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Nasal inoculation of the SARS-CoV-2 S1 protein causes brain inflammation and reduces acetylcholine levels in the mouse brain

In this study, Japanese researchers investigated the effects of nasal inoculation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein on the mouse brain by developing a model that expresses the S1 subunit of the SARS-CoV-2 S protein in the olfactory cavity. 

The neurologic sequelae that affect the central nervous system (CNS) and peripheral nervous system (PNS) are found in almost 30% of COVID-19 patients, including cognitive and memory disorders, headaches, stroke, extrapyramidal and movement disorders, mental disorders, encephalitis or encephalopathy, insomnia, peripheral neuropathy, acute inflammatory polyradiculoneuropathy, orthostatic intolerance, and syncope. It seems that SARS-CoV-2 uses various neuroinvasive strategies and pathways to invade the CNS, such as infection of the nasal olfactory epithelium and axonal transport along the olfactory nerve, retrograde axonal transport, invasion by compromising the blood-brain barrier (BBB), and the use of infected hematopoietic cells as “Trojan horses” (hematogenous route). It is assumed that the olfactory bulb serves as the main gateway for viruses to enter the brain. The cells of the olfactory system express angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease, serine 2 (TMPRSS2), which are essential for viral entry. Interestingly, previous data have shown that olfactory bulbectomy, a surgical destruction of the olfactory bulb, leads to brain inflammation. 

The SARS-CoV-2 S glycoprotein is composed of the S1 and S2 subunits, separated by host cell proteases. S1 is composed of the N-terminal domain (NTD), the receptor binding domain (RBD) with a receptor binding motif (RBM), and two C-terminal domains. The S glycoprotein contains a neurotoxin-like region that has sequence similarities with the HIV glycoproteins, the G-ectodomains of three Rabies lyssavirus (formerly Rabies virus) strains, and snake neurotoxins (α-bungarotoxin from snake Bungarus genera). This neurotoxin-like region is located at the junction of the S1 and S2 segments. Changeux initially suggested that the neurotoxin-like region of the SARSCoV-2 S glycoprotein interacts with the α-subunits of the nicotinic acetylcholine receptors (nAChRs).

Acetylcholine receptors (AChRs) are classified into either metabotropic muscarinic (mAChRs) or ionotropic nicotinic acetylcholine receptors (nAChRs). Pentameric nAChRs are essential for interneuronal communication within the CNS and the autonomic nervous system. They consist of a varying, either homomeric or heteromeric, combination of nine (α2-α10) α subunits and/or three (β2-β4) β subunits. The activation of nAChR leads to fast and nonselective opening of membrane-bound, excitatory cation channels.

Acetylcholine (ACh) is involved in an anti-inflammatory response called the cholinergic anti-inflammatory pathway (CAP). The CAP is a mechanism for suppressing inflammation in the brain and peripheral tissues via the autonomic nerve fibers.

About the study

The authors created a mouse model that expresses the S1 subunit of the SARS-CoV-2 S protein in the olfactory cavity by nasal inoculation with the S1 protein. In brief, they produced a non-proliferative adenovirus vector expressing the S1 protein (S1 Adv) of the original Wuhan strain (Wu strain) and generated the S1 mouse by inoculating the S1 Adv into the nasal cavity.

Results

The behavioral experiments, performed one week after nasal inoculation of the S1 subunit, showed depressive symptoms in the mice expressing the S1 protein in their nasal cavity (S1 mouse), manifested as increased fatigue at the weight-loaded forced swim test and increased immobility at the tail suspension test.

The histopathological examination of the whole brain, excluding the olfactory bulb, demonstrated enhanced expression of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α), and proinflammatory chemokine CCL-2, indicating the inflammation of the brain tissue. The histopathological examination of the olfactory bulb of the S1 mouse demonstrated an enhanced apoptosis. The analysis of neurotransmitter levels showed a decrease of cells positive for the ACh-synthesizing enzyme choline acetyltransferase (ChAT) in the medial septal and diagonal band of Broca, and a decrease of the Ach levels throughout the brain.

The administration of a central cholinergic agent donepezil, an acetylcholine esterase inhibitor, starting from the day of S1 inoculation normalized inflammatory cytokines (IL-6, TNFα), previously increased by the S1 protein. Donepezil also mitigated an enhanced apoptosis in the olfactory bulb and canceled the increase of IL-1β production in the amygdala, induced by the administration of S1. However, donepezil was unable to reverse the decrease in ChAT-positive cells in the medial septal and diagonal bands of Broca. The increased production of inflammatory cytokines in the lungs was also not reduced by donepezil.

Administration of a single lower dose of donepezil, equivalent to the dose for dementia patients, one week after S1 Adv inoculation, tended to reduce the increased expression of IL-6, TNF α, and CCL-2 in the S1 mice brains. According to the authors, these results indicate that donepezil could be used for the treatment of brain inflammation and neurological complications in COVID-19. However, its usefulness needs to be confirmed in clinical trials.

The expression of the S1 mRNA was not found in the brain. The authors stated that the lack of S1 mRNA expression in the mouse brain suggests that brain inflammation is not caused by the SARS-CoV-2 proliferation, but indirectly. In contrast, S1 mRNA expression was found in the lungs of S1 mice and correlated with the production of inflammatory cytokines, showing that the direct action of the S1 protein induced lung inflammation.

In vitro investigation in 3T3 mouse cells and human A549 cells showed that the NTD of the S1 subunit had intracellular calcium-increasing activity. These results are consistent with previous data showing that increased intracellular calcium damages the cells of the olfactory system, especially those in the olfactory bulb.

One week after the S1 inoculation scientists assessed an anti-inflammatory response, called the cholinergic anti-inflammatory pathway (CAP), by intracerebroventricular administration of PNU282987, an agonist of α7nAchRs. After one hour, they measured the expression of inflammatory cytokines in the brains of S1 mice. The results showed normalized or increased expression of inflammatory cytokines, suggesting that brain inflammation was associated with CAP disruption.

Figure from the original paper by Oka N et al.

Finally, to investigate the relationship between systemic and brain inflammation, the scientists enhanced systemic inflammation by intraperitoneal injection of lipopolysaccharide into the S1 mice.  The results showed markedly reduced expression of calbindin, a marker of γ-aminobutyric acid (GABA)-ergic neurons in mouse brains, and the absence of any changes in other neuronal differentiation markers. The authors interpreted this finding as specific to GABA-ergic neurons. 

Conclusion

This study showed that inoculation of the S1 subunit of the SARS-CoV-2 S protein in the olfactory cavity resulted in increased apoptosis of the olfactory system, brain inflammation, and decreased ACh levels in the mouse brain. This animal model, similar to encephalopathy in COVID-19 patients, demonstrated that brain inflammation was caused indirectly and not by direct action of SARS-CoV-2.

In addition, this investigation showed a link between the S1 protein, brain inflammation, and reduced ACh production. These findings could contribute to understanding the pathogenesis and mechanisms of neurological complications associated with COVID-19 and Long COVID syndrome.

This article was published in iScience.

Journal Reference

Oka, N, Shimada K, Ishi A, et al. SARS-CoV-2 S1 protein causes brain inflammation by reducing intracerebral acetylcholine production, iScience 26, 106954, June 16, 2023. (Open Access).  https://doi.org/10.1016/j.isci.2023.106954.

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