Neurexophilin4 is a selectively expressed α-neurexin ligand that modulates specific cerebellar synapses and motor functions

  1. Xiangling Meng
  2. Christopher M McGraw
  3. Wei Wang
  4. Junzhan Jing
  5. Szu-Ying Yeh
  6. Li Wang
  7. Joanna Lopez
  8. Amanda M Brown
  9. Tao Lin
  10. Wu Chen
  11. Mingshan Xue
  12. Roy V Sillitoe
  13. Xiaolong Jiang
  14. Huda Y Zoghbi  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, United States
  3. Howard Hughes Medical Institute, Baylor College of Medicine, United States
7 figures, 1 table and 1 additional file

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 …

https://doi.org/10.7554/eLife.46773.002
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 …

https://doi.org/10.7554/eLife.46773.003
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 …

https://doi.org/10.7554/eLife.46773.005
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…

https://doi.org/10.7554/eLife.46773.006
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 …

https://doi.org/10.7554/eLife.46773.007
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 …

https://doi.org/10.7554/eLife.46773.008
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 …

https://doi.org/10.7554/eLife.46773.009
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 …

https://doi.org/10.7554/eLife.46773.010
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 …

https://doi.org/10.7554/eLife.46773.011
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 …

https://doi.org/10.7554/eLife.46773.012
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. …

https://doi.org/10.7554/eLife.46773.013
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.

https://doi.org/10.7554/eLife.46773.014
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 …

https://doi.org/10.7554/eLife.46773.015
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). …

https://doi.org/10.7554/eLife.46773.016
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 …

https://doi.org/10.7554/eLife.46773.017
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 …

