Neurexophilin4 is a selectively expressed α-neurexin ligand that modulates specific cerebellar synapses and motor functions
- Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
Figures
Figure 1 with 1 supplement
Nxph4 expression marks the components of select brain circuits.
(A–D) β-galactosidase staining of adult Nxph4βgeo/+ mice shows signals in mammillary body-related circuits (A), circumventricular organs (B–C), and cerebellar-vestibular circuits (D). Blue staining …
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Figure 1—source data 1
Brain regions expressing Nxph4.
- https://doi.org/10.7554/eLife.46773.004
Figure 1—figure supplement 1
Generation of Nxph4-βgeo knock-in mouse and characterization of Nxph4 expression by β-galactosidase staining and in situ hybridization.
(A) Targeting scheme to generate Nxph4-βgeo allele. Doted lines indicate homologous recombination sites. ES cells with correct Nxph4-βgeo allele were obtained from KOMP. (B) Genotype analysis of …
Figure 2 with 1 supplement
Nxph4 KO mice displayed multiple neurological deficits.
(A) Plots of weight as a function of age (male n = 13, female n = 16–18; #, difference between HET and KO; * difference between WT and KO). (B) Latency to fall from the accelerating rotarod plotted …
Figure 2—figure supplement 1
Nxph4 KO mice gained less weight but have normal locomotor and righting reflex functions.
(A) RNA in situ hybridization with a probe against Nxph4 only detected Nxph4 signal in WT but not KO mice. Scale bars: 2 mm. (B) RT-qPCR from cerebellar samples with specific primers for Nxph4 (n = 3…
Figure 3 with 2 supplements
Nxph4 is a glycosylated protein that can be detected in the synaptosomes.
(A) A domain model of Nxph4 (adapted from Missler and Südhof, 1998). I: signal peptide; II: a variable domain; III: a conserved domain; IV: a linker region; V: C-terminal domain. Positions of …
Figure 3—figure supplement 1
Generation and characterization of Nxph4-3xFLAG knock-in mice.
(A) A schematic diagram of the strategy to generate an Nxph4-3xFLAG knock-in allele. An sgRNA is designed to excise the coding region corresponding to the C-terminal region of Nxph4 third domain. In …
Figure 3—figure supplement 2
Validation of synaptosomes preparation.
(A) Immunoblot analysis of fractions derived from different steps of synaptosomal preparation. Synapse specific protein PSD-95 showed enriched signal in purified synaptosomes as expected. Somatic …
Figure 4 with 1 supplement
Nxph4 forms a complex with α-neurexin in vivo.
(A) Synaptosomes (tissue used: olfactory bulb, hypothalamus, midbrain, hindbrain, and the cerebellum) from Nxph4-3xFLAG KI or WT (negative control) mice were precipitated with an antibody against …
Figure 4—figure supplement 1
Nxph4 interacts with neurexin1α in vitro and the Nxph4-6A is secreted and glycosylated.
(A) HA-neurexin1α and Nxph4-3xFLAG were co-expressed in HEK293T cells. Cell lysates were precipitated with beads conjugated with an anti-FLAG antibody. Bound proteins as well as input were analyzed …
Figure 5
Nxph4 interacts with GABAARs.
(A,B) Cerebellar synaptosomes from three Nxph4-3xFLAG KI or WT (negative control) mice were precipitated with an antibody against FLAG. Bound proteins as well as 2% input were analyzed by …
Figure 6 with 3 supplements
Loss of Nxph4 reduced inhibition onto cerebellar granule cells.
(A) A simplified diagram illustrating the inhibitory circuit of cerebellar granular layer and single-cell recording experimental design. GC: granule cells; GoC: Golgi Cells; pf: parallel fibers. …
Figure 6—figure supplement 1
Miniature IPSC decay and rise time were not affected in Nxph4 KO mice.
(A–B) Statistical analysis of mIPSC decay (A, t-test) and rise time (B, Mann-Whitney U test). Data are presented as mean ± SEM. n.s., not significant.
Figure 6—figure supplement 2
Deletion of Nxph4 KO did not affect mossy fibers-granule cell EPSC.
(A) Sample traces of granule cell EPSC, evoked by stimulating mossy fibers, exhibited paired-pulse depression at ISI (inter-stimulus interval) 50 ms and ISI 100 ms from WT and KO mice. (B–F) Summary …
Figure 6—figure supplement 3
Nxph4-3xFLAG KI mice showed normal mIPSC.
(A) Miniature IPSCs recorded from the KI and WT cerebellar granule cells. From left to right: representative mIPSC traces, mean mIPSC frequency, amplitude, rise, and decay (n = 11–14 cells; 5mice). …
Figure 7 with 1 supplement
Loss of Nxph4 reduced Golgi-granule inhibitory synapse number.
(A) Gephyrin and vGAT staining in the cerebellar granular layer indicates Golgi-granule inhibitory synapses. (B) Quantification of puncta co-expressing gephyrin and vGAT as an indicator of …
Figure 7—figure supplement 1
Nxph4 KO mice showed normal expression and localization of GABAARs.
