Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

Numerous studies suggest that abnormal upregulation of the complement cascade, a key component of the innate immune system, is involved in the pathogenesis of Alzheimer’s disease (AD), also contributing to synapse elimination in the brain. Several pieces of evidence suggest that recurrent herpes simplex virus-1 (HSV-1) infection reaching the brain is one of the AD risk factors, including those reporting synaptic loss and consequent cognitive deficit following multiple virus replication in the brain. However, the role of complement cascade activation in such events remains unexplored. Murine primary neurons co-cultured or not with microglial BV2 cells and organotypic hippocampal brain slices were used as experimental models of HSV-1 infection. Virus effects on complement cascade activation and synaptic loss were assessed by evaluation of protein and mRNA levels of specific complement components and synaptic markers. Confocal immunofluorescence microscopy was used to analyze microglial phagocytosis of synapses. To evaluate the role of complement cascade activation in such event, experimental models were treated with a neutralizing anti-C3 antibody within the infection. Two-photon imaging and patch-clamp recordings of organotypic hippocampal slices were used to quantify dendritic spine density on secondary apical dendrites of CA1 pyramidal neurons and synaptic transmission. We first found that HSV-1 infection significantly upregulates the expression of components of the classical complement cascade at both mRNA and protein levels, and promotes their localization at synapses. Then, we provide evidence that HSV-1 infection causes increased microglial phagocytosis of synapses, which is partially prevented when the complement cascade is inhibited. Furthermore, by exploiting murine organotypic hippocampal slices, we confirmed that the virus triggers synaptic damage through microglial pruning of synapses via a complement-dependent mechanism. Importantly, in infected CA1 neurons, we detected a significant decrease in spine density, which was paralleled by functional alterations in synaptic transmission. Both events were rescued when infection is performed in the presence of an antibody neutralizing the complement C3 protein. Our data indicate that HSV-1 infection triggers aberrant complement activation and complement-mediated microglial engulfment of damaged synapses, further supporting the role of HSV-1 in neurodegeneration. Herpes simplex virus type 1 (HSV-1), a neurotropic and widespread pathogen, has been suggested as a potential risk factor for Alzheimer’s disease (AD), the most common form of dementia in elderly people. However, the molecular mechanisms triggered by the virus remain to be fully elucidated. Increasing evidence suggests that the innate immune system, particularly the complement cascade, plays a key role in early synapse loss, a hallmark of AD progression. Here, we demonstrate that HSV-1 infection of brain cells induces upregulation of complement components, promoting complement-dependent microglia engulfment, thus contributing to synaptic loss. Our results support a causative role of complement in HSV-1-induced synaptic pruning thus providing a novel understanding of how HSV-1 infection contributes to synaptic loss in AD.