Uncovering the Role of the WASH Complex in Neurological Disorders

Multiple neurodevelopmental and neurodegenerative disorders are driven by disruptions to intracellular trafficking between organelles in neurons. One branch of this trafficking network, the endo-lysosomal pathway, is thought to tightly regulate many neuronal processes, including the dynamic recycling of synaptic receptors and the homeostatic regulation of membrane lipid composition. This idea is supported by numerous accounts of human neurological diseases that develop from the disruption of endo-lysosomal processes. In particular, mutations in subunits of the endosomal WASH complex, Strumpellin and SWIP, are implicated in motor and cognitive disorders, but the cellular etiologies of these associations are unknown (Assoum et al., 2020; De Bot et al., 2013; Elliott et al., 2013; Ropers et al., 2011). This dissertation focuses on dissecting the molecular function of the WASH complex in mouse brain and determining how mutation of the WASH subunit, SWIP, drives neurological dysfunction.To explore the role of the WASH complex in neurons, we first sought to identify the molecular interactors of WASH in mouse brain. We utilized in vivo BioID (iBioID) to delineate the neuronal WASH complex proteome in wild-type mouse brain, revealing a rich network of endosomal proteins. Comparison of this network to previously reported WASH interactors in other cell types demonstrated the conservation of WASH function in endosomal trafficking within the mouse nervous system. Then, to uncover how dysfunction of endosomal SWIP leads to disease, we generated a novel mouse model of a human WASHC4c.3056C>G mutation. We performed quantitative spatial proteomics analysis of SWIPP1019R mouse brain, which demonstrated that this mutation destabilizes the WASH complex and causes significant perturbations in both endosomal and lysosomal pathways. We then utilized cellular and histological analyses to confirm that SWIPP1019R results in an endo-lysosomal disruption in vitro and in vivo. We also found indications of neurodegeneration in SWIPP1019R mice that appeared selective for the motor cortex. To determine if these structural changes produced functional effects in SWIPP1019R brain, we conducted behavioral assays in wild-type and mutant SWIPP1019R littermates at adolescence and adulthood. These studies revealed that SWIPP1019R not only impacts cognition, but also causes significant progressive motor deficits in mice. By analyzing human SWIPP1019R patient data, we found that this WASHC4 mutation produces similar movement deficits in humans. Combined, our research provides the first molecular analysis of WASH complex function in the mammalian nervous system, and supports the model that WASH complex destabilization, resulting from SWIPP1019R, drives cognitive and motor impairments via endo-lysosomal dysfunction in the brain.