Functional role of Protocadherin10 in murine brain development

Autism Spectrum Disorders (ASDs) are a group of debilitating neurodevelopmental disorders that affect millions of patients world-wide. ASD is hypothesized to arise from a complex interplay between genetic and environmental factors that impact the structure and function of the developing brain. A potential cause of the disease pathophysiology is a disrupted excitation/inhibition balance in the central nervous system stemming from abnormal development or connectivity between neurons. One gene linked to the occurrence of ASD in humans is Protocadherin10 (PCDH10). Pcdh10 is a cell adhesion molecule of the transmembrane calcium-dependent adhesion protein (cadherin) superfamily and is highly expressed in the developing embryonic and early postnatal brain, where it is postulated to play a role in the formation and fine-tuning of neural circuits. However, how mutations or copy number variations of PCDH10 result in the development of ASD remains unknown. In addition to its link to ASD, PCDH10 is generally considered a tumor-suppressor gene as the loss of PCDH10 expression has been described in a multitude of cancer types. Here, re-expression of PCDH10 results in decreased proliferation, epithelial-to-mesenchymal transition, migration, angiogenesis and increased apoptosis.

Mouse models that have reduced or lost Pcdh10 expression exhibit ASD-like behaviors including decreased social approach, abnormal anxiety, abnormal fear learning and isolation-induced ultrasonic vocalizations. Moreover, these mice were described to contain a decreased density of dendritic spines in the basolateral amygdala, a brain region involved in fear responses and memory. At last, Pcdh10 was previously linked to synapse elimination in hippocampal neuronal cultures. To further investigate the role and function of Pcdh10 in the development of the brain, we investigated the spatiotemporal expression of Pcdh10 in the mouse. We show that Pcdh10 is highly expressed in brain regions linked to ASD and emotional processing, and at developmental timepoints related to processes of neuronal proliferation, migration, axon guidance and synaptogenesis. Pcdh10 expression is strongest at early postnatal ages, when synapse formation and elimination are fully ongoing. Moreover, Pcdh10 remains relevant in the pre- and postsynapse at adult stages, where it is likely involved in synapse assembly, maintenance and plasticity. At the subcellular level, PCDH10 is preferentially localized at the membrane, although its precleaved intracellular domain construct can localize to the nucleus, where it might be involved in the regulation of gene expression. This intracellular domain of PCDH10 does not affect its adhesive function, and is likely involved in other downstream signaling cascades. In migrating interneurons, the adhesive extracellular domain of PCDH10 is localized at the tip, the branching point and the swelling of the leading process, indicating that PCDH10 is actively involved in this process. To better understand the molecular mechanisms and pathways involved in Pcdh10 function, we investigated near neighbor and direct binding proteins of PCDH10. To achieve this, we performed a proximity analysis in HEK293 cells and generated our own tagged Pcdh10HAV5 mouse line using CRISPR-Cas9, respectively. Analysis of the data revealed that PCDH10 is located within the vicinity of several actin cytoskeleton remodeling proteins in HEK293 cells, and is able to bind proteins involved in synaptic transmission, plasticity and memory and learning in the mouse brain.

To better study the effect of loss-of-function of Pcdh10 in the developing murine brain, we used two conditional knockout lines where Pcdh10 expression is specifically lost in all or a subpopulation of inhibitory interneurons. These mouse models allow us to further investigate whether loss of Pcdh10 in interneurons is sufficient to replicate the ASD-like behaviors previously described. We found that loss of Pcdh10 expression did not affect the axon guidance, the apoptosis or the intrinsic migration capacity of interneurons in the developing forebrain in a general way. Nonetheless, a reduced amount of interneurons was present in the ganglionic eminences, their place of birth, at embryonic day E15.5. Moreover, at embryonic day E17.5, a reduced amount of interneurons was present in the basolateral amygdala. These findings point towards a proliferation or migration defect of interneurons that have lost Pcdh10 expression. At last, conditional and full knockout pups (P7) were tested for their capacity to generate isolation-induced ultrasonic vocalizations (USVs), as a measurement of anxiety. Both full and conditional heterozygous knockout pups exhibited an increased number of USVs, while the conditional homozygous mutant instead showed a decreased number of USVs. Whether the mismatching of Pcdh10 expression in the conditional knockout in interneurons causes the phenotype due to reduced neurotransmission will be further investigated. Additional research is needed to fully understand the underlying neurobiological mechanisms of ASD and the role of PCDH10 herein.