The Role of CASK in Neuronal Morphogenesis and Brain Size in Drosophila: A Genetic Model of Human Intellectual Disability with Microcephaly

Abstract

CASK is a highly conserved gene with major roles in brain development and function. CASK encodes a multi-domain synaptic protein that interacts with numerous binding partners in at least three different subcellular regions. CASK is a member of the MAGUK protein family, defined by its carboxy-terminal end, which includes a guanylate kinase, PDZ and SH3 protein-interaction domains. CASK amino-terminal end, in turn, contains the CaMKinase-like domain, known as a pseudokinase. Highly expressed in neurons, CASK is localized to both pre- and post-synaptic zones, as well as to the nucleus. Mutations in human CASK cause X-linked intellectual disability (ID). There is a phenotypic spectrum of brain-development disorders caused by CASK mutations, that has been divided in two diagnostic categories. Microcephaly with pontine-cerebellar hypoplasia is the most severe, whereas FG syndrome-4 or X-linked ID with or without nystagmus cause the milder phenotype. Both of these phenotypes are accompanied by short stature. Because the CASK-mutant phenotypes in humans, I hypothesized that CASK is essential for neuronal morphogenesis. Therefore, I studied the role of Drosophila CASK in neuronal differentiation and brain development. I used the Drosophila CASK mutation, ∆18, an imprecise-excision allele that eliminates the full-length CASK protein, and the corresponding precise-excision control, Ex33. I examined neuronal morphogenesis in vitro by using primary cultures prepared from the whole CNS of wandering third instar larvae. Morphological parameters of neurite-arbor size and shape were quantified using NeuronMetricsTM software for semi-automated image analysis. CNS neurons lacking full-length CASK grew small arbors in vitro with an altered shape. This phenotype, called “bushy” combines small size (reduced length, higher-order branches, and area) with increased branch density. In addition, I found that CASK has a semi-dominant phenotype, by introducing a transgene with a WT copy of CASK the bushy phenotype was improved. To investigate whether CASK controls brain size, I studied brain morphology of Δ18 homozygous flies by histological examination of serial sections of osmium-stained, plastic-embedded pharate-adult heads. The volumes of brain and head were estimated using Olympus cellSens software. Brain and head estimated total volumes were significantly reduced in Δ18 homozygotes. Reduction in both brain and head indicates that flies have both microencephaly and microcephaly. In addition, I analyzed body size by measuring the pupal case length as a proxy for adult body size. CASK mutants have reduced body size. The small brain and reduced head found in Drosophila CASK mutants provide evidence that this is a good genetic model that parallels the CASK phenotypes in humans. In conclusion, these data suggest that the “small-brain” phenotype, associated with CASK-mutations in flies and microcephaly in children, results from decreased neuronal size and defective formation of dendritic arbors and axonal projections, rather than to a reduced number of neurons. These data strengthen the use of the Drosophila as a genetic model organism for modeling human developmental brain disorders. It would be reasonable to adapt the insights gained to develop new strategies in the field of human medicine, and its special significance regarding human CASK mutations. For instance, by performing a screen for drugs that can normalize disrupted neurons due to CASK disease-causing mutations, potential treatment strategies could be discovered.