SARS-CoV-2 infection of substantia nigra pars compacta induces expression of miR-330-5p at 10 days post-infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been linked to several neurological symptoms in coronavirus disease 2019 (COVID-19) patients; however, the molecular mechanisms underlying virus-induced neuroinflammation are not well identified. For example, the effect of SARS-CoV-2 infection of the substantia nigra pars compacta (SNpc) of the midbrain has not been addressed, in spite of its importance in dopaminergic signalling and neurodegenerative abnormalities. The purpose of this study was to understand the SARS-CoV-2-induced inflammatory response in the SNpc region of the brain. We inoculated (intranasally) transgenic mice expressing human ACE2 under control of the human keratin 18 promoter (K18-hACE-2 mice) with a 4×10³ TCID₅₀ (mild) dose of SARS-CoV-2. Ten days post-inoculation, SARS-CoV-2 was detected in the SNpc of mice, along with increased levels of IL-1β, B1R and ADAM17, and reduced microglial/macrophage occurrence. miR-330-5p expression was significantly reduced in virus-positive SNpc tissue. Luciferase reporter assays supported ADAM17 as a direct target of miR-330-5p. There was no significant difference in miR-330-5p expression levels in the experimental autoimmune encephalomyelitis mice compared to control mice, demonstrating a crucial role for SARS-CoV-2-induced miR-330-5p in brain pathology. Our study uncovers for the first time that SARS-CoV-2 can invade the SNpc and downregulate miR-330-5p expression levels, causing an enhanced ADAM17 expression and possible neuroinflammatory signalling. The results implicate miR-330-5p as a prospective therapeutic target for alleviating midbrain inflammation associated with SARS-CoV-2 infection.

Keywords: long COVID; miR-330-5p; midbrain; neuroinflammation; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Fig. 1.. SARS-CoV-2-inoculated K18-hACE-2 SNpc is positive for viral RNA. (a) A schematic depicting an animal study design. (b) Monitoring SARS-CoV-2 litres in the SNpc of virus-inoculated K18-hACE-2 mice on 10 days post-inoculation by reverse transcriptase quantitative PCR. RNA was extracted from the SNpc of mock and SARS-CoV-2-infected mice. RNA concentrations were measured with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The virus concentration in the specimens was detected by quantitative PCR monitoring nucleocapsid N gene using the SARS-CoV-2 (2019-nCoV) CDC quantitative PCR Probe Assay (Integrated DNA Technologies). The limit of detection for this assay is 50 copies. (c) A representational immunofluorescence image of infected mice (I#3) SNpc positive for S protein of SARS-CoV-2 (arrowheads) compared to SNpcs derived from mock-inoculated mice.

Fig. 2.. SARS-CoV-2 invasion of the brain induces inflammation. (a) Reverse transcriptase quantitative PCR was performed using RNA extracted from the midbrain sections of SARS-CoV-2-inoculated K18-hACE-2 mice on 10 days post-inoculation. We monitored expressions of IL-1β, B1R and ADAM17 using specific primers. Data are the mean±sd (error bars) of three experiments. ANOVA was performed using IBM SPSS v26 (Cary, NC) to determine significant differences between the different treatment and control groups, followed by the Tukey HSD post hoc test for multiple comparisons. ‘****’, ‘***’, ‘**’ and ‘ns’ denote P<0.0001, P=0.0002, P=0.0059 and not significant, respectively. (b) Expression levels of dopamine neurons (TH) and microglia (IBA1) were significantly lowered in SARS-CoV-2-infected SNpc sections compared to mock controls. A representational figure from mouse I#3 is shown.

Fig. 3.. miR-330-5p levels were significantly lowered in SARS-CoV-2-infected brains in a subset (2 of 7) of infected K18-hACE-2 mice. The fold change in the miR-330-5p expression in SARS-CoV-2-infected mice is relative to the average expression of the respective miRNA in mock-inoculated mice, and that is considered to be 1-fold. Expression of miR-93-5p was used as a control. The average±sd of five individual experiments is listed above as the data points. ANOVA was performed using IBM SPSS v.26 (Cary, NC) to determine significant differences between the different treatment and control groups, followed by the Tukey HSD post hoc test for multiple comparisons. ‘****’ and ‘***’ denote P<0.0001 and P=0.0002, respectively.

Fig. 4.. miR-330-5p directly interacts with its target, ADAM17 mRNA. A dual luciferase reporter assay was performed to demonstrate the interactions between miR-330-5p and its putative targets, ADAM17 (a) and FGF7 (b) mRNA (control) in HEK-293T cells. The x-axis indicates the different treatments, while the y-axis denotes the relative luciferase activity. Bars represent average±sd of three individual experiments. ANOVA was performed using IBM SPSS v.26 (Cary, NC) to determine significant differences between the different treatment and control groups, followed by the Tukey HSD post hoc test for multiple comparisons. ‘****’ and ‘ns’ denote P<0.0001 and not significant, respectively.