Structural Analysis Implicates CASK-Liprin-α2 Interaction in Cerebellar Granular Cell Death in MICPCH Syndrome

Abstract

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

Diverse Clinical Phenotypes of CASK-Related Disorders and Multiple Functional Domains of CASK Protein

“…MICPCH/microcephaly is one of the phenotypes of CASK -related disorders that has been extensively examined in animal models. The cerebellar size of cask heterozygous knockout female mice and hypomorphic male mice was shown to be smaller than that in wild-type mice [ 4 , 92 , 94 , 95 ]. Similarly, we reported that the knockout of neurexin, a main binding partner of CASK, from the cerebellar granule cells, induced cell death.…”

“…The importance of the PDZ domain in cerebellar development is supported not only by genetic findings of missense mutations in the PDZ domain but also by our recent report using cerebellar granule cell culture. We demonstrated that the death of cerebellar granule cells from the conditional knockout of CASK can be rescued by the full length of CASK but not by the variants lacking the CaMK, PDZ, or SH3 domains of CASK, indicating that all three domains are essential for the survival of cerebellar granule cells in mice [ 95 ]. Moreover, one of the MICPCH-associated missense mutations (M519T), which could not bind to neurexin in vitro assay [ 63 ], failed to rescue the cell death of cerebellar granule cells from the knockout of the cask gene [ 95 ], indicating that CASK–neurexin binding would be important to cerebellar development.…”

“…We demonstrated that the death of cerebellar granule cells from the conditional knockout of CASK can be rescued by the full length of CASK but not by the variants lacking the CaMK, PDZ, or SH3 domains of CASK, indicating that all three domains are essential for the survival of cerebellar granule cells in mice [ 95 ]. Moreover, one of the MICPCH-associated missense mutations (M519T), which could not bind to neurexin in vitro assay [ 63 ], failed to rescue the cell death of cerebellar granule cells from the knockout of the cask gene [ 95 ], indicating that CASK–neurexin binding would be important to cerebellar development. Although the molecular mechanism of cerebellar granule cell death is still unclear, one possible hypothesis is the neurexin-dependent release of brain-derived neurotrophic factor (BDNF) [ 96 ], which plays an essential role in cerebellar development in mice and primates [ 97 , 98 , 99 ].…”

“…Thus, the CASK–neurexin–Liprin-α tripartite complex may be a molecular mechanism of the development of the cerebellum. Interestingly, a mutation in the CaMK domain, R106P, which was identified from a male patient of MICPCH [ 57 ], did not prevent the cell death of cerebellar granule cells [ 95 ]. The specific residue has been shown to contribute to interactions between CASK and Liprin-α and Mint1 [ 25 ], and an AlphaFold2-prediction of CaMK protein structure revealed that the mutation disrupted the interactions [ 95 ].…”

“…Interestingly, a mutation in the CaMK domain, R106P, which was identified from a male patient of MICPCH [ 57 ], did not prevent the cell death of cerebellar granule cells [ 95 ]. The specific residue has been shown to contribute to interactions between CASK and Liprin-α and Mint1 [ 25 ], and an AlphaFold2-prediction of CaMK protein structure revealed that the mutation disrupted the interactions [ 95 ]. The contribution of CASK–neurexin interactions to cerebellar development is unclear, partially because limited information is available on the molecular structure between CASK and neurexins.…”