COVID-19 Research: Taiwanese Study Finds That SARS-CoV-2 D614 And G614 Spike Variants Impair Neuronal Synapses, Causing Neurological Manifestations

COVID-19 Research: A new study by researchers from the Institute of Molecular Biology, Academia Sinica, Taipei-Taiwan shows that SARS-CoV-2 D614 and G614 spike variants impair neuronal synapses, causing neurological manifestations. The study also showed that these spike variants exhibit differential fusion ability.


 
In the study abstract, the researchers said that the SARS-CoV-2 coronavirus that causes the COVID-19 disease exhibits two major variants based on mutations of its spike protein, i.e., the D614 prototype and G614 variant.
 
Although neurological symptoms have been frequently reported in patients, it is still unclear whether SARS-CoV-2 impairs neuronal activity or function.
 
In this study the researchers show that expression of both D614 and G614 spike proteins is sufficient to induce phenotypes of impaired neuronal morphology, including defective dendritic spines and shortened dendritic length.
 
Utilizing spike protein-specific monoclonal antibodies, the study team found that D614 and G614 spike proteins show differential S1/S2 cleavage and cell fusion efficiency.
 
The study findings provide an explanation for higher transmission of the G614 variant and the neurological manifestations observed in COVID-19 patients.
 
The study findings were published on a preprint server and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.12.03.409763v1
 
The SARS-CoV-2 virus binds to the receptors angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) on host cell surfaces via its spike envelope glycoprotein, triggering membrane fusion for viral entry. https://www.thailandmedical.news/news/breaking-news-covid-19-study-shows-that-sars-cov-2-can-bind-to-human-host-neuropilin-1-receptor-nrp-1,-blocking-certain-pain-signals-in-host
 
In addition to viral entry, SARS2 spike protein also triggers fusion of host cells, giving rise to giant cells harboring multiple nuclei (also known as syncytia) in patients. https://pubmed.ncbi.nlm.nih.gov/32221306/
 
As ACE2 is widely distributed in divergent cell types, including neurons and glial cells, SARS2 can infect multiple organs, including the olfactory and central nervous systems. https://www.biorxiv.org/content/10.1101/2020.04.07.030650v3
 
https://www.biorxiv.org/content/10.1101/2020.06.25.169946v2
 
https://pubmed.ncbi.nlm.nih.gov/32492193/
 
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00172
 
2333487/">https://pubmed.ncbi.nlm.nih.gov/32333487/
 
https://pubmed.ncbi.nlm.nih.gov/32314810/
 
https://www.thelancet.com/pdfs/journals/lanpsy/PIIS2215-0366(20)30287-X.pdf
 
Human brain organoid cultures have further confirmed that SARS2 is able to infect human cortical neurons, evidenced by the presence of respective viral genomes and proteins in neurons. https://www.biorxiv.org/content/10.1101/2020.05.30.125856v1
 
Studies of animal models also support that both SARS and SARS2 can infect neurons of the rodent central nervous system (CNS). https://pubmed.ncbi.nlm.nih.gov/18495771/
 
SARS2 infection results in neurological manifestations and neuropsychiatric illness in both adult and newborn patients, including loss of olfaction and/or taste, dizziness, headache, impaired consciousness, delirium/psychosis, seizures, inflammatory CNS syndromes, and rare cases of ataxia and epilepsy. https://pubmed.ncbi.nlm.nih.gov/32007143/
 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170017/
 
https://pubmed.ncbi.nlm.nih.gov/32275288/
 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249973/
 
https://pubmed.ncbi.nlm.nih.gov/32469387/
 
Neurological symptoms persist in COVID-19 long-haulers, who still have the signs of damage after recovery from infection, indicating long-term impacts of COVID-19 on the nervous system. https://pubmed.ncbi.nlm.nih.gov/32929257/
 
https://www.medrxiv.org/content/10.1101/2020.08.11.20172742v1
 
Neuronal cell death and reduced protein levels of neuronal markers have been reported in SARS2-infected human brain organoids. However, it remains unclear if SARS2 influences neuronal function or activity to cause neurological symptoms. Recent proteomic studies have analyzed protein-protein interactions among SARS2 viral proteins and host proteins and the phosphorylation landscape of SARS2-infected cells. https://www.biorxiv.org/content/10.1101/2020.03.22.002386v3
 
https://www.cell.com/cell/pdf/S0092-8674%2820%2930811-4.pdf
 
These studies indicate that SARS2 viral proteins interact with host proteins, thereby altering cellular status. Mutations in SARS2 spike protein influence viral infectivity. In particular, the D614G mutation of SARS2 spike resulted in a global transition of SARS2 from the D614 prototype originally identified from Wuhan to the G614 variant that became dominant since late May 2020. To date, it is unclear why the D614G mutation enhances SARS2 infectivity.
 
