In one of their previous publications, the authors from the United States and the Netherlands showed the susceptibility and permissivity of dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By contrast, cortical neurons derived from hPSCs did not show susceptibility to SARS-CoV-2 infection. In this article, the same researchers investigated molecular changes in dopaminergic neurons linked to SARS-CoV-2 infection. They also performed postmortem examination of the substantia nigra of diseased COVID-19 patients to investigate whether the changes in DA neurons observed in vitro also occur in the brains of COVID-19 patients.
Dopaminergic neurons
SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins: the spike (S), envelope (E), nucleocapsid (N), and membrane (M) proteins. The S protein is a membrane-bound glycoprotein forming a homotrimer that binds to the membrane-bound angiotensin-converting enzyme 2 (ACE2) in host cells through the receptor binding domain located in the S1 subunit.
Parkinson’s disease (PD) is the second most common neurodegenerative disease, caused by a reduction in the dopaminergic neurons of the substantia nigra, followed by striatal dopamine depletion. PD is one of the α-synucleinopathies characterized by the misfolding of alpha-synuclein (α-Syn) into pathological forms, resulting in neurodegeneration. SNCA, the first gene associated with familial PD, encodes the protein α-Syn. PD is characterized by typical motor symptoms (bradykinesia, rest tremor, and muscle rigidity) and non-motor symptoms (hyposmia, depression, apathy, sleep disorders, and dysautonomia). Viral infections receive more attention as a cause of viral-induced clinical parkinsonism, but the specific mechanism of nigrostriatal dopaminergic neuron degradation after viral infection remains unknown. It is worth noting that a study that employed a computational methodology to predict interactions between human and SARS-CoV-2 proteins revealed that SARS-CoV-2 proteins mimic 43 proteins linked to Parkinson’s disease pathways, including α-Syn. SARS-COV-2 proteins that possibly interact with α-Syn are NSP7 and 3-CL-like (main) protease. (Yapici-Eser H et al. Neuropsychiatric Symptoms of COVID-19 Explained by SARS-CoV-2 Proteins’ Mimicry of Human Protein Interactions. Front. Hum. Neurosci, 23 March 2021.) https://www.frontiersin.org/journals/human-neuroscience/articles/10.3389/fnhum.2021.656313/full
About the Study and Results
The authors differentiated DA neurons from hPSCs. DA neuron identity was validated by immunofluorescence staining with postmitotic DA neuron markers (NURR1: GFP), tyrosine-hydroxylase (TH), MAP2, and FOXA2. Clustering analysis identified four cell populations: three clusters that highly expressed LMO3, a marker of A9 DA neurons, and one cluster that highly expressed CALB1, a marker of A10 DA neurons. These results showed that the population of DA neurons derived from hPSCs was mainly composed of A9 DA neurons (a subtype of DA neurons in the substantia nigra, mostly affected by PD).
The SARS-CoV-2 infection of purified DA neurons derived from hPSCs was confirmed at 24, 48, and 72 hours post-infection (hpi). In TH+ DA neurons, a trend toward increased SARS-CoV-2 infection was observed from 24 to 48 hpi. At 48 hpi, quantitative real-time PCR (rt-qPCR) analysis revealed significant viral replication in infected DA neurons. At 72 hpi, immunostaining of antibodies against the SARS-CoV-2 N protein confirmed infection of approximately 8% of NURR1:GFP+ DA neurons, suggesting that the percentage of infected DA neurons was not very high. By contrast, just a few cortical neurons were positive for SARS-CoV-2 N protein at 72 hpi, with no transcriptional changes after exposure to SARS-CoV-2.
The SARS-CoV-2 viral transcripts, including S, E, N, and M proteins, were highly detected in SARS-CoV-2 infected cells, especially in three clusters that expressed a high level of LMO3, a marker of A9 DA neurons. The SARS-CoV-2 antigens were not detected in a cluster that highly expressed CALB1, a marker of A10 DA neurons.
