ABSTRACT: Headache disorders, including migraine, are common symptoms of COVID-19, which may result from the sensitization of the trigeminal ganglion (TG) and brainstem relay structures. Recent studies suggest that SARS-CoV-2 may invade trigeminal nerve endings in the nasal cavity. However, despite these insights, the precise underlying mechanisms remain poorly understood. Here, we investigated the cellular and molecular changes in the TG and brainstem with a special attention for the spinal trigeminal nucleus in K18-hACE2 mouse model infected with SARS-CoV-2. We first confirmed the expression of the cellular proteins playing a role in SARS-CoV-2 cell entry (ACE2, TMPRSS2 and NRP1) in both structures. In addition, we reported the expression of the viral nucleocapsid (N) and spike (S) proteins in TG and brainstem at 6 days post infection by multimodal approaches (RNA sequencing, RNAscope, and immunohistofluorescence). In the TG, SARS-CoV-2 proteins S and N were detected in nerve fibers as well as in transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) nociceptive neurons. Transcriptomics analysis of the TG from infected K18-hACE2 have revealed significant changes in gene expression, including Ccl2, Atf3, Cxcl10, Saa3, Plin4 genes. Additionally, increased immunoreactivity for ATF3 and Iba1 has been detected in the TG of these mice post-infection. In the brainstem, SARS-CoV-2 protein was exclusively found in neurons, with no detection in astrocytes or microglial cells, the latter exhibiting an activated form in K18-hACE2 infected mice. Bulk RNA-Seq analysis clearly confirmed a cytokine and chemokine storms, inflammasome activation (Gsdmd, Casp4/1, Nlrp3), molecular markers of neuronal activation (Jun, Fos, Fosb), neuronal injury (Atf3) and pain markers (Gfra1, Ntrk1, Ptgs2). We also shown that a significant number of infected neurons were found within, or in close contact to a dense network of CGRP-positive nociceptive nerve fibers in the trigeminal brainstem. Finally, we provided gene interaction networks and identified specific SARS-CoV-2 biomarkers Saa3, Cxcl10, Ccl2, Atf3 and Plin4 in TG and brainstem which could serve as potential indicators of disease severity. In conclusion, this study reports a robust set of transcriptomic and cellular changes triggered by SARS-CoV-2 in the TG and brainstem, offering a potential mechanistic explanation for sensory abnormalities, such as migraine, observed in COVID-19 patients.
