The neurologic sequelae that affect the central nervous system (CNS) and peripheral nervous system (PNS) are found in almost 30% of COVID-19 patients. It seems that severe acute respiratory syndrome coronavirus 2 (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). The Japanese authors in this study investigated how the inoculation of the SARS-CoV-2 S1 protein in the olfactory cavity affects the mouse brain.
It is assumed that the olfactory bulb serves as the main gateway for SARS-CoV-2 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. A recent study demonstrated that intranasal infection with SARS-CoV-2 of K18-hACE2 mice resulted in viral positivity in various parts of the eye, including the retina, but also in increased viral titers in the lungs and brain. https://discovermednews.com/retinal-inflammation-after-intranasal-infection-with-sars-cov-2/
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. Importantly, the S protein contains a neurotoxin-like region, located at the junction of the S1 and S2 subunits, with sequence similarities with three Rabies lyssavirus (formerly Rabies virus) strains and snake neurotoxins (α-bungarotoxin from snake Bungarus genera). Changeux initially suggested that the neurotoxin-like region of the SARS-CoV-2 S glycoprotein interacts with the α-subunits of the nicotinic acetylcholine receptors (nAChRs). https://comptes-rendus.academie-sciences.fr/biologies/item/CRBIOL_2020__343_1_33_0/
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) that suppresses 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. In brief, they produced a non-proliferative adenovirus vector expressing the S1 protein (S1 Adv) of the original Wuhan strain and generated the S1 mouse by inoculating the S1 Adv into the nasal cavity.
The behavioral experiments (weight-loaded forced swim test and the tail suspension test) were performed one week after nasal inoculation of the S1 subunit.
To investigate the relationship between systemic and brain inflammation, the scientists enhanced systemic inflammation by intraperitoneal injection of lipopolysaccharide into the S1 mice.
Results
The behavioral experiments revealed 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, showed enhanced expression of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α), and proinflammatory chemokine CCL-2, indicating the brain tissue inflammation. The histopathological examination of the olfactory bulb demonstrated enhanced apoptosis.
Cells positive for the ACh-synthesizing enzyme choline acetyltransferase (ChAT) were reduced in the medial septal and diagonal bands of Broca, whereas the Ach levels were reduced throughout mice brains.
The administration of donepezil, a central cholinergic agent and acetylcholine esterase inhibitor, starting from the day of S1 inoculation, normalized inflammatory cytokines that were previously elevated (IL-6 and TNFα). Donepezil also mitigated enhanced apoptosis in the olfactory bulb and inhibited the increase of IL-1β production in the amygdala, induced by the administration of S1. However, donepezil did not reverse the decrease in ChAT-positive cells in the medial septal and diagonal bands of Broca. Donepezil also did not reduce the increased production of inflammatory cytokines in the lungs. Administration of a single lower dose of donepezil one week after S1 Adv inoculation reduced the increased expression of IL-6, TNF α, and CCL-2 in the S1 mice brains.
The S1 mRNA was not found in the brain. According to the authors, the lack of S1 mRNA expression in the mouse brain suggests indirect brain inflammation, which is not caused by direct SARS-CoV-2 proliferation. In contrast, S1 mRNA expression was found in the lungs of S1 mice and correlated with the production of inflammatory cytokines, showing that lung inflammation was caused by direct action of the S1 subunit
The researchers then conducted in vitro investigation in 3T3 mouse cells and human A549 cells and showed that the NTD of the S1 subunit had intracellular calcium-increasing activity. These results are consistent with previous data which demonstrated that an increased level of intracellular calcium damages the cells of the olfactory system, especially those in the olfactory bulb.
The anti-inflammatory response called the cholinergic anti-inflammatory pathway (CAP), was assessed by intracerebroventricular administration of PNU282987, an agonist of α7nAchRs, a week after the S1 inoculation. One hour after intracerebroventricular administration of PNU282987, the results showed normalized or increased expression of inflammatory cytokines in the brains of S1 mice, suggesting that brain inflammation was associated with CAP disruption.
Figure from the original paper by Oka N et al.
The enhancement of systemic inflammation by intraperitoneal injection of lipopolysaccharide into the S1 mice showed markedly reduced expression of calbindin, a marker of γ-aminobutyric acid (GABA)-ergic neurons in mouse brains, and the absence of changes in other markers of neuronal differentiation. The authors interpreted this finding as specific for GABA-ergic neurons.
Conclusion
The 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 reduced 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. The administration of donepezil, a central cholinergic agent and acetylcholine esterase inhibitor, normalized inflammatory cytokines that were previously elevated and mitigated enhanced apoptosis in the olfactory bulb. According to the authors, donepezil could be used in the treatment of brain inflammation and neurological complications of COVID-19. However, its usefulness needs to be confirmed in clinical trials.
This investigation also showed a link between the S1 protein, brain inflammation, and reduced ACh production. These findings could contribute to understanding the pathogenesis 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.