In most cases of amyotrophic lateral sclerosis and frontotemporal dementia, the normally nuclear TAR DNA-binding protein 43 (TDP-43) accumulates in the cytoplasm, clumped into stress granules and inclusions of affected cells. While the presence of this cytoplasmic TDP-43 correlates with neurodegeneration, scientists don’t know what makes it toxic. In the February 1 Human Molecular Genetics, researchers led by Marcello Ceci at Tuscia University, Viterbo, Italy, propose that excess TDP-43 harms cells by suppressing global protein synthesis. In cultured cells, overexpressed cytoplasmic TDP-43 bound to ribosomes and squelched translation by half, they found. Meanwhile, inclusions appeared in some cells. When TDP-43 was kept away from ribosomes, however, protein synthesis bounced back to control levels, and fewer inclusions formed. The findings hint that ribosomal TDP-43 may play a role in disease, the authors suggest.

“This interesting study likely reveals a novel function of TDP-43 in the cytoplasm,” Xinglong Wang at Case Western Reserve University, Cleveland, wrote to Alzforum. He was not involved in the work. Wang suggested that future studies look at the effect of disease-causing TDP-43 mutations on protein synthesis.

TDP-43 in the nucleus regulates RNA splicing, stability, and trafficking. The protein’s function in the cytoplasm is less clear, though it has been found to bind various proteins involved in translation (see Freibaum et al., 2010). In addition, recent studies have reported that cytoplasmic TDP-43 can shut down translation of specific proteins (see Coyne et al., 2014Majumder et al., 2016). No one had shown a role in global protein synthesis, however.

To study how TDP-43 relates to the translation machinery, first author Arianna Russo immunostained mouse embryonic hippocampal cells for TDP-43 and a ribosomal scaffolding protein called receptor activated C kinase 1 (RACK1). In neurites and cell bodies, TDP-43 frequently appeared next to RACK1, suggesting that it formed part of the ribosomal translation machinery. The authors then used neuroblastoma cells to express a mutant version of RACK1 that lacked the ribosomal binding site but still bound TDP-43. In cells that expressed this mutant RACK1, the amount of TDP-43 bound to ribosomes dropped by about two-thirds. The data suggest that TDP-43 may interact with the translational machinery through RACK1, the authors noted.

Would this change in a disease state? To mimic the effects of ALS in the neuroblastoma cells, the scientists overexpressed a mutant TDP-43 that lacks the nuclear localization signal and accumulates in cytoplasm. Global protein synthesis dropped by nearly half, and about 20 percent more cells died than in control cultures. Overexpressing wild-type RACK1 in these cultures rescued protein synthesis and cell viability to control levels, perhaps by boosting ribosomal function to compensate for increased TDP-43. Intriguingly, overexpressing mutant RACK1 also restored translation and viability, but had an additional benefit in that it halved the number of inclusions containing TDP-43. It may be that ribosomal TDP-43 seeds inclusions, hence keeping the protein away from the translation machinery prevents this, the authors speculated.

Does any of this relate to disease? The authors examined motor neurons in spinal cord sections from ALS patients and controls. In patients, they found RACK1 present in about half of the TDP-43 inclusions, suggesting this ribosomal protein could contribute to aggregation. They did not examine cases with mutant TDP-43.

Paul Taylor at St. Jude Children’s Research Hospital, Memphis, Tennessee, noted that the findings fit nicely with the new hypothesis that alterations in assemblies of RNA-binding proteins may drive ALS pathogenesis (see Oct 2015 webinarOct 2016 news; Taylor et al., 2016). Low-complexity sequence domains in these proteins allow them to associate together and affect RNA metabolism through fluid, dynamic mechanisms, he noted. Disease-causing mutations in TDP-43 and other RNA-binding proteins, on the other hand, have the effect of congealing these assemblies, stifling translation and triggering fibril formation (see Sep 2015 newsOct 2015 newsSep 2016 news). “It naturally follows that we will find modifiers of toxicity among the constituents of these higher-order assemblies, particularly among the direct interactome of TDP-43,” Taylor wrote to Alzforum (see full comment below). The authors could not be reached for comment.—Madolyn Bowman Rogers

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  1. Many if not all aspects of RNA metabolism take place in the setting of higher order, dynamic assemblies that arise through phase transitions. In the past year we have learned that low-complexity sequence domains (LCDs) in RNA-binding proteins (such as TDP-43, hnRNPA1 and FUS) participate in building these assemblies (Molliex et al., 2015; Lin et al., 2015; Patel et al., 2015). Indeed, this is likely essential to the influence exerted by RNA-binding proteins on RNA utilization and metabolism. The function of these LCDs is altered by disease-causing mutations. In the best documented cases, the mutations reduce the dynamism of the assemblies and promote fibrillization (Molliex et al., 2015; Patel et al, 2015; Murakami et al., 2015; Conicella et al., 2016). Importantly, an adverse consequence of poly-dipeptides produced from mutant C9ORF72 is to interact with LCDs and reduce dynamism, resulting in the same consequence as mutations in the LCDs themselves (Lee et al., 2016). These discoveries are at the core of the hypothesis that a primary driver of ALS is altered dynamics of higher-order assemblies composed of RNA-binding proteins such as TDP-43, hnRNPA1 and FUS. Our proposed mechanism is that the biology that normally takes place within higher-order mRNP assemblies is impaired at multiple levels, including translation, and simultaneously promotes the assembly of stable TDP-43 fibrils. It naturally follows that we will find modifiers of toxicity among the constituents of these higher-order assemblies, particularly among the direct interactome of TDP-43. Thus, this paper reveals further detail that is to be expected by our proposed hypothesis.

