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Jun 7
2021

Packard team identifies function of major ALS-linked protein

A team led by Packard scientist Jiou Wang, a neuroscientist at Johns Hopkins University, has identified the function of an ALS-linked gene

A team led by Packard scientist Jiou Wang, a neuroscientist at Johns Hopkins University, has identified the function of an ALS-linked gene. A mutation in the gene C9orf72 is the most common genetic cause of ALS and frontotemporal dementia (FTD), but its normal function has remained unclear. In a paper in Cell Metabolism, Wang and his team showed that the C9orf72 protein helps to regulate energy production in the mitochondria, the cell’s powerhouse.

“It is surprising to find that the C9orf72 protein is localized to the mitochondria and plays an important role in the assembly of respiratory machineries. Considering the key functions of the mitochondria in controlling energy supplies and cell death, the identification of the C9orf72 function in the organelle will contribute to our understanding of the origin of the pathologies in ALS/FTD and other neurodegenerative disorders,” said Wang.

Since an international team of scientists first uncovered that as many as 45% of those with an inherited form of ALS carry a repeat expansion in C9orf72, researchers have been trying to identify how the gene contributes to disease. Scientists have identified two major ways in which the expansion can create problems: by disrupting the normal function of the C9 protein and by the production of harmful RNAs and proteins. To date, most of the work has focused on the toxicity of the expansion’s byproducts rather than how the expansion interferes with the protein’s normal function.

Wang’s previous work showed patients with the C9orf72 expansion had lower levels of the C9 protein, and that the loss of this protein makes cells more sensitive to stress, creating motor deficits. In search of the molecular functions of the C9 protein in the cell, Wang and colleagues observed that the C9 protein interacts with many mitochondrial proteins, prompting them to hypothesize that the C9 protein plays a role in energy production in the mitochondria.

The scientists examined mouse embryonic fibroblasts and found wild-type C9 protein in the mitochondria. To locate the C9 protein within the organelle, the researchers used soundwaves and chemicals to break down the outer membrane of the mitochondria. These experiments, combined with immuno-electron microscopy of isolated mitochondria, showed that the C9 protein is localized to the space between the inner and outer mitochondrial membranes. A series of six cysteine amino acids that are evolutionarily conserved within the C9 protein directs it to a complex of proteins that imports it to the mitochondrial intermembrane space.

When the C9 protein was removed from the mouse embryonic fibroblasts, Wang and colleagues found that the most downregulated pathway was oxidative phosphorylation, the biochemical process by which cells make energy in the form of ATP. The researchers stressed the cells by feeding them galactose, which forces cells to use oxidative phosphorylation to make ATP. Cells lacking the C9 protein had a higher rate of death than those with the intact C9 protein, and they had less oxidative phosphorylation activity and produced less ATP. These results indicate that C9 is crucial to the production of ATP via oxidative phosphorylation. Further experiments showed that the C9 functions by stabilizing one of the proteins that helps to assemble one of the key protein complexes that makes up the oxidative phosphorylation protein chain. Indeed, the loss of C9 protein leads to a reduction of the key respiratory chain complex in multiple types of cells and tissues including stem cell-derived motor neurons and spinal cord tissues from C9 ALS/FTD patients.

Cells with reduced oxidative phosphorylation capacity need an alternative source of energy, especially if they don’t have ready access to glucose. After being grown in a galactose medium, C9-knockout cells also had increased rates of glycolysis, the process that breaks down glucose for energy. Neurons in particular have high energy needs when firing, which requires a functional oxidative phosphorylation pathway to produce the necessary ATP. By studying the neurons from C9 ALS/FTD patients, the researchers found that the oxidative phosphorylation pathway and ATP deficiencies in these cells likely contributes to their degeneration and death.

This newly identified function for the C9orf72 protein shows how energetic stressors can lead to cell death in the absence of functional C9 protein, and that the protein plays a key role in the cell’s energy buffering capacity. The results further cement the role of energy homeostasis in ALS/FTD and the impacts of the aging process on mitochondrial function.