22 Apr 2016

Step out of the limelight for a minute, C9ORF72—there’s a new ALS gene in town. Called CCNF, it encodes the cyclin F protein, which is integral to the ubiquitin-proteasome system. As reported April 15 in Nature Communications, carriers of CCNF variants dot the family trees of kindreds with a history of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or both. Scientists led by Ian Blair, Macquarie University, Sydney, Australia, zeroed in on these mutations by performing genetic linkage analysis and exome sequencing in these families. They found CCNF mutations in sporadic ALS, as well. Their frequency in ALS comes close to that of TDP-43 and FUS mutations, but follow-up studies in larger cohorts will be needed to get a better idea of how common they are in FTD, said Blair. The results add weight to the idea that neuronal recycling systems jam up in these diseases, he told Alzforum.

“This interesting study nicely demonstrates the power of combining family-based studies with whole-exome sequencing to quickly identify interesting variants,” wrote Steven Finkbeiner, Gladstone Institute of Neurological Disease, San Francisco, California, to Alzforum. “It’s fascinating that [CCNF] appears to be another example of a genetic mutation that can cause ALS or FTD.” Finkbeiner was not involved in the study.

Mutations in several genes, including SOD1, TARDBP encoding TDP-43, FUS, and C9ORF72 (also see on AlzPedia) explain about two-thirds of familial and 5 percent of sporadic ALS. The remainder carry no known mutations. Up to 15 percent of ALS patients—including those with a C9ORF72 mutation—develop FTD as well. The two diseases can even show up in the same family. Both are marked by clumps of ubiquitinated proteins in the cytoplasm, which include misfolded TDP-43.

A spotty history. Ten members of one family have either ALS (black fill) or FTD (gray). All 10, and four kin who are still normal, have the S621G mutation in CCNF. [Williams et al., 2016.]

In search of new disease-causing mutations, first author Kelly Williams and colleagues analyzed the genomes of members from an extended Australian family in which 10 people had either ALS or FTD but no known disease-associated polymorphisms. Williams found that all the affected people had a variation on chromosome 16 (see image above). By sequencing the exomes in four affected people, the group found a missense variant at position 1,861 in the CCNF gene, also called FBXO1. A guanine took the place of the usual adenosine nucleotide in codon 621 of cyclin F, yielding glycine instead of serine in the protein. More gene sequencing revealed that all afflicted members of the same family carried that S621G mutation. Four relatives, three much younger than and one near the expected age of onset, have the mutations as well. To confirm that variants in that gene cause disease, the researchers searched through genetic information from hundreds of ALS and FTD families from Australia, Europe, North America, and Japan. Among those kindreds, five other protein-altering mutations in CCNF cropped up. Analyzing sporadic cases of ALS uncovered 19 more (seven never documented before), for a total of 25 different variants in cyclin F that associate with disease. 

What does CCNF do? While its name might suggest a role in the cell cycle, cyclin F actually makes up part of the ubiquitin-protease system (UPS), one of several means cells use to dispose of unwanted proteins. Specifically, cyclin F sticks a ubiquitin onto target proteins, flagging them for destruction in the proteasome. By using a florescent reporter designed to be devoured by the proteasome, the group confirmed that the mutation mucks up this system. In motor neuron-like cells carrying mutant CCNF, the reporter built up, causing the cells to glow green. 

Proteasome activity assays indicated the protease complex itself worked just fine, hinting that aberrant ubiquitin tagging or poor transfer of tagged proteins to the proteasome were responsible for the accumulation of the reporter, Blair said. Neuronal cells with the mutation accumulated ubiquitinated RRM2—a target of cyclin F—as well as ubiquitinated TDP-43. Scientists have wondered why ubiquitinated TDP-43 accrues in ALS and some FTD cases. “This [work] points to a mechanism through which TDP-43 could be accumulating,” Blair said.

Blair thinks the mutated protein over-ubiquitinates its targets, perhaps even healthy proteins that are not normally destined for degradation. Then, because the proteasome can’t keep up, these proteins accumulate and aggregate, he proposed. His group is now examining whether CCNF mutations affect a specific cadre of proteins, including TDP-43, or if these mutations have more general impact on protein homeostasis. Other ALS-linked variants—namely UBQLN2, SQSTM1/p62, and VCP—all relate to ubiquitin or the proteasome in some way. “We’re gathering a body of evidence that increasingly points to abnormal proteostasis in ALS and FTD,” Blair said.

