An army of geneticists scouted for disease genes in the DNA of people with amyotrophic lateral sclerosis. They found a TANK. TANK-binding kinase 1, to be precise, or TBK1 to its allies. The gene, which mobilizes in autophagy and neuroinflammation, cropped up in one of the largest exome-sequencing projects yet, noted Tim Harris of Biogen Idec in Cambridge, Massachusetts. Harris is the senior author of a paper describing the finding in the February 19 Science Express. He and collaborators scoured the protein-coding DNA of 2,874 ALS and 6,405 control genomes. While Harris estimates the new gene underlies fewer than 2 percent of ALS cases, he said it still offers reason to pursue therapeutic targets in autophagy and neuroinflammation. 

 

Alternate Spellings. Variants in TBK1 occur across the protein, in both ALS cases (red lines) and controls (blue lines). [Image courtesy of Science/AAAS.]

“It is an impressive genetics study and quite convincing,” commented Randal Tibbetts of the University of Wisconsin in Madison, who was not involved in the work.

ALS genes have been coming fast and furious lately. TBK1 is the sixth potential risk gene to be identified in the past two years, following on the heels of TUBA4ACHCHD10matrin 3, TREM2, and ErbB4.

Harris and co-senior authors Richard Myers of the HudsonAlpha Institute for Biotechnology in Huntsville, Alabama, and David Goldstein of Columbia University in New York collected those thousands of DNA samples from collaborators across the globe. A positive control, the known ALS gene SOD1, was significantly associated with the disease in their data set. Joint first authors Elizabeth Cirulli of the Duke University School of Medicine in Durham, North Carolina, and Brittany Lasseigne of the HudsonAlpha Institute discovered a few dozen variants in the TBK1 gene, occurring all across the sequence and in both ALS cases and controls (see image above). TBK1 sequences differed in nearly 1.5 percent of ALS genomes, compared to 0.2 percent of the control samples, and the gene easily passed statistical tests indicating some of those variants are associated with the disease, Harris said. Because the variants are rare, it required a study of this size to find the ALS link, he added.

TBK1 contains an amino-terminal kinase domain, a ubiquitin-like domain in the middle, and a leucine zipper at the carboxyl terminus. With the variants sprinkled throughout the coding region of the gene, the genetic data offer no easy hint as to what the problem might be in people carrying those mutations. Goldstein said he and others are already experimenting with the mutant proteins to determine which domains might be affected in ALS.

TBK1’s functions offer some clues as to how the protein might function in motor neurons. In fact, it participates in two processes already known to be altered in ALS. A recurrent theme in this and other neurodegenerative diseases has been defects in the disposal of worn-out or aggregated proteins (see Jul 2010 newsJul 2014 news). TBK1 promotes autophagy by phosphorylating two other proteins linked to ALS, optineurin and sequestosome 1 (also known as p62) (Morton et al., 2008; Pilli et al., 2012; and Related Papers below). Researchers have found TBK1 and optineurin co-localizing together with protein aggregates in HeLa human cervical cancer cultures (Korac et al., 2013). 

ALS researchers have also been pursuing the role of immune cells in the disease; sometimes they protect neurons and other times they attack them (see Oct 2008 news; Sep 2009 news; May 2011 Webinar). TBK1 plays a complex role in immunity. It inhibits kinases that activate the NF-κB complex, thus checking production of pro-inflammatory cytokines such as interferon-β. However, it also activates the transcription factor IRF-3, which promotes interferon transcription (see image below; Clark et al., 2011; Fitzgerald et al., 2003; Liu et al., 2015; and Related Papers). Optineurin and sequestosome 1 also inhibit NF-κB-based inflammation (Zhu et al., 2007; Zhang et al., 2014). “TBK1 may play an important role in neuroinflammatory response,” commented Hideshi Kawakami of Hiroshima University in Japan, who discovered that optineurin was an ALS gene (see May 2010 news). Kawakami played no role in the current work.

