C9ORF72 Function: Is the ALS Protein a Membrane Traffic Cop?

C9ORF72, made by the ALS-linked gene of the same name, finally has a job description: According to two recent studies, it regulates membrane trafficking.

Bioinformatics handiwork placed C9ORF72 in the DENN class of proteins. Short for differentially expressed in normal and neoplasia, these proteins regulate membrane events such as vesicle fusion. One of the studies, published December 13, 2012, in Frontiers in Genetics online, comes from the laboratory of L. Aravind at the National Center for Biotechnology Information in Bethesda, Maryland. Timothy Levine and colleagues at University College London, U.K., reported their findings in the January 16 Bioinformatics online.

The implications of this computational biology research for neurobiology and disease are uncertain at this point. A hexanucleotide expansion in the gene is the most common mutation in families with amyotrophic lateral sclerosis or frontotemporal dementia (FTD). Although many scientists ascribe pathology to an accumulation of toxic C9ORF72 mRNA, the normal function of the protein could yield additional clues and may be important for understanding side effects of potential therapeutics. “C9ORF72 is likely to be involved in some trafficking event of membranes within the cell,” Aravind said. Future ALS therapies targeting the C9 expansion in mRNA might diminish those functions.

“I think these are intriguing theoretical papers,” commented Jeffrey Rothstein of Johns Hopkins University in Baltimore, Maryland, who was not part of either study. But to prove a role for C9ORF72 in membrane biology, researchers will need better tools than those currently available. Rothstein has observed C9ORF72 on membranes with an antibody his lab made, but he is not confident the antibody binds only C9ORF72. “All the antibodies that exist lack specificity,” he said. Similarly, researchers are struggling to develop mouse models, because the repeat region itself is unstable, Rothstein said (see ARF related news story). Worms missing a portion of the gene slither at half-normal speed, indicating the protein has an important function, Levine noted (see ARF related news story).

Meet the DENN Family
Who are the DENNs, anyway? All proteins in this family regulate Rab GTPases—switches that control membrane-based processes such as vesicle budding, cell movement, and membrane fusion. DENNs are GEFs, aka GDP-GTP exchange factors. These turn on inactive GTPases by removing their GDP, leaving room for a new GTP to enter and reactivate the enzyme. DENNs clasp the enzyme and squeeze out the GDP, Aravind said.

Aravind is not an ALS researcher. With first author Dapeng Zhang, he pursued DENN-like protein structures to better understand eukaryotic evolution. The proliferation of membranous organelles in early eukaryotes was linked to an explosion in GTPases which regulate those membranes, Aravind said. Levine, in turn, was piqued by C9ORF72’s unknown function. When he comes across a protein like that, he immediately boots up his favorite tool, HHpred. This structure prediction software identifies proteins that share predicted arrangements of α-helices and β-sheets, even if their amino acid sequences diverge. Using HHpred, both teams identified C9ORF72 as a new DENN.

“C9ORF72 has all the essential features of the DENN,” Aravind said. “We predict that when its crystal structure is solved, it will look just like a garden-variety DENN.” Moreover, C9ORF72 is an ancient DENN. It likely dates back to the last common ancestor of all eukaryotes, meaning it arose before neurons ever existed. That implies it must have a conserved function in many cell types, said Aravind.

Levine proposes rechristening the C9ORF72 gene DENNL72, for DENN-like 72. The original moniker merely denoted the open reading frame’s position on chromosome nine. “Anything is better than that name,” commented Ralph Nixon of the Nathan Kline Institute on Orangeburg, New York. At least DENNL72 rolls off the tongue a bit easier, said Nixon, who was not involved in either study. Indeed, ALS researchers are playing with what to call this important gene (see ARF related news story).

That C9ORF72 might act in membrane trafficking jibes with defects in this area affecting neurons in particular, because neurons must shuttle vesicles up and down axons. “I cannot think of a system that would be more vulnerable to something that even slightly impairs it, or slows it down,” Nixon said. Identifying the protein as a GEF provides some focus for future research, Nixon said, but he added that the large number of different Rab GTPases still makes it difficult to predict which specific membrane events C9ORF72 might direct.

Aravind speculated, nonetheless. He noted that while the earliest eukaryote possessed a C9ORF72 homologue, certain lineages—fungi and plants among them—have since lost the gene. These kingdoms have also lost cilia, membranous organelles that are a bit like axons and dendrites in neurons. He reasoned, then, that C9ORF72 might regulate cilia-related functions, which would potentially make it a regulator of Rab5. In neurons, this GEF regulates endocytosis and axonal transport, among other things (reviewed in Ng and Tang, 2008). Axon traffic becomes jammed in a number of neurodegenerative conditions (see ARF related news story). Nixon’s lab has linked Rab5 to amyloid-β precursor protein processing (see ARF related news story).

C9ORF72 in Disease
If C9ORF72 is confirmed to be a DENN, then it will join a growing company of membrane regulators implicated in neurodegenerative disease. ALS genes include ALS2, which contains GEF domains (see ARF related news story on Hadano et al., 2001, and Yang et al., 2001), and VAPB, short for vesicle-associated membrane protein (VAMP)-associated membrane protein B. Mutations in the endosomal protein CHMP2B cause FTD (Skibinski et al., 2005). Defects in membrane-related proteins such as Rabs and the molecular motors that transport vesicles have also been linked to hereditary spastic paraplegia, spinocerebellar ataxia, and the peripheral neuropathy Charcot-Marie-Tooth disease (reviewed in Gissen and Maher, 2007). Some evidence suggests that amyloid precursor protein participates in vesicle transport (see ARF related news story and Suzuki et al., 2006), as does AlzGene Hit Number 2, Bin1. There are hints that another DENN, DENN/MAPK activating death domain, influences neuronal sensitivity to amyloid-β (see ARF related news story on Del Villar et al., 2004; Mo et al., 2012). “You cannot dismiss that kind of concentration of genetic information in one system,” Nixon said. Medicines that affect Rabs, he suggested, deserve some attention in these sorts of conditions.

On the other hand, the prevailing theory of C9ORF72 pathology suggests the function of the protein may not matter much. The disease-linked expansion appears in an intron, so it should not alter the structure of the protein product. Many scientists believe that the extended mRNA itself, which forms inclusions, causes problems in affected cells. Supporting this idea, researchers found that dissolving this RNA normalizes gene expression in cells from people with C9ORF72-based ALS (see ARF related news story).

Even if C9ORF72’s real contribution to ALS stems from its mRNA, researchers will need to look out for negative effects from any treatment that curtails that mRNA and, thus, the protein. “In all cases where we find genes for neurologic disease, I think it is going to be key to understanding the function of the gene,” commented John Hardy of University College London in an e-mail to Alzforum. “These papers are the first pointers to the functions of C9ORF72, or DENNL72, as we may call it now,” Hardy wrote.

Zhang D, Iyer LM, He F, Aravind L. Discovery of novel DENN proteins: Implications for the evolution of eukaryotic intracellular membrane structures and human disease. Front Genet. 2012;3:283. Abstract

Levine TP, Daniels RD, Gatta AT, Wong LH, Hayes MJ. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics. 2013 Jan 16. Abstract

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