A significant number of individuals with COVID-19 or the postacute phase of COVID-19 exhibit symptoms of central nervous system (CNS) disorders. The rise in the number of patients with neurological sequelae underscores the importance of identifying the mechanisms of the CNS infection and CNS regions implicated in the pathogenesis of neuroCOVID. The authors from the United Kingdom conducted this study to investigate whether a wild-type form of SARS-CoV-2 (Wuhan strain) and five variants of concern/interest, the Alpha, Beta, Delta, Eta, and Omicron differently affect cells of the CNS and modulate CNS infection.
The neurological symptoms in COVID-19 patients range from mild symptoms such as headaches, confusion, insomnia, peripheral neuropathy, orthostatic intolerance, and syncope to more severe disorders such as stroke, extrapyramidal and movement disorders, encephalitis or encephalopathy, seizures, acute disseminated encephalomyelitis, Guillain Barre syndrome, or hemorrhagic myelitis. https://discovermednews.com/hemorrhagic-myelitis-after-the-sars-cov-2-infection/
The neuroinvasion has been documented for almost all the β-CoVs, including SARS-CoV, MERS-CoV, and SARS-CoV-2, resulting in a similar spectrum of symptoms. The viruses enter the CNS via different pathways, including the olfactory pathway by which viruses reach the olfactory bulb via the olfactory nerves, the BBB, blood-cerebrospinal fluid (CSF) barrier, retrograde axonal transport, and the use of infected hematopoietic cells as “Trojan horses”. The viruses can use the olfactory and the hematogenic pathways simultaneously. As clinical studies failed to detect significant levels of viral RNA in the CSF, CSF as a major transportation route for SARS-CoV-2 has been questioned. The olfactory bulb is thought to be the main gateway for viral entry into the brain. Studies have found that the olfactory bulb is the site of viral entry into the CNS after intranasal inoculation. The viral antigens were detected in CNS regions that possess first- or second-order neural connections with the olfactory bulb, such as the cerebral cortex, basal ganglia, the midbrain, and the hypothalamus. Intranasal infection with SARS-CoV-2 in K18-hACE2 mice led to viral positivity in various parts of the eye, including the retina, but also in increased viral titers in the brain and lungs. https://discovermednews.com/retinal-inflammation-after-intranasal-infection-with-sars-cov-2/ Some authors think that viruses mainly enter the CNS via olfactory sensory neurons and peripheral sensory nerves. https://discovermednews.com/susceptibility-neurons-pns-cns-to-infection-sarscov2/
The BBB and blood-CSF barrier are highly complex networks that protect the CNS parenchyma from harmful elements, including viruses. Certain viruses can overcome these obstacles by infecting vascular endothelial cells. This facilitates the direct passage of viruses through these barriers to the CNS. It should be noted that certain CNS structures, such as the choroid plexus and the circumventricular organs including the hypothalamus, are not completely protected by the BBB and can serve as virus entry points. https://discovermednews.com/the-involvement-of-hypothalamic-circuits-in-sars-cov-2-infection-of-the-central-nervous-system/ The principal components of the BBB are microvascular endothelial cells, but, pericytes and astrocytes are the ultimate control of the barrier phenotype. Two host-cell factors are important for SARS-CoV-2 viral entry into many cell types: angiotensin-converting enzyme 2 (ACE2), which is bound by the SARS-CoV-2 spike (S) protein, and transmembrane protease, serine 2 (TMPRSS2), which cleaves the S-protein, allowing this binding to take place.
About the Study and Results
In this in vitro study, the researchers used primary human pericytes, fetal astrocytes, endothelial cells, and microglial cell lines to investigate the impact of SARS-CoV-2 on their functional activities. Cells and an in vitro 3D model of BBB were infected with the wild-type Wuhan strain or the Alpha, Beta, Delta, Eta, and Omicron variants.
ACE2 receptors were expressed only by astrocytes and microglia, and to a lesser extent by endothelial cells, but pericytes did not express ACE2. As only the Omicron variant replicated in pericytes which did not express ACE2 receptors, it indicates that the virus utilizes an alternative receptor to enter those cells.
The serine protease TMPRSS2 was expressed in all cell types, although pericytes exhibited a lower expression level. Astrocytes, endothelial cells, pericytes, and microglia expressed CD147 and neuropilin-1 (NRP-1) which interacts with the SARS-CoV-2 S protein as a co-receptor for viral entry. According to the authors, ACE2, TMPRSS2, CD147, and NRP1 expression on astrocytes and microglia suggests a potential SARS-CoV-2 entry into those cells.
The results further revealed that susceptibility and permissivity of brain cells to SARS-CoV-2 infection were dependent on the SARS-CoV-2 variant. The wild-type Wuhan strain, and the Alpha, Beta, Delta, and Omicron variants led to a productive infection of brain cells. The infections caused by the Alpha, Beta, Delta, and Omicron variants were more extensive than those caused by the wild-type Wuhan strain.
The viability of pericytes was more affected by viruses, compared to the viability of other analyzed cells. The Alpha and Beta variants were cytopathic only for pericytes. The Wuhan wild-type strain was cytopathic for pericytes and microglia. The Omicron variant was cytopathic for pericytes and endothelial cells. The Wuhan strain disrupted the endothelial barrier, showing excessive and potentially lethal stress to those cells and decreased microglial metabolic activity. The Delta and Eta variants decreased the metabolic activity of astrocytes.
All SARS-CoV-2 variants increased the mitochondrial activity in endothelial cells and pericytes, either temporarily (wild-type Wuhan strain) or continuously (Alpha, Beta, Delta, Eta, and Omicron variants). The Alpha and Beta variants caused a slight increase in astrocyte mitochondrial activity, and only the Alpha variant caused a slight increase in microglial mitochondrial activity. According to the authors, these findings suggest stress of those cells upon exposure to SARS-CoV-2.
All SARS-CoV-2 variants, except the Beta, altered the extracellular concentration of glutamate, the main excitatory neurotransmitter in the mammalian CNS. The Omicron decreased, whereas other variants increased extracellular concentration of glutamate ( the Delta continuously and the Wuhan wild-type, Alpha, Eta transiently).
In vitro BBB model
The Wuhan wild-type strain, and to a lesser extent, the Omicron variant, disrupted the integrity of the BBB. The other SARS-CoV-2 variants did not exhibit any effect. All SARS-CoV-2 variants, except the Eta variant, had a negative impact on one or more tight or adherens junction proteins, which play a crucial role in maintaining the integrity and selective permeability of the BBB. The most affected junctional protein was VE-cadherin which was significantly decreased by the wild-type Wuhan strain, the Alpha and Delta variants.
Conclusion
This study showed that the wild-type Wuhan strain and five variants Alpha, Beta, Delta, Eta, and Omicron, carrying specific mutations that modulate their infectivity and transmissibility, affect the brain cells and the BBB differently.
This article was published in the Journal of Neuroinflammation.
Journal Reference
Proust et al. Differential effects of SARS-CoV-2 variants on central nervous system cells and blood-brain barrier functions. Journal of Neuroinflammation (2023) 20:184. (Open Access) https://doi.org/10.1186/s12974-023-02861-3
