Gao, Tianhao; (2025) Functional Analysis of Cholesterol 25-hydroxylase in Neuroinflammatory Conditions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The brain, the most cholesterol-enriched organ, contains nearly a quarter of the body's total cholesterol. About 70% of brain cholesterol is in the myelin sheath. Damage or loss of myelin, known as demyelination, is associated with several neurological diseases such as multiple sclerosis (MS). In demyelinating diseases, microglia are critical for repair after demyelination and support remyelination through a series of processes: phagocytosis of myelin debris, secretion of regenerative factors and regulation of the extracellular matrix. Cholesterol 25-hydroxylase (CH25H), a multi- transmembrane endoplasmic reticulum oxidoreductase, is the essential enzyme that catalyses the conversion of cholesterol to 25-hydroxycholesterol (25-OHC). Interestingly, RNA sequencing data show that Ch25h is highly expressed in activated microglia and macrophages during neuroinflammatory conditions such as lipopolysaccharide (LPS) treatment. However, it remains unclear whether CH25H plays a crucial role in demyelinating diseases or other neuroinflammatory conditions. In this thesis, a Ch25h-specific RNA probe was developed and applied to multiple CNS inflammatory models. High expression of Ch25h was also observed in activated microglia in LPC- and cuprizone-induced demyelination models, particularly during the acute phase. In MS models, Ch25h-deficient mice exhibited decreased microglial recruitment and activation, and further induced increased lipid droplet accumulation. Ch25h-deficient mice also impair oligodendrocyte progenitor cell (OPC) proliferation and delay remyelination, especially in the LPC models. Similar phenotypes were observed in the cuprizone model, where early-stage OPC proliferation was reduced. In vitro assays revealed that Ch25h and its enzymatic product 25-OHC enhanced microglial migration but did not directly impact phagocytosis of myelin debris. Transcriptomic profiling via RNA-seq of Ch25h-deficient primary microglia revealed downregulation of disease-associated microglia (DAM) genes and significant alterations in lipid metabolism pathways. Overall, the findings of the present thesis show that Ch25h is crucial for regulating microglial lipid metabolism and supporting early remyelination in acute demyelinating conditions. Further investigation using chronic demyelination models or tissues from MS patients may provide a more comprehensive understanding of CH25H function in disease progression. This thesis provides foundational insights into Ch25h as a potential therapeutic target in MS, linking cholesterol metabolism, immune activation, and myelin repair.

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