See Part 1.
25 November 2011. At RNA-Binding Proteins in Neurological Disease, a satellite of the annual meeting of the Society for Neuroscience, held 10-11 November 2011 in Arlington, Virginia, researchers reported big new discoveries in ALS genetics. Among the presenters who fingered potential new genes, many described a biochemical approach, examining disease-linked aggregates for promising candidates. It may be that a good way to find ALS genes is to take these aggregates and ask what is in them, said Robert Brown of the University of Massachusetts Medical School in Worcester. Gene variants that associate with ALS may illuminate the cause of related diseases, Brown added. Many labs are now beginning to tease apart aggregates to look for mutants that might be risk factors for disease.
Brown’s ideas bore out as the satellite meeting progressed and speakers rolled out a whole host of new proteins involved in these diseases (see also Part 1). Ian Mackenzie of the University of British Columbia in Vancouver, Canada, discussed one such related disease—frontotemporal lobar dementia. Mackenzie described an FTLD pathological aggregate that included new proteins related to ALS-linked Fused-in-Sarcoma (FUS): FUS’s close relatives TAF15 (TAT box binding protein (TBP)-associated factor 15) and EWS (Ewing sarcoma protein). Together with first author Manuela Neumann of the University Hospital Zurich, Switzerland, Mackenzie and colleagues reported the work in the September issue of the journal Brain. In another publication in the November Archives of Neurology, first author Faisal Fecto and senior author Teepu Siddique, of the Northwestern University Feinberg School of Medicine in Chicago, Illinois, looked beyond RNA binding proteins and reported that p62, a ubiquitin-binding protein involved in proteasomal and autophagic protein degradation, is mutated in some ALS cases. Paul Taylor of St. Jude’s Children’s Research Hospital in Memphis, Tennessee, who co-chaired the meeting with Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, reported how a protein-based approach led him to new genes involved in human inclusion body myopathy with Paget’s frontotemporal dementia (IBMPFD), a disease that shares genetic risk factors, and sometimes symptoms, with ALS. Nicholas Seyfried of the Emory School of Medicine in Atlanta, Georgia, reported on a newly ALS-linked protein, PTB-associated splicing factor (PSF), which could provide fodder for a future gene hunt.
Mackenzie and Neumann theorized that if FUS appears in FTLD inclusions, then TAF15 and EWS might do the same. FUS, EWS, and TAF15 make up the FET family that shares homology and roles in RNA transcription, processing, and transport (Law et al., 2006; Tan and Manley, 2009; Kovar, 2011). And indeed, Mackenzie reported that cytoplasmic TAF15 inclusions appeared in postmortem tissue taken from both brain and spinal cord of patients who had FTLD with FUS pathology. EWS occasionally appeared in aggregates as well. TAF15 and EWS were absent from inclusions in the brain or spinal cord of ALS cases, Mackenzie said. All three proteins tended to be insoluble in FTLD-FUS cases, compared to healthy tissue, although for EWS, the difference was not statistically significant. TAF15 and EWS represent good candidates for FTLD genes, Mackenzie suggested. TAF15 has already been shown to be mutated in ALS (Ticozzi et al., 2011).
Further, Mackenzie proposed that defects in nuclear import of FET proteins could force them into the cytoplasm, causing disease. This protein family shares a proline-tyrosine (PY)-rich nuclear localization signal that is recognized by transportin, which ferries proteins into the nucleus. Mutations in the FUS PY region cause ALS. When the researchers transfected HeLa cervical cancer cells with a transportin inhibitor called M9M, TAF15 and EWS were forced into the cytoplasm, as happens for FUS (see ARF related news story). Barred from the nucleus, the TAF15 and EWS redistributed into stress granules, mimicking disease.
For their part, Fecto and Siddique also looked to previously implicated genes and proteins to form their hypothesis about p62. This protein is encoded by the gene sequestosome 1 (SQSTM1) and appears in inclusions in Alzheimer’s disease, Parkinson’s disease, ALS, and other neurodegenerative conditions (Kuusisto et al., 2001; Zatloukal et al., 2002; Gal et al., 2007; Mizuno et al., 2006). SQSTM1 is mutated in some cases of Paget’s disease of bone (Laurin et al., 2002). Given that so many inclusion body proteins—such as TAR DNA binding protein 43 (TDP-43) and FUS—have turned out to be genetically implicated in disease, p62 was a natural suspect, Fecto told ARF.
The researchers sequenced the gene from 546 people with ALS and 738 control subjects. They discovered 10 novel mutations among the ALS cases, including nine missense mutations and one deletion. The finding shows p62 is no mere innocent bystander that gets swept up into aggregates, but a real player in the disease, said Fecto, who speculated that the mutations might alter ubiquitin binding or make p62 more aggregation prone. Overall, genetic studies seem to be converging on two pathways, Fecto said. They are the RNA binding proteins and the protein degradation system. Perhaps not coincidentally, these pathways are intertwined, he noted. For example, p62 and ubiquilin 1 regulate TDP-43 stability and aggregation (Brady et al., 2011; Kim et al., 2009).
Both ALS and IBMPFD share mutations in p62. Another gene the two diseases have in common is valosin-containing protein (VCP; see ARF related news story on Johnson et al., 2010). VCP participates in a wide variety of cellular processes, including transcription and autophagy. It separates ubiquitinated proteins from complexes so they can be destroyed. At the meeting, Taylor presented new mutations in two RNA-binding proteins—the details are not yet available—that account for IBMPFD in two families. Like TDP-43 and FUS in ALS neurons, these mutant proteins exit the nucleus for the sarcoplasm of muscle cells.
Researchers at the University of Pennsylvania in Philadelphia have predicted that RNA-binding proteins with prion-like domains are also involved in ALS (see Part 1). Taylor’s two new IBMPFD proteins fit this profile. James Shorter of UPenn, who also spoke at the meeting, collaborated with Taylor to discover, via a computer algorithm, that the new proteins contain prion-like sequences. His lab also found that the proteins aggregate in vitro, especially when they contain the disease-linked mutations. I think more and more of these RNA-binding proteins with prion-like domains will be revealed as crucial in neurodegenerative disease. Paul’s dataare extremely compelling, especially with the support from our prion-domain algorithm and pure protein studies, Shorter wrote in an e-mail to ARF.
Finally, Seyfried presented PSF, which might be another potential candidate for a gene in ALS or related conditions. He reported that there is more PSF in insoluble fractions of frontal cortex from people who died of FTLD than from neurologically healthy controls. That protein functions in RNA transcription, splicing, and transport, Seyfried said. He found that PSF remained mostly nuclear, even in FTLD tissue, but occasionally filled the cytoplasm of oligodendrocytes. His team is now considering what role the protein might have in that cell type.
Fecto F, Yan J, Vemula SP, Liu E, Yang Y, Chen W, Zheong JG, Shi Y, Siddique N, Arrat H, Donkervoort S, Ajroud-Driss S, Sufit RL, Heller SL, Deng HX, Siddique T. SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch Neurol. 2011 Nov. 68(11):1440-1446. Abstract
Neumann M, Bentmann E, Dormann D, Jawaid A, DeJesus-Hernandez M, Ansorge O, Roeber S, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ang LC, Bilobao J, Rademakers R, Haass C, Mackenzie IR. FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations. Brain. 2011 Sep. 134(Pt 9):2595-2609. Epub 2011 Aug. 19. Abstract
This is Part 2 of a two-part series. See Part 1.
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