Investigating the role of HDL and PCSK9 as pharmacological targets in neurodegenerative disorders: focus on Multiple Sclerosis and Alzheimer's disease

Neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer’s disease (AD) pose significant challenges to global health. However, the underlying molecular mechanisms of these diseases remain only partially elucidated and the currently available pharmacological treatments are unable to effectively counteract the progression of neurodegeneration. A large body of evidence has highlighted that alterations of cerebral cholesterol homeostasis represent a crucial factor in the onset and progression of these disorders. Specifically, the importance of an adequate cholesterol supply to neurons for proper brain development and functionality has already been reported, and this is ensured by brain astrocyte-to-neuron cholesterol transport. The present doctoral research project aimed to investigate the contribution of cholesterol metabolism dysregulation to neurodegeneration by integrating ex vivo and in vitro approaches, with particular focus on HDL and PCSK9 as interrelated molecular determinants. In the first section, a clinical case-control study evaluated HDL functionality in the serum and cerebrospinal fluid (CSF) of MS patients. HDL function, specifically the cholesterol efflux capacity (CEC) from astrocytes, the first step of cerebral cholesterol transport, was markedly reduced in the CSF of MS patients. This impairment mirrored observations previously reported in AD, suggesting a shared alteration of CNS cholesterol transport across neurodegenerative diseases. Notably, defective HDL functionality was more considerable in MS patients with oligoclonal bands, a marker of neuroinflammation, indicating that inflammatory processes may further disrupt HDL-mediated cholesterol transport within the CNS. In contrast, peripheral HDL function remained unaltered, suggesting that cholesterol dysregulation in MS is primarily central rather than systemic. The acute phase protein serum amyloid A (SAA) was significantly increased in MS serum but not in CSF, reinforcing its role as a peripheral inflammatory biomarker. Additionally, elevated PCSK9 levels in the CSF, but not in serum, of MS patients further supported the relationship of this protein with neurodegeneration, as already observed in AD. Collectively, these data support the hypothesis that HDL dysfunction and PCSK9 upregulation are pivotal elements contributing to MS and other neurodegenerative pathologies. The second part of this work examined the molecular interplay between ApoE4 isoform and PCSK9 in the context of AD neurodegeneration. ApoE4, the strongest genetic risk factor for AD, was shown to exert detrimental effects on cholesterol uptake, exacerbating the PCSK9-induced negative effect, confirming the isoform-specific negative impact of ApoE4 on neuronal cells. Moreover, mechanistic investigations revealed that ApoE4 induces PCSK9 expression and secretion, whereas ApoE3 exerts no effect. These findings suggest a transcriptional relationship in which ApoE4 upregulates PCSK9, amplifying neurodegenerative cascades by reducing neuronal cholesterol availability and compromising neuronal survival. Based on these insights, the third section examined the potential of PCSK9 inhibition as a therapeutic approach for mitigating neurodegenerative processes. A series of novel small-molecule PCSK9 inhibitors was synthesised and tested in neuronal models and patient-derived astrocytes. Importantly, new compounds, after demonstrating good PCSK9 inhibition activity on protein expression and secretion, modestly increased LDL receptor expression and improved neuronal viability following amyloid-β₁₋₄₂ (Aβ₁₋₄₂)-induced toxicity, displaying a concentration-dependent neuroprotective effect. These compounds also dampened NF-κB pathway activation, indicating a reduction in PCSK9-mediated neuroinflammatory signalling. Among the tested molecules, MR-3 emerged as the most promising candidate to be tested also in patient-derived astrocytes. In this cell model, MR-3 selectively reduced PCSK9 expression in AD-derived astrocytes without showing cytotoxicity, suggesting preferential inhibitory activity under pathological conditions. While the precise mechanisms require further validation, these outcomes highlight PCSK9 inhibition as a promising pharmacological strategy counteracting apoptotic and inflammatory processes implicated in AD pathogenesis. Overall, this study delineates a mechanistic framework linking neurodegenerative disorders, namely MS and AD, and dysregulation in lipid metabolism. In fact, it was demonstrated the negative impact of HDL dysfunctions in MS, and the unfavourable and synergistic effect of ApoE4 and PCSK9 on neuronal cholesterol availability and survival. Moreover, it provides compelling preclinical evidence that pharmacological inhibition of PCSK9 confers neuroprotective and anti-inflammatory effects, thus representing a promising therapeutic approach against neurodegeneration. These findings open new perspectives in the field of neuropharmacology, positioning the modulation of lipid-related pathways as a multitarget strategic frontier in the development of treatments for neurodegenerative disorders.