Enterovirus-A71 preferentially infects and replicates in human motor neurons, inducing neurodegeneration by ferroptosis

Abstract

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe²⁺ and peroxidated lipids. Notably, the Fe²⁺ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Keywords: EV71; Ferritin; Ferroptosis; Human motor neurons; Pluripotent stem cells; scRNA-Seq.

Figure 1.

Human ESC-derived motor neurons are susceptible to EV-A71 infection. A. hESC were differentiated for 28 days into motor neurons and then were infected with EV-A71 S41. B. Immunodetection of EV-A71 VP2 viral capsid protein. White arrows indicate cells where VP2 and ISL1 staining are colocalized. C. Percentage of VP2⁺ cells and D. VP2⁺ ISL1⁺ MNs, detected at multiplicities of infection (MOI) 1 and 5, n = 6. Quantification at 72 hpi might be inaccurate due to the presence of floating dead cells. E. Immunostaining for non-structural viral protein (EV-A71 3D) and dsRNA. F. Virus titers in the supernatant was determined by plaque assay using RD cells, n = 3.

Figure 2.

scRNA-Seq analysis to study host gene expression during EV-A71 infection reveal highest viral RNA in MNs within a heterogeneous culture. A. Annotated Seurat plot showing the presence of motor neurons, spinal interneurons, early neurons and neural progenitor cells within the human ESC-derived spinal neural culture. B. Distribution of expressed markers in each population. C. EV-A71 infection has no global effect on composition of the neural culture at 6 hpi. D. Expression level of EV-A71 S41 RNA in each cell population. E. Percentage of “virus-high” cells in each population. “Virus-high” cells are defined by cells having viral RNA that is higher than the mean expression level. F. Immunostaining of neural cultures infected with EV-A71 using antibodies against EV-A71 VP2, revealing higher percentage of EV-A71 infected ISL1⁺ MNs than SOX1⁺ NPCs. n = 12, t-test, ****p < 0.0001. Yellow arrows indicate double-positive cells. G. Higher double-stranded RNA (dsRNA) signals and percentage of dsRNA⁺ cells within ISL1⁺ MNs as compared to SOX1⁺ NPCs. n = 12, t-test, ****p < 0.0001. Yellow arrows indicate double-positive cells. H. Immunostaining of infected spinal cord organoids. I. Magnified images of a rosette of the infected spinal cord organoids. Quantification of the EV-A71 VP2 intensity within the SOX1⁺ neural rosette and outside the rosette. n = 8, paired t-test, ***p < 0.001. J. Expression level of SCARB2 RNA in each cell population (mock). K. Expression level of ANXA2 RNA in each cell population (mock).

Figure 3.

DEGs of the infected cells suggest mitochondrial dysfunction. A. Top up- and down-regulated genes for motor neurons after EV-A71 infection as compared to mock-infected cells. B. Gene Ontology enrichment analysis of the significantly upregulated genes after EV-A71 infection in MNs. C. Gene Ontology enrichment analysis of the significantly downregulated genes after EV-A71 infection in MNs. D. JC-1 assay was conducted to validate occurrence of mitochondrial dysfunction after infection. n = 15, One-way ANOVA, ****p < 0.0001. E. Effects of treatment with elamipretide (SS31) on mitochondrial function and EV-A71 infection. n = 15, One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F. SS31 treatment did not lead to significant changes of infection rate. n = 6, Student’s t-test.

Figure 4.

Ferroptosis in human motor neurons after EV-A71 infection. A. Expression levels of FTL and FTH1 after infection in each population. B. Expression levels of FTL and FTH1 after infection in each population with high viral RNA load (High) and low viral RNA load (Low). C. Free irons within the cells were measured by Ferro-Orange. D. Number of cells that were positive for Ferro-Orange in the EV-A71-infected and mock-infected cultures. E. Ferro-Orange intensity in each cell at 6 h after EV-A71 or mock infection. n > 1900 cells from 3 cultures, Mann-Whitney Test, ****p < 0.0001. F. Lipid peroxidation measured by BODIPY C11 581/591. G. Number of cells displaying oxidized C11 in EV-A71-infected and mock cultures. H. Mean oxidized C11 intensity in each cell at 24 h after EV-A71 or mock infection. n > 2300 cells from 3 cultures, Mann-Whitney Test, ****p < 0.0001.

Figure 5.

Ferroptosis inhibitors restore mitochondrial defects, promote survival, and decrease infection rates in EV-A71 infected MNs. A. Mitochondrial membrane potential in EV-A71-infected or mock cultures treated with ferroptosis inhibitors, ferrostatin-1 and deferoxamine (DFO) at 24 hpi. n = 12, Two-way ANOVA. B. DAPI counts at 48 hpi normalized to mock-infected control. n = 12, One-way ANOVA. C. Immunodetection of VP2 in MNs treated with ferroptosis inhibitors at 48 hpi. D. Percentage of VP2⁺ cells. n = 9, One-way ANOVA. E. Viral titers determined by plaque assay in culture supernatants collected at 48 hpi. n = 3, One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.