Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK-neurexin interaction

Abstract

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASK^M519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASK^G659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASK^W919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

Figure 1.. CASK^M519T and CASK^G659D phenotypic characteristics.

A) Magnetic resonance imaging brain scan of Subject 1 with the heterozygous missense mutation, CASK^M519T. Sagittal and coronal views of Subject 1 (T1-weighted images). In addition to microcephaly, global hypoplasia of the cerebellum, including the cerebellar vermis, is noted (arrows). B) Phenotype and genotype of Subject 1. C) Magnetic resonance imaging brain scan of Subject 3 with heterozygous missense mutation, CASK^G659D. B) Phenotype and genotype of Subject 3. C) The five major domains of CASK, with the locations of M519T, G659D and W919R mutations indicated in the linear structure. Note: in some CASK conformations, the SH3 and GUK domains are integrated into a single structural entity.

Figure 2.. CASK^M519T disrupts CASK-neurexin interaction.

A) Images of HEK293FT cells transfected with GFP-CASK without neurexin (A) and (B,C) GFP-CASK or GFP-CASK^M519T plasmid DNA each co-expressed with neurexin-1β-FLAG. After 48 hours, cells were fixed, permeabilized and immunostained for neurexin. Scale bar = 5μm. C) Colocalization analysis performed from 22 different images collected from 3 separate experiments. Results are plotted as mean±SD. * indicates p < 0.05. E) GST pull-down using either GST or GST-neurexin cytosolic tail fusion protein (GST-NX) from HEK293FT cell lysates transfected with GFP-CASK^M519T. ‘Start’ indicates the cell lysate. The precipitated proteins were immunoblotted for CASK. * indicates CASK endogenously expressed in HEK293FT cells.

Figure 3.. Structural impacts of the G659D mutation.

A) Homology model of CASK’s integrated SH3-GUK domain structure (LaConte et al. 2014) showing the location of native residues G659 and W919 (cyan), as well as mutant side chains (G659D, magenta) and associated contacts (dotted lines). SH3 region, yellow. Hinge region, orange. GUK region, gray. B) Representative images of HEK293FT cells expressing GFP-CASK plasmid as indicated. Note aggregation of protein (indicated by arrows) for both GFP-CASK^W919R and GFP-CASK^G659D. Scale bar = 10μm. C) Quantitation of percentage of cells displaying protein aggregates. The data is represented as mean ± S.E.M., n = 8. * indicates p < 0.05. D) Representative blot showing immunoprecipitation of FLAG-tagged Mint1. HEK293FT cells were transiently transfected with cDNA for GFP-CASK (wild-type and mutants) and FLAG-Mint1 as indicated. FLAG-Mint1 was precipitated from solubilized cells using the M2 beads and blotted for indicated antigens.

Figure 4.. CASK do not translocate to nucleus upon Tbr1 co-expression.

Images of A) HEK293FT cells and B) cortical neurons co-transfected with GFP-CASK and mCherry-Tbr1. C) Images of HEK293FT cells co-transfected with CASK and Tbr1 and immunostained with antibodies for CASK and Tbr1. Scale bars = 5μm. D) Blot showing immunoprecipitation of GFP-CASK using GFP-trap beads. Transfected cDNA is indicated at the top, and antigens for which immunoblotting has been performed is indicated on the right.

Figure 5.. Molecular dynamics simulations predict increased mobility of CASK’s hinge region and disruption of the SH3 domain.

A) B-factors (Ų) calculated from RMS fluctuations of α-carbons in a homology model of CASK’s SH3-GuK (CASK-WT, blue; CASK^G659D, red) domain during 100 ns of molecular dynamics simulations at each residue. Points are the average B-factor at a given position, and lines are upper and lower SEM boundaries (n = 3 trajectories). The residues that compose the hinge region are indicated by gray bar. Inset, hinge region. B) Most populated CASK^WT (left) and CASK^G659D (right) structures during three 100 ns molecular dynamics trajectories based on cluster analysis. SH3 domain, yellow. Hinge region, orange. GUK domain, gray. β strand containing site of G659D mutation, green. Black arrow indicates disrupted β-barrel structure.

Figure 6.. CASK^G659D disrupts CASK-neurexin interaction.

A) Homology model of the CASK PSG supramodule (left), based on PALS1-Crumbs PSG supramodule structure (right; 4wsi.pdb). PDZ domain, purple. SH3 domain, yellow. Hinge region, orange. CASK GuK domain, gray. PALS1 GuK domain, lavender. β strand containing site of G659D mutation, green. Crumbs, cyan. B) GST pull-down using either GST or GST-neurexin cytosolic tail (GST-NX) fusion protein from HEK293FT cell lysates transfected with cDNA indicated at the top. ‘Start’ indicates the cell lysate. The precipitated proteins were immunoblotted for CASK. * indicates CASK endogenously expressed in HEK293FT cells.

Figure 7.. CASK^G659D but not CASK^W919R disrupts CASK-neurexin interaction.

Images of HEK293FT cells transfected with GFP-CASK^W919R or GFP-CASK^G659D plasmid DNA co-expressed with neurexin-1β-FLAG (A,B). After 48 hours, cells were fixed, permeabilized and immunostained for neurexin. Scale bar = 5μm. C) Co-localization analysis of neurexin and CASK performed from 22 different images collected from 3 separate experiments. Results are plotted as mean±SD. * indicates p < 0.05.