Titolo della tesi: Herpes Simplex Virus -1 (HSV-1) induces complement activation in brain cells: possible trigger of synaptic deficits
Background: Several pieces of evidence suggest that recurrent herpes simplex virus-1 (HSV-1) infection reaching the brain is one of the risk factors for Alzheimer's disease (AD). Numerous studies suggest that abnormal upregulation of the complement cascade, a key component of the innate immune system, is involved in the pathogenesis of AD, also concurring to synaptic elimination in the brain. Hence, we investigated if HSV-1 triggers complement cascade activation in brain cells likely leading to synaptic loss through microglia phagocytosis.
Methods: Primary neuronal cultures were isolated from the brain of rat or mouse embryos (E17). For triple co-culture experiments, the murine microglial BV2 cell line was added to neuron/glial co-cultures in a ratio of 1:5 (microglia: neurons) before HSV-1 infection. Cells were analyzed for the expression of complement components, PSD-95 and synaptophysin (postsynaptic and presynaptic markers, respectively) at protein and mRNA levels with western blotting (WB), confocal immunofluorescence (IF) analyses, and RT-PCR. ELISA assay was performed to detect C3 in supernatants. An engulfment assay was set up to study microglial phagocytosis of synaptic material, exploiting IF. IF quantification of synaptic material inside microglial lysosomes (labeled with anti-CD68 antibody) was performed in triple co-culture experiments to study synaptic pruning. Neutralization assay was performed by infecting the co-cultures in the presence or absence of an anti-C3 antibody or compstatin, a C3 convertase inhibitor.
Results: We first checked the effect of HSV-1 infection on the intracellular expression of C1q, C3 and C4 in the primary neuron/glia co-cultures. Real-time PCR and WB analyses of cell lysates revealed that HSV-1 infection caused a significant increase of C1q, C3, and C4 at both mRNA and protein levels, whereas ELISA assay showed an increased amount of C3 in the supernatant of infected cells. Interestingly, we found that C1q and C4 localized at the synaptic level upon HSV-1 infection, suggesting that they may take part in HSV-1-driven synaptic damage. To explore this possibility, we infected cells in the presence or absence of agents able to inhibit complement activation, such as anti-C3 antibody or compstatin, and monitored PSD-95 and synaptophysin expression levels. WB analyses revealed that HSV-1 infection significantly downregulated both the synaptic proteins in untreated cultures, whereas a partial rescue was observed when the infection was performed in the presence of complement inhibitors. Results from the engulfment assay and IF analyses indicate that HSV-1 infection triggers increased microglial phagocytosis of synaptic materials, which was partially inhibited when the complement cascade is inhibited.
Conclusion: Overall our data provided evidence of a novel mechanism that may underlie the HSV-1-induced synaptic damage in murine primary neuronal cells. We indeed demonstrated that HSV-1 infection triggers in cultured neurons the upregulation of key components of the complement cascade that in turn cooperate with microglia for the phagocytosis of the synapses damaged by the virus. Our findings, by uncovering a novel mechanism through which the virus could trigger synaptic damage, further support the role of HSV-1 in neurodegeneration.