Synthetic viral RNA mimetics induce neuronal loss through microglial phagocytosis.

BACKGROUND: Microglial Toll-like receptor (TLR) 3 and 7 signalling is induced during active viral inflammation and emerging evidence suggests that it may persist in post-infectious state due to continued presence of free viral RNA. In addition, TLR3 and TLR7 can recognize self-derived RNA, leading to sustained microglial activation. TLR3 and TLR7 activation have also been implicated in neurodegenerative processes in major neurodegenerative disorders. However, the link between chronic TLR3- and TLR7-mediated neuroinflammation and progressive neuronal damage remains unclear.

METHODS: Neuronal-glial co-cultures and pure microglial cell cultures isolated from Wistar rat brain tissue were treated with synthetic TLR3 and TLR7 receptor activators-poly(I:C) and loxoribine. Neuronal density and viability were evaluated by double nuclear staining with Hoechst 33342 and propidium iodide. Microglial cell number and area were assessed by isolectin-IB4 labelling. Phosphatidylserine exposure on neuronal membranes was detected by Annexin V-Cy3 labelling. Evaluation of microglia activation (Iba-1 and CD68 expression), caspase-3 activation, synaptic density (synapsin-1/PSD-95 colocalization), neuronal (NeuN-positive cells) detection inside microglia were carried out by immunostaining. Levels of TNF-α, lactadherin and galectin-3 in cell culture medium were quantified by ELISA, while nitric oxide production was determined using Griess assay. Microglial phagocytic activity was assessed by measuring the uptake of phosphatidylserine-coated microparticles.

RESULTS: In this study, we show that poly(I:C) and loxoribine induced loss of viable neurons without any increase in apoptosis (chromatin condensation/caspase-3 negative) or necrosis (propidium iodide negative) in neuronal-glial co-cultures. Neuronal loss was associated with alterations in microglial number and morphology, as well as enhanced production of pro-inflammatory factors and upregulated Iba-1 and CD68 expression. In co-cultures, poly(I:C) and loxoribine induced phosphatidylserine exposure on neuronal plasma membranes acting as 'eat-me' signal and opsonin lactadherin and galectin-3 production promoting phosphatidylserine recognition. NeuN-positive neurons exhibiting normal chromatin distribution were found inside of microglial cells, indicating their removal through phagocytic uptake. Under exposure to poly(I:C) and loxoribine, microglial cells showed enhanced phagocytic activity, evidenced by increased uptake of phosphatidylserine-covered polystyrene beads. Moreover, poly(I:C) and loxoribine were shown to induce microglia-dependent synaptic loss, indicating that TLR3 and TLR7 associated neuroinflammation contributes to both synaptic and neuronal pathology.

CONCLUSION: Overall, our data suggest that synthetic TLR3 and TLR7 ligands poly(I:C) and loxoribine cause microglia-mediated neuronal and synaptic loss in neuronal-glial co-cultures through phagocytic uptake.