https://doi.org/10.7554/eLife.46773.018

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyMouse monoclonal anti-FLAG M2Sigma-AldrichCat# F1804 RRID:AB_262044(1:1000)
AntibodyRabbit polyclonal anti-pan Neurexin-1MilliporeCat# ABN161 RRID:AB_10917110(1:1000)
AntibodyMouse monoclonal anti-HA1.1BioledgendCat# 901513, RRID:AB_2565335(1:5000)
AntibodyMouse monoclonal anti-VinculinSigma-AldrichCat# V9131, RRID:AB_477629(1:10000)
AntibodyRabbit polyclonal anti-GABAARα1Synaptic SystemCat# 224 203, RRID:AB_2232180(1:1000)
AntibodyRabbit polyclonal anti-GABAARα6Synaptic SystemCat# 224 603, RRID:AB_2619945(1:1000)
AntibodyMouse monoclonal anti-Vglut1MilliporeCat# MAB5502,
RRID:AB_262185
(1:1000)
AntibodyGuinea pig polyclonal anti-vGATFrontier Institute co. LtdCat#: VGAT-GP-Af1000, RRID: AB_2571624(1:1000)
AntibodyMouse monoclonal anti-beta ActinAbcamCat#: ab20272
RRID:AB_445482
(1:2000)
AntibodyRabbit polyclonal anti-TGF beta 1AbcamCat#: ab92486, RRID:AB_10562492(1:1000)
AntibodyRabbit polyclonal anti-MeCP2Zoghbi Lab#0535(1:1000)
AntibodyMouse monoclonal anti-GephyrinSynaptic systemCat#: 14701,
RRID:AB_887717
(1:1000)
AntibodyRabbit polyclonal anti-
PSD-95
Cell signalingCat # 2507,
RRID:AB_561221
(1:1000)
Recombinant DNA reagentpLenti-mCherryAddgeneCat# 36084,
RRID:Addgene_36084
Recombinant DNA reagentpLenti-Nxph4-3xFLAG-mCherryThis paperN/AResults subsection‘Nxph4 is a secreted glycoprotein’
Recombinant DNA reagentpLenti--Nxph4-4Q-HA-mCherryThis paperN/AResults subsection ‘Nxph4 is a secreted glycoprotein’
Recombinant DNA reagentpLenti--Nxph4-6A-HA-mCherryThis paperN/AResults subsection ‘Nxph4 is a secreted glycoprotein’
Recombinant DNA reagentpCMV-Nxph4-HAThis paperN/AResults subsection‘Nxph4 is a secreted glycoprotein’
Recombinant DNA reagentpCMV-Nxph4-4Q-HAThis paperN/AResults subsection‘Nxph4 is a secreted glycoprotein’
Recombinant DNA reagentpCMV-Nxph4-6A-HAThis paperN/AResults subsection‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCAG-HA-Nrxn1β S4(+)AddgeneCat# 59410,
RRID:Addgene_59410
Recombinant DNA reagentpCAG-HA-Nrxn1β S4(-)AddgeneCat# 59409,
RRID:Addgene_59409
Recombinant DNA reagentpCAG-HA-Nrxn1αAddgeneCat# 58266,
RRID:Addgene_58266
Recombinant DNA reagentpCAG-HA-Nrxn1α-LNS1This paperN/AResults subsection‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCAG-HA-Nrxn1α-LNS2This paperN/AResults subsection‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCAG-HA-Nrxn1α-LNS3This paperN/AResults subsection‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCAG-HA-Nrxn1α-LNS4This paperN/AResults subsection‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCAG-HA-Nrxn1α-LNS5This paperN/AResults subsection ‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCMV-GABARα1N-HAThis paperN/AResults subsection ‘Nxph4 interacts with α-neurexin in vivo’
Recombinant DNA reagentpCMV-GABARα6N-HAThis paperN/AResults subsection ‘Nxph4 interacts with α-neurexin in vivo’
Chemical compound, drugX-GalThermo FisherCat# 15520034
Chemical compound, drugLipofectamine 2000Thermo FisherCat# 11668500
Commercial assay, kitAntigen Unmasking Solution (citrate based)Vector LaboratoriesCat# H-3300
Commercial assay, kitAvidin/Biotin
Blocking Kit
Vector LaboratoriesCat# SP-2001
Commercial assay, kitTSA KitInvitrogenCat# T-20932
Commercial assay, kitPapain Dissociation System KitWorthington Biochemical Corp.Cat# LK003150
Commercial assay, kitPNGase FNEBCat# P0704S
Commercial assay, kitmiRNeasy Mini KitQiagenCat#: 217004
Commercial assay, kitM-MLV Reverse TranscriptaseThermo FisherCat#: 28025013
Commercial assay, kitiTaq Universal SYBR Green SuperMixBIO-RADCat#: 1725124
Cell line (Homo sapiens)HEK293T cellsATCCRRID:CVCL_0063
Strain, strain background
(Mus musculus)
Mouse: FVBThe Jackson LaboratoryStock No: 001800
Strain, strain background
(Mus musculus)
Mouse: Nxph4 βgeo+/-This paperN/AThe Jackson Laboratory: 033791
Strain, strain
background
(Mus musculus)
Mouse: Nxph4 FLAG/FLAGThis paperN/AThe Jackson Laboratory: 033792
Sequence-based reagentNxph4βgeo+/- genotyping forward primer: 5’-AAAGACTAGCAGACGCAGCAThis paperN/AMaterials and methods subsection ‘Nxph4-βgeo mouse’
Sequence-based reagentNxph4βgeo+/- genotyping reverse primer: 3’-CCCTAACTCCCCCAAACAGAThis paperN/AMaterials andmethods subsection ‘Nxph4-βgeo mouse’
Sequence-based reagentNxph4 FLAG/FLAGgenotyping forward primer: 5’-TCAAGTTCTCGCTGTTGGTGThis paperN/AMaterials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’
Sequence-based reagentNxph4 FLAG/FLAG genotypingreverse primer: 3’-TTCCACGTGGCAATTAAAAGThis paperN/AMaterials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’

Additional files

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