CNTNAP2 stabilizes interneuron dendritic arbors through CASK

Abstract

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2’s C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

CNTNAP2 is targeted to endosomes by the polarity protein PAR3

“…GluA1 (synaptic membrane protein) was highly enriched in the crude membrane (S5), and synaptic (S6) fractions while PSD95 (synaptic cytosolic protein) was only enriched in the S6 fraction; β‐tubulin (nonsynaptic cytosolic protein) was absent from either S5 or S6 fractions (Figure b, bottom). We found both PAR3 and CNTNAP2 in the S6 (synaptic) fraction, consistent with previous publications (Chen et al, ; Gao et al, ; Lin et al, ; Oiso et al, ). We also validated previous studies and showed strong colocalization of PAR3 with PSD95 (Figure S1).…”

“…We performed a yeast two‐hybrid screen of a mouse brain cDNA library with the entire CNTNAP2 intracellular domain (ICD) as bait (amino acids 1284–1331) to find intracellular interactors of CNTNAP2 (Figure a). Out of three million candidates screened, we identified 41 unique clones (Gao et al, ), with PAR3 being one of the most intriguing, given its role as a spatial organizer (Ahmed & Macara, ; Duman et al, ; Zhang & Macara, ). We confirmed this positive result by demonstrating yeast growth on high stringency plates only when expressing both bait/prey plasmids, indicating a direct physical interaction (data not shown).…”

“…Previous results from our laboratory (Gao et al, ) show that CNTNAP2 also interacts with calcium/calmodulin‐dependent serine protein kinase (CASK) at the plasma membrane. As both CASK and PAR3 appear to regulate CNTNAP2 localization via PDZ domains and because CNTNAP2 has only one PDZ‐binding region, there may be competitive binding that serve to localize CNTNAP2 to specific regions in a context‐dependent manner.…”

Association between CNTNAP2 polymorphisms and autism: A family‐based study in the chinese han population and a meta‐analysis combined with GWAS data of psychiatric genomics consortium

“…CNTNAP2 is highly expressed in the frontal and temporal lobes of the developing human brain, and the striatal circuits and frontal cortex of the adult brain. Functional studies indicated that CNTNAP2 was involved in dendrite stabilization, neuron migration, subsequent laminar organization, and connectivity of specific neural circuits [Gao et al, ; Poliak et al, ; Strauss et al, ; Zerbi et al, ]. Cntnap2 knockout neurons showed impaired axonal growth and synaptic abnormalities, which might play important roles in autism [Canali et al, ; Spooren, Lindemann, Ghosh, & Santarelli, ; Varea et al, ].…”

HNRNPU: Key to Neurodevelopmental Disorders such as Intellectual Delay, Epilepsy, and Autism

“…Probably CNVs and SNVs of the HNRNPU gene may produce distinct phenotypic outcomes. In view of the plethora of possibly pathogenic molecular mechanisms, there is a clear need for in vitro and in vivo functional studies to assess the effects of CNVs and the different SNVs in HNRNPU, as has been done for CNTNAP2 SNVs [Brumback et al, 2017;Scott et al, 2017;Canali et al 2018;Gao et al, 2018;Liska et al, 2018;Vogt et al, 2018;Zerbi et al, 2018]. The outcome of such studies may help us understand the phenotypic variability in patients with CNVs and SNVs affecting HNRNPU .…”