Finkbeiner agreed. “The connection to protein homeostasis is a prominent theme in ALS/FTD as well as neurodegenerative disease more generally,” he wrote. Family studies similar to this one should help explain why motor neurons take a hit in some people, causing ALS, and cortical neurons in others, leading to FTD, he added. At the same time, the growing number of ALS genes and rare variants combined with a lack of strong environmental links is prompting a rethink about the nature of “sporadic” ALS, he continued. It may turn out that sporadic forms combine rare genetic variants that impinge on critical pathways required for motor/cortical neuron survival.

Meanwhile, now that these scientists have identified the disease-associated allele in the original family, relatives have the option of finding out their genetic status, and potentially using preimplantation genetic diagnosis to avoid passing on the allele to their children, said Blair (see Jul 2014 series).—Gwyneth Dickey Zakaib

Comments

1. • Wataru Araki
     National Institute of Neuroscience, NCNP
   • Posted: 25 Apr 2016

   Williams et al. revealed the novel ALS/FTD-associated gene CCNF, which encodes cyclin F protein, a component of the SCF E3 ubiquitin-ligase complex. I find this paper particularly intriguing for two reasons. First, the paper clearly indicates that the ubiquitin-proteasome system is intimately involved in the pathological mechanism of ALS/FTD. Second, the frequencies of CCNF mutations are relatively high and comparable to those of TARDBP.

   The study provided evidence that various mutations of CCNF impair ubiquitin-mediated proteasomal degradation, which is suggested to be due to abnormal ubiquitination or transport to the proteasome. Previous studies, including ours, have shown that full-length TDP-43 is primarily degraded via the proteasomal pathway (Araki et al., 2014; Scotter et al., 2014). It remains to be elucidated whether cyclin F-SCF complex is mechanistically involved in the pathological accumulation of TDP-43, and what pathological features are observed in the patients with CCNF mutations.

   References:

   Araki W, Minegishi S, Motoki K, Kume H, Hohjoh H, Araki YM, Tamaoka A. Disease-Associated Mutations of TDP-43 Promote Turnover of the Protein Through the Proteasomal Pathway. Mol Neurobiol. 2014 Jan 30; PubMed.

   Scotter EL, Vance C, Nishimura AL, Lee YB, Chen HJ, Urwin H, Sardone V, Mitchell JC, Rogelj B, Rubinsztein DC, Shaw CE. Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci. 2014 Mar 15;127(Pt 6):1263-78. Epub 2014 Jan 14 PubMed.

   View all comments by Wataru Araki

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References

Alzpedia Citations

1. TDP-43
2. FUS
3. C9ORF72

Series Citations

1. Preimplantation Genetic Diagnosis: A Family Choice 3 Jul 2014

External Citations

1. SOD1
2. TARDBP 
3. C9ORF72 

Further Reading

Papers

1. Nakamura R, Sone J, Atsuta N, Tohnai G, Watanabe H, Yokoi D, Nakatochi M, Watanabe H, Ito M, Senda J, Katsuno M, Tanaka F, Li Y, Izumi Y, Morita M, Taniguchi A, Kano O, Oda M, Kuwabara S, Abe K, Aiba I, Okamoto K, Mizoguchi K, Hasegawa K, Aoki M, Hattori N, Tsuji S, Nakashima K, Kaji R, Sobue G, Japanese Consortium for Amyotrophic Lateral Sclerosis Research (JaCALS). Next-generation sequencing of 28 ALS-related genes in a Japanese ALS cohort. Neurobiol Aging. 2016 Mar;39:219.e1-8. Epub 2015 Dec 7 PubMed.
2. Coady TH, Manley JL. ALS mutations in TLS/FUS disrupt target gene expression. Genes Dev. 2015 Aug 15;29(16):1696-706. Epub 2015 Aug 6 PubMed.
3. Alves CJ, Dariolli R, Jorge FM, Monteiro MR, Maximino JR, Martins RS, Strauss BE, Krieger JE, Callegaro D, Chadi G. Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration. Front Cell Neurosci. 2015;9:289. Epub 2015 Aug 4 PubMed.
4. Sabatelli M, Marangi G, Conte A, Tasca G, Zollino M, Lattante S. New ALS-Related Genes Expand the Spectrum Paradigm of Amyotrophic Lateral Sclerosis. Brain Pathol. 2016 Mar;26(2):266-75. PubMed.

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5. Brain Imaging Gets the Scoop on Eating Disorders in FTD 4 Mar 2016