Rosina Giordano-Santini

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca ²⁺ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

Significance: Ramón y Cajal’s neuron doctrine, which states that neurons are individual cells that do not share any membrane or cytoplasmic continuity between them, has underpinned our view of modern neuroscience. However, there is considerable evidence that fusogens, specialized proteins essential and sufficient for the fusion of cells in other tissues, are expressed in the nervous system of several species in response to viral infection, stress conditions, and neurological disease. By manipulating the expression of fusogens in the chemosensory neurons of Caenorhabditis elegans , our results provide conclusive evidence that deregulation of fusogen expression causes neuronal fusion and can have deleterious effects on neural circuitry and behavioral outputs, revealing a possible novel underlying cause of neurological disorders.

Following a transection injury to the axon, neurons from a number of species have the ability to undergo spontaneous repair via fusion of the two separated axonal fragments. In the nematodeCaenorhabditis elegans, this highly efficient regenerative axonal fusion is mediated by epithelial fusion failure-1 (EFF-1), a fusogenic protein that functions at the membrane to merge the two axonal fragments. Identifying modulators of axonal fusion and EFF-1 is an important step toward a better understanding of this repair process. Here, we present evidence that the small GTPase RAB-5 acts to inhibit axonal fusion, a function achieved via endocytosis of EFF-1 within the injured neuron. Therefore, we find that perturbing RAB-5 activity is sufficient to restore axonal fusion in mutant animals with decreased axonal fusion capacity. This is accompanied by enhanced membranous localization of EFF-1 and the production of extracellular EFF-1-containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion inC. eleganshermaphrodites and the first regulator of EFF-1 in neurons.SIGNIFICANCE STATEMENTPeripheral and central nerve injuries cause life-long disabilities due to the fact that repair rarely leads to reinnervation of the target tissue. In the nematodeCaenorhabditis elegans, axonal regeneration can proceed through axonal fusion, whereby a regrowing axon reconnects and fuses with its own separated distal fragment, restoring the original axonal tract. We have characterized axonal fusion and established that the fusogen epithelial fusion failure-1 (EFF-1) is a key element for fusing the two separated axonal fragments back together. Here, we show that the small GTPase RAB-5 is a key cell-intrinsic regulator of the fusogen EFF-1 and can in turn regulate axonal fusion. Our findings expand the possibility for this process to be controlled and exploited to facilitate axonal repair in medical applications.

Following a transection injury to the axon, neurons from a number of species have the ability to undergo spontaneous repair via fusion of the two separated axonal fragments. In the nematode C. elegans , this highly efficient regenerative axonal fusion is mediated by Epithelial Fusion Failure-1 (EFF-1), a fusogenic protein that functions at the membrane to merge the two axonal fragments. Identifying modulators of axonal fusion and EFF-1 is the next step towards harnessing this process for clinical applications. Here, we present evidence that the small GTPase RAB-5 acts to inhibit axonal fusion, a function achieved via endocytosis of EFF-1 within the injured neuron. Consequently, we find that perturbing RAB-5 activity increases the capacity of the neuron to undergo axonal fusion, through enhanced membranous localization of EFF-1 and the production of extracellular EFF-1-containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion and the first regulator of EFF-1 in neurons.