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Eukaryon

Stick with me, TDP-43!

Grace Dodis
Lake Forest College
Lake Forest, Illinois 60045

Explore how TDP-43, an important RNA binding protein (RBP) of the Purkinje cell, helps the rescue of spinocerebellar ataxia type 31 in this review of “Regulatory role of RNA chaperone TDP-43 for RNA misfolding and repeat-associated translation in SCA31.”

 

Dodis Fig 1

Spinocerebellar ataxia can strike anyone at any age. It is a progressively debilitating, autosomal dominant neurodegenerative disease that, more often than not, leads to death. Roughly 50%-80% of families carry gene mutations that result in this disease (7). There are many types of SCAs, all the way up to type 44 and counting. While each type may have symptoms specific to their pathology, many all-embracing symptoms may include incoordination of movement and speech, unsteady gait, eye-movement limitations, muscle spasticity, as well as cognitive and/or behavioral difficulties (6).

 

Delving further into the molecular basis of SCA, it is known that most prevalent types are caused in part by a repeat expansion in the Purkinje cell’s DNA – specifically, a glutamine-encoding CAG re-peat. These repeated CAG nucleotides translate into an expanded polyglutamine tract in the protein, causing degeneration of the proteins and eventually leading to cell death (6). Some other SCA types have different repeat expansions within the cell as part of their pathogenesis. For example, SCA type eight has a CTG trinucleotide repeat. Each type also has a specific range of proteins or mutations as the focus: a main protein of type one is homer-3, type seven is centered upon the mutant ataxin-7 protein (8), and many more types are being closely studied for answers to the molecular pathways of all SCAs. Taro et al. (5) dove into researching SCA type 31.

 

Before starting their studies, the authors knew that RNA foci2 characterize microsatellite expansion disorders, which are repeat-associated disorders. By focusing their studies on TDP-43 — an RNA binding protein that belongs to the heterogeneous nuclear ribonucleoprotein family and is involved in parts of RNA (3) — and its interactions with UGGAA repeats, they are able to show RNA foci in SCA31. The function of TDP-43 is to act as an RNA chaperone, suppressing the toxicity of expanded UGGAA. An RNA chaperone, seen in the figure, helps RNA through the folding process, keeping it from getting stuck in a misfolded secondary structure. They work by inhibiting the barrier that prevents misfolded RNA from unfolding in order to fix its shape. (4). This function is crucial to inhibiting RNA foci aggregation.

 

Controlling buildup of RNA foci, TDP-43 regulates UGGAA RNA folding. However, Taro et al. did not know this at first, and had yet to identify the specific protein with which this mechanism was controlled. Their first job was to express a human SCA31 repeat tract in Drosophila, or fruit flies, in which they successfully created five lines with approximately 80-100 TGGAA repeats, and one line in which there was spontaneous contraction to 22 TGGAA repeats. After this was done, the authors were able to show that the expression of UG-GAAexp caused toxicity and RNA foci in vivo, dependent upon both gene dosage and repeat length. The SCA31 and control repeats were expressed in the compound eyes of the flies for easier visual assesment of the effects. Using a fluorescence in situ hybridization (FISH) test3, they showed that the controls and the short UGGAA22 had no significant impact on the morphology of the eye, as expected. However, the data showed that UGGAAexp(s) (strongly expressed UGGAAexp) had much more eye morphology distortion, whereas UG-GAAexp(w) (weakly expressed UGGAAexp) caused less damage to the eyes. After highlighting the distortion caused by UGGAAexp, the authors then completed a screen for potential UGGAAexp-binding proteins. This is when they found the protein TDP-43, along with many other RBPs. Although later in their research, the authors confirmed their results with TDP-43 using these other proteins, their main focus at first was the effects of TDP-43 on UGGAA re-peats.

