. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med. 2018 Mar;24(3):313-325. Epub 2018 Feb 5 PubMed.


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  1. I think this is a great paper, and makes a convincing argument that haploinsufficiency of C9orf72 can be detrimental to the function of neurons, in addition to similar findings previously shown in myeloid cells. I think it adds further evidence that the disease cannot be simply looked at from the perspective of one potential toxic mechanism (RNA granules, dipeptide repeats) and rather should be looked at as resulting from a combination of gain- and loss-of-function effects within and across cell types in the nervous system.

    View all comments by Robert Baloh
  2. It has long been known that the hexanucleotide expansions in a non-coding region of the C9ORF72 gene that cause ALS and FTD are accompanied by a reduction in C9ORF72 expression (van Blitterswijk et al., 2015; DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012; Belzil et al., 2013). However, while considerable attention has focused on toxic gain-of function pathogenic mechanisms directly arising from the repeat expansions, the contributions of partial loss of the normal C9ORF72 protein to disease have been difficult to pin down. For example, C9ORF72 knockout mice do not develop ALS or FTD-like neurodegenerative phenotypes (O'Rourke et al., 2016; Burberry et al., 2016; Atanasio et al., 2016; Koppers et al., 2015). However, it has been unclear whether this reflects a limitation of the model system or a genuine lack of contribution of C9ORF72 protein deficiency to disease mechanisms.

    This new paper from Shi et al. builds on the extensive use of human iPSC-derived motor neurons and data derived from them to propose the provocative new idea that endosome and lysosome dysfunction arising from C9ORF72 haploinsufficiency renders neurons more sensitive to glutamate excitotoxicity due to defects in glutamate receptor traffic. Interestingly, they also found that inhibition of PIKFYVE, a lipid kinase of the endolysosomal pathway, protects C9ORF72-deficient neurons from glutamate-induced excitotoxicity.

    These observations refocus attention on the possible disease relevance of C9ORF72 haploinsufficiency and draw attention to the fact that even though recent years have brought many new insights into possible functions for the C9ORF72 protein, there is still considerable uncertainty and a lack of consensus concerning the direct biochemical function or functions of C9ORF72.

    Shi and colleagues present evidence that C9ORF72 localizes to and presumably functions on early endosomes but that late endosomes and lysosomes are abnormal in neurons lacking C9ORF72. These findings parallel the lysosome defects that have previously been observed in selected hematopoietic cell types in C9ORF72 KO mice and a human C9ORF72 KO cell line (O'Rourke et al., 2016; Sullivan et al., 2016; Amick et al., 2016). As early endosomes and lysosomes are connected to one another by membrane traffic along the endocytic pathway, it is reasonable to expect that endosomal functions of C9ORF72 could initiate a cascade of changes that ultimately affects lysosomes.

    As the field currently stands, there is increasing confidence that C9ORF72 has a function that is somehow relevant to the endolysosomal pathway, but there is also considerable confusion about its precise direct function or functions. For example, in contrast to the neuronal early endosome localization for C9ORF72 that was observed by Shi et al., C9ORF72 was found to very specifically and selectively localize to late endosomes and lysosomes in non-neuronal human cells (Amick et al., 2016). Likewise, in contrast to the early endosome/Rab5 focus presented in the new study, C9ORF72 has also been proposed to act as a guanine nucleotide exchange factor for Rab8a and Rab39b (Sellier et al., 2016; Yang et al., 2016), small GTPases of recycling endosomes and the Golgi (Amick and Ferguson, 2017; Ang et al., 2003; Giannandrea et al., 2010). Interactions between C9ORF72 and Rab1a (Webster et al., 2016), a GTPase with well-characterized functions at the endoplasmic reticulum (Tisdale et al., 1992) have also been reported. Beyond Rab-dependent membrane trafficking functions, C9ORF72 localization to the nuclear envelope via interactions with nuclear pore proteins has also been reported (Xiao et al., 2015). Meanwhile, interactions with the ULK1 complex have suggested a role in the regulation of autophagy (Sullivan et al., 2016; Sellier et al., 2016; Webster et al., 2016). 

    It has been exciting to see this flood of new insights. However, there is a requirement for greater integration between cell biological, genetic and biochemical approaches to decipher which of these observations are most physiologically relevant and directly connected to the fundamental function of C9ORF72 and the larger SMCR8- and WDR41-containing protein complex in which it resides. Identifying direct functions for the C9ORF72 protein could also help to identify common vulnerabilities that are shared by neurons carrying mutations in the growing list of endolysosomal genes whose mutations cause ALS-FTD that is accompanied by TDP-43 pathology.

