Ablation of SNX6 leads to defects in synaptic function of CA1 pyramidal neurons and spatial memory
- Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- Jinling Hospital, School of Medicine, Nanjing University, China
- Nanjing University, China
Figures
Figure 1 with 1 supplement
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 …
Figure 1—figure supplement 1
Expression and subcellular distribution of SNX6 in the CNS.
(A) Western blotting of wild-type mouse tissue lysates. (B) A schematic showing the relative positions of coronal sections in (D) and (E) in sagittal view. (C–E) Immunohistochemical analysis of SNX6 …
Figure 2
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), …
Figure 3
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 …
Figure 4 with 1 supplement
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 …
Figure 4—figure supplement 1
Colocalization analysis of superresolution images of triple-stained neurons captured by 3D-SIM (15 neurons for each immunostaining experiment).
Unlike colocalization analysis of 2D confocal images of double-stained neurons in other figures, we adopted the methodology developed by Fletcher et al. to measure voxel-based colocalization between …
Figure 5 with 2 supplements
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 …
Figure 5—figure supplement 1
Immunoblotting analysis of Homer1b/c and PSD95 in hippocampi from Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice.
β-actin serves as loading control. Shown are samples from three pairs of littermates. Data represent mean ± SEM.
Figure 5—figure supplement 2
Ablation of SNX6 causes decrease in spine density of CA1 but not CA3 neurons.
(A) Hippocampal neurons in dissociated culture were transfected with pLL3.7.1 on DIV14, fixed on DIV18 and immunostained with antibodies to CTIP2, a transcription factor specifically expressed in DG …
Figure 6 with 2 supplements
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 …
Figure 6—figure supplement 1
Overexpressed mCherry-SNX6 and mEmerald-Homer1c recapitulate the distribution of endogenous proteins in hippocampal neurons.
(A) Hippocampal neurons in dissociated culture were transfected with construct expressing SNX6 on DIV14, digitonin extracted and fixed on DIV16 and immunostained with antibodies to EEA1, Homer1b/c, …
Figure 6—figure supplement 2
SNX6-, Homer1c-positive puncta move bidirectionally in the dendritic shaft.
(A) SNX6-, Homer1c-positive puncta in proximal dendrite move away from the cell body. Shown are the first frames of motile SNX6-, Homer1c-positive puncta in boxed areas (Left panel, black arrow …
Figure 7
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 …
Figure 8
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 …
Figure 9 with 4 supplements
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 …
Figure 9—figure supplement 1
Cre-mediated knockout of SNX6 in cultured neurons.
Hippocampal neurons in dissociated culture from Snx6fl/fl mice were infected with AAVs expressing pAOV-CaMKIIα-EGFP-2A-3Flag or pAOV-CaMKIIα-EGFP-2A-Cre on DIV12, fixed on DIV18 and immunostained …
Figure 9—figure supplement 2
Ablation of SNX6 causes an increase in surface expression of mGluR5 in hippocampal neurons, which is rescued by SNX6 or Homer1c.
(A) DIV13 neurons were 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-expressed …
Figure 9—figure supplement 3
Homer1a overexpression increases surface mGluR5 expression.
This experiment serves as a control for Figure 9—figure supplement 2 and is in agreement with previous reports (Ango et al., 2002). (A) Hippocampal neurons were co-transfected with pLL3.7.1 and …
Figure 9—figure supplement 4
Ablation of SNX6 causes a decrease in the number of clathrin positive synapses.
(A) Hippocampal neurons in dissociated culture from Snx6fl/fl and Nestin-Cre; Snx6fl/fl littermates were immunostained with antibodies to clathrin heavy chain and PSD95. Dendritic spines are …
Figure 10 with 2 supplements
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 …
Figure 10—figure supplement 1
VPS35 is efficiently knocked down in HEK293 cells.
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 hr. The relative amount of VPS35 was …
Figure 10—figure supplement 2
Colocalization of Homer1b/c with VPS35 does not require SNX6 and vice versa.
(A) Hippocampal neurons in dissociated culture from Snx6fl/fl and Nestin-Cre; Snx6fl/fl littermates were transfected with pLL3.7.1 on DIV14, immunostained with antibodies to Homer1b/c and VPS35 on …
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 …
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).
Videos
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
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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
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 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 …
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 …
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 …
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 …
Tables
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).
| voxel colocolization values (%) / | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| EEA1-SNX6-Homer1b/c | |||||||||||||||
| EEA1 with SNX6 and Homer1b/c | 15.68/ | 12.05/ | 17.48/ | 15.18/ | 16.92/ | 16.48/ | 15.33/ | 18.86/ | 15.76/ | 11.96/ | 21.87/ | 15.98/ | 14.58/ | 16.76/ | 19.04/ |
| SNX6 with EEA1 and Homer1b/c | 18.56/ | 13.12/ | 17.28/ | 13.96/ | 17.44/ | 12.36/ | 15.92/ | 22.64/ | 15.08/ | 14.32/ | 16.34/ | 19.92/ | 15.12/ | 15.44/ | 17.56/ |
| Homer1b/c with EEA1 and SNX6 | 14.24/ | 13.16/ | 13.64/ | 10.22/ | 16.28/ | 16.84/ | 13.24/ | 13.92/ | 14.84/ | 14.52/ | 18.56/ | 15.36/ | 13.92/ | 13.44/ | 17.63/ |
| p150Glued-SNX6-Homer1b/c | |||||||||||||||
| p150Glued with SNX6 and Homer1b/c | 15.63/ | 8.13/ | 10.06/ | 10.06/ | 10.06/ | 10.06/ | 10.06/ | 10.06/ | 10.06/ | 7.62/ | 16.38/ | 9.35/ | 8.85/ | 9.97/ | 9.21/ |
| SNX6 with Homer1b/c and p150Glued | 14.57/ | 12.89/ | 10.41/ | 13.02/ | 10.18/ | 12.67/ | 18.49/ | 12.33/ | 9.51/ | 9.54/ | 23.55/ | 11.24/ | 9.39/ | 9.18/ | 10.7/ |
| Homer1b/c with SNX6 and p150Glued | 10.32/ | 8.27/ | 8.33/ | 7.15/ | 9.66/ | 9.8/ | 13.16/ | 8.49/ | 8.73/ | 8.69/ | 15.07/ | 9.17/ | 8.24/ | 8.43/ | 9.03/ |
| DIC-SNX6-Homer1b/c | |||||||||||||||
| DIC with SNX6 and Homer1b/c | 9.21/ | 9.65/ | 20.05/ | 7.9/ | 13.41/ | 15.25/ | 9.91/ | 10.88/ | 9.41/ | 8.53/ | 10.61/ | 9.64/ | 9.78/ | 10.98/ | 16.12/ |
| SNX6 with DIC and Homer1b/c | 12.00/ | 13.19/ | 14.91/ | 12.02/ | 13.07/ | 15.49/ | 11.32/ | 9.81/ | 10.1/ | 16.07/ | 10.79/ | 12.48/ | 11.77/ | 9.86/ | 23.11/ |
| Homer1b/c with DIC and SNX6 | 11.43/ | 9.81/ | 13.78/ | 9.82/ | 10.71/ | 10.86/ | 9.77/ | 9.82/ | 9.22/ | 10.36/ | 9.75/ | 7.87/ | 9.73/ | 10.21/ | 17.17/ |
