Cholesterol is an essential molecule in central nervous system physiology and cell signalling, with a wide range of roles, such as being an essential component of cell membranes, lipid rafts and myelin sheets, or serving as a precursor for neurosteroids. The brain relies mainly on de novo cholesterol synthesis and eliminates cholesterol through its conversion to 24(S)-hydroxycholesterol (24OHC), which readily crosses the blood brain barrier into circulation. The enzyme that catalyzes this reaction is the neuronal-specific cholesterol 24-hydroxylase (CYP46A1). Knockout-mice for Cyp46a1 exhibit inactivation of brain cholesterol synthesis, which is accompanied by deficiencies in learning and memory. It has been suggested that the cognitive deficits might be due to a reduction in the levels of intermediaries of the mevalonate pathway, namely isoprenoids. However, it has never been shown if modulation of CYP46A1 expression could effectively affect prenylation, a post-translation modification critical for membrane association of signalling proteins with fundamental roles in cell biology, such as small guanosine triphosphate-binding proteins (sGTPases). Hence, we started by assessing the effect of CYP46A1 expression on the activation of sGTPses in neuronal cells. We observed that increased expression of CYP46A1 enhanced prenylation and activation of sGTPases of the Rho and Rab family, and that this effect was dependent on the activation of the mevalonate pathway. Since sGTPases control a wide variety of functions in the cell, a great number of cellular pathways might be modulated by CYP46A1. Indeed, we have shown that CYP46A1 overexpression leads to a decrease in Liver X Receptor (LXR) transcriptional activity and in mRNA levels of LXR-target genes involved in cholesterol efflux, in a prenylation-dependent manner. These results highlight a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons enhance isoprenoid synthesis and sGTPase prenylation. Moreover our results also showed that CYP46A1 triggers an increase in neuronal dendritic outgrowth and dendritic protrusion density, and elicits in vitro and in vivo increase of synaptic proteins in crude synaptosomal fractions. Strikingly, in neurons, these effects were abolished by pharmacological inhibition of geranylgeranyl transferase I (GGTase-I) activity. Furthermore, CYP46A1 expression increased tropomyosin-related kinase (Trk) receptors phosphorylation, its interaction with GGTase-I, and the activity of GGTase-I. This interaction Trk-GGTase-I was shown to be crucial for the enhanced dendritic outgrowth mediated by CYP46A1. Cholesterol supplementation studies indicated that cholesterol reduction by CYP46A1 is the necessary trigger for these effects. Taking into account the role of CYP46A1 in cholesterol elimination and neuronal function, we hypothesized that it could be a potential therapeutic target in Niemann-Pick type C disease (NPC), a lysosomal storage disorder, characterized by cholesterol accumulation in the late endosomes/lysosomes (LE/L) compartment, leading to progressive neurodegeneration. Upon ectopic expression of CYP46A1 in human NPC fibroblasts, we observed a reduction in cholesterol accumulation in the LE/L compartment, which was accompanied by partial normalization of the expression levels of several genes involved in cholesterol homeostasis. We used the chemical compound U18666A to mimic the NPC phenotype in neuronal cells, and observed that CYP46A1 overexpression protects and reverts cholesterol accumulation induced by U18666A, but also inhibits the increase in reactive oxygen species and lipid peroxidation. Further experiments led us to conclude that CYP46A1 induces the expression of the antioxidant enzyme heme-oxygenase-1 (HO-1), and that the activity of this enzyme contributes to CYP46A1-mediated protection against oxidative stress. This work contributes to a further understanding of how cholesterol influences the brain, namely how the cholesterol-metabolizing enzyme, cholesterol 24S-hydroxylase affects neuronal development and function, and highlights the role of this enzyme as a therapeutic target in NPC, a disorder that has very limited therapeutic options.

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016