While TBK1 was the only definite find in the screen, the authors detected a handful of other genes that looked more tentatively like they might be associated with ALS. Of these, the highest scorer on statistical tests was NIMA-related kinase 1 (NEK1), which sat right near the line for  significance. NEK1 participates in cell-cycle regulation, a process not previously linked to ALS. Indeed, most neurons are post-mitotic, meaning they have long halted their cell division processes. “NEK1 mystifies me,” said Goldstein. On one hand, some in vitro research supports a role for this gene in ALS. Study collaborator Wade Harper of the Dana-Farber/Harvard Cancer Center in Boston found that NEK1 co-immunoprecipitated from human kidney cells with two ALS-linked proteins, Alsin and VAPB. On the other hand, NEK1 variants are common among the general population. If those variants do confer risk for ALS, they must do so in only some carriers, said Goldstein.

TANK in a Dish.

Motor neurons derived from the iPS cells of a person with a TBK1 mutation may help researchers learn more about ALS. [Image courtesy of the laboratory of Tom Maniatis, Columbia University, New York.]

That Harris and colleagues analyzed nearly 3,000 exomes and found only rare ALS mutations suggests that there are no more common variants to find, at least no simple ones, commented Stuart Pickering-Brown of the University of Manchester in the United Kingdom, who was not involved with the work. Pickering-Brown pointed out that this kind of study would not detect expansions, such as the hexanucleotide repeats in the C9ORF72 gene (see full comment below). Harris plans to continue sequencing more samples, probably entire genomes, he told Alzforum. Goldstein said he is working on a TBK1 mutant mouse, and others are developing a stem cell line from a person with one of the mutations (see image above left).

The TBK1 finding raises lots of questions for ALS researchers to tackle, commented Tibbetts. How do mutations affect its kinase function? Does TBK1 turn up in aggregates in the spinal cord of people with the disease? How about TBK1 mutations in other neurodegenerative diseases, such as frontotemporal dementia? “It is a lot of food for thought,” Tibbetts said.—Amber Dance

Comments

Make a Comment

Comments on News and Primary Papers

  1. This paper tells us there are probably no unidentified common genes for ALS, because the frequency of mutations found in this study are generally less that 1 percent. However, this does not exclude the possibility there is a common gene with a mutation like the C9orf72 expansion, because a variant such as this would not be picked up by this type of analysis.

    I think this paper also tells us that to get the most out of exome sequencing we have to really analyze large numbers of exomes.  We are at a similar stage to where we were with genome-wide association studies 10 years ago.  It's only when large numbers of cases were analyzed that the real genes came through.

    There are many reports in the literature of putative ALS genes.  I think this study is very useful in helping to work out what is and what is not a true ALS gene.

     

    View all comments by Stuart Pickering-Brown
  2. This is a very important and comprehensive study in which exome sequencing of 2,874 ALS patients and 6,405 controls has been used to identify genes contributing to ALS predisposition using a population of predominantly sporadic ALS cases. In particular, the major gene revealed from this study, but not evident in any earlier studies using exome sequencing or GWAS, was TBK1, a protein involved in autophagy and directly responsible for the phosphorylation of optineurin and p62/sequestosome, already identified as familial ALS (FALS) genes.

    The size of the multicenter cohort and the analytical procedure used (gene-based collapsing analysis), where DNA variants satisfying defined criteria qualified for inclusion in a gene-based analysis, significantly contributed to the success of the study. After the initial discovery phase, 51 genes were selected for a replication study in a further 1,318 cases and 2,371 controls using the same analytical approach, and when both studies were combined they yielded a high level of significance for the association of DNA variants with disease and an overall prevalence of DNA variants in TBK1 of 1.1 percent in cases and 0.2  in controls.

    It was also of great interest to see that using the same methodology to study the association between clinical features and DNA variants in known genes, including TBK1, only one gene reached statistical significance and that was D-amino acid oxidase (DAO), an enzyme that degrades D-serine, an obligate co-agonist at the N-methyl-D-aspartate subtype of glutamate receptor. This is highly relevant in the light of accumulating data that expression of DAO mutations in motor neuron cell lines or primary motor neuron cultures leads to ubiquitinated protein aggregate formation, increased autophagy, and apoptotic cell death, key processes occurring universally in ALS pathogenesis.

