Influence of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) on brain cholesterol metabolism and potential impact on Alzheimer's Disease pathogenesis

Abstract

Alzheimer’s Disease (AD) represents the most common and burdening form of dementia against which, currently, no drug is effective in slowing or counteracting the disease. The most challenging aspect is represented by the fact that the etiopathological processes leading to AD development are not yet fully clarified. Among other hypotheses, the involvement of lipid metabolism has been postulated. Cholesterol, indeed, actively participates in several relevant processes for brain homeostasis and disturbances in its metabolism have been described in several neurodegenerative diseases, including AD. Brain relies on in situ cholesterol biosynthesis, due to the presence of BBB; moreover, adult neurons strongly depend on cholesterol provided by astrocytes, as their endogenous cholesterol biosynthetic rate is limited. Hence, brain cholesterol transport from astrocytes to neurons, specifically mediated by apoE-containing lipoparticles, represents a crucial step for neuronal homeostasis. Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9), one of the major regulators of plasma LDL-C levels, whose involvement in different processes relevant for CNS homeostasis has been described, may possibly impact directly on brain cholesterol transport by degrading cerebral lipoprotein receptors. However, the currently available evidence of an involvement of PCSK9 in AD, possibly by disturbing brain cholesterol transport, is still limited and controversial. This research work stems from the previous observation by our research group of increased PCSK9 concentrations in cerebrospinal fluid (CSF) of AD subjects as compared to controls. The main objective of this work is the investigation of the potential involvement of PCSK9 in brain cholesterol dysregulation, which represents a central aspect of AD, aiming in clarifying its role in the disease etiopathogenesis and potentially identify PCSK9 as a possible disease-related biomarker and a pharmacological target. To this aim, a translational study was set up, following three different experimental approaches including in vitro studies on cultured astrocytes and neuronal cell models, and ex vivo analyses upon samples isolated from both animal models of the disease and patients with different degrees of cognitive impairment. In the first part of the present research work, the impact of PCSK9 on the most relevant steps of brain cholesterol transport has been explored. In cultured astrocytes, PCSK9 induced an increase in endogenous cholesterol biosynthesis, which, however, wasn’t able to counterbalance the impairment of cholesterol endocytosis due to its degrading effect on lipoprotein receptor, leading to an overall reduction in astrocyte cholesterol content, furtherly lowered by the co-incubation with Aβ1-42 fibrils. The observed impairment of astrocyte cholesterol homeostasis, namely reduced intracellular cholesterol, may eventually translate into reduced supply to neurons. Consistently, in cultured neurons PCSK9 furtherly promoted cellular cholesterol depletion by impairing both the endocytic process as well as by reducing intracellular cholesterol biosynthesis, possibly resulting in compromised neuronal viability. In the second part of the present research work, the impact of PCSK9 on cholesterol homeostasis was investigated in a transgenic mouse model of AD lacking PCSK9. Specifically, the PCSK9 heterozygous and, even more, the homozygous mutation in mice led to a significant decrease in both serum and cerebral cholesterol content. Interestingly, transgenic AD mice were characterized by an overall reduction of total circulating as well as cerebral cholesterol levels, more exacerbated in mice bearing PCSK9 homozygous or heterozygous deletion. This evidence supports the involvement of PCSK9 in cerebral cholesterol homeostasis, thus reinforcing the previously described in vitro evidence. In the third part of the present research work, a case-control study has been performed to explore PCSK9 as well as the main lipid levels in 83 subjects with different degrees of cognitive impairment, namely subjects with stable MCI, patients with MCI that furtherly degenerated to AD at follow-up, and patients with clinically diagnosed AD. CSF and serum PCSK9 levels were similar among the three considered groups, but CSF PCSK9 concentration was higher as compared to that of a control group previously analysed. This evidence suggests that CSF PCSK9 levels may start to increase during the prodromal phases of the disease. AD subjects carrying at least one apoE Ɛ4 allele showed higher PCSK9 concentrations as compared to non-carriers. Furthermore, a positive correlation between serum and CSF PCSK9 levels was highlighted in AD patients, suggesting that exchange between peripheral and central PCSK9 levels may occur, possibly as a consequence of an increased BBB permeability, that has been previously reported. In conclusion, the results of this translational study, performed upon in vitro as well as ex vivo experimental approaches, indicate the involvement of PCSK9 in AD pathogenesis, possibly through the modulation of brain cholesterol homeostasis. Altogether, these data support the possibility to recognize PCSK9 as a pharmacological target in AD, for which effective therapies are still lacking. Hence, the development of small lipophilic molecules inhibiting PCSK9 and being able to cross BBB may represent an interesting future pharmacological perspective to restore brain cholesterol homeostasis, thus providing a potential future innovative therapy for AD.