A team at Stanford University School of Medicine has used CRISPR/Cas9 technology to gain new insights into the genes that might represent new targets for the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Led by Aaron Gitler, Ph.D., professor of genetics, and Michael Bassik, Ph.D., assistant professor of genetics, the team applied CRISPR/Cas9 knockout (KO) screens in human cells and in primary mouse neurons to identify genes that promote or prevent toxicity of the abnormal ALS protein aggregates that are believed to cause the death of neurons. They suggest such genes could potentially represent promising new drug targets for ALS.

“These toxic protein aggregates are what’s likely driving the pathology in the disease, but no one really knows how they cause neuronal cell death,” Prof. Gitler notes. “That’s really what we wanted to probe in this study.” The researchers report their findings in Nature Genetics, in a paper entitled “CRISPR–Cas9 Screens in Human Cells and Primary Neurons Identify Modifiers of C9orf72 Dipeptide-Repeat-Protein Toxicity.”

ALS causes progressive loss of motor neurons from the brain and spinal cord, which leads to muscle weakness, paralysis, and death, usually within 2 to 5 years. Mutations in one gene, C9orf72, are understood to represent the most common cause of ALS. The mutated gene contains a huge repeated section of bases, resulting in production of dipeptide-repeat proteins (DPRs) that clump together and are believed to be the main cause of toxicity and cell death. Mutations in C9orf72 are also the most common cause of the neurodegenerative disease frontotemporal dementia (FTD), which is the second most common cause of dementia in patients younger than 65 years of age. 

“In a healthy person, you might see 10 to 20 of these DNA repeats,” comments Michael Haney, a graduate student and co-lead author of the team’s published paper. “But in ALS, they expand to hundreds or even thousands of repeated segments, and that’s the template for the production of these toxic proteins.” It’s these toxic DPRs are believed to play a direct role in ALS progression, the authors point out. “Studies in flies and human cells have suggested that DPRs may be the main drivers of neuronal toxicity.”

To try and understand the genetic basis of DPR toxicity, the team used CRISPR/Cas9 technology to carry out comprehensive genome-wide knockout screens in human cells. The aim was to identify genetic modifiers of C9orf72 DPR toxicity—effectively to try and find which genes might either promote or prevent the ability of DPR proteins to lead to cell death. 

Rather than test each gene one at a time, genome-wide CRISPR knockout screens use libraries of single-guide RNAs (sgRNAs) to effectively knock  out tens of thousands of human genes simultaneously. “We used a lentiviral sgRNA library comprising ten sgRNAs per gene and targeting ~20,500 human genes, along with ~10,000 negative-control sgRNAs,” they explain. To interpret the results, the researchers then used deep sequencing techniques to track the effect—whether protective, sensitizing, or neutral—of each sgRNA in a pooled population of the knockout cells. “That is, sgRNAs that protected cells from DPR toxicity were enriched, and those that sensitized cells were depleted form the pooled population of cells,” they continue. The aim was to identify genes that might either enhance or inhibit DPR toxicity. If knocking out one particular gene reduced the toxicity of the DPR repeats, then that gene may represent a drug target. 

The initial screens identified about 200 genes, each of which, when absent, either protected the cells from toxic DPRs or increased toxicity.  Secondary screens in mouse primary cortical neurons were then used to validate the most promising protective genes.

Among the top hits was a gene, known as RAB7A, which is involved in endosomal trafficking, and the endoplasmic reticulum (ER) protein Tmx2. Knocking out the Tmx2 gene in the primary mouse cortical neurons completely protected the cells from a synthetic cause of neurotoxicity that was otherwise almost always fatal to the cells.

To test the potential effects of knocking out Tmx2 in more disease-relevant human cells, the team generated induced motor neurons (iMNs) from induced pluripotent stem cells (iPSCs) taken from patients with C9orf72 ALS.  They then used two different short hairpin RNAs to demonstrate that knocking down the Tmx2 protein significantly increased the proportion of surviving C9orf72 iMNs in two different cell lines. The researchers suggest that while further studies will be needed to verify their findings in additional patient cell lines and ALS models, “these results suggest that our screening strategy may be useful to identify potent modifiers of ALS-related phenotypes…the gene KOs that mitigated toxicity, such as TMX2, may serve as future therapeutic targets.”

Scientists aren’t yet completely clear on the role of Tmx2 in the endoplasmic reticulum, but the protein is thought to be involved in the cell’s responses to environmental stresses, and particularly those that trigger cell death. “Several lines of evidence have suggested a potential role for ER stress in C9orf72 pathogenesis,” the authors write. Their results with Tmx2 also suggest “a role for ER stress in DPR-mediated toxicity,” they note. “These results suggest that decreased Tmx2 may be protective by modulating the ER-stress response elicited by the DPRs.”

“We’re still in the early phases, but I think figuring out exactly what Tmx2 normally does in a cell is a good place to start—that would hint at what functions are disturbed when these toxic species kill the cell, and it could point to what pathways we should look into,” study team member Nicholas Kramer suggests.

“We could imagine that Tmx2 might make a good drug target candidate,” Haney comments. “If you have a small molecule that could somehow impede the function of Tmx2, there might be a therapeutic window there.”

The Stanford team suggests that their study is the first, to their knowledge, to use this genome-wide CRISPR human KO screen to look at the genetic basis of a neurodegenerative disorder. “Here, we used comprehensive CRISPR–Cas9 KO screens in human cells with further validation screens in primary neurons to discover modifiers of C9orf72 DPR toxicity,” they conclude. “We identified endosomal trafficking, confirming the results from previous studies in model organisms, and also identified new genes that suggest that ER function and ER stress are important in c9FTD/ALS pathogenesis….Together, our results demonstrate the promise of CRISPR–Cas9 screens in defining mechanisms of neurodegenerative disease,” they write. “We anticipate that CRISPR screens in human cells and primary neurons will be a powerful addition to the experimental toolbox to study mechanisms of neurodegenerative disease.”

Drs. Gitler and Bassik are now using the same technology to help understand other causes of ALS and other neurological disorders, such as Hungington’s, Parkinson’s, and Alzheimer’s diseases, which involve toxic proteins. “I think it’s a really exciting application for CRISPR screens, and this is just the beginning,” Bassik states.