function of SNX6 in the CNS excitatory neurons.”

ii) In the second paragraph, in addition to discussion about the link between Homer1b/c expression in CA1 neurons and impairment in the CA3-CA1 pathway-dependent brain function of SNX6 knockout animals, we discussed the possibilities that 1) it could function via interaction with neuronal proteins other than Homer1 and 2) SNX6 could function in synaptic structure and function of neurons in the cortex:

“…Whether there are other SNX6-interacting proteins that are also required for CA1 neuron function awaits further investigation. As spatial learning and memory involve not only the hippocampus but also other cortical areas such as the entorhinal cortex and the medial prefrontal cortex (Jo et al., 2007; Nagahara et al., 1995; Nakazawa et al., 2004; Steffenach et al., 2005; Zhou et al., 1998), it also remains to be determined whether and how ablation of SNX6 affects the synaptic structure and function of neurons in other parts of the cortex.”

iii) In the third paragraph, we discussed the possibility that SNX6 functions via mechanism(s) distinct from dynein-driven transport to regulate dendritic distribution of Homer1, and proposed alternative mechanisms such as diffusion, cotransport with other proteins and active transport driven by different motor/adaptor.

“…We found that the motility of Homer1c-associated vesicles in dendritic shaft requires SNX6, and that ablation of SNX6 or inhibition of dynein-dynactin activity causes reduction in the amount of Homer1b/c in distal dendrites. Previously imaging assays and quantitative modeling have established that dynein-driven bidirectional transport contributes to polarized targeting of dendrite-specific cargo (Kapitein et al., 2010). Therefore, lack of dynein‒dynactin-driven transport in the dendritic shaft provides a possible mechanism for the Homer1b/c distribution phenotype in Snx6-/- neurons. However, since the majority of Homer1c puncta are immobile in dendrites of steady-state neurons, and little is known about the cellular functions of SNX6 apart from its role as dynein cargo adaptor, it is also possible that SNX6 regulates the distribution of Homer1b/c in dendrites via mechanism(s) distinct from dynein‒dynactin-driven transport. Moreover, ablation of SNX6 does not cause complete loss of Homer1b/c from distal dendrites, indicating that mechanism(s) other than SNX6-mediated transport contributes to its localization to dendritic shaft far from the cell body. Since disruption of the secretory pathway does not affect Homer1b/c localization to dendritic shaft and spines, alternative mechanisms for its distribution in dendrites include diffusion of free protein molecules, cotransport with proteins other than the PSD95-GKAP-Shank complex or vesicular transport mediated by different motor(s) and/or adaptor(s).”

iv) In the fourth paragraph, first we described the observation that the spine:shaft ratio of Homer1b/c did not change in distal dendrites in SNX6 KO neurons, then we presented possible mechanisms for translocation of Homer1b/c from shaft to spines, finally we discussed our data indicating that entry of Homer1b/c into spines via SNX6-independent protein diffusion.

“Notably, in Snx6-/- neurons, although there was a decrease in the amount of Homer1b/c in both shaft and spines of distal dendrites (Figure 5F), the spine:shaft ratio of its signals remained constant throughout the dendrite (Figure 8B), indicating that once in dendrite, Homer1b/c could enter the spines via SNX6-independent mechanism(s). In dendrites, several mechanisms exist for transfer of postsynaptic components from shaft to synaptic sites in spines, including cytosolic diffusion, exocytosis of transmembrane proteins at the plasma membrane and their lateral diffusion to synaptic sites, and active transport by molecular motors. The AMPARs enter dendritic spines via both lateral diffusion and actin-based, Myosin V-driven transport of recycling endosomes (Adesnik et al., 2005; Correia et al., 2008; Makino and Malinow, 2009; Wang et al., 2008; Yudowski et al., 2007). Since Homer1b/c is a scaffolding protein, its entry into spines might rely on diffusion of free molecules, possibly released from endosomal carriers or from the cytosolic pool in the shaft, or transport of the vesicular cargo directly from the shaft by a different motor. Our results from live imaging, FRAP and quantitative analyses show that direct spine entry of Homer1c puncta is an extremely rare event, and that the dynamic turnover of Homer1c in spines is not affected by ablation of SNX6. Collectively these data indicate that in steady-state neurons, Homer1b/c enters spines by cytosolic diffusion, and SNX6 is not required for its spine localization.”

v) In the last paragraph, we discussed the findings that SNX6 does not require retromer activity to regulate dendritic distribution of Homer1b/c as well as the potential roles of SNX family members in the CNS. We replaced “SNX6-mediated dendritic transport of Homer1b/c” with “SNX6-regulated distribution of Homer1b/c in dendritic shaft”.

6) Corresponding changes in subtitles in Results are listed below:

– “SNX6 associates with Homer1b/c on vesicles in dendritic shaft” is replaced with “SNX6 directly interacts with Homer1b/c”.

– “Dendritic vesicular transport of Homer1b/c requires SNX6” is replaced with “Ablation of SNX6 causes decrease in distribution of Homer1b/c in distal dendrites”.

– “SNX6-mediated dendritic transport of Homer1b/c is driven by dynein−dynactin” is replaced with “Active transport of a fraction of Homer1b/c molecules in the dendritic shaft requires SNX6”.

– “The SNX6-mediated Homer1b/c transport pathway is distinct from other trafficking pathways in dendrite” is replaced with “The Homer1b/c trafficking pathway is distinct from the PSD95 and secretory trafficking pathways in dendrite”.

– “Translocation of Homer1b/c from dendritic shaft to spines is SNX6-independent” is replaced with “Translocation of Homer1b/c from dendritic shaft to spines is SNX6- and vesicular transport-independent”.

