The enzyme ADM-4 as an essential fusogen required for nerve repair


Researchers at The University of Queensland have identified a molecule essential for regulating the repair of injured nerves, which could help people recover from nerve damage.

The finding was made using the nematode worm C. elegans which has long been studied by researchers for its ability to self-repair nerve cells.

Professor Massimo Hilliard and his team at UQ’s Queensland Brain Institute (QBI) have identified that the enzyme ADM-4 is an essential protein regulating the molecular glue, or fusogen, needed for nerve repair.

“We have shown that animals lacking ADM-4 cannot repair their nerves by fusion,” Professor Hilliard said.

“ADM-4 must function within the injured neuron to stabilize the fusogen EFF-1 and allow the membranes of the separated nerves to merge.

“An exciting part of this discovery is that ADM-4 is similar to a mammalian gene, opening up the possibility that one day we may harness this process in humans.”

Study first author, Dr. Xue Yan Ho, said the nematode provided a great platform for these studies.

“Our goal is to uncover the molecules and understand their role in nerve repair in C. elegans,” Dr. Ho said.

“If we can understand how to control this process, we can apply this knowledge to other animal models.

“The hope is that one day, we can induce the same mechanical process in people who have had a nerve injury.

“We are still a long way from this goal, but the discovery of ADM-4’s role is an important step forward.”

Nerve cells communicate using long, cable-like structures called axons.

As they are long and thin, they are very susceptible to breaking, which stops nerve cells from communicating and leads to issues like paralysis.

A few years ago, Professor Hilliard and his team discovered that C. elegans could spontaneously re-join two separated axon fragments, a process called axonal fusion.

QBI’s Associate Professor Victor Anggono helped the team define the molecular mechanisms of this process.

“Using neurosurgery to stitch together damaged nerves has limited success,” A/Professor Anggono said.

“A different approach using gene technology to directly provide the molecular glue, or activate the fusogen regulator ADM-4, or using pharmacology to activate these components, may facilitate complete regeneration.”

The axon, the longest neuronal process, is essential for the transmission of electrochemical signals to other neurons or muscles. Damage to axons in the central or peripheral nervous system can result in lifelong disabilities. Functional recovery is achieved when the axon of the injured neuron regenerates and, either directly or indirectly, reinnervates its original target. A highly efficient method of functional axonal repair, known as axonal fusion, has been observed in many invertebrate species, including the nematode Caenorhabditis elegans (1–6).

Axonal fusion occurs when the proximal axonal fragment, which is still attached to the cell body, regrows, reconnects, and fuses with its own severed distal axonal fragment, reestablishing the original axonal tract (7) and restoring neuronal function (8, 9).

Using the two C. elegans mechanosensory PLM (posterior lateral microtubule) neurons as a model system, we have previously shown that molecules of the apoptotic clearance machinery regulate axonal fusion (7). During apoptotic cell corpse engulfment, apoptotic cells expose the lipid phosphatidylserine (PS) on the outer leaflet of their plasma membrane, which functions as an “eat me” signal for the engulfing phagocyte (10).

Analogously, we have shown that, following axonal injury, PS exposure on the outer membrane of the distal fragment functions as a “save me” signal for recognition by the regrowing proximal fragment (7). This specific recognition of the distal fragment by the proximal fragment is mediated by PS-binding molecules, such as the PS receptor PSR-1 and the secreted transthyretin TTR-52 (7). Last, physical merging between the two membranes of the separated axonal fragments is mediated by the nematode fusogen epithelial fusion failure-1 (EFF-1), which we, and others, have shown to relocalize to the axonal membrane following axotomy, restoring both membrane and cytoplasmic continuity (4, 7). Overexpression of EFF-1 within the injured neuron can compensate for the lack of PSR-1 and TTR-52, revealing a key and sufficient role for EFF-1 in the axonal fusion process (7).

EFF-1 was initially identified in C. elegans through genetic screens for cell-cell fusion failure phenotypes and was found to be required for correct epidermis, vulva, and pharynx formation during development (11, 12). It is a type I single-pass transmembrane glycoprotein with structural and functional similarity to class II viral fusion proteins (13).

Expression of EFF-1 is able to induce cell fusion in many organisms and cell types, including C. elegans embryos (14, 15), cultured insect cells (16), and mammalian cells (13, 17). Fusion mediated by EFF-1 has been shown to require the presence of EFF-1 on both opposing cell membranes (12, 16–18). A trans-trimerization model for EFF-1–driven membrane fusion has been proposed, whereby formation of a trans-trimer made of two monomeric EFF-1 molecules on one membrane and a single EFF-1 molecule on the other membrane leads to conformational changes in EFF-1 (13). 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.

Once postulated to be fusogens themselves, the ADAM (a disintegrin and metalloprotease) protein family are type I single-pass transmembrane metalloproteases that contain three signature extracellular domains: the metalloprotease, disintegrin, and cysteine-rich domains. ADAM1, ADAM2, and ADAM3 were initially thought to be fusogens required for membrane fusion between egg and sperm in mice (20). However, it was later found that these ADAM proteins might be indirectly involved in the fertilization process and that cell-cell fusion is not dependent on their fusogenic potential (21). ADAM metalloproteases have also been shown to cleave regulators of axonal regeneration in both mammalian systems and C. elegans (22, 23).

Four ADAM proteins are present in the C. elegans genome, UNC-71, ADM-2, ADM-4, and SUP-17. Here, we show that ADM-4, an ortholog of human ADAM17/TACE (tumor necrosis factor–α–converting enzyme), is essential for EFF-1–mediated axonal fusion. We reveal that ADM-4 mediates axonal fusion cell-autonomously within the PLM neurons and that its overexpression promotes axonal fusion above wild type (WT) levels. We further demonstrate that ADM-4 metalloprotease activity and its binding to PS are required for its function within the axonal fusion context. Last, we show that ADM-4 binds EFF-1 and stabilizes this fusogen after injury. Thus, we propose a model in which ADM-4 promotes axonal fusion in the regrowing axon by stabilizing monomeric EFF-1, and in turn driving the fusogenic EFF-1 trans-trimer.

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Source: University of Queensland


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