A new research study by researchers at the ALS Therapy Development Institute sheds light on the way abnormal proteins created because of mutations in the C9orf72 gene cause toxicity in motor neurons. The study, published on April 17, 2023 in the journal Frontiers in Cellular Neuroscience and titled “Reduced C9orf72 Expression Exacerbates polyGR Toxicity in Patient iPSC-Derived Motor Neurons and a Type I Protein Arginine Methyltransferase Inhibitor Reduces that Toxicity” details research led by Director of Cell Biology, Kyle Denton, PhD and largely executed by Associate Scientist III, Therese Dane in collaboration with other members of ALS TDI’s Cell Biology Team. 

Summary of Research

Dr. Denton, Ms. Dane and colleagues conducted experiments that helped shed light on the ways abnormal proteins resulting from mutations in the C9orf72 gene cause toxicity in motor neurons. They also demonstrated further evidence that type I PRMT inhibitors, a class of molecules that are currently under investigation as a potential ALS treatment, may help rescue cells from these harmful effects. 

In any healthy human cell, there are two copies of the C9orf72 gene. People with C9orf72-related ALS have a mutation in one or, in rare cases, both copies of this gene, known as a C9orf72 repeat expansion mutation. To advance our understanding of how these mutations might cause damage to motor neurons, Dr. Denton and his team used induced Pluripotent Stem Cells (iPSCs) to create four lines of motor neurons for testing.

  • The first line, which served as a control, was from a healthy person with two normal copies of the C9orf72 gene.
  • For the second and third cell lines, Therese used gene editing techniques to remove one copy of the C9 gene in the former and both copies in the latter. This resulted in one cell line with half the regular levels of the C9 protein produced by the gene, and one with no C9 protein.
  • The fourth line was derived from samples provided by a participant in ALS TDI’s ALS Research Collaborative Program with C9orf72-related ALS.

Therese then exposed these cells to polyGR – one of the toxic proteins known to be produced by cells with C9 repeat-expansion mutations. 

Research Findings

When measuring the effects of the polyGR protein on each cell line, Therese and her team found that the first (healthy control) cell line showed the most resistance to the toxic effects of polyGR, while the second and third each suffered progressively more damage. This could imply that cells with lower levels of the C9 protein are more susceptible to damage from polyGR.

However, the cells derived from a person with C9orf72-related ALS showed similar levels of damage as the cell line with no C9 genes at all, even though they contained one healthy C9 gene and one with a repeat-expansion mutation. This finding could indicate that both the lack of normal C9 protein and the presence of toxic proteins created by a mutated C9 gene are both involved in causing motor neurons to die in cases of ALS.

Additionally, the team found that treatment with a Type 1 PRMT Inhibitor significantly reduced the damage to cells treated with polyGR.  Some of these findings were previously presented at the International ALS/MND Symposium in December 2022. In early 2023, the team repeated these experiments with cells taken from a volunteer with C9orf72-related ALS – finding a similar rescue effect as in the previous tests in control cells. This further supported Type-1 PRMT inhibitors' importance in finding a treatment for C9orf72-related ALS.

The ALS TDI Cell Biology team plans to explore additional experiments to further investigate how type I PRMTs interact with C9orf72. They also plan to explore inhibition of these enzymes in models of other forms of ALS in order to assess their potential to treat forms of ALS beyond C9orf72.

Key Take-Aways:

  • Researchers at ALS TDI studied how proteins resulting from mutations in the C9orf72 gene cause toxicity in motor neurons.
  • They found that cells with lower levels of normal C9orf72 protein were more susceptible to damage from polyGR, a toxic protein produced by cells with C9orf72 repeat-expansion mutations.
  • The lack of normal C9orf72 protein and the presence of toxic proteins created by a mutated C9orf72 gene may both be involved in causing motor neurons to die in cases of ALS and C9orf72-mutation mediated frontotemporal dementia.
  • Treatment with a Type 1 PRMT inhibitor significantly reduced the damage to control cells treated with polyGR, providing further support for their importance in finding a treatment for C9orf72-related ALS.
  • The ALS TDI cell biology team plans to explore additional experiments to further investigate how type I PRMTs interact with C9orf72 and to explore inhibition of these enzymes in models of other forms of ALS.

To learn more about ALS TDI’s Research to end ALS, click here.

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