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SARS-CoV-2 can infect and replicate in human motor neurons differentiated from induced pluripotent stem cells (in vitro study)

 

A significant proportion of patients experience neurological and neuromuscular symptoms during and after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Apart from the central nervous system (CNS), the peripheral nervous system (PNS) is also affected. In this study, the Italian authors investigated the effects of SARS-CoV-2 on the expression of the main SARS-CoV-2 receptors and infectability of human motor neurons (MNs) differentiated in vitro from induced pluripotent stem cells (iPSC-MNs).

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins, namely the spike (S), envelope (E), nucleocapsid (N), and membrane (M) protein. 

It seems that SARS-CoV-2 uses various neuroinvasive strategies and entry pathways to invade the nervous system, such as infection of the nasal olfactory epithelium and axonal transport along the olfactory nerve, retrograde axonal transport, invasion by compromising the blood-brain barrier, retrograde virus spread from the lungs to the CNS via the vagus nerve, and the use of infected hematopoietic cells as “Trojan horses” (hematogenous route). It remains unknown whether observed neuromuscular manifestations of SARS-CoV-2 infection are caused by a direct viral invasion of motor neurons, and/or are a collateral injury resulting from an uncontrolled innate immune response. Damage to motor neurons leads to the deterioration of muscle function, manifested as muscle weakness, atrophy, or paralysis.

Two host-cell factors are important for SARS-CoV-2 viral entry into many cell types: angiotensin-converting enzyme 2 (ACE2), which is bound by the S protein, and transmembrane serine protease 2 (TMPRSS2), which cleaves the S protein, allowing this binding to take place. The expression level of ACE2 is low in the human brain. In contrast, the expression of neuropilin 1 (NRP1), CD147, TMPRSS2, and furin is higher and broader than ACE2, indicating that they may be putative mediators of SARS-CoV-2 entry into human nervous system cells. Accordingly, the authors of the present study assumed that infection of human motor neurons with SARS-CoV-2 mainly relies on CD147 and/or NRP1 binding.

 

About the study

The authors used an in vitro model of human motoneurons differentiated from induced pluripotent stem cells (iPSC-MNs) to investigate the expression of the main SARS-CoV-2 receptors and the infectability of iPSC-MN by SARS-CoV-2. The iPSCs from three healthy donors (1 male and 2 females, aged between 37 and 49 years) differentiated into motor neurons that expressed both, neuronal (bIII-tubulin and SMI-312) and motoneuronal (ChAT, HB9) markers.

To verify the infectability of iPSC-MNs by SARS-CoV-2, VeroE6 cells were exposed to supernatants collected from iPSC-MNs infected with SARS-CoV-2. The expression of viral ORF7A, ORF3A, ORF8, RDRP, S, E, and N genes in uninfected (mock) and infected iPSC-MNs was analyzed.

In addition, the researchers assessed the gene expression of the main SARS-CoV-2 receptors (ACE2, CD147, NRP1) and peptidases (TMPRSS2, furin) in iPSC-MNs infected with SARS-CoV-2 and the A549-hACE2 cells, as a positive control. They also analyzed the expression of 46 genes related to inflammatory, apoptotic, and antiviral pathways.

Results

The iPSC-MNs expressed the main receptors used by the virus (ACE2, CD147, NRP1, TMPRSS2, furin), but at different levels. Compared to the A549-hACE2 cell line, infected iPSC-MNs expressed lower levels of ACE2 and furin genes and higher levels of CD147 and TMPRSS2 genes. The expression of the NRP1 gene was comparable between iPSC-MNs and A549-hACE2 cells. The immunofluorescence assay for ACE2, CD147, and NRP1 markers confirmed these results.

To validate the infectability of iPSC-MNs, VeroE6 cells were exposed to supernatants collected from infected iPSC-MNs at different time points. The supernatants collected from infected iPSC-MNs were able to re-infect VeroE6 cells. 

A gene expression assay detected the expression of SARS-CoV-2 specific genes (N, E, spike, ORF3A, ORF8, and ORF7A) exclusively in infected iPSC-MNs. The expression of N1, S1, S2, and E2 in infected iPSC-MNs was subsequently confirmed by rt-PR. The immunofluorescence assay detected N protein exclusively in infected iPSC-MNs, mainly at the perinuclear level, in the soma, and along the neurite extensions, but the percentage of infected cells was very low. The rt-PCR that analyzed the expression of viral N1 and N2 genes in supernatants from iPSC-MN cultures, confirmed that SARS-CoV-2 can infect and replicate in human motor neurons, but the levels of viral replication were lower than in VeroE6 cells. Viral replication in infected iPSC-MNs was not accompanied by a cytopathic effect as assessed by the crystal violet assay.

SARS-CoV-2 infection altered the expression of 10 genes involved in cell survival, metabolism, antiviral, and inflammatory response in iPSC-MNs. The virus upregulated the expression of BCL2, BCL2 -associated X protein, caspase 8, CD147, proinflammatory cytokine interleukin-6, MX1, and sphingosine-1-phosphate receptor 1 genes, and down-regulated the expression of factors involved in the antigen processing and presentation, like human leukocyte antigen-A and endoplasmic reticulum aminopeptidase 1. The expression of angiogenin, which exerts neurotrophic and neuroprotective functions, acts as a circulating protein in inflammation and contributes to systemic responses to infection was also down-regulated.

An increased ratio between the anti-apoptotic BCL2 and pro-apoptotic BAX genes suggested that programmed cell death is somehow prevented in neuronal cells after the infection.

 

Conclusion

According to the authors, this study has shown, for the first time, that SARS-CoV-2 can infect and replicate in iPSC-derived human motor neurons, although viral replication and percentage of infected cells were lower than in VeroE6 cells, susceptible to SARS-CoV-2.

 

This article was published in Frontiers in Cellular Neuroscience.

 

Journal Reference

Cappelletti G, Colombrita C, Limanaqi1 et al. Human motor neurons derived from induced pluripotent stem cells are susceptible to SARS-CoV-2 infection. Front. Cell. Neurosci, Sec. Cellular Neuropathology. 05 December 2023. Volume 17, 2023. (Open Access)  https://doi.org/10.3389/fncel.2023.1285836

 

 

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