Use of a human cell model of tuberous sclerosis complex (TSC) for study of neuroinflammation and neuronal dysfunction in dementia

Tuberous sclerosis complex (TSC) is caused by mutations in the genes TSC1 or TSC2 and is a rare genetic disorder. These mutations result in loss of function of the respective gene, thus leading mammalian target of rapamycin complex 1 (mTORC1) overactivity. TSC is a multi-organ disorder with known comorbidities such as glia tumours, epilepsy, autism spectrum disorder (ASD) or psychiatric disorders. Despite this, it is still unknown what molecular and developmental changes are occurring in the patient brain. Alzheimer's Disease (AD) is neurodegenerative disease and the most common form of dementia. Literature research suggests that similar molecular pathways are dysregulated in both AD and TSC and this project is aiming to identify commonalities in the developmental, autophagy and inflammation related markers. Human induced pluripotent cells (iPSCs) enable human astrocytes and neurons to be cultured in vitro, thereby circumventing developmental and genetic differences of an animal model compared to humans, and they allow the generation of neurons and astrocytes; cell types which are otherwise inaccessible from humans due to major ethical concerns unless post-mortem. By using two CRISPR-based techniques CRISPR-Cas9 and CRISPR-Cas13a systems, both knockout and knockdown models for TSC1 were generated. These cell lines underwent neuronal differentiation to identify changes in developmental and autophagy markers similar to AD and a database analysis of TSC patients identified further target genes/pathways which were common between both disorders. The TSC1 cell models were used to generate neurons to investigate the effect of TSC1 loss on neuronal development. While the knockout model creates a chronic loss of TSC1, the knockdown model enabled controlled reduction of TSC1 mRNA at different timepoints of development for brief periods. Expression of developmental, inflammation, autophagy and urokinase pathway marker genes were tested at different timepoints of the neuronal development. Astrocytes generated from Cas13 cells, where the TSC1 knockdown was induced at different levels, demonstrated excitotoxicity as well as dysregulated calcium signalling. The dysregulation of the analysed marker genes seen in both TSC1 models is consistent to that previously reported in both TSC and AD, strengthening the overlap between both disorders. Overall, both TSC1 cell models displayed significant dysregulation of markers throughout the development, demonstrating the importance of TSC1 expression for neuronal differentiation as acute loss caused significant dysregulation for weeks after the treatment. As these dysregulated markers align with observed AD pathology, this strengthens the hypothesis of that the TSC pathway will be an interesting target for future AD research and potential treatment.