Unleashed From the Nucleus, TDP-43 Wreaks Havoc in Mitochondria

In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the usually nuclear TDP-43 wanders out into the cytoplasm, causing neurons to wither and die. What makes cytoplasmic TDP-43 so toxic? A report in the June 27 Nature Medicine suggests that the protein invades mitochondria, where it binds the organelle’s mRNAs, preventing their translation and disrupting a protein complex that produces energy. Scientists led by Xinglong Wang at Case Western Reserve University, Cleveland, report that keeping TDP-43 out of mitochondria prevents this toxicity and rescues motor deficits in mice expressing mutant versions of the protein. “Mitochondria might be major accumulation sites of TDP-43 in the cytoplasm of dying neurons,” wrote Wang to Alzforum. “Targeting mitochondrial TDP-43 could be a novel therapeutic approach for ALS and other TDP-43-linked neurodegenerative diseases.” 

Running Amok. In healthy human motor neurons (top), TDP-43 (red) resides in the nucleus. In ALS (bottom), it co-localizes with the mitochondrial marker TOM20 (green). [Courtesy of Xinglong Wang, Nature Medicine.]

TDP-43 contains two domains that bind RNA. In the nucleus, these help the protein regulate RNA splicing, transportation, and translation (Buratti and Baralle, 2012). What effect might those domains have when the protein ends up in the cytoplasm in ALS, and in other neurodegenerative diseases such as frontotemporal dementia and Alzheimer’s (Neumann et al., 2006)? 

To find out, co-first authors Wenzhang Wang, Luwen Wang, and Junjie Lu, who is at Brigham & Women’s Hospital in Boston, wanted to know where cytoplasmic TDP-43 concentrated. They examined spinal cord neurons from patients with ALS and frontal cortex neurons from patients with FTD. In those cells, cytoplasmic TDP-43 co-localized with mitochondrial markers (see image above). They used fractionation experiments and immunoelectron microscopy to see where exactly it settled in mitochondria—which sport outer and inner membranes surrounding a gel-like matrix. Both techniques revealed TDP-43 on the matrix side of the inner mitochondrial membrane, where the mitochondrial RNAs are found. Mitochondria from patients with ALS or FTD contained more TDP-43 than those from age-matched controls. To see if mutations affected localization to mitochondria, the researchers overexpressed wild-type or TDP-43 with the G298S, A315T, or A382T mutations associated with ALS in human embryonic kidney cells. Cells harboring the mutants accumulated more of the protein in mitochondria than did those expressing wild-type protein. Interestingly, the minuscule amount of TDP-43 that was found in the cytoplasm of control cells also ended up in mitochondria.

Once there, what did TDP-43 do? Immunoprecipitation experiments suggested that it bound two mRNAs in particular—those that code for ND3 and ND6, subunits of complex I, the first of the respiratory chain complexes that establish the ATP-producing proton gradient across the inner mitochondrial membrane. Binding those mRNAs reduced their translation and diminished complex I function. Overexpressing wild-type TDP-43 in HEK293 cells lowered the mitochondrial membrane potential, the rate of oxygen consumption, and ATP levels, and caused mitochondria to break apart. Mutant TDP-43 proved even more disruptive. However, if the researchers deleted a TDP-43 motif that seemed to act as a mitochondrial localization signal, the cells appeared normal.

To confirm that TDP-43 messed with mitochondria in vivo, Wang and colleagues injected a virus encoding either wild-type or mutant TDP-43 into the cerebral cortices of normal lab mice. Mitochondria malfunctioned, broke up, and neurons died—more so with mutant TDP-43. However, by taking out the mitochondrial localization signal from the TDP-43 constructs, the researchers prevented these effects. The researchers also synthesized a small inhibitory peptide called PM1, which competes with TDP-43 for mitochondrial import. Treating TDP-43 A315T mice with PM1 prevented the loss of neurons and synapses in the cortex and spinal cord, averted astrogliosis and TDP-43 inclusions, and restored normal gait and motor behavior. Gastrointestinal problems appear to bring on some of the motor deficits and shorten survival time in this mouse model. However, Wang pointed out that the loss of skeletal muscle mass and motor/cortical neurons is not likely related to GI distress. He also said that his group has another study in review, reporting a protective effect of PM1 in another TDP-43 transgenic mouse without any GI problems. Robert Baloh, Cedars-Sinai Medical Center in Los Angeles, whose lab developed the TDP-43 A315T mouse model, agreed that while behavioral findings in these mice are hard to interpret, the histological abnormalities seen in neurons, and their rescue by PM1, are quite convincing.

