A key function of C9ORF72 is to regulate initiation of autophagy, according to a study published June 22 online in the EMBO Journal. The findings suggest that hexanucleotide repeat expansions in the C9ORF72 gene, linked to ALS and frontotemporal dementia (FTD), may contribute to disease through loss-of-function mechanisms that impair autophagy, in addition to previously described gain-of-function toxicity caused by RNA foci and dipeptide repeat proteins (DPRs). The results may also help explain the appearance of pathologic correlates of autophagy dysfunction in patients with C9ALS/FTD.
“Results from this paper suggest there could be both a loss-of-function and gain-of-function in ALS/FTD,” commented Udai Pandey, of Children’s Hospital of Pittsburgh, PA, who was not involved in the study, “and they may interact with each other,” if accumulating DPRs cannot be effectively processed by the protein quality control system.
Reduced autophagy has been implicated in C9ALS/FTD, based on the presence of inclusions positive for ubiquitin and p62, a sign of impaired autophagy (Cooper-Knock, J et al., 2012; Mackenzie et al., 2014). At the same time, patient tissue indicates that the levels of C9ORF72 mRNA and protein are reduced (DeJesus-Hernandez et al., 2011; Gijselink, I et al., 2012) . These observations prompted the investigators to ask whether the two observations were connected. Co-senior authors Andrew Grierson and Kurt De Vos of Sheffield Institute for Translational Neuroscience (SITraN) at the University of Sheffield, UK, with first co-authors Christopher Webster and Emma Smith and colleagues, tested this hypothesis by first reducing C9ORF72 expression using RNA interference in cell culture, and monitoring the initiation of autophagy by visualizing the level of a reporter protein in response to an autophagy trigger, Torin1. Under control conditions, Torin1 treatment led to an increase in the number of autophagosomes, however, when levels of C9ORF72 expression were reduced using small interfering RNAs (siRNAs), this prevented the Torin1-induced increase and hinted at impaired induction of autophagy. This finding was confirmed through additional experiments in cell lines and rat primary cortical neurons, which also demonstrated reduced autophagosome production in response to silencing expression of C9ORF72.
Induction of autophagy requires the formation of a complex between Unc-51-like kinase 1 (ULK1), autophagy-related 13 (ATG13) protein, and FAK family kinase-interacting protein of 200 kDa (FIP200), which is necessary for initiation of autophagy. When the investigators reduced FIP200 with siRNAs, this prevented autophagy induction by C9ORF72 expression, indicating that C9ORF72 acts through the ULK1 complex. Further biochemical analyses, including co-immunoprecipitation, proximity ligation, and in-vitro binding assays, all confirmed the interaction of the C9ORF72 protein directly with ULK1 and other members of the complex. Overexpressing C9ORF72 potentiated translocation of the ULK1 complex to the phagophore to initiate autophagosome formation. C9ORF72 knockdown prevented this process, indicating it is required for complex translocation. Interestingly, knocking down Rab1a, a regulator of ULK1 complex translocation, prevented C9ORF72 from promoting ULK1 translocation to the phagophore. Together, De Vos said, these results show that C9ORF72 regulates the initiation of autophagy, and that by binding to Rab1a, it promotes translocation of the ULK1 complex to phagophore.
The team further showed that reduction in C9ORF72 expression increased p62-positive puncta in cells, reminiscent of pathology in patients, and that reintroducing it reduced the number of puncta. Finally, providing additional evidence for an effect of C9ORF72 mutations on autophagy in patients, the team found reduced baseline levels of autophagy triggered by bafilomycin A1 treatment in induced neurons (iNeurons) derived from two C9ALS/FTD patients as compared to control iNeurons.
Together, these results imply that autophagy is reduced in C9ALS/FTD, but is that likely to contribute to the disease? “It is pretty clear that most people find a reduction in C9ORF72 mRNA in patient tissue,” De Vos said, although the case for reduction in protein isn’t as tight yet. The presence of p62-positive inclusions in patients is also suggestive. “A defect in autophagy could explain that. But we don’t have direct proof of this. When we knock out the protein, we can mimic the pathology, but whether that is the cause in the patients, we don’t know.”
On the other hand, he said, C9ORF72 knock-out mice don’t develop a strong ALS phenotype (see Jun 2015 news): “Not having the protein doesn’t seem to be enough to cause disease.” Perhaps, he said, both a loss of function and gain of function is needed for the full disease.
“If you have an aggregating protein,” such as the DPRs, “in normal circumstances the cell will get rid of it. But if you are deficient in autophagy, you could exacerbate the defect, and get into a negative loop,” in which aggregation reduces autophagy and impaired autophagy exacerbates protein aggregation.
“This study tells us that C9ORF72 operates through the autophagy pathway,” Pandey commented, “and this is important because there are drugs available to modify this pathway. This is the most exciting part of the study.” The most important next step is to try the same experiments using the C9 mutation. “This paper helps us understand the basic biology of the protein–they showed that beautifully—but there is a lot of work left to do.”
Recent results of C9ORF72 knockouts have mainly indicated an immune system regulation, noted Fenghua Hu, of Cornell University (see Mar 2016 news). “It is not clear how this is connected to autophagy-perhaps it is a different function of C9, or it may be that autophagy is regulating inflammation, for instance.”
Whatever the mechanism, defects in autophagy are a common theme in both ALS and FTD, Hu pointed out, with more than half the genes involved in the two diseases involved in the pathway, including TBK1 (see Feb 2015 news), CHMP2B (see Sep 2015 news), UBQLN2, progranulin, P62, OPTN and TMEM106B. “Autophagy and lysosomal dysfunction appear to be central to the two diseases,” she said.
Christopher P Webster, Emma F Smith, Claudia S Bauer, Annekathrin Moller, Guillaume M Hautbergue, Laura Ferraiuolo, Monika A Myszczynska, Adrian Higginbottom, Matthew J Walsh, Alexander J Whitworth, Brian K Kaspar, Kathrin Meyer, Pamela J Shaw, Andrew J Grierson, Kurt J De Vos. The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. EMBO J. 2016 Aug 1;35(15):1656-1676. epub Jun 22. [Pubmed].
Home page image: Webster et al., 2016, under CC-BY-4.0.