In vitro study: SARS-CoV-2 induces the fusion of neurons, affecting neuronal activity

A new in vitro study, by researchers from Australia and Finland, found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces the fusion between neurons and between neurons and glia, disrupting their communication.

The SARS-CoV-2 is primarily a respiratory disease, but growing evidence suggests the presence of viral RNA and proteins in the brain. Angiotensin-converting enzyme 2 (hACE2), which serves as the main receptor for SARS-CoV-2, is expressed in both neuronal and glial cells within the human central nervous system. The spectrum of neurological symptoms in patients with neuro long COVID persist for months following the acute SARS-CoV-2 infection and results in a long-term impairment of functional ability.

Other viruses can cause encephalitis and meningitis, which are characterized by multiple neurological symptoms. Some viruses don’t kill their host cells. Therefore, other neuropathological mechanisms must be responsible for the progression of infection, which leads to brain dysfunction. Certain viruses use molecules called fusogens to fuse with host membranes and enter cells in non-neuronal tissues. Viral fusogens are involved in the recognition, binding, and entrance of viruses into their host cells. These viruses then hijack the cellular machinery in order to produce viral components, with newly synthesized viral fusogens. This leads to the formation of multinucleated syncytia, which allow viruses to spread “from within,” without the necessity for virion release into the extracellular space.

About the study

The goal of this in vitro study was to investigate whether the SARS-CoV-2 infection causes neuronal fusion and the formation of syncytia, which permanently alters the neuronal circuitry and function.

The fluorescence fusion assay was performed in murine brain cells. After 72 hours of infection, confocal fluorescence microscopy showed the presence of fused neurons. Using the SARS-CoV-2 S protein and the p15 fusogen, the researchers examined whether and how the presence of viral fusogens on the surface of host cells affects the nervous system cells. The p15 protein has been isolated from the baboon orthoreovirus which causes meningoencephalomyelitis in these primates. The results confirmed that expression of this small transmembrane fusogen is the only viral protein required by the baboon orthoreoviruses to form a syncytium. 

In contrast to p15, the S protein must bind to its specific receptor hACE2 to trigger fusion, and both the S protein and hACE2 must be expressed in order to promote fusion of brain cells. The expression of either the S protein or hACE2 alone did not generate any fusion events. The S protein (or the p15) promoted fusion between neurons, but also between neurons and glia and between glia and glia when the fusogens were expressed in these cells.

The researchers also used a variation of fluorescence fusion assay to determine whether neuronal fusion implies a temporary or permanent diffusion of cytoplasmic material between cells. The findings substantiated the existence of an active cytoplasmic bridge between the S protein/hACE2–fused neurons, and p15-fused neurons. The fused neurons retained their morphology and remained viable for a long period of time.

Researchers also used two different neuronal systems, cortical neurons differentiated in 2D cultures and 3D brain organoids derived from human embryonic stem cells, in order to examine the possibility of fusion in human-derived neurons expressing the S protein. Three days after the expression of the fusogens, neuronal cultures were inspected, and clusters of interconnected neurons were found. There was no cell fusion in the absence of fusogens or in the presence of S-6P, an inactive mutant of the S protein.

Syncytia are normally formed in most tissues at the level of the cell bodies. However, SARS-CoV-2 infection of nerve cells results in fusion not only between the somas, but also between neurites distant from the somas, which are important for cell-cell communication. Fusion driven by the S protein resulted in formation of fusion bridges that could extend over hundreds of micrometers. The formation of neuronal bridges allows the exchange of small proteins and large mitochondria between interconnected neurons.

The image from the original article of Martínez-Mármol et al, mouse neurons (in magenta) fuse (yellow) when they express the SARS-CoV-2 protein and its receptor.

Most of the fused neurons (~90%) fired simultaneously, whereas the remaining 10% showed a complete loss of neuronal activity. The neurons that lost their activity were fused tightly at the level of their somas. A variable pattern of neuronal activity was observed in nonfused neurons, ranging from highly synchronized to completely asynchronized. The loss in neuronal activity was complete for every neuron that fused with glial cell. Regardless of the synchronization between fused neurons, the frequency of neuronal activity was not altered. Using Ca2+imaging, the researchers demonstrated severely compromised neuronal activity in fused neurons. The concentration of intracellular Ca2+ increased within the neuronal bridge between neurons, creating peaks of Ca2+ that reflected the patterns of neuronal activity.

According to the authors, viral infections which drive the expression of viral fusogens have the potential to initiate an irreversible fusion of brain cells, causing alteration in neuronal communication. The ability of infected neurons to fuse with neighboring neurons or glial cells results in the sharing of large molecules and organelles, and in impaired neuronal activity.

Given a variety of structures that can be transported through these viral fusogen-mediated cellular structures, it is tempting to speculate that viruses and other toxic aggregates may also utilize these pathways to spread to neighboring cells. It could also represent a mechanism of viral spreading that escapes the immune system.

These findings suggest a possible pathomechanism of neuronal malfunction caused by SARS-CoV-2 infection. The effect on neuronal fusion will depend on the viral load in the brain and the areas that are infected. The authors also noted that fused neurons remain viable despite their altered circuitry and function. This event, which is difficult-to-detect, could explain some of the neurological manifestations of viral infection. In addition, neuronal fusion may be more important for other viruses, like the rabies viruses, which infect more neurons than SARS-CoV-2.

The authors noted that the current versions of the Moderna, Pfizer-BioNTech, and Johnson & Johnson anti SARS-CoV-2 vaccines encode the full-length S protein with two mutations that stabilize the prefusion conformation and inactivate its fusogenicity. They utilized the identical mutant form of spike S-2P as a negative control, thereby demonstrating the complete absence of fusogenicity. Nevertheless, they emphasized that the fusogenic potential of any future vaccines containing viral fusogens must be considered.

This article was published in Science Advances.

Journal Reference

Martínez-Mármol et al., SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity. Sci. Adv. 9, eadg2248 (2023) 7 June 2023 (Open Access).