SARS-CoV-2 infection exacerbates the cellular pathology of Parkinson's disease in human dopaminergic neurons and a mouse model

Abstract

While an association between Parkinson's disease (PD) and viral infections has been recognized, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on PD progression remains unclear. Here, we demonstrate that SARS-CoV-2 infection heightens the risk of PD using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons and a human angiotensin-converting enzyme 2 (hACE2) transgenic (Tg) mouse model. Our findings reveal that SARS-CoV-2 infection exacerbates PD susceptibility and cellular toxicity in DA neurons pre-treated with human preformed fibrils (hPFFs). Additionally, nasally delivered SARS-CoV-2 infects DA neurons in hACE2 Tg mice, aggravating the damage initiated by hPFFs. Mice infected with SARS-CoV-2 display persisting neuroinflammation even after the virus is no longer detectable in the brain. A comprehensive analysis suggests that the inflammatory response mediated by astrocytes and microglia could contribute to increased PD susceptibility associated with SARS-CoV-2. These findings advance our understanding of the potential long-term effects of SARS-CoV-2 infection on the progression of PD.

Keywords: COVID-19 sequalae; DA neuron; PD; Parkinson’s disease; SARS-CoV-2; disease modeling; dopaminergic neuron; hACE2 transgenic mouse; neuroinflammation; neurological sequelae.

Graphical abstract

Figure 1

Pathology of SARS-CoV-2 infection with hPFF-induced PD in hESC-derived DA neurons (A) Schematic experimental summary of DA neurons infected with SARS-CoV-2 (0.1 MOI) infection and treated with hPFFs. (B) Representative western blot bands of nucleocapsid protein (NP) and TH levels in hDA neurons infected with SARS-CoV-2 and then treated with hPFFs. (C) Quantification of NP and TH levels. n = 6, biological repeat. (D) Representative western blot expression of α-Syn and p-α-Syn in hDA neurons treated with SARS-CoV-2 and/or hPFFs. (E) Quantification of α-Syn western blot levels. n = 5, biological repeat. (F) Quantification of p-α-Syn western blot levels. n = 5, biological repeat, values are mean ± SEM, one-way ANOVA. (G) Representative immunofluorescence image of p-α-Syn (green) and TH (red). (H) Representative immunofluorescence image of p-α-Syn (green) and NP (red). (I) Quantification of NP. n = 5, biological repeat, values are mean ± SEM, unpaired two-tailed Student’s t tests. (J) The neuritic p-α-Syn intensity. The intensity excluding the somatic region of p-α-Syn was measured using ImageJ. n = 5, biological repeat. (K) The percentage of somatic p-α-Syn contrasted with DAPI. For the somatic p-α-Syn-positive cells, manual counting was conducted, considering only cells positive for both DAPI and p-α-Syn staining. n = 5, biological repeat. (L) Representative image of TUNEL staining of DA neurons infected with SARS-CoV-2 and treated with hPFFs. (M) Quantification of TUNEL-positive DA neuron cells. n = 6, biological repeat. (N) A graph showing the difference in cell viability measured with Alamar Blue. n = 6, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance; N.D., non-detection. See also Figures S1–S3.

Figure 2

Molecular pathogenesis of SARS-CoV-2 infection with hPFF-induced PD progression in hESC-derived DA neurons (A) Venn diagram indicating upregulated genes related to hPFF-induced PD progression or hPFF-induced PD progression with SARS-CoV-2 (0.1 MOI) infection. (B) Results of Gene Ontology analysis using genes upregulated in hPFF-induced PD progression and hPFF-induced PD progression with SARS-CoV-2 infection. (C) Results of KEGG pathway analysis using genes upregulated in hPFF-induced PD progression and hPFF-induced PD progression with SARS-CoV-2 infection. (D) Venn diagram illustrating genes involved in the pathogenesis of hPFF-induced PD progression that are exacerbated by SARS-CoV-2 infection. (E) The results of KEGG pathway analysis employing genes that are exacerbated by SARS-CoV-2 infection among genes involved in hPFF-induced PD progression. (F) Validation of neuronal-apoptosis-related gene expression using quantitative real-time PCR. n = 3, biological replicates. (G) Validation of PD-associating autophagy-related gene expression using quantitative real-time PCR. n = 3, biological repeat. (H) Validation of PD pathogenesis-associated gene expression using quantitative real-time PCR n = 3, biological repeat. (I) Validation of DA neuron functional gene expression levels using quantitative real-time PCR. n = 3, biological repeat, values are mean ± SD, one-way ANOVA. (J) Representative western blot images of LC3A/B and SQSTM1/p62 to analyze autophagic function in human DA neurons. (K) Quantitative graphs of LC3A/B and SQSTM1/p62 from (J). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (L) Representative western blot images of voltage-dependent anion channel (VDAC), succinate dehydrogenase (SDHA), and prohibitins (PHBs) to analyze mitochondrial function in cells. (M) Quantitative graph of VDAC, SDHA, and PHBs from (L). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (N) Representative Western blot images of HSP60, cytochrome c oxidase (COX) IV, and cytochrome c (Cyto C) to analyze mitochondrial function in human DA neurons. (O) Quantitative graphs of HSP60, COX IV, and Cyto C from (N). (P) Fluorescent microscopy images of the lysosomal intracellular activity assay. (Q) Quantitative graph corresponding to the assay results from (P). n = 9, biological repeat, values are mean ± SEM, one-way ANOVA. (R) Results data from the 20S proteasome assay. n = 7, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance.