Interestingly, SARS-CoV-2 infection decreased the expression of midbrain DA neuron markers NR4A2, FOXA2, and LMX1A, especially A9 markers LMO3 and DKK3. Quantitative RNA in situ hybridization confirmed a decreased expression of the A9 marker LMO3 (but not the A10 marker CALB1), and the loss of A9 DA neurons after SARS-CoV-2 infection. It appears that A9 DA neurons are particularly vulnerable to SARS-CoV-2, similar to A9 DA neurons in the substantia nigra which are most affected in PD.
The ACE2 blocking prevented virus infection, suggesting that SARS-CoV-2 infection of DA neurons derived from hPSCs depends on ACE2 receptors.
Gene analysis revealed that the cell cycle, DNA replication, chemokine/cytokine transcripts, inflammation, and senescence pathways were the top-upregulated pathways in SARS-CoV-2-infected DA neurons. Further analysis confirmed that SARS-CoV-2 triggered cellular senescence in DA neurons, and upregulated senescence-associated genes, such as CCL2, CCL20, CSF1, CXCL11, GDF15, IGF2R, IL1B, IL6ST, IQGAP1, and TNFRSF11B. SARS-CoV-2 also upregulated other senescence-associated markers in DA neurons, such as lysosomal senescence-associated β-galactosidase (SA-β-gal) and lipofuscin. DA neurons infected with SARS-CoV-2 displayed senescence-associated phenotypes, including increased accumulation of lysosomes, mitochondrial dysfunction, and increased protein oxidation.
By contrast, senescence-associated genes were not upregulated in cortical neurons derived from hPSCs, which is consistent with previous data showing that cortical neurons are not susceptible to SARS-CoV-2 infection. Also, the senescence pathway was not upregulated in lung organoids, pancreatic cells, liver organoids, and cardiomyocytes infected with SARS-CoV-2.
The FDA Food and Drug Administration (FDA)-approved drugs riluzole, metformin, and imatinib were found to block SARS-CoV-2-mediated DA neuron senescence. Riluzole, metformin, and imatinib reduced β-gal activity in a dose-dependent manner without cytotoxicity. They also down-regulated the genes involved in the senescence pathway and decreased viral RNA in DA neurons infected with SARS-CoV-2. Metformin has already been identified as a potential COVID-19 therapeutic agent, and its use was associated with reduced mortality in COVID-19 patients with obesity and type 2 diabetes. Two randomized, placebo-controlled studies found that metformin significantly reduced the viral load and the incidence of long COVID syndrome in outpatients infected with SARS-CoV-2. https://discovermednews.com/beneficial-effect-of-metformin-in-individuals-infected-with-sars-cov-2/
Finally, the authors examined autopsy samples of the substantia nigra from six COVID-19 patients and three age-matched controls to determine whether the selective vulnerability of hPSC-derived DA neurons and the resulting senescence and inflammation were reflected in the brains of COVID-19 patients. SARS-CoV-2 transcripts were detected in all six substantia nigra samples. Remarkably, the same transcriptional signatures identified in SARS-CoV-2 infected DA neurons in vitro were found in autopsy samples, including the induction of chemokine/cytokine, inflammation, and senescence-associated genes. Low viral RNA levels were identified in frozen tissue samples from other brain regions.
Conclusion
This study demonstrated the selective in vitro vulnerability to SARS-CoV-2 infection of purified DA neurons derived from hPSCs and the associated inflammatory and cellular senescence response. The findings of a comparable inflammatory and senescence signature in brain samples from COVID-19 patients suggest that these results may have clinical relevance. But, the authors noted the possibility that other cell types, such as astrocytes or microglia, or other pathological changes, such as a hypoxic state, could contribute to the inflammatory and senescence signatures in the postmortem substantia nigra samples. They, however, recommended that COVID-19 patients should be closely monitored for an increased risk of developing PD-related symptoms in the coming years.
This article was published in Cell Stem Cell.
Journal Reference
Yang, L., Kim TW, Han Y et al. SARS-CoV-2 infection causes dopaminergic neuron senescence. Cell Stem Cell 2024; 31, 1–16. https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(23)00442-3
