Identification of novel regulatory mechanisms to control cholesterol homeostasis: a focus on brain cells

Cholesterol plays several structural and metabolic roles that are pivotal for the human body. Various experimental evidence highlights that the homeostatic maintenance of cholesterol metabolism is essential in the brain, especially for differentiation, synaptogenesis and neurotransmission. The most part of cholesterol in the central nervous system (CNS), occurs through de novo synthesis, which specifically occurs in brain cells. Differently from embryogenesis, when neurons autonomously provide for their cholesterol needs, during adulthood neurons abandon the biosynthesis and the requirement of this sterol is supplied by ApoE-rich lipoproteins derived from astrocytes, which remains metabolically active. Several data evidenced that epigenetic factors can play a key role in the control of metabolic processes, including cholesterol metabolism. In this regard, the involvement of Bromodomain and Extra-Terminal domain (BET) proteins is still unclear. Thus, the initial objective of this PhD project was to investigate the role of BET proteins in the modulation of lipid homeostasis. Since cholesterol homeostasis is particularly active in hepatic cells, we employed human liver-derived HepG2 cells, a widely accepted experimental model to study lipid metabolism. The role of BET proteins was unraveled by using a loss-of-function approach, by treating HepG2 cells with JQ1, a powerful and selective BET inhibitor. The main results show that BET blockade determined a significant modulation of the proteins involved in lipid biosynthesis, uptake and intracellular trafficking. Once ascertained that BET blockade by JQ1 modulates cholesterol metabolism, the effects of BET inhibition were evaluated on neuronal differentiation, an important physiological process that leads to the formation of new neurons from neural stem cells (NSC). Interestingly, this process is strongly based on cholesterol metabolism. Therefore, we assessed whether BET inhibition could stimulate neuritogenesis through the modulation of cholesterol homeostasis. To reach this aim, different neuronal-derived cell lines were used as experimental model. Data collected displayed that JQ1-mediated BET inhibition induces neuronal differentiation through a remarkable modulation of cholesterol metabolism. In addition, we found that BET blockade-mediated effects are accompanied by the activation of specific signaling pathways and by autophagy induction. To strengthen the validity of these results, the pro-differentiating effects of JQ1 were confirmed in isolated NSC and on in vivo adult neurogenesis in mice. The attention was then focused on the study of signaling factors putatively involved in the control of cholesterol homeostasis in brain cells. In this context, neurotrophins constitute a class of growth factors capable of influencing metabolism of different cell types, although they were initially identified as signal molecules closely involved in neuronal survival and differentiation. Specifically, we examined the impact of nerve growth factor (NGF) in cholesterol homeostasis. Since this metabolic process is highly regulated in astrocytes, we investigated whether NGF could influence cholesterol metabolism in glial cells. To reach this objective, an astrocyte-derived cell line, U373, was employed. Our results show that proteins implicated in cholesterol metabolism network increase following NGF administration in U373 cells. Furthermore, NGF considerably enhances ApoE secretion and extracellular cholesterol content in the culture medium. Since ApoE has been reported to increase the resilience of neurons to oxidative insult, we evaluated the impact of NGF on survival under conditions of oxidative stress. Co-cultures and U373-conditioned medium experiments revealed that NGF treatment efficiently contrasts redox dysbalance in N1E-115 neurons. On the contrary, neuroprotective effects mediated by NGF are abolished when N1E-115 are co-cultured with ApoE-silenced U373 cells. Thereby, these data suggest that NGF modulates cholesterol metabolism in astrocytic cells and exhibits neuroprotection against oxidative stress by promoting glial ApoE secretion. Collectively, the data collected in this PhD thesis provide new hints about the molecular mechanisms linking the regulation of cholesterol metabolism in brain cells. Clarifying the molecular mechanisms that induce, regulate and maintain brain processes is not only fundamental to better understand the CNS physiology, but also to develop new therapeutic strategies useful for counteracting most neurodevelopmental and neurodegenerative diseases associated to defects in cholesterol metabolism.