Figure 3

Brain mapping of mice infected with SARS-CoV-2 (A and B) Brain mapping of mice (A) 3 and (B) 10 days after 10³ TCID₅₀ SARS-CoV-2 infection. Ipl, internal plexiform layer of the olfactory bulb (OB); Mi, mitral cell layer of the OB; eci, ependymal and subendymal layer/olfactory ventricle; E/OV, anterior commissure, intrabulbar part; M1, primary motor cortex; M2, secondary motor cortex; CPu, caudate putamen (STR); fi, fimbria of the hippocampus (HIP), Pri, piriform cortex; V2ML, secondary visual cortex, mediolateral area; V2MM, secondary visual cortex, mediomedial area; RSA, retrosplenial agranular cortex; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; Crus, crus of the ansiform lobule; Lve, lateral vestibular nucleus; MvePC, medial vestibular nucleus, parvicellular; MveMC, medial vestibular nucleus, magnocellular. (C) NP infection/DAPI ratio cell count graph in OB, STR, HIP, VM, and cerebrum (CB)/brain stem (BS) regions on day 3. (D) NP infection/DAPI cell count ratio graph in the OB, STR, HIP, VM, and CB/BS regions on day 10. (E) Comparison graph between day 3/10 NP infection/DAPI cell count ratio graphs in the OB, STR, HIP, VM, and CB/BS regions. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance.

Figure 4

SARS-CoV-2 increased hPFF-induced PD-like pathology (A) Schematic diagram of the SARS-CoV-2 (10³ TCID₅₀) infection schedule in ACE2 Tg mice treated with hPFF. (B) Representative immunofluorescence image of TH (green) and NP (red). (C) NP/DAPI ratio of (B). (D) Representative immunofluorescence image of TH (green) and p-α-Syn (red). (E) Quantification of (D) p-α-Syn intensity in VM at 60 days. n = 5, biological repeat, values are mean ± SEM, one-way ANOVA. (F) Representative TH immunohistochemistry images of the VM region of ACE2 Tg mice. (G) Quantification of (F) TH intensity. n = 6, biological repeat, values are mean ± SEM, one-way ANOVA. (H) Representative western blot bands of DAT, TH, α-Syn, and β-actin in VM tissue. (I) Quantification of DAT, TH, and α-Syn levels from (H). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance; N.D., non-detection. See also Figure S4.

Figure 5

Infection with SARS-CoV-2 sustained the expression of GFAP and Iba-1 for 60 days (A) Representative GFAP immunochemistry images of the VM region of ACE2 Tg mice infected with SARS-CoV-2 (10³ TCID₅₀). (B) Quantification of GFAP in the SNpc region. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (C) Representative Iba-1 immunochemistry images of the VM region of ACE2 Tg mice infected with SARS-CoV-2. (D) Quantification of Iba-1 in the SNpc region. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (E) Representative western blot bands of GFAP and Iba-1 in the VM. (F) Quantification of GFAP and Iba-1 western blot bands in the VM. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (G) Representative TH immunochemistry images of the VM region of ACE2 Tg mice infected with SARS-CoV-2. (H) Quantification of GFAP in the SNpc region. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (I) Representative western blot bands of NP, TH, DAT, and α-Syn in VM after infection with SARS-CoV-2 for 7 and 60 days. (J) Quantification of (I). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance. See also Figures S5 and S6.

Figure 6

SARS-CoV-2 infection upregulates hPFF-induced Iba-1 and GFAP expression in the VM, and this effect persists even after 60 days (A) Representative Iba-1 IHC images of the VM SNpc and SNr regions. (B) Quantification of Iba-1 levels. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (C) Representative western blot band of Iba-1 in the VM. (D) Quantification of Iba-1 expression levels from (C). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (E‒G) qPCR analysis of the VM in hPFF-injected mice treated with vehicle or SARS-CoV-2 for Il1a, Tnf, and C1qa. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (H) Representative GFAP immunochemistry images of the SNpc and SNr regions. (I) Quantification of GFAP levels. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (J) Representative western blot band of GFAP in the VM. (K) Quantification of GFAP expression levels from (J). n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. (L) qPCR analysis of the VM in hPFF-injected mice treated with vehicle or SARS-CoV-2 for A1-specific transcripts. n = 3, biological repeat, values are mean ± SEM, one-way ANOVA. n.s., non-significance. See also Figures S7–S10.