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Loss of C9orf72, gene linked to ALS, affects motor skills in early studies

Reduced activity in the C9orf72 gene linked to amyotrophic lateral sclerosis (ALS) affected the neuromuscular junction – where nerve and muscle cells connect – in a zebrafish model, establishing the model as a tool for research into disease and the gene’s role in symptoms of ALS.

Zebrafish in this C9orf72 The loss-of-function model showed “motor defects, muscle atrophy, loss and death of motor neurons in the early stages of larvae and adults,” the scientists reported. As such, they expect the model to be useful for testing drugs aimed at restoring communication between nerve cells and muscles.

The study, “Reduced C9orf72 function leads to defective synaptic vesicle release and neuromuscular dysfunction in zebrafish, ”Was published in Communications biology.

ALS is characterized by the narrowing and death of the motor neurons that control voluntary muscle movement. A defective C9orf72 gene, which results in an abnormal protein of the same name, is the most common genetic cause of the disease, occurring in about 50% of hereditary cases and up to 10% of sporadic cases.

Analysis of tissues and cells isolated from patients with ALS indicates that this genetic defect results in loss of C9orf72 function, but the exact mechanism leading to the disease remains poorly understood.

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Studies in ALS patients and animal models suggest that this loss of function alters the synapses that connect the ends of motor nerve cells and muscle cells at the neuromuscular junction (JNM) – the place where nerves and muscle cells communicate.

“This synaptic dysfunction is observed in all people with the disease and occurs before the death of motor neurons,” said Zoé Butti, first author of the study and doctoral student at the National Institute for Scientific Research (INRS), in a statement. .

To further explore these mechanisms, Butti and a research team based at the INRS-Center Armand-Frappier Santé Biotechnologie in Quebec, Canada, examined the impact of the reduction C9orf72 gene activity on the neuromuscular system of zebrafish.

It is worth noting that the zebrafish is a standard research model. They have a similar genetic structure to humans, are easy to maintain, and their eggs develop outside the mother’s body, making it easier to study early development.

After establishing the ALS zebrafish model – which was about half the normal C9orf72 gene activity – researchers found that most embryos died within weeks of fertilization. Compared to healthy zebrafish, SLA fish had a survival rate of 2-5% after 15 days. Those who survived without physical defects showed a significant decrease in motor activity.

An examination of their neuromuscular junctions at the time when poor motor activity became evident (six days after fertilization, commonly abbreviated by scientists as dpf) revealed a significant reduction in synaptic markers in the ALS fish, which had not been observed at earlier times.

“While the synaptic structures of the NMJ develop properly and are preserved in the early stages of larvae in [ALS fish], they start to degenerate from 6 dpf, ”the team wrote.

Other measurements showed a decrease in the frequency and strength of electrical nerve signals in the connection of muscle cells at the neuromuscular junction.

The build-up and agglutination of a protein called TDP-43 in nerve and muscle cells is a hallmark of ALS. Typically, TDP-43 is found in the nucleus, but muscle cells in the fish model ALS had this protein primarily outside the nucleus, in the cytoplasm of the cells.

However, when the mutant fish were given the genetic instructions to make a healthy C9orf72 protein, most of these alterations were rescued, becoming similar to a healthy group of control fish, which provided “strong evidence that C9orf72 silence in zebrafish recapitulates a [disease-causing] characteristic of ALS, ”the scientists wrote.

“In about 97% of patients with ALS, the TDP-43 protein is depleted from the nucleus and forms aggregates in the cytoplasm rather than being in the nucleus, as is the case in healthy people,” said Kessen Patten, PhD, study leader. author and specialist in ALS at the INRS center. “We want to further investigate this relationship between the two proteins.”

SLA fish that survived to adulthood showed impaired swimming behavior and swam with weak movements, showing signs of paralysis before dying. These fish also showed alterations in their NMJ, smaller mature motor neurons in the spinal cord, and severe narrowing (atrophy) of muscle fibers, “consistent with the pathology of motor neuron degeneration in patients with ALS,” wrote Researchers.

Analysis of spinal cord motor neurons in adult zebrafish with ALS revealed that TDP-43 was depleted in the cell nucleus, but aggregated in the cytoplasm.

To understand the molecular mechanisms underlying C9orf72 deficiency, the researchers also looked at protein production in ALS and monitored the fish in the early embryonic stages.

Of the 24 proteins differentially produced in ALS fish and control fish, several were proteins necessary for the proper functioning of synapses. The top protein was SV2a, which is also downregulated in the neurons of patients with C9orf72-related ALS. The experiments confirmed that SV2a interacted with the C9orf72 protein.

The SV2a protein is known to be involved in the release of synaptic vesicles, which carry chemical messengers (neurotransmitters) in the NMJ from nerve cells to muscle cells. Analysis showed that this process was significantly reduced in ALS fish, suggesting that C9orf72 plays a key role in regulating the release of synaptic vesicles from nerve cells.

The researchers also found that production of the Rab3a protein, essential for transporting synaptic vesicles to nerve cell synapses, was lower in ALS fish.

“In conclusion, we generated a stable C9orf72-LOF model in zebrafish that summarized some of the main features of ALS and improved our understanding of the pathogenesis of ALS,” the researchers wrote. “It is important to note that our results demonstrate that the loss of C9orf72 function impairs synaptic function at the level of NMJs and leads to motor deficits.”

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