Greta Maiellano

Ligand-gated acetylcholine receptors (AChRs) are pentameric transmembrane proteins that display extensive molecular diversity through combinatorial assembly of subunits. In the nematode Caenorhabditis elegans, this diversity is exceptionally expanded, with nearly 60 AChR subunit genes, yet the composition and function of many receptor subtypes remain poorly defined. At neuromuscular junctions (NMJs), L-AChRs are heteromeric AChRs sensitive to levamisole, a nematode-specific L-AChR agonist, that play a crucial role in locomotion. Forward genetic screens for mutants resistant to levamisole have identified the L-AChR canonical subunits (LEV-1, UNC-29, LEV-8, UNC-63, and UNC-38) along with key biosynthetic regulators. ACR-8 is an alternative L-AChR subunit that has been poorly characterized, largely because acr-8 mutants remain sensitive to levamisole. Using genetic approaches and in vivo imaging, we show that ACR-8-containing L-AChRs (ACR-8*) are present at the NMJ and, surprisingly, they are more abundant than the well-characterized LEV-8-containing L-AChRs (LEV-8*). Although both LEV-8* and ACR-8* are enriched at synapses and rely on the same clustering machinery, they segregate into distinct postsynaptic nanodomains. The 2 populations play a redundant function in worm locomotion, and in mutant conditions, 1 receptor population can compensate for the loss of the other. Interestingly, ACR-8* are already present at high levels at the NMJ from early developmental stages, whereas LEV-8* levels gradually increase throughout development. Our work sheds new light on the molecular landscape of AChRs of the C. elegans NMJ.

Protocadherins, a family of adhesion molecules with a crucial role in cell–cell interactions, have emerged as key players in neurodevelopmental and psychiatric disorders. In particular, growing evidence links genetic alterations in the protocadherin 9 ( PCDH9 ) gene with autism spectrum disorder and major depressive disorder. 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’s role in the male mouse hippocampus at the ultrastructural, biochemical, transcriptomic, electrophysiological, and network levels. 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 in the CA1. Synapse overgrowth is sustained by the widespread upregulation 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 in increased excitatory postsynaptic currents (mEPSC) and reduced network activity in the CA1 of Pcdh9 KO mice. In conclusion, our work uncovers Pcdh9 ’s pivotal role in shaping the morphology and function of CA1 excitatory synapses, thereby modulating glutamatergic transmission within hippocampal circuits.

Protocadherins are cell adhesion molecules with crucial role in cell-cell contacts, whose mutations or altered expression have been implicated in multiple brain disorders. In particular, growing evidence links genetic alterations in Protocadherin 9 ( PCDH9 ) gene with Autism Spectrum Disorder (ASD) and Major Depression 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 underlying Pcdh9 physiological function and its involvement in brain pathology remain largely unknown. To this aim, we conducted a comprehensive investigation of PCDH9 role in the 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 broad up-regulation of synaptic genes and the dysregulation of key drivers of synapse morphogenesis, as revealed by single-nucleus RNAseq. Synaptic and transcriptional defects are accompanied by increased EPSC frequency and disturbances in the hippocampal network activity of Pcdh9 KO mice. In conclusion, our work indicates that Pcdh9 regulates the morphology and function of excitatory synapses in the CA1, thereby affecting glutamatergic transmission in hippocampal circuitries.

Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.