COVID-19 and Parkinson's Disease: Shared Inflammatory Pathways Under Oxidative Stress

Abstract

The current coronavirus pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a serious global health crisis. It is a major concern for individuals living with chronic disorders such as Parkinson's disease (PD). Increasing evidence suggests an involvement of oxidative stress and contribution of NFκB in the development of both COVID-19 and PD. Although, it is early to identify if SARS-CoV-2 led infection enhances PD complications, it is likely that oxidative stress may exacerbate PD progression in COVID-19 affected individuals and/or vice versa. In the current study, we sought to investigate whether NFκB-associated inflammatory pathways following oxidative stress in SARS-CoV-2 and PD patients are correlated. Toward this goal, we have integrated bioinformatics analysis obtained from Basic Local Alignment Search Tool of Protein Database (BLASTP) search for similarities of SARS-CoV-2 proteins against human proteome, literature review, and laboratory data obtained in a human cell model of PD. A Parkinson's like state was created in 6-hydroxydopamine (6OHDA)-induced differentiated dopamine-containing neurons (dDCNs) obtained from an immortalized human neural progenitor cell line derived from the ventral mesencephalon region of the brain (ReNVM). The results indicated that SARS-CoV-2 infection and 6OHDA-induced toxicity triggered stimulation of caspases-2, -3 and -8 via the NFκB pathway resulting in the death of dDCNs. Furthermore, specific inhibitors for NFκB and studied caspases reduced the death of stressed dDCNs. The findings suggest that knowledge of the selective inhibition of caspases and NFκB activation may contribute to the development of potential therapeutic approaches for the treatment of COVID-19 and PD.

Keywords: 6OHDA; Parkinson’s disease; SARS-CoV-2; apoptosis; caspase; inhibitors; nuclear factor kappa B (NFκB); oxidative stress.

Figure 1

The effect of 6-hydroxydopamine (6OHDA) treatment on NFκB expression in differentiated dopamine-containing neurons (dDCNs). The 6OHDA-treated dDCNs were exposed to 100 µm 6OHDA for 2 h to induce stress, as mentioned in Section 2.4. (A) Figure shows positive staining for TH (green), a marker for dopaminergic neurons, in control and 6OHDA-treated dDCNs, indicating that cells are dopamine-containing neurons. Positive staining for the p65-NFκB (red) was found in control and 6OHDA-treated dDCNs. The white arrow indicates positive staining for TH and p65-NFκB in the same dDCN in both control and 6OHDA-treated dDCNs. Scale bars a and b = 50 µm, while c and d = 100 µm. (B) The graph illustrates the proportion of the p65-NFκB expressed in TH positive control and 6OHDA-treated dDCNs. Increased expression of the p65-NFκB was observed in dDCNs following exposure to 6OHDA, indicating that 6OHDA-induced toxicity enhanced NFκB activity in dDCNs. Means of three experiments ± SEM are shown, p < 0.05. (C) The effect of 6OHDA treatment on p65NFκB level was measured and compared with control and 6OHDA-treated dDCNs. Briefly, cell extracts from both control and 6OHDA-treated dDCNs were subjected to Western immunoblotting. Membranes were probed with antibodies for p65NFκB and housekeeping protein GAPDH. (D) Densitometric analysis showed a significant increase in p65-NFκB level in dDCNs that were treated with 6OHDA. The (+) and (-) signs indicate with or without treatment respectively. Densitometric value represents mean ± SEM of five experiments. Table of densitometry values and statistical analysis can be found in supplementary Table S1 and S2 for B and D, respectively.

