Gamma protocadherins are required for synaptic development in the spinal cord - PubMed (original) (raw)

Gamma protocadherins are required for synaptic development in the spinal cord

Joshua A Weiner et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Fifty-eight cadherin-related protocadherin (Pcdh) genes are tandemly arrayed in three clusters (alpha, beta, and gamma) on mouse chromosome 18. The large number of clustered Pcdh family members, their presence at synapses, and the known binding specificities of other cadherin superfamily members all suggest that these Pcdhs play roles in specifying synaptic connectivity. Consistent with this idea, mice lacking all 22 genes of the Pcdh-gamma cluster have decreased numbers of spinal cord synapses and are nearly immobile. Interpretation of this phenotype was complicated, however, by the fact that Pcdh-gamma loss also led to apoptosis of many spinal interneurons. Here, we used two methods to circumvent apoptosis and neurodegeneration in Pcdh-gamma mutant mice. First, we analyzed mutants lacking both Pcdh-gamma proteins and the proapoptotic protein Bax. Second, we generated a hypomorphic allele of Pcdh-gamma in which apoptosis was minimal. In both cases, spinal interneurons were preserved but the mice bore dramatically decreased numbers of spinal cord synapses and exhibited profound neurological defects. Moreover, synaptic function was compromised in neurons cultured from the hypomorphs. These results provide evidence for a direct role of gamma-Pcdhs in synaptic development and establish genetic tools for elucidating their contribution to synaptic specificity.

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Figures

Fig. 1.

Fig. 1.

Loss of Bax rescues spinal interneuron apoptosis in _Pcdh_-γ deletion mice. (A) Schematic of the targeted _Pcdh_-γ genomic locus with loxP sites (orange triangles) upstream of the first variable exon and within constant exon 3. The locus was deleted by transfection of Cre to generate the _Pcdh_-γdel allele. This line was crossed with Bax mice to produce _Pcdh_-γdel/del;_Bax_-/- double mutants. (B_–_E) In contrast to _Pcdh_-γdel/del single mutants (B), which exhibit spinal cord hypoplasia caused by apoptosis of interneurons and loss of axonal tracts (stained for neurofilaments, red), double mutant neonates are morphologically normal (C), exhibit no loss of interneurons (D; neurons stained for NeuN), and lack evidence of apoptosis (E; staining for cleaved caspase-3). (F) Neuron number is increased in _Bax_-/- mice, because apoptosis is blocked; the _Pcdh_-γ mutation has no significant effect on neuron number in this background. Bars show mean ± SEM of 7–14 microscope fields from three animals per genotype. *, P < 0.02, compared to _Pcdh_-γ+/del;Bax+/-. (Bar: 100 μm.)

Fig. 2.

Fig. 2.

Reduced synaptic density in the absence of neurodegeneration in _Pcdh_-γdel/del;Bax-/- double mutant spinal cord. (A) Immunostained synaptic puncta in sections through the intermediate gray of control, _Pcdh_-γdel/del and _Pcdh_-γdel/del;Bax-/- neonatal spinal cords. (Bars: 12 μm.) (B) Both _Pcdh_-γdel/del and _Pcdh_-γdel/del;Bax-/- mutants exhibit significant reductions in the density of excitatory (anti-PSD-95+) and inhibitory [anti-gephyrin+ and anti-glycine receptor+ (glyR+)] synapses. Bars show mean ± SEM of 7–14 microscope fields from three animals per genotype and are expressed as percent of control (_Pcdh_-γ+/del;Bax+/-). *, P < 0.05. Data from _Pcdh_-γdel/del single mutants, recalculated from data in Wang et al. (17), are shown for comparison.

Fig. 3.

Fig. 3.

A hypomorphic, C-terminally truncated allele of _Pcdh_-γ (_Pcdh_-γtr). (A) The targeted _Pcdh_-γtr allele contains an internal ribosome entry site (IRES)-GFP/LacZ fusion cassette inserted into constant exon 3, followed by a loxP site (triangle) left behind by excision of a neo selection marker. Cassette insertion results in a 57-aa truncation of the common _Pcdh_-γ C terminus. (B) Duplicate Northern blots of brain RNA hybridized with probes against the _Pcdh_-γ constant exon 3′ UTR and the LacZ gene. In _Pcdh_-γtr/tr mice, transcription of the inserted cassette is observed in place of the _Pcdh_-γ 3′ UTR. (C) Western blots of lysates from brains of WT and _Pcdh_-γtr or _Pcdh_-γdel mice probed with a rabbit antiserum against the _Pcdh_-γ common C terminus. Levels of γ-Pcdh proteins (arrow) are reduced in _Pcdh_-γtr/tr brains. Specificity of the antiserum is demonstrated by its recognition of recombinant protein in lysates of COS cells transfected with a full-length _Pcdh_-γ B2 cDNA and by the absence of the appropriate band in _Pcdh_-γdel/del brains. The upper band is nonspecific. (D) Duplicate Western blots of lysates from COS cells transiently transfected with constructs encoding either full-length or C-terminally truncated _Pcdh_-γ C4/GFP fusion proteins, probed with anti-_Pcdh_-γ and anti-GFP antisera. Similarity of the two blots suggests that C-terminally truncated protein retains major anti-_Pcdh_-γ epitopes. Difference in intensity between lanes within each blot is caused by a difference in the number of transfected cells between cultures.

