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., 2014; Majumder 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 October 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 September 2015 news; September 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. The authors could not be reached for comment.
Freibaum BD, Chitta RK, High AA, Taylor JP. 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.
Coyne AN, Siddegowda BB, Estes PS, Johannesmeyer J, Kovalik T, Daniel SG, Pearson A, Bowser R, Zarnescu DC. 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.
Majumder P, Chu JF, Chatterjee B, Swamy KB, Shen CJ. Co-regulation of mRNA translation by TDP-43 and Fragile X Syndrome protein FMRP. Acta Neuropathol. 2016 Nov;132(5):721-738. PubMed.
Taylor JP, Brown RH Jr, Cleveland DW. Decoding ALS: from genes to mechanism. Nature 2016 Nov 9;539(7628):197-206. PubMed.
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