(A) In the cerebellar granular layer, vGAT marks the Golgi cell inhibitory terminals and vGlut1 marks the mossy fiber endings. Scale bar: 20 μm. (B) Immunoblotting analysis of GABAARα1/6 in the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-FLAG M2 | Sigma-Aldrich | Cat# F1804 RRID:AB_262044 | (1:1000) |
| Antibody | Rabbit polyclonal anti-pan Neurexin-1 | Millipore | Cat# ABN161 RRID:AB_10917110 | (1:1000) |
| Antibody | Mouse monoclonal anti-HA1.1 | Bioledgend | Cat# 901513, RRID:AB_2565335 | (1:5000) |
| Antibody | Mouse monoclonal anti-Vinculin | Sigma-Aldrich | Cat# V9131, RRID:AB_477629 | (1:10000) |
| Antibody | Rabbit polyclonal anti-GABAARα1 | Synaptic System | Cat# 224 203, RRID:AB_2232180 | (1:1000) |
| Antibody | Rabbit polyclonal anti-GABAARα6 | Synaptic System | Cat# 224 603, RRID:AB_2619945 | (1:1000) |
| Antibody | Mouse monoclonal anti-Vglut1 | Millipore | Cat# MAB5502, RRID:AB_262185 | (1:1000) |
| Antibody | Guinea pig polyclonal anti-vGAT | Frontier Institute co. Ltd | Cat#: VGAT-GP-Af1000, RRID: AB_2571624 | (1:1000) |
| Antibody | Mouse monoclonal anti-beta Actin | Abcam | Cat#: ab20272 RRID:AB_445482 | (1:2000) |
| Antibody | Rabbit polyclonal anti-TGF beta 1 | Abcam | Cat#: ab92486, RRID:AB_10562492 | (1:1000) |
| Antibody | Rabbit polyclonal anti-MeCP2 | Zoghbi Lab | #0535 | (1:1000) |
| Antibody | Mouse monoclonal anti-Gephyrin | Synaptic system | Cat#: 14701, RRID:AB_887717 | (1:1000) |
| Antibody | Rabbit polyclonal anti- PSD-95 | Cell signaling | Cat # 2507, RRID:AB_561221 | (1:1000) |
| Recombinant DNA reagent | pLenti-mCherry | Addgene | Cat# 36084, RRID:Addgene_36084 | |
| Recombinant DNA reagent | pLenti-Nxph4-3xFLAG-mCherry | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
| Recombinant DNA reagent | pLenti--Nxph4-4Q-HA-mCherry | This paper | N/A | Results subsection ‘Nxph4 is a secreted glycoprotein’ |
| Recombinant DNA reagent | pLenti--Nxph4-6A-HA-mCherry | This paper | N/A | Results subsection ‘Nxph4 is a secreted glycoprotein’ |
| Recombinant DNA reagent | pCMV-Nxph4-HA | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
| Recombinant DNA reagent | pCMV-Nxph4-4Q-HA | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
| Recombinant DNA reagent | pCMV-Nxph4-6A-HA | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCAG-HA-Nrxn1β S4(+) | Addgene | Cat# 59410, RRID:Addgene_59410 | |
| Recombinant DNA reagent | pCAG-HA-Nrxn1β S4(-) | Addgene | Cat# 59409, RRID:Addgene_59409 | |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α | Addgene | Cat# 58266, RRID:Addgene_58266 | |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS1 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS2 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS3 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS4 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS5 | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCMV-GABARα1N-HA | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
| Recombinant DNA reagent | pCMV-GABARα6N-HA | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
| Chemical compound, drug | X-Gal | Thermo Fisher | Cat# 15520034 | |
| Chemical compound, drug | Lipofectamine 2000 | Thermo Fisher | Cat# 11668500 | |
| Commercial assay, kit | Antigen Unmasking Solution (citrate based) | Vector Laboratories | Cat# H-3300 | |
| Commercial assay, kit | Avidin/Biotin Blocking Kit | Vector Laboratories | Cat# SP-2001 | |
| Commercial assay, kit | TSA Kit | Invitrogen | Cat# T-20932 | |
| Commercial assay, kit | Papain Dissociation System Kit | Worthington Biochemical Corp. | Cat# LK003150 | |
| Commercial assay, kit | PNGase F | NEB | Cat# P0704S | |
| Commercial assay, kit | miRNeasy Mini Kit | Qiagen | Cat#: 217004 | |
| Commercial assay, kit | M-MLV Reverse Transcriptase | Thermo Fisher | Cat#: 28025013 | |
| Commercial assay, kit | iTaq Universal SYBR Green SuperMix | BIO-RAD | Cat#: 1725124 | |
| Cell line (Homo sapiens) | HEK293T cells | ATCC | RRID:CVCL_0063 | |
| Strain, strain background (Mus musculus) | Mouse: FVB | The Jackson Laboratory | Stock No: 001800 | |
| Strain, strain background (Mus musculus) | Mouse: Nxph4 βgeo+/- | This paper | N/A | The Jackson Laboratory: 033791 |
| Strain, strain background (Mus musculus) | Mouse: Nxph4 FLAG/FLAG | This paper | N/A | The Jackson Laboratory: 033792 |
| Sequence-based reagent | Nxph4βgeo+/- genotyping forward primer: 5’-AAAGACTAGCAGACGCAGCA | This paper | N/A | Materials and methods subsection ‘Nxph4-βgeo mouse’ |
| Sequence-based reagent | Nxph4βgeo+/- genotyping reverse primer: 3’-CCCTAACTCCCCCAAACAGA | This paper | N/A | Materials andmethods subsection ‘Nxph4-βgeo mouse’ |
| Sequence-based reagent | Nxph4 FLAG/FLAGgenotyping forward primer: 5’-TCAAGTTCTCGCTGTTGGTG | This paper | N/A | Materials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’ |
| Sequence-based reagent | Nxph4 FLAG/FLAG genotypingreverse primer: 3’-TTCCACGTGGCAATTAAAAG | This paper | N/A | Materials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’ |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46773.019