 

To show how TDP-43 acts on UGGAA RNA, the authors needed to prove that the two interact. Again, they used a FISH test where they highlighted the specific location of TDP-43 and UGGAAexp RNA, this time in human Purkinje cells. When the two separate images were merged together, they showed that TDP-43 does bind to UGGAA RNA, because TDP-43 and UGGAAexp RNA in the Purkinje cell were both localized to the nucleus of the cell. The next step taken by the scientists was to prove that TDP-43 and its RNA recognition motifs (RRMs) are responsible for suppressing UGGAAexp-mediated toxicity. The flies expressing UGGAAexp and human wild-type TDP-43 (TDP-43 WT) showed dramatic restoration in pigment and morphology of the eyes. However, when the scientists altered the RRMs on the TDP-43 protein, RNA foci aggregation showed because the protein could no longer find the misfolded RNA and bind to it. Then the authors knocked down the entire TDP-43 protein, again resulting in heightened aggregation. This information supported the idea that TDP-43 functions as an RNA chaperone, because without it, the accumulation of RNA foci was tremendous. TDP-43 functioning as an RNA chaperone was a central discovery in the authors’ research.

 

Now that the importance of TDP-43 has been highlighted, it is possible to relate the research to possible treatments. If a safe and feasible method of injection can be found, inserting more RBPs into a diseased individual may help with the misfolding of RNA so that it does not form RNA foci. Aside from treatments, another related topic for future studies may be the mutations that cause the molecular pathogenesis of other SCA types. Some researchers are focusing studies on finding treatments. For example, one idea to treat SCA types two and three is to reduce polyglutamine by developing drugs that bind to mutated proteins. There are also many centrally acting drugs such as physostigmine, L-5-hydroxytryptophan, and buspirone currently undergoing clinical trials (1). Looking back at Taro et al.’s published work, it is clear that the authors did not put much time into thinking of possible topics for future studies. Other than this one small critique, the scientists did a wonderful job with their research and explanations. Before their work was done, it was assumed that all SCA types had a responsible gene or protein that caused the disease. Now, because of Taro et al., the scientific community knows to look in the nucleus for possible RNA foci as well.

 

References:

 

  1. Bezprozvanny, I., & Klockgether, T. (2009). Therapeutic pro-spects for spinocerebellar ataxia type 2 and 3. Drugs of the Future, 34(12), 10.1358/dof.2009.034.12.1443434. http:// doi.org/10.1358/dof.2009.034.12.1443434

 

  1. Bhardwaj, A., Myers, M. P., Buratti, E., & Baralle, F. E. (2013). Characterizing TDP-43 interaction with its RNA tar-gets. Nucleic Acids Research, 41(9), 5062–5074. http:// doi.org/10.1093/nar/gkt189

 

  1. Buratti, E., & Baralle, F. E. (2001). Characterization and Func-tional Implications of the RNA Binding Properties of Nuclear Factor TDP-43,  a  Novel  Splicing  Regulator  ofCFTRExon

 

  1. Journal of Biological Chemistry,276(39), 36337-36343. doi:10.1074/jbc.m104236200

 

  1. Herschlag, Daniel. (1995). RNA Chaperones and the RNA Fold-ing Problem. The Journal of Biological Chemistry, 270(36), 20,871-20,874. doi:10.1074/jbc.270.36.20871

 

  1. Ishiguro, T., Sato, N., Ueyama, M., Fujikake, N., Sellier, C., Kanegami, A., … Ishikawa, K. (2017). Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31. Neuron, 94(1), 108-124. doi:10.1016/j.neuron.2017.02.046

 

  1. Jayadev, S., & Bird, T. D. (2013). Hereditary ataxias: Over-view. Genetics in Medicine,15(9), 673-683. doi:10.1038/ gim.2013.28

 

  1. Stevanin, G., Bouslam, N., Thobois, S., Azzedine, H., Ravaux, L., Boland, A., … Brice, A. (2003). Spinocerebellar ataxia with sensory neuropathy (SCA25) maps to chromosome 2p. Annals of Neurology,55(1), 97-104. doi:10.1002/ ana.10798

 

  1. Zander, C. & Takahashi, J. (2001). Similarities between spino-cerebellar ataxia type 7 (SCA7) cell models and human brain: Proteins recruited  in  inclusions  and  activation  of  caspase-

 

  1. Human Molecular Genetics,10(22), 2569-2579. doi:10.1093/hmg/10.22.2569

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