Figure 2

The effect of 6OHDA treatment on the expression of caspases in NFκB-positive dDCNs. (A) Figure shows positive staining for p65-NFκB (green) and for caspase-2, -3 and -8(red) in control and 6OHDA-treated dDCNs. Caspase-2 expression was observed in the majority of p65-NFκB positive control dDCNs. A higher expression of caspase-3 was observed in p65-NFκB positive 6OHDA-treated dDCNs when compared to control dDCNs. Caspase-8 was found in low levels in control dDCNs, but an increased expression of caspase-8 was observed in p65-NFκB positive 6OHDA-treated dDCNs. White arrow indicates the positive staining of some dDCNs stained with the p65-NFκB and caspase-2 (upper panel), p65-NFκB and caspase-3 (middle panel) and p65-NFκB and caspase-8 (lower panel) in both control and 6OHDA-treated dDCNs. Scale bar = 100 µm. (B) Graph shows the proportion of the p65-NFκB positive cells that expressed caspases-2, -3 and -8 in control and 6OHDA-treated dDCNs. The proportion of caspases-2, -3 and -8 was expressed in less than half of p65-NFκB positive control dDCNs. A significant increased proportion of caspases-2, -3 and -8 in p65-NFκB positive cells was observed after 6OHDA treatment. The (+) and (-) signs indicate with or without treatment respectively. Means of three experiments ± SEM are shown. A table of values and statistical analysis can be found in supplementary Table S3.

Figure 3

6OHDA stimulates the classical NFκB pathway in dDCNs. (A) Control and 6OHDA-treated dDCNs were treated with 70 μM Inhibitor of NFκB kinase (IKK for 2 h (see Section 2.3). Cell extracts were subjected to WB immunoblotting and membranes were probed with p65-NFκB and Gylceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibodies. The absence of NFκB was observed in IKK-treated dDCNs, suggesting that the NFκB classical pathway is involved in death of dDCNs. Illustrative examples of p65-NFκB and housekeeping protein GAPDH in control, IKK, 6OHDA, IKK + 6OHDA-treated dDCNs are shown. (B) Densitometric analysis showed a significant increase in p65-NFκB levels in 6OHDA-treated dDCNs compared to control dDCNs (p < 0.01). (C) Control and 6OHDA-treated dDCNs were treated with zVADfmk and IKK. Results showed that combined treatments significantly decreased death of 6OHDA-treated dDCNs. However, both inhibitors synergistically could not provide complete inhibition of cell death induced by 6OHDA toxicity in dDCNs. The proportion of cells surviving was determined by MTT absorbance at 570 nm. The (+) and (-) signs indicate with or without treatment respectively. Means of three experiments ± SEM are shown in B and C. A table of densitometry values and statistical analysis can be found in supplementary Tables S4 and S5 for B and C, respectively.

Figure 4

6OHDA triggered apoptotic death in dDCNs. To determine if 6OHDA stimulated death of dDCN is via the apoptotic or necrotic route, the TUNEL assay was used. Various inhibitors such as IKK, zVADfmk, zVDVADfmk, and zIETDfmk were used with 6OHDA to determine if the inhibitor decreased 6OHDA-mediated death of dDCNs. 6OHDA triggered death of dDCNs via the apoptotic route. The result illustrates that all studied inhibitors reduced apoptotic death of dDCNs at various rates. The result is shown as the relative percentage of cell death compared with control. The (+) and (-) signs indicate with or without treatment respectively. Means of three experiments ± SEM are shown. A table of values and statistical analysis can be found in supplementary data S6.

Figure 5

Illustration describing inflammation and apoptosis mechanisms in SARS-CoV-2 infection, suggesting similarity with those in Parkinson’s disease under oxidative stress. The SARS-CoV-2 entry in the cell requires interaction with the surface molecules angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), leading to activation of NFκB via pattern recognition receptors (PRR), which is further amplified by STAT-1, STAT-3 and oxidative stress, similar to 6OHDA-induced toxicity in treated dDCNs showing increased p65-NFκB expression. This NFκB stimulation induces caspase-2 and -8 expression in 6OHDA-treated dDCNs, which are the upregulated caspases from prolonged STAT-1 activation, NFκB and membrane-associated ORF3a viral protein in SARS-CoV-2 infected cells, leading to apoptotic cell death through the extrinsic pathway, thus promoting the development of PD and COVID-19. (BioRender software was used to create this figure).