SARS-CoV-2 Infection Causes Dopaminergic Neuron Senescence

Abstract

COVID-19 patients commonly present with neurological signs of central nervous system (CNS)^1-3 and/or peripheral nervous system dysfunction⁴. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

Keywords: SARS-CoV-2; midbrain dopamine; neuron senescence.

Extended Data Figure 1.. hPSC-derived dopaminergic cells can be infected by SARS-CoV-2 pseudo-entry virus.

a, Representative confocal images of hPSC-derived DA neurons stained with antibodies recognizing Nurr1-GFP, TH or FOXA2. Scale bar=50μm. b, Representative confocal images of hPSC-derived DA neurons stained with ACE2 antibody. Scale bar=50μm. c, Luciferase activity in lysates from hPSC-derived DA neurons at 24 hpi following exposure to SARS-CoV-2-entry virus at MOI=0.01. d, Representative confocal images of hPSC-derived DA neurons infected with SARS-CoV-2-entry virus (MOI=0.01) at 24 hpi using antibodies against luciferase and DA markers. Scale bar=50μm. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. ***P < 0.001.

Figure 1.. hPSC-derived DA neurons, and in particular A9 DA neurons, are permissive to SARS-CoV-2 infection.

a, Schematic for in vivo infection. b, Representative confocal image of DA neuron xenograft stained with antibodies against ACE2 and Nurr1-GFP. Scale bar=50μm. c, Representative confocal image of DA neuron xenograft at 24 hpi stained with antibodies against Luc and Nurr1-GFP. Scale bar=60μm. d. qRT-PCR analysis of total RNA extracted from hPSC-derived DA neurons at 48 hpi of SARS-CoV-2 infection (MOI=0.2) for viral N sgRNA. The graph depicts the mean sgRNA level normalized to ACTB. e, Representative confocal images of hPSC-DA neurons infected with SARS-CoV-2 (MOI=0.1) at 72 hpi using antibodies against SARS-CoV-2 Nucleocapsid protein (SARS-N) and markers for DA neurons. Scale bar=50μm. f, Transmission electron microscope (TEM) images of DA neurons at 72 hpi of SARS-CoV-2 (MOI=1.0). Arrows point to SARS-CoV-2 viral particles. Right panel: Zoom in images. Scale bar=1μm. g, RNA-seq read coverage of the viral genome in infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). The schematic below depicts the SARS-CoV-2 genome and was created using BioRender. h, PCA plot of gene expression profiles from mock infected and SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). i, Clustering analysis of mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). j, Heatmap of DA neurons and A9 DA neuron marker genes expression levels in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). k, l, Fluorescence in situ hybridization (k) and quantification (l) of A9 DA marker, LMO3, in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2) Scale bar=10μm. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. **P < 0.01.

Figure 2.. SARS-CoV-2 infection induces senescence of DA neurons.

a, Volcano plot indicating differentially expressed genes in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). Differentially expressed genes (p-adjusted value < 0.05) are indicated in red. Non-significant differentially expressed genes with a log2 (Fold Change) > 0.5 are indicated in black. b, c, Heatmap of chemokine/cytokines (b) and inflammation associated genes (c) in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). d, IPA analysis of differentially expressed genes in a. e, Heatmap of senescence associated genes in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). f, g, Beta-Gal staining (f) and quantification (g) of mock or SARS-CoV-2 infected hPSC-derived DA neurons at 72 hpi (MOI=0.1). Scale bar=75μm. h. qRT-PCR analysis of senescence related genes of mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). i. TEM images of mock or SARS-CoV-2 infected hPSC-derived DA neurons at 72 hpi (MOI=1.0). Scale bar=2μm. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.. Riluzole, metformin, and imatinib rescue SARS-CoV-2 induced senescence of DA neurons.

a, Primary screening results. X-axis is the compound number. Y axis is the Z-score. Red line is Z-score <−2, which means the luminescent signal is lower than average-2xSTDEV. b-d, Chemical structures of riluzole (b), metformin (c), and imatinib (d). e-g, Efficacy and cytotoxicity curves of riluzole (e), metformin (f), and imatinib (g). h, i, Beta-Gal staining (h) and the quantification (i) of DMSO or drug candidate-treated hPSC-derived DA neurons at 72 hpi upon SARS-CoV-2 infection (MOI=0.1). Scale bar=100μm. j, qRT-PCR analysis of senescence related genes of DMSO or drug candidate-treated hPSC-derived DA neurons at 48 hpi upon SARS-CoV-2 infection (MOI=0.1). k, qRT-PCR analysis of total RNA extracted from DMSO or drug candidate-treated hPSC-derived DA neurons at 48 hpi upon SARS-CoV-2 infection (MOI=0.1) for viral N sgRNA. The graph depicts the mean sgRNA level normalized to ACTB. l, m, Representative confocal images (l) and quantification (m) of DMSO or drug candidate-treated hPSC-derived DA neurons at 72 hpi upon SARS-CoV-2 infection (MOI=0.1) using antibodies against SARS-CoV-2 Nucleocapsid protein (SARS-N) and markers for DA neurons. Scale bar=100μm. n, o, PCA plot of gene expression profiles (n) and clustering analysis (o) of DMSO or drug candidate-treated hPSC-derived DA neurons at 48 hpi upon SARS-CoV-2 infection (MOI=0.1). p, Heatmap of senescence associated genes of DMSO or drug candidate-treated hPSC-derived DA neurons at 48 hpi upon SARS-CoV-2 infection (MOI=0.1). N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.. SARS-CoV-2 is detected in autopsy substantia nigra samples of COVID-19 patients.

a-c, Heatmap of chemokine/cytokine (a), inflammation associated genes (b) and senescence associated genes (c) in the autopsy substantia nigra sections of COVID-19 patients versus non-COVID-19 patients. (N=6 COVID-19 patients; N=3 non-COVID-19 patients). d, Heatmap of viral transcripts in autopsy substantia nigra sections of COVID-19 patients.