Dottorato in MALATTIE INFETTIVE, MICROBIOLOGIA E SANITA' PUBBLICA

Titolo della tesi: GLIA-NEURON CROSSTALK DURING HSV-1 INFECTION AND ITS ROLE IN NEURONAL DAMAGE

Herpes simplex virus type 1 (HSV-1) causes a contagious infection that affects approximately 60% to 95% of adults worldwide. HSV-1 is associated mainly with infections of the mouth, pharynx, face, eye, and central nervous system (CNS). Its replication has been associated with the induction of neurodegenerative processes typical of Alzheimer’s disease (AD). Previous data show that in vitro HSV-1 infection enhances AD hallmarks’ neuronal appearance: production/accumulation of neurotoxic fragments of Amyloid Precursor Proteins (APP) and hyperphosphorylation of Tau protein; this data was recently confirmed in an in vivo recurrent HSV-1 infection model. It is known that CNS is populated not only by neurons but also by glia and microglia, and it is possible to hypothesize that, during HSV-1 infection, these cells surrounding neurons may undergo activation and expression of proinflammatory genes, enhancing neuronal damage. Microglia and astroglia are consistently found surrounding amyloid plaques in AD brains. Aβ deposition causes a microglial-mediated inflammatory response. Proinflammatory molecules are involved in pathways of neuronal apoptosis. Even though we already know something about the role of glia during HSV-1 infection of the brain, it is still unknown whether glial activation is protective or harmful or, more likely, whether this depends on the disease stage. The present Ph.D. thesis aims to investigate the glia-neuron crosstalk during HSV-1 induced neuronal damage and the role of microglial cells in it. In order to investigate the glia-neuron crosstalk during HSV-1 infection, mixed-neuronal-glia co-cultures were infected with HSV-1 (m.o.i. 1) and after 24h the conditioned media (sup) was treated with UV to inactivate the neo-formed virions and transferred onto neurons or mixed neuronal-glia co-cultures (pretreatment) for 24 h (viral titer were checked before and after UV by standard plaque assay). Then, the cellular monolayers were infected with HSV-1 (m.o.i. 3). After 20h, the viral titer was quantified by standard plaque assay: HSV-1 infection resulted to be inhibited in pretreated co-cultures (about 1.5/2 log) respect to unpretreated one. These data suggested that sup from HSV-1-infected Mixed Neuron-Glial co-cultures contains some cytokines, as well as interferon type 1 (INF-I), know to induce an antiviral program, and/or IL-6, know to trigger inflammatory genes (including INF-I) transcription via Nf-kB or STAT3. Previous data from our lab demonstrated also Abeta peptides release in the sup of HSV-1 mixed cells and Abeta is known to induce STAT3 activation to enhance INF-I gene transcription. Then, the subcellular distribution of p65 (a NF-kB family member) was studied. We observed by WB and confocal analysis that during HSV-1 infection (with or without pretreatment) p65 moves from cytoplasm to nucleus, and there its phosphorylation state was significatively increased in pretreated HSV-1-infected samples respect to mock-infected and unpretreated HSV-1-infected cells. These results suggest a clear activation of the transcription factor Nf-kB and its target genes, including neuroinflammatory genes, by the protein content of the sup. The low viral titer of pretreated HSV-1-infected samples could be supported by further activation of INF-I gene via NF-KB. We also investigated the effects of the presence of microglial cells on HSV-1-induced Tau phosphorylation and APP amyloidogenic processing. We found out that the presence of microglia influenced tau hyperphosphorylation (particularly at Thr 205 residue). Phospho-tau level was determined by confocal microscopy demonstrating that the presence of microglial cells in neuroglial co-cultures induced the level of tau hyperphosphorylation (Anti-Tau (phospho T205)) ratio in two times during HSV-1 infection (MOI 3). At the same time the level of ß-amyloid (Aß 1-42) was significantly less (two times less) the co-cultures with the presence of microglial cells. The involvement of HSV1 infection in the induction of a neuroinflammation condition suggests the use of antiviral molecules with anti-inflammatory activity. Good candidates may be resveratrol analogs, recently synthesized from “Università degli Studi della Tuscia”. These molecules were screened on Vero cells, highly permissive to HSV-1 infection, and the greatest molecule resulted GT 2.9. This molecule was tested on HSV-1-infected Vero cells and mixed-neuronal-glia co-cultures and was able to inhibit viral replication when was added after the adsorption phase of the virus life cycle. The analysis of the GT 2.9-induced inhibition mechanism revealed a reduced activation of two transcription factors, involved in several pathways including inflammatory ones: Nf-kB and Nrf2. Further studies are in progress. Overall, the results obtained from this experimental thesis demonstrate that microglial cells play a complex role in HSV-1 induced neuroinflammation and the development of neurodegenerative diseases participating in tau processes hyperphosphorylation and ß-amyloid accumulation. Moreover, HSV-1 induced microglia may impact the activation of several important inflammation signaling pathways such as STAT3, NRF2, and NFKB. Also it was demonstrated that the resveratrol derivative GT 2.9 has effective anti-HSV-1 properties, also in synergy with acyclovir, and lay the foundations for further in vitro and in vivo studies to support his candidacy as a potential anti-herpetic drug.