Fig. 4.

Fig. 4.

Reduced synaptic density in the absence of neurodegeneration in _Pcdh_-γtr/tr spinal cord. (A_–_C) Immunostained sections through the spinal cords of neonatal control and _Pcdh_-γtr/tr mice. _Pcdh_-γtr/tr spinal cords are morphologically normal and exhibit little or no evidence of neurodegeneration (A), neuronal loss (B, quantitation in D), or apoptosis (C). However, _Pcdh_-γtr/tr spinal cords exhibit significant reductions in the density of both excitatory and inhibitory synapses [micrographs in E (stained as in Fig. 2), quantitation in _F_]. (Bars: 100 μm, A_–_C; 12 μm, E.) Bars in D and F show mean ± SEM of 21 microscope fields from three animals per genotype. *, P < 0.0001, compared with controls.

Fig. 5.

Fig. 5.

Normal differentiation but abnormal synaptic development in cultured _Pcdh_-γtr/tr spinal interneurons. Cultures were fixed and stained at 9 days in vitro. (A_–_C) Mutant neurons differentiate normally in vitro as in vivo, as assessed by MAP2 (A), class III β-tubulin (B), and NeuN (C) expression. Apoptotic mutant neurons are only rarely observed (C, arrow); most cleaved caspase-3+ cells in the cultures are glia. (D) Neither neuronal density (NeuN+) nor somato-dendritic area (MAP2+) differed between control and _Pcdh_-γtr/tr cultures. (E_–_H) Synapses formed in mutant cultures, as detected with antibodies to presynaptic terminals [synaptophysin for all terminals (E); VGlut for excitatory terminals (G); GAD for inhibitory terminals (H)] and to PSDs [PSD-95 for excitatory and gephyrin for inhibitory (F)]. (Bars: 30 μm in A_–_C, G, and H; 10 μm in E and F.) (I) The number of excitatory (VGlut+) and inhibitory (GAD+) terminals, and the average size of excitatory terminals, are decreased in mutants. Bars in I show mean ± SEM for 24–30 microscope fields obtained from three separate cultures including five control and four mutant embryos. Number of puncta and puncta area are shown normalized to MAP2+ area [which did not differ between genotypes (D)]. *, P < 0.001, compared with controls.

Fig. 6.

Fig. 6.

Reduced synaptic current amplitude in _Pcdh_-γtr/tr neurons. (A) Action potentials (Upper) and spontaneous synaptic events (Lower) were recorded from control (Left) and _Pcdh_-γtr/tr (Right) neurons. Vm, membrane resting potential; Vh, holding potential. (B) Mean synaptic amplitude (Left) was significantly lower in _Pcdh_-γtr/tr (29 ± 2 pA) than in control (58 ± 5 pA) neurons. *, P < 0.05; _n_ = 30–39 cells per genotype. The mean synaptic frequency (_Right_) was similar in control (1.78 ± 0.45 Hz) and _Pcdh_-γtr/tr (1.57 ± 0.39) neurons. (_C_) Pharmacological isolation of spontaneous excitatory (upper trace) and inhibitory (lower trace) synaptic events. Note the fast (<5 ms) and slow (>10 ms) decay times of the excitatory (•) and inhibitory (○) synaptic events respectively. n = 7–8 cells per pharmacological condition. CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; APV, 2-amino-5-phosphonovaleric acid. (D and E) Cumulative probability histograms demonstrate the reduction in excitatory (D) and inhibitory (E) synaptic amplitudes in _Pcdh_-γtr/tr neurons. ▴, Pchdtr/tr; □, control. (Insets) The mean amplitudes for control and _Pcdh_-γtr/tr excitatory (D) and inhibitory (E) synaptic events are shown. n = 15–20 neurons each genotype. *, P < 0.05.

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References

    1. Yamagata, M., Sanes, J. R. & Weiner, J. A. (2003) Curr. Opin. Cell Biol. 15, 621-632. - PubMed
    1. Scheiffele, P. (2003) Annu. Rev. Neurosci. 26, 486-508. - PubMed
    1. Washbourne, P., Ditayetev, A., Scheiffele, P., Biederer, T., Weiner, J. A., Christopherson, K. S. & El-Husseini, A. (2004) J. Neurosci. 24, 9244-9249. - PMC - PubMed
    1. Takai, Y., Shimuzu, K. & Ohtsuka, T. (2003) Curr. Opin. Neurobiol. 13, 520-526. - PubMed
    1. Yamagata, M., Weiner, J. A. & Sanes, J. R. (2002) Cell 110, 649-660. - PubMed

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