SVELARE I MECCANISMI MOLECOLARI DELLA NEURODEGENERAZIONE SIMILE ALLA MALATTIA DI ALZHEIMER INDOTTA DA HSV-1

Alzheimer's Disease (AD) is the main cause of dementia, characterized by neuronal damage and cognitive decline. While aging remains the principal risk factor for AD, various genetic and environmental elements contribute to its development. Among these, the neurotropic virus Herpes Simplex Virus 1 (HSV-1) has emerged as a significant factor in the infectious hypothesis of AD. This thesis explores the role of HSV-1 in AD pathology through an in-depth analysis, aiming to identify the mechanisms underlying this connection. We first examined the impact of HSV-1 infection on neuronal cells in both in vivo and in vitro models. Results obtained via immunofluorescence (IF) and Western Blot (WB) demonstrate that during the persistent infection of the enteric nervous system, HSV-1 induces the accumulation of significant AD biomarkers (amyloid-beta (Aβ), its precursor APP and hyperphosphorylated Tau). Moreover, qRT-PCR and WB analysis revealed that viral infection increases the amyloidogenic processing of APP. HSV-ΔICP27, incapable of replication and expressing only two immediate-early (IE) genes, caused an identical phenotype. These data suggest that specific viral proteins, ICP0 and ICP4, are crucial in neurodegeneration. To further investigate their role, we developed a piggyBac/Tet-On system for stable and inducible expression of HSV-1 genes in neuronal cell lines. We initially characterized the platform by expressing a fluorescent reporter gene. Vitality tests confirmed that the system is non-toxic to cells; it can be precisely tuned with varying inducer concentrations. Additionally, we observed minimal background expression in the absence of induction. All these results were confirmed when expressing HSV-1 genes of interest. Therefore, we provide a robust tool to timely study the effect of a single viral gene in terms of inducing a neurodegenerative phenotype. Our results indicated that IE genes, in particular ICP4, can trigger the appearance of AD-related markers. Our research also highlights the role of oxidative stress in HSV-1-induced neurodegeneration. We observed elevated production of reactive oxygen species (ROS) in response to HSV-1 and HSV-ΔICP27 in our infection models. Antioxidant treatment reduced virus-induced ROS levels and prevented Aβ accumulation, reinforcing the involvement of oxidative stress in the HSV-1-AD relationship. We investigated the role of inflammasome activation. Our findings reveal that HSV-1 and HSV-ΔICP27 infection activates NLRP3 and IFI16 inflammasomes in neuronal cells, leading to the activation of caspase 1 and IL-1β production. Moreover, caspase 1 inhibition led to a significant decrease in Aβ and hyperphosphorylated Tau levels. Interestingly, ICP4 expression induced NLRP3 and IFI16 inflammasome activation, indicating its fundamental role in triggering an inflammatory response in neurons during HSV-1 infection. Moreover, HSV-1 induced lipid droplet (LDs) accumulation and an increase in peroxidized lipids. qRT-PCR analysis of LDs-related genes revealed significant alterations in lipid metabolism in response to infection. Through a neural network approach applied to this dataset, we identified several proteins belonging to pathways altered in AD, linking HSV-1 and LDs to the pathology. Interestingly, co-culturing neuronal cells with glial cells led to the restoration of alterations induced by HSV-1 infection. This finding highlights the critical protective role of glial cells in mitigating virus-induced neuronal damage. In summary, this thesis provides mechanistic evidence supporting the role of HSV-1 in neurodegeneration through oxidative stress, inflammasome activation, and lipid dysregulation, with an insight into the role of ICP4. These results pave the way for future research and therapeutic strategies aimed at mitigating HSV-1-related neurological complications and potentially preventing AD.