SARS-CoV-2 main protease mpro causes microvascular brain pathology

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Clinical Pharmacology and Toxicology, Center of Brain, Behavior and Metabolism (CBBM), University of Lübeck, Lübeck-Germany has alarmingly found that the SARS-CoV-2 main protease mpro causes microvascular brain pathology cleaving NEMO (The essential modulator of nuclear factor-κB) in brain endothelial cells

The study findings were published in the peer reviewed journal: Nature Neuroscience. 

https://www.nature.com/articles/s41593-021-00926-1

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A considerable proportion of patients, up to 84% of those with severe COVID-19, show neurological signs and symptoms including anosmia, epileptic seizures, strokes, loss of consciousness and confusion1,2,3. Typically, COVID-19 can present with the clinical picture of encephalopathy2.

Beyond 4 weeks after onset, the post-acute COVID-19 syndrome includes cognitive impairment and a range of psychiatric symptoms and may affect up to 76% of patients4. Although a direct infection of the brain remains a matter of debate, SARS-CoV-2 viral genomes were detected in the brain and cerebrospinal fluid (CSF) of some patients, supporting the notion that SARS-CoV-2 gains access to the brain3,5,6.

Viral RNA has been found in blood and virus-like particles or viral proteins in brain endothelial cells5,7,8,9,10, suggesting that SARS-CoV-2 reaches the brain by a hematogenous route. In line with a vascular infection, endothelial cells in other organs have been identified as targets of SARS-CoV-2 infection11,12.

In patients with COVID-19, magnetic resonance imaging detected lesions that are compatible with a cerebral small-vessel disease and with a disruption of the blood–brain barrier (BBB)13,14,15. Autopsy studies have confirmed this interpretation15,16,17,18,19. However, the microvascular pathology and the underlying mechanisms in COVID-19 are still unclear.

In brains of SARS-CoV-2-infected patients, as well as mouse and hamster models, we found an increase in empty vascular basement membrane tubes, so-called string vessels, reflecting microvascular pathology. The SARS-CoV-2 genome encodes two viral proteases that are responsible for processing the viral polyproteins into the individual components of the replication and transcription complexes.

We found that one of them, SARS-CoV-2 Mpro (also called Nsp5 or 3CLpro)20, cleaves the host protein nuclear factor (NF)-κB essential modulator (NEMO). NEMO is involved in signaling cascades that regulate the transcription of numerous genes, including the antiviral type I interferons and other immune genes21. Beyond gene regulation, NEMO modulates cell survival and prevents apoptosis and necroptosis22.

The ablation of NEMO in brain endothelial cells induced microvascular pathology in mice that was reminiscent of what we observed in brains of patients with COVID-19. Of note, the widespread death of endothelial cells, rarefaction of capillaries, disruption of the BBB and neuroinflammation due to NEMO ablation were prevented by deleting receptor-interacting protein kinase 3 (Ripk3), a protein kinase that is essential for regulated cell death.

Importantly, a pharmacological inhibitor of RIPK signaling prevented the microvascular pathology induced by Mpro. These data suggest a potential therapeutic option to interfere with the neurological consequences of COVID-19.

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To date, it has been found that a considerable proportion of patients, up to 84% of those with severe COVID-19, show neurological signs and symptoms including anosmia, epileptic seizures, strokes, loss of consciousness and confusion. Typically, COVID-19 can present with the clinical picture of encephalopathy.

Beyond 4 weeks after onset, the post-acute COVID-19 syndrome includes cognitive impairment and a range of psychiatric symptoms and may affect up to 76% of patients. https://pubmed.ncbi.nlm.nih.gov/32294339/
 
https://pubmed.ncbi.nlm.nih.gov/32637987/
 
https://pubmed.ncbi.nlm.nih.gov/32882182/
 
https://pubmed.ncbi.nlm.nih.gov/33753937/
 
Though a direct infection of the brain remains a matter of debate, SARS-CoV-2 viral genomes were detected in the brain and cerebrospinal fluid (CSF) of some patients, supporting the notion that SARS-CoV-2 gains access to the brain.
https://pubmed.ncbi.nlm.nih.gov/32314810/
 
https://pubmed.ncbi.nlm.nih.gov/32402155/
 
The viral RNA has been found in blood and virus-like particles or viral proteins in brain endothelial cells, suggesting that SARS-CoV-2 reaches the brain by a hematogenous route. In line with a vascular infection, endothelial cells in other organs have been identified as targets of SARS-CoV-2 infection.

In patients with COVID-19, magnetic resonance imaging detected lesions that are compatible with a cerebral small-vessel disease and with a disruption of the blood–brain barrier (BBB) Autopsy studies have confirmed this interpretation. However, the microvascular pathology and the underlying mechanisms in COVID-19 are still unclear.
 
The NeuroCOVID study team describes structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology.
 
In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, the study team found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries.
 
The study team obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. They have also found alarmingly confims that Mpro of SARS-CoV-2 cleaves host cell NEMO with high efficiency.

In infected cells, Mpro is located in the cytosol and nucleus, where NEMO is also present. This may benefit the virus by preventing the induction of antiviral type I interferons that depends on NEMO. Indeed, levels of type I interferons are low or absent in the peripheral blood of patients with COVID-19.
 
Cleaving NEMO is also a strategy used by other viruses.
https://pubmed.ncbi.nlm.nih.gov/26656704/
 
https://pubmed.ncbi.nlm.nih.gov/27984784/
 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019732/
 
However, the tropism of SARS-CoV-2 is likely to limit NEMO inactivation to specific cell types. Accordingly, some NF-κB-dependent cytokines, such as TNF and IL-6, are highly upregulated, indicating that the cells of origin have escaped NEMO inactivation.

In addition to its central role in immunity, NEMO supports the survival of some but not all cell types. While neurons, glia and endothelial cells of peripheral vessels seem to resist NEMO deficiency or are even protected by it against inflammatory stimuli, the survival of other cells, including brain endothelial cells, is supported by NEMO.
 
The study findings suggest that, in COVID-19, brain endothelial cells are at disproportionate risk when being infected by SARS-CoV-2 because of their dependence on NEMO activity for survival.
 
It should be noted that Cleavage of NEMO by Mpro mimics the genetic disease incontinentia pigmenti that is caused by inactivating mutations in the NEMO (IKBKG) gene. In the latter condition, patients suffer from a mix of neurological symptoms, such as encephalopathy, stroke and seizures that resemble neurological manifestations of COVID-19.
 
The absence of NEMO in mice induced a loss of endothelial cells and microvascular pathology. Subsequently, patchy hypoxia developed in the brain and the BBB became leaky. In parenchymal cells, a prominent upregulation of GFAP indicated the activation of astrocytes, in line with the finding that GFAP concentrations are elevated in the blood of patients with COVID-19. An increased BBB permeability and astrocyte activation may cause epileptic seizures in patients with COVID-19 as in incontinentia pigmenti.
 
Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology.
 
The researchers believe that RIPK is a potential therapeutic target to treat the neuropathology of COVID-19.


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