Probing cholesterol homeostasis in models of neurodevelopmental disorders

Neurodevelopmental disorders (NDDs) are complex heterogenous brain disorders caused by impaired development or maturation of the central nervous system. NDDs affect about 1% of the population and result from genetic and/or environmental factors. NDDs comprise intellectual disability (ID), autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), amongst others (APA, 2013). ASD is the most studied neurodevelopmental disorder with hundreds of risk genes and several molecular pathways identified as involved in disease pathology. One relatively unexplored pathway which could be implicated in ASD and other NDDs is cholesterol metabolism. Indeed, alterations in cholesterol metabolism have been reported in Fragile X Syndrome (Berry-Kravis, 2015) and Rett’s Syndrome (Buchovecky, 2013), two monogenic syndromes where a substantial fraction of individuals meets diagnostic criteria for ASD. Cholesterol is a major lipid in mammalian membranes and is particularly enriched in the brain, where it can affect synaptic activity and plasticity (Korinek et al., 2020, Djelti et al., 2015, Li et al., 2022). However, detecting cholesterol alterations in the brain is technically difficult and complicated by the localized synthesis and transport of cholesterol across cell types and sub-cellular compartments.
In this thesis, I report the development of a ratiometric, genetically-encoded probe that can be applied to monitor cholesterol distribution and levels in a cell-type-specific manner. I validated this probe for in vivo use by probing cholesterol distribution in NPC1KO mice, a genetic model for a lysosomal storage disorder with cholesterol transport deficits that is characterized by developmental delay and neurodegeneration. I further applied these probes to examining cholesterol distribution in Ptchd1KO mice. Patched-domain containing 1 (Ptchd1) is an X-chromosomal risk gene for both ASD and ID. Alterations in excitatotory / inhibitory (E/I) balance and behavior have been reported in Ptchd1KO mouse models, but the function of Ptchd1 protein remains unknown. Due to the presence of a sterol-sensing domain (SSD), we hypothesized that Ptchd1 could be involved in cholesterol transport or homeostasis similarly to other SSD-containing proteins. We looked for cholesterol-related phenotypes in Ptchd1-expressing neuronal populations in a global Ptchd1KO mouse model, previously established and characterized by our lab (Tora et al., 2017). We did not observe any significant changes in cholesterol levels and distribution using the cholesterol D4H probe or lipidomic analysis. Furthermore, no changes were detected in the peripheral blood of the Ptchd1KO animals. The lack of cholesterol phenotype in this model seems to indicate that Ptchd1 is not essential for maintenance of cholesterol homeostasis in mice. I further probed Ptchd1 sub-cellular localization and identified a novel interacting protein. My data suggests that Ptchd1 is localized to early endosomes, interacts with the retromer complex, as well as a the neddylation enzyme Uba3. In sum, this work deepens our understanding of the Ptchd1 protein and the pathophysiology associated with Ptchd1 mutations, and provides a novel genetically-encoded probe for examining cholesterol homeostasis in vivo.