Disruption of the autism-associated Pcdh9 gene leads to transcriptional alterations, synapse overgrowth, and defective network activity in the CA1

Abstract

Protocadherins, a family of adhesion molecules with crucial role in cell-cell interactions, have emerged as key players in neurodevelopmental and psychiatric disorders. In particular, growing evidence links genetic alterations in Protocadherin 9 (PCDH9) gene with Autism Spectrum Disorder (ASD) and Major Depressive Disorder (MDD). Furthermore, Pcdh9 deletion induces neuronal defects in the mouse somatosensory cortex, accompanied by sensorimotor and memory impairment. However, the synaptic and molecular mechanisms of PCDH9 in the brain remain largely unknown, particularly concerning its impact on brain pathology. To address this question, we conducted a comprehensive investigation of PCDH9 role in the male mouse hippocampus at the ultrastructural, biochemical, transcriptomic, electrophysiological and network level. We show that PCDH9 mainly localizes at glutamatergic synapses and its expression peaks in the first week after birth, a crucial time window for synaptogenesis. Strikingly, Pcdh9 KO neurons exhibit oversized presynaptic terminal and postsynaptic density (PSD) in the CA1. Synapse overgrowth is sustained by the widespread up-regulation of synaptic genes, as revealed by single-nucleus RNA-seq (snRNA-seq), and the dysregulation of key drivers of synapse morphogenesis, including the SHANK2/CORTACTIN pathway. At the functional level, these structural and transcriptional abnormalities result into increased excitatory postsynaptic currents (mEPSC) and reduced network activity in the CA1 of Pcdh9 KO mice. In conclusion, our work uncovers Pcdh9 pivotal role in shaping the morphology and function of CA1 excitatory synapses, thereby modulating glutamatergic transmission within hippocampal circuits.Significance statement Converging evidence indicates that genetic alterations in Protocadherin 9 (PCDH9) gene are associated with Autism Spectrum Disorder (ASD) and Major Depressive Disorder (MDD). However, our understanding of PCDH9 physiological role and molecular mechanisms in the brain, as well as its connection to synaptic dysfunction and brain pathology, remains limited. Here we demonstrate that Pcdh9 regulates the transcriptional profile, morphology and function of glutamatergic synapses in the CA1, thereby tuning hippocampal network activity. Our results elucidate the molecular and synaptic mechanisms of a gene implicated in neurodevelopmental and psychiatric disorders, and suggest potential hippocampal alterations contributing to the cognitive deficits associated with these conditions.

Figure 1.

PCDH9 is enriched at glutamatergic synapses, and its expression peaks in the first postnatal weeks in the hippocampus. A, Representative PCDH9 Western blot of brain regions and body organs from 2-month-old wild-type mice. Two PCDH9 isoforms at ∼130 and ∼180 kDa are detected, as previously reported (Bruining et al., 2015). OB, olfactory bulb; PFC, prefrontal cortex. B, Quantifications of PCDH9 levels from A. Protein levels were normalized to GAPDH. n = 4. Kruskal–Wallis test, no significant differences. n.d., not detected. C, Representative PCDH9 Western blot on hippocampus from wild-type mice at different ages. E18, embryonic day 18; P3–P180, postnatal day 3–180. D, Quantifications of PCDH9 levels from C. Protein levels were normalized to GAPDH. Mean with SEM is represented. n = 3. Kruskal–Wallis test, **p < 0.01. E, Representative PCDH9 Western blot on cortex from 2-month-old wild-type mice. n = 2. S2, supernatant 2 (cytosol-enriched fraction); P2, pellet 2 (membrane-enriched fraction). PSD95 and ACTIN are used as markers of P2 and S2, respectively. F, Representative confocal images of DIV18 rat hippocampal neurons coimmunostained for PCDH9 and synaptic markers VGLUT1, VGAT, PSD95, and GEPHYRIN. Insets show higher magnification of a dendrite. n = 3 independent cultures. G, Manders’ coefficient quantification of PCDH9 puncta colocalizing with presynaptic (top) and postsynaptic (bottom) markers analyzed in F. VGLUT1, VGAT, n = 13 neurons. Mann–Whitney test, ****p < 0.0001. PSD95, HOMER1, GEPHYRIN, n = 17–27 neurons. Kruskal–Wallis test, *p < 0.05. H, Pearson’s coefficient quantification of PCDH9 signal colocalizing with presynaptic (top) and postsynaptic (bottom) markers analyzed in F. VGLUT1, VGAT, n = 13 neurons. Mann–Whitney test, ***p < 0.001. PSD95, HOMER1, GEPHYRIN, n = 17–27 neurons. Postsynaptic markers, Kruskal–Wallis test, ****p < 0.0001. See Extended Data Figure 1-1 for more details.

Figure 2.

Pcdh9 deletion leads to aberrant presynaptic and postsynaptic compartments in the CA1. A, Representative electron micrographs of pyramidal neuron synapses from the dorsal CA1 of 2-month-old WT and Pcdh9 KO mice. Scale bar, 100 nm. B, Quantifications of synaptic features from A. n = 30–50 synapses from two mice per genotype. The horizontal solid line across the violin plot represents the median, and the two dotted lines indicate the quartiles. Mann–Whitney test, *p < 0.05, **p < 0.01. C, Representative electron micrographs of pyramidal neurons synapses from the vmPFC layers 5–6 of 2-month-old WT and Pcdh9 KO mice. Scale bar, 100 nm. D, Quantifications of synaptic features from C. n = 50–70 synapses from two mice per genotype. The horizontal solid line across the violin plot represents the median, and the two dotted lines indicate the quartiles. Mann–Whitney test, no significant differences. E, Representative western blot (left) and corresponding quantification (right) of synaptic markers levels on hippocampus from 2-month-old WT and Pcdh9 KO mice. n = 8–15 mice per genotype. Mann–Whitney test, no significant differences. F, Representative Western blot (left) and corresponding quantification (right) of synaptic markers levels on cortices from 2-month-old WT and Pcdh9 KO mice. n = 6–9 mice per genotype. Mann–Whitney test, no significant differences. See Extended Data Figures 2-1 and 2-2 and Extended Data Table 2-1 for more details.