– “Changes in synaptic transmission and receptor trafficking in Snx6-/- neurons” is replaced with “Ablation of SNX6 causes impairment of AMPAR-mediated synaptic transmission and decrease in AMPAR surface expression”.

– “SNX6-mediated dendritic vesicular transport of Homer1b/c is retromer-independent” is replaced with “Activity of the retromer core complex is not required for SNX6-regulated dendritic distribution of Homer1b/c”.

7) Corresponding changes in subtitles for the Figures are listed below:

Figure 5. “SNX6 mediates vesicular transport of Homer1b/c to synaptic sites in dendrites” is replaced with “Partial loss of Homer1b/c from distal dendrites of Snx6-/- neurons”.

Figure 6. “SNX6-mediated vesicular transport of Homer1b/c requires dynein−dynactin activity” is replaced with “SNX6 is required for motility of Homer1b/c vesicles in dendritic shaft and their association with dynein−dynactin”.

Figure 8. “Translocation of Homer1b/c from dendritic shaft to spines does not require SNX6” is replaced with “Homer1b/c enters spines by SNX6-independent protein diffusion”.

Figure 9. “Altered synaptic structure and function in SNX6-deficient neurons” is replaced with “Impaired synaptic transmission and decreased surface AMPAR levels of Snx6-/- neurons”.

Figure 10. “Long-range vesicular transport and synaptic localization of Homer1b/c is retromer-independent” is replaced with “The retromer core complex is not required for SNX6-regulated dendritic distribution of Homer1b/c”.

https://doi.org/10.7554/eLife.20991.045

Article and author information

Author details

  1. Yang Niu

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    3. Graduate School, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    YN, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Zhonghua Dai and Wenxue Liu
    Competing interests
    The authors declare that no competing interests exist.
  2. Zhonghua Dai

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    Contribution
    ZD, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Yang Niu and Wenxue Liu
    Competing interests
    The authors declare that no competing interests exist.
  3. Wenxue Liu

    1. Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China
    2. State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
    3. MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, Nanjing, China
    4. Model Animal Research Center, Nanjing University, Nanjing, China
    Contribution
    WL, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Yang Niu and Zhonghua Dai
    Competing interests
    The authors declare that no competing interests exist.
  4. Cheng Zhang

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    Contribution
    CZ, Formal analysis, Investigation, Visualization, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  5. Yanrui Yang

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    Contribution
    YY, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  6. Zhenzhen Guo

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    3. Graduate School, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    ZG, Data curation, Formal analysis, Investigation, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  7. Xiaoyu Li

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. Graduate School, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    XL, Data curation, Formal analysis, Investigation, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  8. Chenchang Xu

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    3. Graduate School, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    CX, Formal analysis, Investigation, Methodology, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  9. Xiahe Huang

    State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    Contribution
    XH, Data curation, Formal analysis, Investigation, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  10. Yingchun Wang

    State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    Contribution
    YW, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  11. Yun S Shi

    1. State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
    2. MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, Nanjing, China
    3. Model Animal Research Center, Nanjing University, Nanjing, China
    Contribution
    YSS, Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    yunshi@nju.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
  12. Jia-Jia Liu

    1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
    Contribution
    J-JL, Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    jjliu@genetics.ac.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6099-1059

Funding

National Natural Science Foundation of China (31530039)

  • Yang Niu
  • Zhonghua Dai
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Jia-Jia Liu

Ministry of Science and Technology of the People's Republic of China (2014CB942802)

  • Yang Niu
  • Zhonghua Dai
  • Wenxue Liu
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Yun S Shi
  • Jia-Jia Liu

State Key Laboratory of Molecular Developmental Biology, China (2014-MDB-TS-01)

  • Yang Niu
  • Zhonghua Dai
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Xiahe Huang
  • Yingchun Wang
  • Jia-Jia Liu

National Natural Science Foundation of China (31325017)

  • Yang Niu
  • Zhonghua Dai
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Jia-Jia Liu

National Natural Science Foundation of China (31471334)

  • Yang Niu
  • Zhonghua Dai
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Jia-Jia Liu

Ministry of Science and Technology of the People's Republic of China (2016YFA0500100)

  • Yang Niu
  • Zhonghua Dai
  • Wenxue Liu
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Yun S Shi
  • Jia-Jia Liu

Ministry of Science and Technology of the People's Republic of China (2014CB942804)

  • Yang Niu
  • Zhonghua Dai
  • Wenxue Liu
  • Cheng Zhang
  • Yanrui Yang
  • Zhenzhen Guo
  • Xiaoyu Li
  • Yun S Shi
  • Jia-Jia Liu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. Michael Davidson, Johannes Hell, Connie Cepko, A Kimberley McAllister, Vivek Malhotra, Li Yu and Hong Tang for providing reagents, Dr. Jian-Jun Yang (Jinling Hospital, School of Medicine, Nanjing University, China) for technical advices and Dr. You-ming Lu (Huazhong University of Science and Technology, China) for valuable advice in carrying out the Morris water maze tests. This work was supported by funding from the National Natural Science Foundation of China (31530039, 31325017 and 31471334 to J-J Liu), the Ministry of Science and Technology of China (2014CB942802 and 2016YFA0500100 to J-J Liu and 2014CB942804 to YS Shi) and State Key Laboratory of Molecular Developmental Biology (2014-MDB-TS-01 to J-J Liu).

Ethics

Animal experimentation: This study was performed in compliance with the guidelines of the Animal Care and Use committee of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Permit Number: AP2013001). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

Reviewing Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Publication history

  1. Received: August 28, 2016
  2. Accepted: January 28, 2017
  3. Accepted Manuscript published: January 30, 2017 (version 1)
  4. Version of Record published: February 23, 2017 (version 2)

Copyright

© 2017, Niu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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