Innate Immune Control of Neurotransmission

Abstract Neuroinvasive flavivirus infections, such as those caused by West Nile virus (WNV) and Zika virus (ZIKV), lead to severe neurological damage due to aberrant synaptic activity and excitotoxicity. However, despite their growing threat to public health, no approved vaccines or specific treatments for these infections exist. Emerging evidence suggests that innate immune signaling within neurons can modulate neurotransmission, offering potential avenues for neuroprotection. Our preliminary studies reveal that Receptor-interacting protein kinase 3 (RIPK3), an immune kinase traditionally associated with necroptotic cell death, plays an unexpected role in preserving neuronal survival during flavivirus infection by suppressing excitatory neurotransmission. RIPK3 activation diminishes sensitivity to glutamatergic stimulation, reduces calcium influx through N-methyl-D- aspartate receptors (NMDARs), and requires the activity of Ca²⁺/calmodulin-dependent protein kinase II alpha (CaMKIIα). In this proposal, we hypothesize that RIPK3 suppresses excitatory neurotransmission through modulation of synaptic phosphoprotein activity, promoting neuronal survival during viral encephalitis and excitotoxic stress. To test this, we will pursue two specific aims: Aim 1 will define the mechanisms by which RIPK3 controls neuronal excitability using chemogenetics, electrophysiology, and in vivo calcium imaging to assess how RIPK3 activation impacts neuronal firing and synaptic function. Aim 2 will elucidate how RIPK3's regulation of the phosphoproteome confers neuroprotection by identifying binding partners and mapping phosphorylation events downstream of RIPK3 signaling, as well as evaluating the roles of the RIPK3 targets CaMKIIα and calcineurin in neuroprotection. This research will advance our understanding of neuroimmune regulation of neurotransmission, potentially uncovering new therapeutic targets for neuroinvasive viral infections and providing fundamental new insights into the interplay between immune signaling and neuronal function.