    Overall, this study has clearly demonstrated the value of exome sequencing for defining genes that underlie predisposition to ALS, most importantly relevant to sporadic cases of ALS, but with strong implications for familial cases as well. This opens up the field to a greater understanding of ALS predisposition and pathogenesis.

    Please note that exome sequencing data has been contributed to this study from our Imperial College FALS cohort, through the FALS sequencing Consortium.

    View all comments by J. de Belleroche
  3. I think this is a really exciting paper. A few people from our group were involved (Jan Veldink and Leonard van den Berg), so I was already aware of the findings. In ALS there is a discussion about where the missing heritability is and it has been suggested that it could be in rare variants. Therefore, it has been proposed that large whole-exome or whole-genome sequencing studies could be the way forward. This paper seems to confirm that view. It is reassuring that they find SOD1 and TARDBP as major hits (positive controls).

    The statistics for the TBK1 gene are convincing and it is even more intriguing that it points toward previously implicated pathways and genes. The fact that most aggregates that are found in the motor neurons of ALS patients stain positive for OPTN and SQSTM1 suggests that these genes (or their pathways) are highly relevant to ALS, despite the fact that mutations in these genes are rare. Similarly, the frequency of mutations in TBK1 is low, but the fact that the gene again implicates autophagy and inflammation is exciting. This suggests that these are pathways on which we should be focusing. Larger and collaborative next generation sequencing are underway and hopefully these will reveal more pieces of the ALS puzzle.

    View all comments by Michael van Es

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Mutant SOD1 Inflames If Not Quenched by Autophagy
  2. Can Autophagy Protect ALS Cell Models from Mutant TDP-43?
  3. Microglia in ALS: Helpful, Harmful, or Neutral?
  4. ALS: T Cells Step Up
  5. Optineurin Mutations Cause ALS, If Not Glaucoma

Webinar Citations

  1. Grasping the Shadow Force: Immune Cells in ALS

Paper Citations

  1. . Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. FEBS Lett. 2008 Mar 19;582(6):997-1002. Epub 2008 Feb 26 PubMed.
  2. . TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity. 2012 Aug 24;37(2):223-34. PubMed.
  3. . Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates. J Cell Sci. 2012 Nov 23; PubMed.
  4. . Novel cross-talk within the IKK family controls innate immunity. Biochem J. 2011 Feb 15;434(1):93-104. PubMed.
  5. . IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 2003 May;4(5):491-6. PubMed.
  6. . Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science. 2015 Jan 29; PubMed.
  7. . Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP. Curr Biol. 2007 Aug 21;17(16):1438-43. PubMed.
  8. . RNA-Seq and ChIP-Seq Reveal SQSTM1/p62 as a Key Mediator of JunB Suppression of NF-κB-Dependent Inflammation. J Invest Dermatol. 2014 Dec 16; PubMed.

External Citations

  1. TANK-binding kinase 1
  2. TUBA4A
  3. CHCHD10
  4. matrin 3
  5. TREM2
  6. ErbB4
  7. SOD1
  8. optineurin 
  9. sequestosome 1
  10. NIMA-related kinase 1
  11. Alsin 
  12. VAPB
  13. C9ORF72 

Further Reading

Papers

  1. . Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science. 2011 Jul 8;333(6039):228-33. Epub 2011 May 26 PubMed.
  2. . Polyubiquitin binding to optineurin is required for optimal activation of TANK-binding kinase 1 and production of interferon β. J Biol Chem. 2011 Oct 14;286(41):35663-74. Epub 2011 Aug 23 PubMed.
  3. . Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003 May 16;300(5622):1148-51. Epub 2003 Apr 17 PubMed.
  4. . The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. J Exp Med. 2004 Jun 21;199(12):1641-50. PubMed.
  5. . Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol Cell. 1998 Mar;1(4):507-18. PubMed.

Primary Papers

  1. . Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science. 2015 Mar 27;347(6229):1436-41. Epub 2015 Feb 19 PubMed.