Screens in diverse cell models in lab ID 2 new ALS therapeutic targets

Proteins PIKFYVE and SYF2 may be useful molecular targets: Study

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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A scientist conducts tests in a lab using petri dishes and vials of blood.

A team of U.S. researchers have identified two proteins in the lab — PIKFYVE and SYF2 — that may each be a useful therapeutic target for treating amyotrophic lateral sclerosis (ALS).

Their findings on these potential molecular targets were published in a pair of studies earlier this month.

“Our discoveries bring us closer to achieving our big picture goal: finding treatments that can be broadly effective for all patients who suffer from ALS,” Jason Ichida, an associate professor of stem cell biology and regenerative medicine at the University of Southern California (USC), said in a university press release.

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ALS is caused by the death and dysfunction of motor neurons, the nerve cells responsible for controlling movement. To identify potential therapeutic targets for the disease, a team of scientists, led by Ichida, conducted a screen in a disease model using motor neurons derived from induced pluripotent stem cells (iPSCs).

Simply put, this cellular model involves collecting easily accessible cells, like skin and blood cells, from people with ALS. Then, through a series of biochemical manipulations, the cells are reprogrammed in a way that allows them to grow into motor neurons.

For these studies, the researchers used motor neurons derived from people with familial ALS — when the disease affects more than one person in the same family — with a known disease-associated mutation. They also used cells from sporadic ALS patients without any known mutation.

“A minority of patients have a variety of genetic causes of ALS that can be inherited within families, and a majority have what is known as ‘sporadic’ disease because its causes are unknown. This makes it a difficult challenge to find one treatment that will work for all patients with ALS,” said Gabriel Linares, PhD, a postdoctoral researcher in Ichida’s lab and co-author of both studies.

In one study, titled “SYF2 suppression mitigates neurodegeneration in models of diverse forms of ALS” and published in Cell Stem Cell, the researchers described disease-associated processes in these neurons and conducted a screen of hundreds of compounds to identify any potential therapeutic target for ALS.

As anticipated, analyses showed that motor neurons derived from ALS, whether sporadic or familial, showed defects compared with motor neurons derived from people without neurological disease.

In particular, ALS neurons died more quickly in cellular experiments, and also had abnormal clumps of proteins such as TDP-43. Clumps of TDP-43 are characteristic of ALS, found in nearly all cases of the disease, and are thought to be involved in driving nerve cell death.

Using this cellular model, the researchers conducted a screen of more than 1,900 molecules, looking for compounds that could rescue the disease-associated features of ALS motor neurons.

The results identified 50 compounds that were effective for most cells from familial ALS patients, including the approved ALS therapies Radicava (edaravone) and riluzole (sold as Rilutek and other formulations). However, many of these therapies, including Radicava and riluzole, showed only modest efficacy in cells from sporadic ALS patients.

The variability in response for different patients “underscored the importance of identifying broadly effective targets for ALS,” the researchers said.

Experiments using the iPSC-derived motor neurons showed that the compound apilimod could improve nerve cell survival and reduce the toxic accumulation of TDP-43 and other proteins in ALS motor neurons. Apilimod is known to block the activity of the protein PIKFYVE.

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In the study “PIKFYVE inhibition mitigates disease in models of diverse forms of ALS,” published in Cell, the research team conducted detailed assessments to investigate blocking PIKFYVE with apilimod as a potential therapy for ALS.

Treatment with apilimod or genetic strategies to reduce PIKFYVE activity led to a reduction in disease-typical death in motor neurons derived from people with sporadic and familial forms of ALS, the results showed. In addition, it extended survival in multiple models of ALS, including mice and fruit fly models.

“We show that PIKFYVE kinase inhibition is broadly efficacious in models of diverse forms of ALS,” the team concluded.

Mechanistic experiments illustrated that blocking PIKFYVE led to an increase in exocytosis — a process in which molecular cargo is wrapped in a membrane, shipped to the surface of the cell, and then dumped outside of the cell. By activating exocytosis, PIKFYVE inhibition prompted ALS motor neurons to get rid of toxic TDP-43 by moving it outside the cell.

The team noted that this effect was seen only in ALS motor neurons, not in motor neurons from healthy controls.

“We were able to pinpoint precisely how PIKFYVE inhibition mitigates neurodegeneration, which is important for informing the development of new targeted treatments,” said Shu-Ting (Michelle) Hung, a PhD student at USC and study co-author.

In the original iPSC screen, many of the other compounds identified as effective in both familial and sporadic ALS motor neurons are known to activate the androgen receptor. This protein receptor normally activates in response to certain hormones, most notably testosterone, which leads to changes in cells’ genetic activity.

Because activating the androgen receptor itself is likely to cause unwanted side effects, especially for women, the researchers conducted a series of experiments looking for other targets that could induce similar effects. The data identified SYF2 as a likely candidate. This protein is involved in RNA splicing, a process that help to control how certain genes are read to produce proteins.

The researchers showed that blocking SYF2 reduced TDP-43 clumping in ALS motor neurons from familial and sporadic patients, and it extended survival in mice engineered to form these toxic protein aggregates.

“Our … findings identify SYF2 and [related mechanisms that regulate RNA splicing] as important modulators of ALS pathology in patient-derived neurons and mice,” the scientists concluded.