Role of NRP1/HDAC4/CREB/RIPK1 Axis in SARS‐CoV2 S1 Spike Subunit‐Induced Neuronal Toxicity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is associated with neurological symptoms, but the molecular mechanisms have not yet been identified. Since the S1 subunit (S1) of the envelope of the SARS-CoV2 Spike glycoprotein can reach the CNS, we studied whether S1 could cause neuronal death in a direct manner. Transfection of the S1 plasmid in SH-SY5Y cells reduces cell survival in a time-dependent manner, whereas the overexpression of the S2 subunit does not. Notably, isoform 4 of histone deacetylases (HDAC4) is involved in S1-induced cell toxicity, whereas, among the different cell death drug inhibitors, only the necroptosis blocker Necrostatin-1 counteracted the neurodetrimental effect of S1. Coherently, an increase of the necroptosis marker receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and a reduction of its transcriptional repressor cAMP response element-binding protein (CREB) occur in S1-overexpressing cells. Noteworthy, HDAC4 interacts with CREB determining its protein reduction and the consequent increase of RIPK1. Importantly, we found that S1 recombinant protein (S1rp), through the internalization of the surface receptor Neuropilin 1 (NRP1), but not via Angiotensin-Converting Enzyme 2 (ACE 2) receptor, enters the cytoplasm causing cell death in differentiated SH-SY5Y cells. Finally, in accordance with other papers demonstrating that COVID-19 patients had more severe ischemic strokes with worse outcomes, we found that S1rp increased oxygen glucose deprivation/reoxygenation-induced toxicity in an additive manner, via the NRP1/HDAC4/CREB/RIPK1 pathway. In conclusion, this is the first report identifying the molecular determinants involved in Spike S1-induced neurotoxicity.