Dissecting the intricacies of sterol dysregulation in Huntington’s disease

Background: Huntington’s disease (HD) is a devastating neurodegenerative condition, caused by an expanded polyglutamine tract in the Huntingtin (Htt) protein. HD is manifested by personality changes, movement disorders and/or dementia. Prior evidence exists for sterol dysregulation in HD models, observed both in neurons and astrocytes. In the brain the cholesterol is produced in situ, as the blood brain barrier prevents the cholesterol metabolized in the periphery to be used by the brain cells. The consensus stablished supports the idea that neurons synthesize the cholesterol needed in the soma, but rely on the cholesterol delivered by astrocytes in the form of apolipoproteins at synapses. A key player studied to be responsible to communicate the cholesterol status of neurons to astrocytes is the 24S-hydroxycholesterol (24S-OHC) also knows as the cerebrosterol (the brain oxysterol). Aim: Prior results from the lab showed cholesterol accumulation in primary striatal neurons infected with lentiviral expression vectors encoding a mutant Htt fragment (Htt171­82Q) compared to cells expressing a wild-type Htt fragment (Htt171­18Q). Following on these results and the previous published work from different laboratories, the aim of this thesis was to investigate the sterol status of HD striatal neurons in order to better understand this process and the consequences for the neurons and astrocytes’ health in the context of HD. Methods: Filipin staining, cholesterol oxidase assay, isotope-dilution gas chromatography­mass spectrometry, NeuN positive staining cell count – neuronal cell death assay, Mitosox assay, Fluo-4 AM imaging, extracellular multi-electrode array recordings, immunocytochemistry and real-time quantitative PCR. Results: I observed sterol accumulation in primary striatal neurons expressing mutant Htt, which one release into the medium significantly increased levels of 24S-OHC. The cerebrosterol is toxic to striatal neurons and the mechanism elucidated was by inducing increased superoxide levels in the mitochondria. Besides the toxicity effect, 24S-OHC was observed to sensitize N-Methyl-D-aspartate (NMDA) receptor function, measured by increased calcium influx in the presence of NMDA and increased amplitude and frequency of firing in the presence of glutamate, modulating NMDA receptor activity. In addition, the studies collected support that 24S-OHC induces sterol dysregulation in astrocytes by downregulating SREBP2 target genes and the purposed mechanism is by blocking the transcription factor’s migration into the nucleus, in this way promotes inhibition of cholesterol and its precursors synthesis. In parallel 24S-OHC up-regulates LXR target genes aiming to increase cholesterol efflux. Conclusion: The results part of this thesis conveyed with others’ published work and brought novelty into the field. Like others I observed sterol accumulation at the plasma membrane and in lysosomes and stablished a link with the sterol dyshomeostasis observed in my model with HD hallmarks – mitochondrial stress and excitotoxicity, caused by the increased of 24S­OHC efflux. In addition, I was also able to link the sterol dysregulation in neurons with astrocyte’s cholesterol dyshomeostasis, as evidenced by a different group. This thesis provides new insights into the potential aetiopathogenic mechanisms of HD and reconcile some apparently disparate previous findings regarding sterol disposition in this disorder.