In this research, the study team used primary cultured neurons and three different cell lines to investigate biochemical and cell biology properties of spike D614 and G614 variants. They unexpectedly found that both D614 and G614 spike proteins alter neuronal morphology. Moreover, these two spike variants have differential properties in terms of S1/S2 proteolysis and cell fusion, which may account for variant infectivity of SARS-CoV-2 variants.
 
The study finds show that expression of SARS2 spike protein is sufficient to impair neuronal morphology. Both the D614 and G614 spike variants cause defects in the dendritic spines of mature neurons and reduce the dendritic length of developing neurons. Since neuronal morphology is highly relevant to neuronal function, the morphological defects caused by SARSCoV-2 spike protein imply that infected neurons function abnormally, which is likely relevant to the neurological and neuropsychiatric symptoms presented by COVID-19 patients.
 
To confirm altered neuronal activity upon SARS2 infection, it is imperative to perform electrophysiological recordings in the future.
 
It is unclear how spike proteins control neuronal morphology. Since spike protein distribution overlaps with that of F-actin and given that F-actin cytoskeletons are critical regulators of neuronal morphology, spike proteins may control F-actin dynamics or rearrangement via an unknown mechanism to influence neuronal morphology. It will be intriguing to investigate the mechanism by which spike proteins might target to F-actin and regulate F-actin dynamics. It has been questioned if SARS2 can infect neurons by binding to ACE2 because ACE2 expression levels are low in the nervous system..
 
The study team postulates three possible mechanisms by which SARS2 can infect neurons.
 
First, though low, ACE2 transcripts are still detectable in neurons . Low-level ACE2 expression may be sufficient for infection when viral titers are high.
 
Secondly, infection may be mediated by another spike receptor, NRP1, which is highly expressed in neurons.
 
Thirdly, membrane fusion may be mediated by digested S2 fragments, since such fragments are present on virion surfaces and SARS2 spike protein-expressing cells (presumably virus-infected cells in patients).
 
Consequently, neurons may be infected by locally high virion dosages released from proximal infected tissues or upon fusing with neighboring infected cells.
 
Certainly, these three possibilities are not mutually exclusive and further investigations are required to elucidate the detailed infection pathways of SARS2 in vivo.
 
Utilizing a series of biochemical and cell biological studies, the study team reports three differences between D614 and G614 spike proteins. First, the results echo a previous cryo-EM study that spike proteins are readily processed into S1 and S2 fragments in host cells.
 
S1/S2 cleavage of G614 spike is more pronounced compared to that of the D614 prototype. Second, perhaps due to having higher amounts of S2 fragments, G614 spike protein-expressing cells exhibited more efficient cell fusion than D614-expressing cells. Third, G614 and D614 spike proteins exhibit slightly different mobilities on SDS-PAGE gels, indicating that the D614G mutation may influence posttranslational modifications to modulate spike protein activity.
 
In terms of proteolytic processing of spike variants, the conclusions of several studies are conflicting. https://www.biorxiv.org/content/10.1101/2020.06.14.151357v1
 
https://www.biorxiv.org/content/10.1101/2020.06.20.161323v2
 
https://www.cell.com/cell/fulltext/S0092-8674(20)31229-0
 
Utilizing pseudotyped virus bearing SARS2 spike protein, some researchers did not observe distinct proteolytic cleavage between D614 and G614 variants.
 
But when producing pseudotyped virus, the furin cleavage site and ER-retention signal of spike protein tends to be mutated or removed in order to obtain high concentration virion titers. https://pubmed.ncbi.nlm.nih.gov/32221306/
 
Certain structural studies have also used the same strategy to generate high yields of homogenous spike proteins. https://www.cell.com/cell/fulltext/S0092-8674(20)31229-0
 
As a result, this kind of modification may alter spike protein properties, contributing to discrepancies among results.
 
The study team concluded, “Though infection mechanisms in virus-infected cells are expected to be more complex than for expression of spike protein alone and some key conclusions should be further validated in SARS2-infected cells, our study sheds light on the properties of SARS2 spike protein in host cells. Our findings also provide possible explanations for why the D614G mutation enhances infectivity and how SARS2 infection impairs neuronal function.”
 
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