Figure 3.

Pcdh9 deletions induces the dysregulation of key synaptic genes and the broad upregulation of the synaptic transcriptome in the CA1. A, UMAP plots of snRNA-seq data. A total of 16,833 nuclei from WT and Pcdh9 KO hippocampi were grouped into 46 different clusters and colored based on the attributed cell type. n = 2 mice per genotype. B, UMAP plots of snRNA-seq data, with nuclei colored based on the combined expression levels of CA1 markers. The numbers of the clusters identified as CA1 are in blue. C, Heatmap of CA1 markers fold changes among the different clusters. Clusters identified as CA1 populations are enclosed in a rectangle. D, Volcano plots showing the KO/WT fold change and p-value distributions of genes in CA1 Clusters 5 (top), 6 (center), and 13 (bottom). Upregulated and downregulated DEG are colored in green and red, respectively. E, Network visualization of the combined 31 DEG found in CA1 Clusters 5, 6, and 13. Each node represents a gene and is colored based on its function. A line connecting two nodes indicates a physical or functional interaction, as analyzed with STRING. The line thickness indicates the strength of data support. F, The four GO categories (biological processes) identified with g:Profiler on CA1 DEG are represented. The green and red indicate up- and downregulated DEG, respectively. For each GO category, the statistical significance and the associated genes are represented (brown square, inferred from experiments; orange square, inferred from sequences). G, The three synaptic GO categories identified with SynGO on CA1 DEG. For each GO category, the statistical significance and the associated genes are indicated. H, Representative confocal images of CA1 neurons from 3-month-old WT and Pcdh9 KO mice, immunostained for MAP2 (neuronal marker) and CORTACTIN or SHANK2. Scale bar, 20 µm. I, Quantification of CORTACTIN (top) or SHANK2 (bottom) puncta fluorescence intensity from H. n = 8–12 sections from 3–4 mice per genotype. Mann–Whitney test, *p < 0.05. J, The effect of Pcdh9 deletion on the expression levels of nonsynaptic, presynaptic, and postsynaptic genes in Clusters 5 (top left), 6 (top rigth), and 13 (bottom) is shown. The horizontal line across the violin plot represents the median. Kruskal–Wallis test, ****p < 0.0001. See Extended Data Figures 3-1 and 3-2 and Extended Data Tables 3-1, 3-2, 3-3, 3-4, and 3-5 for more details.

Figure 4.

Pcdh9 deletion leads to increased frequency of EPSCs and reduced network activity in the CA1. A, Representative traces of mEPSCs recorded from WT and Pcdh9 KO CA1 pyramidal neurons. B, Quantification of amplitude, decay time, area, and frequency of mEPSCs from A. Mean with SD is represented. n = 16–17 recorded neurons from five independent animals per genotype. Mann–Whitney test, *p < 0.05. C, Representative traces of mEPSCs recorded from vmPFC pyramidal neurons from WT and Pcdh9 KO mice. D, Quantification of amplitude, decay time, area, and frequency of mEPSCs from C. Mean with SD is represented. n = 21–22 recorded neurons from five independent mice per genotype. The horizontal line across the plot represents the mean. Mann–Whitney test, no significant differences. E, Quantification of paired-pulse ratio experiments in CA1 pyramidal neurons from WT and Pcdh9 KO mice. n = 5 independent mice per genotype. Mann–Whitney test, no significant differences. F, Quantification of CA1 fEPSP slope before and after HFS from WT and Pcdh9 KO mice. n = 5 independent mice per genotype. Mann–Whitney test, no significant differences. G, Representative MEA recordings from WT and Pcdh9 KO hippocampal slices. H, Representative raster plots showing CA1 electrical activity recorded from 100 channels over 2 min (top) and representative traces recorded from a single channel positioned in the CA1 (bottom), showing reduced firing activity in Pcdh9 KO mice. I, Quantification of firing rate, burst activity, number of spikes per burst, and burst duration from MEA recordings for each hippocampal subregion in WT and Pcdh9 KO genotypes. The horizontal line across the box plot represents the mean. n = 4–6 independent mice per genotype. Mann–Whitney test, **p < 0.01. J, Synchronization index of the CA1 subregion in WT and Pcdh9 KO genotypes. The mean with SEM is represented. n = 3–6 independent mice per genotype. Mann–Whitney test, *p < 0.05. See Extended Data Figure 4-1 for more details.

Figure 5.

Pcdh9 ablation leads to an aberrant transcriptional profile, morphology and function of CA1 excitatory synapse, and defective CA1 network activity. Pcdh9 deletion causes synaptic dysregulation at multiple levels, transcriptional (key synaptic genes alterations and widespread upregulation of the synaptic transcriptome), morphological (presynaptic terminal and PSD overgrowth), and functional (increased mEPSCs frequency) and results in impaired network activity in the CA1.