Wang lab research reveals role of C9 in nutrient and metabolic stress response
In order to understand how a repeat expansion mutation in the gene C9orf72—the most common genetic cause of ALS—leads to disease, scientists also need to understand the protein’s normal function. Since this hexanucleotide repeat expansion causes a loss of this normal function, Yon Ji, PhD, a postdoctoral fellow in the lab of Jiou Wang, MD, PhD, associate professor in the Department of Biochemistry and Molecular Biology, has been working to understand the normal role of the C9 protein and what happens when it is lost.
In a new paper in PLOS Genetics, Ji and colleagues identified a new mechanism through which C9orf72 may cause disease. Using a Caenorhabditis elegans nematode worm model, the researchers showed that the C9 protein helps to control the function of a key regulator of cellular homeostasis under nutrient stress. The loss of normal C9 function caused by the repeat expansion may lead to metabolic problems that cause neuronal degeneration.
“We see a clearer manifestation of defects from the loss of C9 in cells under stress,” Wang said. “A better understanding of C9orf72 function is needed for the development of an optimal therapeutic strategy.” “Fortunately, C9orf72 has an orthologue in C. elegans known as ALFA-1, providing us with an opportunity to investigate the conserved function of this protein,” said Ji.
Using a C. elegans worm strain that carried a mutation in alfa-1, the researchers tracked the newly hatched worms through the larval stage. If food is abundant, this stage only takes a few hours; with no food, the larval stage can stretch out for several weeks. Compared to the wild-type strain, the worms carrying a mutation in alfa-1 showed significantly decreased survival of larvae under nutrient stress.
“Following this clue, we found that the worms lacking C9 orthologue have less fat and then uncovered a conserved role of C9 in the regulation of nutrient metabolism,” said Ji.
Survival under nutrient stress at the larval stage requires a metabolic change to preserve the energy needed for long-term survival during starvation. C. elegans stores fat for energy in its intestines and digests it in lysosomes. The alfa-1 mutant worms showed fewer lipids in their lysosomes than controls, indicating a problem with lipid metabolism in lysosomes that could explain the worms’ premature deaths.
To understand the molecular ripple effects of alfa-1 loss, Ji and colleagues compared the transcriptomes (the messenger RNA molecules) of mutant and control C. elegans. The scientists found unusually high levels of transcripts involved in lipid metabolisms in the mutants. Subsequently, they found that a transcription factor protein called HLH-30, which regulates lysosome formation and lipid metabolism in C. elegans, is abnormally activated in the nuclei of mutant worms. Further experiments revealed that HLH-30 promotes the digestion of lipids for energy in lysosomes, and that this protein is essential for the lipid metabolism that allows for survival during starvation.
The mammalian version of HLH-30, known as TFEB, is regulated by mTOR, a protein that regulates a wide range of cellular processes. In experiments done in cultured human cells, the scientists identified an mTOR-related enzyme through which the C9 protein regulates TFEB. Known as Rag GTPases, these proteins recruit TFEB to the lysosome and inhibit its translocation to the nucleus. The C9 protein binds to Rag GTPases, which allows TFEB to be recruited at the lysosome. Once TFEB is recruited on the lysosome membrane, it is phosphorylated by mTOR allowing TFEB to stay in the cytoplasm. In human cells lacking a functional C9 protein, the amount of Rag GTPases are decreased. In turn, less amount of TFEB is recruited at the lysosome and therefore more TFEB remains in the nucleus. This amplified cellular stress under starvation conditions.
These findings are important, Wang explains, because they provide a mechanism through which the C9 protein does its job. Understanding how C9 helps to regulate metabolic homeostasis, especially under nutrient stress, will provide researchers with better insights into the development of age-dependent neurodegenerative diseases as well as improved strategies for designing therapies that can target this process in ALS patients.
“We expect that our findings of the C9 protein function will help better understand the disease processes and suggest new ways of developing therapies,” said Ji.
This post is adapted from a Packard Center Research Byte.