Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein - RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway

Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.—Wang, Y., Meriin, A. B., Zaarur, N., Romanova, N. V., Chernoff, Y. O., Costello, C. E., Sherman, M. Y. Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery