NEUROPILIN-1 MEDIATES SARS-CoV-2 INFECTION of ASTROCYTES PROMOTING NEURON DYSFUNCTION

ABSTRACT

Background: SARS-CoV-2 primarily infects the lung but may also damage other organs including the brain, heart, kidney, and intestine. Central nervous system (CNS) disorders include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Methods: We used iPSC-derived brain organoids and primary human astrocytes from cerebral cortex to study direct SARS-CoV-2 infection, as confirmed by Spike and Nucleocapsid immunostaining and RT-qPCR. siRNAs, blocking antibodies, and small molecule inhibitors were used to assess SARS-CoV-2 receptor candidates. Bulk RNA-seq, DNA methylation seq, and Nanostring GeoMx digital spatial profiling were utilized to identify virus-induced changes in host gene expression. Results: Astrocytes were robustly infected by SARS-CoV-2 in brain organoids while neurons and neuroprogenitor cells supported only low-level infection. Based on siRNA knockdowns, Neuropilin-1, not ACE2, functioned as the primary receptor for SARS-CoV-2 in astrocytes. The endolysosomal two-pore channel protein, TPC, also facilitated infection likely through its regulatory effects on endocytosis. Other alternative receptors, including the AXL tyrosine kinase, CD147, and dipeptidyl protease 4 (DPP4), did not function as SARS-CoV-2 receptors in astrocytes. SARS-CoV-2 infection dynamically induced type I, II, and III interferons, and genes involved in Toll-like receptor signaling, MDA5 and RIG-I sensing of double-stranded RNA, and production of inflammatory cytokines. Genes activating apoptosis were also increased. Down-regulated genes included those involved in water, ion and lipid transport, synaptic transmission, and formation of cell junctions. Epigenetic analyses revealed transcriptional changes related to DNA methylation states, particularly decreased DNA methylation in interferon-related genes. Long-term viral infection of brain organoids resulted in progressive neuronal degeneration and death. Conclusion: Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. Deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.