ActivatingRAC1variants in the switch II region cause a developmental syndrome and alter neuronal morphology

Abstract

RAC1 is a highly conserved Rho GTPase critical for several cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Some of these variants have been previously shown to have a dominant negative effect. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. Here we describe eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria, and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblasts spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborisation neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause developmental syndrome. Our findings reveal that these variants cause altered downstream signalling resulting in abnormal neuronal morphology and reveal the WAVE regulatory complex/Arp2/3 pathway as a possible therapeutic target for activating RAC1 variants. These insights also have the potential to inform the mechanism and therapy for other disorders caused by variants in genes encoding other Rho GTPases, their regulators and downstream effectors.

A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association

“…The structural modeling studies also support our hypothesis that the p.Tyr40His variant inactivates RAC3-PAK1 signaling. Previous functional analyses of variants in the Switch II region showed constitutive activation of downstream signals in the RAC1-PAK1 axis 14 , and that variants adjacent to and in the Switch I region (p.Tyr40His and p.Asn39Ser) inactivate the signal. p.Tyr40His appeared to affect RAC3-PAK1 binding more strongly than p.Asn39Ser from the molecular modelling (Fig.…”

“…All previously reported cases with RAC1 variants located in Switch I or II regions involved altered GTP/GDP-bound states 13 , 14 , 23 . In the Online Mendelian Inheritance in Man ( https://omim.org/ ), de novo RAC1 variants have been found in intellectual developmental disorder (MIM#617751) with the highly variable phenotype, such as neurodevelopmental delay with abnormal brain magnetic resonance imaging findings, epilepsy, facial dysmorphisms, cardiovascular malformations and poor feeding.…”

“…In this study, we describe a case with VACTERL association and a novel de novo variant of RAC1 [NM_018890.4:c.118T > C p.(Tyr40His)] at the RAC1-PAK1 binding site, which is adjacent to the Switch I region. From the previous study, RAC1 missense variants in the Switch II region can change GTP/GDP-bound states and cause the constitutive activity of downstream pathways including PAK family kinases 13 , 14 . However, effects of other RAC1 missense variants are still undetermined.…”

DOCK3-Associated Neurodevelopmental Disorder—Clinical Features and Molecular Basis

“…De novo, heterozygous variants in RAC1 are associated with a distinct NDD that includes dysmorphic features, moderate/severe intellectual disability, seizures, hypotonia, CNS anomalies including cerebellar dysplasia, hypoplasia of the corpus callosum, enlarged ventricles, mega cisterna magna, a thin brainstem, white matter abnormalities, and polymicrogyria [41,55]. In a study, 14 cases with de novo, heterozygous RAC1 mutations were reported [41,55]. Of the six cases that had functional studies, four were shown to be gain-of-function type and two had a dominant-negative effect.…”

Presynaptic Rac1 in the hippocampus selectively regulates working memory

“…Dysregulation of Rac1 leads to abnormalities in synaptogenesis (Wiens et al, 2005), spine morphology (Nakayama et al, 2000), and synaptic plasticity (Haditsch et al, 2009), each of which has been proposed to be a contributing factor for various developmental brain disorders, including autism spectrum disorder (ASD) and schizophrenia. For example, altered Rac1 activity is a factor in ASD-like behaviors in the DOCK4-deficient mouse model of ASD (Guo et al, 2021), and a mutation in the switch II region of RAC1 is implicated in neurodevelopmental disorders (Banka et al, 2022). Additionally, mutations in the Rac-GEF Trio are associated with autism and intellectual ability and impair neuroligin-1 induced synaptogenesis (Fingleton & Roche, 2023; Tian et al, 2021).…”