Together, the results suggest that in the cytoplasm, TDP-43 becomes toxic because it inappropriately binds mitochondrial mRNA. The trace amount of mitochondrial TDP-43 in healthy cells hints that this is a normal function of TDP-43 that goes overboard in disease, Wang wrote. He is now investigating how the protein functions in normal neurons and why TDP-43 accumulates in mitochondria in ALS.

“Overall, the data is very clean and robust; they have done a beautiful job of looking at this from various angles,” said Giovanni Manfredi, Weill Cornell Medical College, New York. “The idea that TDP-43 gets into the mitochondrial matrix and causes complex I deficiency through an RNA-binding mechanism is very intriguing and novel.” He noted that these authors and others have found TDP-43 on the outside of mitochondria, where it seems to disrupt interactions with the ER, but said that this is the first paper to show that TDP-43 reaches the core of the organelle (Wang et al., 2013; Stoica et al., 2014). The findings need to be replicated, he said, but seem to imply that mitochondrial effects are central to TDP-43 toxicity and could be a therapeutic target. That inclusions of the protein are found in genetic and sporadic forms of disease hint that this mechanism applies to both, Manfredi added.

“We have always assumed that the link between TDP-43 toxicity and mitochondrial dysfunction was indirect,” said Baloh. While he echoed the need for replication, he said the extensive work in human tissue, cell lines, and multiple mouse models makes a definitive direct connection between the two. “I would say they’ve done about as much as a single lab can do to make the point that TDP-43 is actively localized to mitochondria and that that has something to do with toxicity of TDP-43.” The challenge now, he said, is to put this in context of other proposed mechanisms for TDP-43 toxicity and figure out which is most important for disease (Aug 2015 news; Feb 2014 news).—Gwyneth Dickey Zakaib 

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References

Research Models Citations

  1. TARDBP (A315T) (congenic)

News Citations

  1. Does New Role for ALS-Linked Protein Help Explain Neurodegeneration?
  2. Escort Service: A Cytoplasmic Role for TDP-43

Paper Citations

  1. Buratti E, Baralle FE. TDP-43: gumming up neurons through protein-protein and protein-RNA interactions. Trends Biochem Sci. 2012 Jun;37(6):237-47. PubMed.
  2. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. PubMed.
  3. Wang W, Li L, Lin WL, Dickson DW, Petrucelli L, Zhang T, Wang X. The ALS disease-associated mutant TDP-43 impairs mitochondrial dynamics and function in motor neurons. Hum Mol Genet. 2013 Dec 1;22(23):4706-19. Epub 2013 Jul 4 PubMed.
  4. Stoica R, De Vos KJ, Paillusson S, Mueller S, Sancho RM, Lau KF, Vizcay-Barrena G, Lin WL, Xu YF, Lewis J, Dickson DW, Petrucelli L, Mitchell JC, Shaw CE, Miller CC. ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nat Commun. 2014 Jun 3;5:3996. PubMed.

Further Reading

Papers

  1. Onesto E, Colombrita C, Gumina V, Borghi MO, Dusi S, Doretti A, Fagiolari G, Invernizzi F, Moggio M, Tiranti V, Silani V, Ratti A. Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts. Acta Neuropathol Commun. 2016 May 5;4(1):47. PubMed.
  2. Magrané J, Cortez C, Gan WB, Manfredi G. Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models. Hum Mol Genet. 2014 Mar 15;23(6):1413-24. Epub 2013 Oct 23 PubMed.
  3. Sasaguri H, Chew J, Xu YF, Gendron TF, Garrett A, Lee CW, Jansen-West K, Bauer PO, Perkerson EA, Tong J, Stetler C, Zhang YJ. The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo. Brain Res. 2016 Sep 15;1647:57-64. Epub 2016 May 4 PubMed.
  4. Buratti E, Baralle FE. Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease. Front Biosci. 2008 Jan 1;13:867-78. PubMed.
  5. Nonaka T, Suzuki G, Tanaka Y, Kametani F, Hirai S, Okado H, Miyashita T, Saitoe M, Akiyama H, Masai H, Hasegawa M. Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43. J Biol Chem. 2016 Mar 11;291(11):5473-83. Epub 2016 Jan 14 PubMed.
  6. Woerner AC, Frottin F, Hornburg D, Feng LR, Meissner F, Patra M, Tatzelt J, Mann M, Winklhofer KF, Hartl FU, Hipp MS. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA. Science. 2016 Jan 8;351(6269):173-6. Epub 2015 Dec 3 PubMed.

Primary Papers

  1. Wang W, Wang L, Lu J, Siedlak SL, Fujioka H, Liang J, Jiang S, Ma X, Jiang Z, da Rocha EL, Sheng M, Choi H, Lerou PH, Li H, Wang X. The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity. Nat Med. 2016 Aug;22(8):869-78. Epub 2016 Jun 27 PubMed.

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