    An additional finding reported by Shi et al. that is certain to stimulate discussion is the observation that inhibiting PIKFYVE, a lipid kinase of the endolysosomal pathway, protects C9ORF72-deficient neurons from glutamate excitotoxicity. As there is a long history of investigation of glutamate excitotoxicity as an ALS disease mechanism, it would be interesting to know how riluzole or similar drugs compare to PIKFYVE inhibition in the protection of C9ORF72 mutant neurons from glutamate.

    The neuroprotective effects of PIKFYVE inhibition are somewhat surprising given that PIKFYVE inhibition interferes with lysosome function by blocking the delivery and maturation of newly made lysosomal hydrolases (Gayle et al., 2017). On the surface, this runs counter to a widespread belief that enhancing lysosome function should be neuroprotective. Likewise, loss-of-function mutations in FIG4, a key binding partner of PIKFYVE, actually cause severe neurodegeneration in mice (Chow et al., 2007) and ALS in humans (Chow et al., 2009). Nonetheless, the PIKFYVE inhibitor known as apilimod was found to be well-tolerated in human clinical trials and is currently under investigation in humans for the treatment of non-Hodgkin lymphoma (Gayle et al., 2017). Therefore, there is precedent for therapeutic efforts to target this pathway in the context of human disease.

    While the idea that PIKFYVE inhibition might have therapeutic benefits through modulation of glutamate receptor trafficking is very intriguing, significant follow-up is required to determine whether the beneficial effects observed by Shi et al. in relatively acute assays in very specific models can translate into significant therapeutic benefits in human disease that would exceed those seen with existing glutamate blockers such as riluzole. A major challenge for addressing this issue is the difficulty in faithfully modeling the full range of human ALS-FTD pathology in either transgenic mouse models or human iPSC-derived neurons. Further progress in this area is required to understand the relative contributions of RNA, dipeptide repeat protein, and C9ORF72 protein haploinsufficiency-based mechanisms to ALS-FTD pathology and the ability of candidate therapies to reverse the most deleterious defects.


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    View all comments by Shawn Ferguson
  3. This study clearly demonstrates that C9ORF72 haploinsufficiency plays a role in disease and that likely all three mechanisms—C9 haploinsufficiency, RNA repeat toxicity, and dipeptide repeat toxicity—contribute to the disease progression. It is interesting that both isoforms of C9ORF72 can rescue motor neuron survival and that inhibiting PI(3,5)P2 synthesis can rescue the phenotype as well. In addition, they show that C9ORF72 is localized to early endosomes and C9 deficiency leads to defects in lysosomal biogenesis. This indicates endolysosomal system could be a novel therapeutic target for C9-ALS.

    View all comments by Fenghua Hu
  4. The paper by Shi et al. indicates the significance of normal function of C9ORF72 and its involvement in the lysosome/autophagosome pathway. This study reports that neurodegeneration in C9ORF72 carriers could be a result of the combined action of gain-of-function and loss-of-function mechanisms. 

    One of the key implications of this study is that increasing C9ORF72 activity in motor neurons could mitigate disease. Notably, our recent study is in agreement with such a possibility (McGoldrick et al., 2018). We reported a mosaic C9ORF72 carrier with upregulated C9ORF72 expression, in which the presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology. Specifically, we investigated a neurologically normal individual at age 90 carrying a 70-repeat C9ORF72 allele in blood that expanded to a pathogenic range in offspring (~1,750 repeats). The 70-repeat allele is not methylated (in contrast to DNA methylation of large expansions leading to transcriptional silencing of C9ORF72 (Xi et al., 2015). Remarkably, RNA foci and DPR burdens were similar or even significantly increased in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology or signs of neurodegeneration. His CNS tissues had different degrees of mosaicism of small (70-repeat) and large (>1,000 repeats) expansions. Surprisingly, in each mosaic tissue, C9ORF72 mRNA levels were significantly increased compared to an ALS-affected daughter (e.g., by 51 percent in cerebellum, 45 percent in frontal cortex, 325 percent in cervical spinal cord, and 106 percent in thoracic spinal cord). Increased C9ORF72 expression correlated with higher levels of the 70-repeat allele (McGoldrick et al., 2018). 

    Of interest, the highest C9ORF72 expression among CNS tissues of unaffected individuals was detected in the cerebellum (Renton et al., 2011) with high abundance of DPRs in C9ORF72 cases but no degeneration (Mackenzie et al., 2015). 

    Small expansions (e.g., 70 repeats) might be considered ‘‘pre-mutations’’ to reflect their propensity to expand in the next generation. Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues (associated with different levels of C9ORF72 expression). Follow-up studies might help explain the high frequency of ALS- or FTD-affected individuals with an expansion but without a familial history (e.g., 21 percent among Finnish ALS subjects).


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    View all comments by Janice Robertson

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  1. Lack of C9ORF72 Protein Renders Neurons More Vulnerable to Degeneration