Disruption of RAB-5 Increases EFF-1 Fusogen Availability at the Cell Surface and Promotes the Regenerative Axonal Fusion Capacity of the Neuron

Abstract

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 Caenorhabditis 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-1containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion in C. elegans hermaphrodites and the first regulator of EFF-1 in neurons.Peripheral and central nerve injuries cause life-long disabilities due to the fact that repair rarely leads to reinnervation of the target tissue. In the nematode Caenorhabditis 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 axonalfragmentsbacktogether.Here,weshowthatthesmallGTPaseRAB-5isakeycell-intrinsicregulatorof 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.

Fusogen-mediated neuron−neuron fusion disrupts neural circuit connectivity and alters animal behavior

“…The effects of fusogen overexpression in neurons could go beyond neuronal cell−cell fusion and associated behavioral defects. In C. elegans, fission during endocytosis and phagocytosis can be mediated by AFF-1 and EFF-1, respectively (54,55), and EFF-1−mediated axonal fusion following injury has been shown to be regulated by the GTPase RAB-5 (56). It is therefore possible that the expression of fusogens in neurons can not only result in neuronal fusion but also impact on fission events that could affect neuronal morphology, repair, and function.…”

The metalloprotease ADM-4/ADAM17 promotes axonal repair

“…These molecules have been shown to control the presence of EFF-1 to fusion sites in larval hypodermal cells. In the context of axonal repair, we have previously shown that RAB-5 regulates fusion by controlling the levels of EFF-1 in C. elegans ( 19 ). There was a notable increase in the amount of EFF-1 with cotransfection of ADM-4 in HEK293T cells.…”

“…As a result, EFF-1 transmembrane segments anchored in the two opposing membranes are brought into contact, pulling the two membranes into one ( 13 ). Previous studies have shown that monomeric EFF-1 is metastable ( 13 ) and that EFF-1 internalization from the cell membrane can prevent axonal fusion ( 19 ), whereas trimeric EFF-1 is stable and irreversible ( 13 ). However, the molecules that regulate the formation of functional EFF-1 trimers from EFF-1 monomers across opposing membranes to allow axonal fusion after injury remain unknown.…”

Femtosecond laser microdissection isolating regeneratingC. elegansneurons for single cell RNA sequencing

“…The transparent body of C. elegans further allows analysis of such phenotypes, including axon regrowth rate, guidance, and fusion at a single neuron resolution in vivo with fluorescence microscopy. Past nerve regeneration studies in C. elegans have led to the discovery of many conserved nerve regeneration pathways [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] , notably the dual leucine zipper kinase (DLK-1/p38) signaling cascade, whose role in neural development, regeneration, and degeneration has been confirmed in numerous vertebrate and invertebrate species 7,[27][28][29][30][31] .…”