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Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain- hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder. Author summary During brain development, localized gene expression is crucial for the formation and function of specific brain regions. Various groups of proteins are known to regulate segmentation through controlled gene expression, among them, the Rho GTPase regulator family. In this study, we identified a frameshift mutation in the Rho guanine nucleotide exchange factor 2 gene (ARHGEF2) in two children presenting with intellectual disability, mild microcephaly, and a midbrain- hindbrain malformation. This phenotype is also observed in Arhgef2 mutant mice, highlighting the importance of ARHGEF2 across development of distinct mammalian species. We show that loss of Arhgef2 affects neurogenesis and also cell migration. In addition, we extended the current knowledge of ARHGEF2 expression and its role in early central nervous system development, with special reference to the formation of the precerebellar system. In addition to extensive literature on ARHGEF2, we now provide evidence for its significant role in neuronal migration in brain development and link the gene to human neurodevelopmental disease.

The O-GlcNAc post-translational modification involves the attachment of single N-acetyl-D-glucosamine residues to serine and threonine of nucleocytoplasmic proteins of multicellular eukaryotes. The microtubule associated-protein tau has been suggested to be extensively O-GlcNAc modified and to compete with tau phosphorylation. Tau becomes hyperphosphorylated in Alzheimer’s disease (AD) which leads to the formation of the characteristic aggregates seen in the brains of AD patients. The reciprocal relationship between tau O-GlcNAcylation and tau phosphorylation suggests that if O-GlcNAc levels rise then phosphorylation levels should decrease. If O-GlcNAc levels can be increased in vivo, hyperphosphorylation of tau might be, therefore, antagonized by the increased levels of O-GlcNAc. One way to increase O-GlcNAcylation of tau is to inhibit the enzyme which removes O-GlcNAc, a glycoside hydrolase referred to as OGA. Existing OGA inhibitors were unsuitable for use in vivo. For this reason, a potent, selective, and stable inhibitor, termed Thiamet-G, was developed in the laboratory. Treatment with Thiamet-G increased O-GlcNAc levels in both cells as well as in the mouse brain. These substantial increases in O-GlcNAc, resulted in decreased levels of tau phosphorylation at sites of phosphorylation that are implicated in AD. Collaboratively, I also found that Thiamet-G treatment results in prevention of neurodegeneration in the JNPL3 mouse model and further observed that this effect does not appear to arise by prevention of hyperphosphorylation but more likely by blocking the aggregation of tau. This proposal is further supported by experiments showing that O-GlcNAc modification of tau impairs the aggregation of truncated and full length human tau in vitro. In order to study the site-specific effects of tau O-GlcNAcylation several sites of tau O-GlcNAc were mapped collaboratively and the major site of tau O-GlcNAc was found to be Ser400. I developed a site-specific Ser400 O-GlcNAc tau antibody and find that this site of glycosylation plays a key role in the aggregation of tau in vitro. The tools described herein should prove useful in further clarifying the role of O-GlcNAc in tau biology and also may serve to clarify OGA as a target with therapeutic potential.