    References:

    . Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell. 2015 Sep 24;163(1):123-33. PubMed.

    . Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. Mol Cell. 2015 Oct 15;60(2):208-19. Epub 2015 Sep 24 PubMed.

    . A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell. 2015 Aug 27;162(5):1066-77. PubMed.

    . ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function. Neuron. 2015 Nov 18;88(4):678-90. Epub 2015 Oct 29 PubMed.

    . ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure. 2016 Sep 6;24(9):1537-49. Epub 2016 Aug 18 PubMed.

    . C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell. 2016 Oct 20;167(3):774-788.e17. PubMed.

  2. The article by Marcello Ceci’s group provides an important advance in our understanding of the biology of TDP-43. The mechanisms of aggregation of TDP-43 are clearly important for understanding the pathophysiology of ALS, but such understanding is always informed by a broader understanding of the biology of the protein. In the current manuscript (of which I am a co-author), Marcello’s group has elegantly demonstrated that TDP-43 interacts with the ribosome, and that this interaction is mediated by RACK1. The manuscript demonstrates this interaction using multiple independent approaches, but I think one of the most powerful approaches is Marcello’s use of ribosomal profiling, which fractionates ribosomal proteins and allows investigation of the biochemical association of TDP-43 with the ribosome, which occurs through the interaction with RACK1. An important caveat for this work is the need to examine the results using gene deletion rather than overexpression. Nonetheless, these results are important because many RNA binding proteins are known to regulate translation, but the role played by TDP-43 in translation is poorly understood. The current manuscript clearly demonstrates a strong potential for a generalized role of TDP-43 in translation.

    The field has been captivated by the potential role that RNA granules, particularly (but not exclusively) stress granules, might play in the pathophysiology of ALS (Kedersha and Anderson, 2007Protter and Parker, 2016).  RNA binding proteins, such as TDP-43, control the localization and utilization of RNA through a process of “reversible aggregation”, which can sequester particular RNA binding proteins and associated transcripts and thereby regulate their translation (Ash et al., 2014; Liu-Yesucevitz et al., 2010; Johnson et al., 2009; Kim et al., 2013). The biology of these proteins is exemplified in studies using recombinant proteins, where the biophysical properties of individual proteins are highlighted. This process is termed liquid-liquid phase separation (LLPS) (Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015; Murakami et al., 2015). These studies also show the tendency this LLPS process to go awry and irreversibly form amyloids. This process is thought to contribute to disease, but much work remains to be done before we fully understand the extent to which LLPS contributes to human disease.

    A meaningful translation to disease is to look for clues in the pathological specimens. The current study by Ceci and colleagues makes an important contribution to our understanding of disease by showing that RACK1 associates with ALS pathology in the spinal cord of patients with ALS, and also showing that other stress granule proteins, such as PABPC, are associated with TDP-43 pathology. Thus, there clearly is some pathological connection between all of these proteins. Taken together, this work provides a significant advance in our understanding of the basic biology of TDP-43, and also supports a role for RNA granules/stress granules in the pathophysiology of ALS.

    References:

    . Mammalian stress granules and processing bodies. Methods Enzymol. 2007;431:61-81. PubMed.

    . Principles and Properties of Stress Granules. Trends Cell Biol. 2016 Sep;26(9):668-79. Epub 2016 Jun 9 PubMed.

    . Pathological stress granules in Alzheimer's disease. Brain Res. 2014 Oct 10;1584:52-8. Epub 2014 Aug 7 PubMed.

    . Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One. 2010;5(10):e13250. PubMed.

    . TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity. J Biol Chem. 2009 Jul 24;284(30):20329-39. Epub 2009 May 22 PubMed.

    . Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature. 2013 Mar 28;495(7442):467-73. PubMed.

    . Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. Mol Cell. 2015 Oct 15;60(2):208-19. Epub 2015 Sep 24 PubMed.

    . Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell. 2015 Sep 24;163(1):123-33. PubMed.

    . A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell. 2015 Aug 27;162(5):1066-77. PubMed.

    . ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function. Neuron. 2015 Nov 18;88(4):678-90. Epub 2015 Oct 29 PubMed.

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References

Alzpedia Citations

  1. TDP-43

Webinar Citations

  1. Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?

News Citations

  1. ALS Research ‘Gels’ as Studies Tie Disparate Genetic Factors Together
  2. ALS Protein Said to Liquefy, Then Freeze en Route to Disease
  3. FUS Phase Transitions: Liquids and Gels
  4. Helical Tail Holds Sway Over TDP-43 Packaging

Paper Citations

  1. . Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. J Proteome Res. 2010 Feb 5;9(2):1104-20. PubMed.
  2. . Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis. J Neurosci. 2014 Nov 26;34(48):15962-74. PubMed.
  3. . Co-regulation of mRNA translation by TDP-43 and Fragile X Syndrome protein FMRP. Acta Neuropathol. 2016 Nov;132(5):721-738. Epub 2016 Aug 12 PubMed.
  4. . Decoding ALS: from genes to mechanism. Nature. 2016 Nov 9;539(7628):197-206. PubMed.

Further Reading

Primary Papers

  1. . Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes. Hum Mol Genet. 2017 Apr 15;26(8):1407-1418. PubMed.