In a recent study posted to the bioRxiv* preprint server, researchers used preclinical mouse models and human post-mortem samples to study the presence and distribution pattern of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein in the skull-meninges-brain (SMB) axis, including the recently discovered skull-meninges connection (SMC).
Study: SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19. Image Credit: SciePro/Shutterstock.com
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Despite many technical challenges, several studies have investigated how the central nervous system (CNS) remains engaged in coronavirus disease 2019 (COVID-19)-related symptoms.
A few studies even detected SARS-CoV-2 in brain tissues and widespread immune activation signals in the brain. This led scientists to believe that virus-shed proteins circulated in the bloodstream and promoted an inflammatory response.
However, the highly immunogenic SARS-CoV-2 S protein, also used in COVID-19 vaccines, emerged as an ideal candidate for investigating COVID-19-dependent and independent effects in the human brain.
About the study
In the present study, researchers used optical tissue clearing technology to identify all tissue targets that accumulated SARS-CoV-2 S1 protein in mice and investigated the distribution of S1 in post-mortem samples from COVID-19 patients.
Additionally, the team assessed if S1 alone induced brain pathologies in the absence of other viral proteins. (Note that the S1 protein with N501Y mutation infects wild-type (WT) mice through binding with mouse angiotensin-converting enzyme 2 (ACE2)).
For animal experiments, they used two-month-old wild-type C57Bl6/J mixed-sex mice housed on 12/12 hours of light & dark cycles with random access to food and water.
They obtained S1 proteins with N501Y mutation and labeled them with the fluorescent dye Alexa Fluor 647. Next, they microinjected spike S1 (N501Y) directly intravenously into the skull marrow of test animals under general anesthesia using a 1 mL syringe with a 28-gauge needle.
After three days of administering intravenous injections, the team collected each animal's skull, meninges, and brain tissues for proteomic analysis and mapped the distribution of fluorescently labeled spike S1. Next, they collected the mouse brain cortex tissue at days and 28 days to evaluate cell death and neuronal damage.
The researchers also performed mass spectrometry (MS)-based label-free quantitative proteomics analysis of brain region-matched human tissues from non-COVID-19 patients (controls).
It helped them detect whether S protein was persistently present in the skulls of patients who died from non-COVID-related reasons during the first two years of the pandemic.
Further, the team characterized the protein expression of SARS-CoV-2 infected skull tissues from post-mortem human samples with MS-based proteomics.
The optical clearing SHANEL protocol helped them analyze the histopathological profile of human skull samples with the dura mater.
Study findings
The researchers observed a substantial accumulation of SARS-CoV-2 S protein in the niches of human skull marrow, meninges, SMC, and brain parenchyma in both mouse and human-origin samples.
In healthy mice, direct microinjections of S into skull marrow niches triggered brain proteome alterations and parenchymal cell death. It also demonstrated the immunogenicity of SARS-CoV-2 S in the absence of other viral components. However, additional data is needed to show active replication of SARS-CoV-2 in these tissues.
Nevertheless, the observed accumulation of SARS-CoV-2 and its S glycoprotein at the CNS peripheries in the skull samples of some people suggested a likely mechanism for the neurological effects of SARS-CoV-2 infection.
This occurred even in those who recovered from illness but died later due to non-COVID-19-related reasons.
Proteomics analysis on the brain cortex of eight COVID-19 patients and 11 controls helped the researchers identify 7,138 proteins, gene set enrichment analysis (GSEA) of which correlated these dysregulated proteins to biological processes modulated in response to SARS-CoV-2 infection.
Further, this proteomics data showed perturbations in the complement and blood coagulation cascades, neutrophils-related pathways, including neutrophil extracellular traps (NETs) formation, and an upregulation of pro-inflammatory proteins, such as interferon-alpha/beta (IFN-α/β).
GSEA revealed differential expression of 76 proteins, with up and downregulation of 49 and 27 proteins, respectively.
Accordingly, the researchers found upregulation of the pyrin domain and a caspase recruitment domain (PYCARD), and NOD-like receptor family CARD domain containing 3 (NLRC3), all implicated with the assembling of inflammasomes.
Among others, they identified the Bone Marrow Stromal Cell Antigen 2 (BST2) pathway, which restricted the viruses from infecting nearby cells. In addition, they found activated myeloid cells with enlarged cell body morphologies in brain tissues of COVID-19 cases.
Studies have detected SARS-CoV-2 S in patient immune cells more than 12 months after the infection and in their plasma up to a year post-recovery.
Indeed, it invades the brain from cerebral circulation and persists in the human body. Yet, only a few studies have shown a link between the brain-related pathologies apparent in severe COVID-19 and the host’s brain proteome.
However, studies have reported that S triggers the expression of inflammatory cytokines and chemokines in macrophages and lung epithelial cells, thereby compromising endothelial functions.
In this study, it also activated an immune response in the SMB axis, potentially via recruiting and more neutrophils.
Though the researchers could not distinguish the direct effects of S from the systemic effects of COVID-19. It acted as an inflammatory stimulus that led to the development of immune response in the brain and increased the expression of pro-inflammatory proteins, e.g., calprotectin and pro-platelet basic protein (PPBP), a thromboses-related protein.
In the meninges, this inflammatory state led to the upregulation of neutrophil degranulation proteins, e.g., PDGFA-associated protein 1 (PDAP1) that might be linked to higher levels of NETs in the SMB axis and the invasion of neutrophils into the meninges.
NETs also triggered tissue damage, including endothelium damage, thereby altering coagulation processes and rationalizing why some COVID-19 patients develop mini-infarcts in the brain parenchyma. In fact, in the brain, proteins engaged in neurodegeneration pathways were highly dysregulated.
Conclusion
The study data suggested a mechanism for SARS-CoV-2 entry into the CNS. However, alternatively, SARS-CoV-2 S could reach the brain through other routes.
For example, it could traverse the cerebrovascular to reach the brain parenchyma or immune cells, e.g., phagocytic cells could carry it too. So, future studies should investigate other possible routes for entry of SARS-CoV-2 into the brain.
Furthermore, the researchers identified several proteins which previously showed no association with COVID-19, e.g., proteins implicated with neurological diseases, e.g., ras-related protein Rab-8B and EF-hand domain-containing protein D2.
Further, the panel of these proteins found in the patient's plasma might help with the early prognosis of COVID-19-induced brain-related complications.
These molecules and those involved in other commonly perturbed immune and other pathways could be leveraged as therapeutic targets.
In conclusion, all future efforts should be directed towards characterizing these proteins as biomarkers and therapeutic targets to prevent and treat COVID-19-related neuro-complications.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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