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

Abstract

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.

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

“…In neurodegenerative disease, increasing evidence has shown that cytoplasmic TDP-43 localizes to stress granules (SGs) to form protein aggregates. [27][28][29] SGs are cytoplasmic aggregates of stalled translation initiation complex. 30 Here we used a construct expressing EGFP-tagged G3BP1, a core component of SGs, to monitor the formation of SGs.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…Within the nucleus, TDP43 has essential roles in RNA transcription, splicing, and stability [13,14,[47][48][49][50], and transports select mRNA to localized sites of translation within the cytoplasm [20,51,52]. In addition to its function in RNA trafficking, cytoplasmic TDP43 also helps regulate RNA translation and homeostasis by sequestering transcripts in RNA granules [53][54][55][56]. Thus, the loss of TDP43 function stemming from inadequate nuclear protein, a gain of function arising from the accumulation of cytoplasmic TDP43, or both, might result in RNA misprocessing and subsequent neurodegeneration.…”

“…Prion-like domains are also found in mammalian proteins whose function requires reversible selfaggregation, including proteins that are involved in the formation of stress granules [89], RNA-dense cytoplasmic particles that sequester nonessential mRNAs and prevent their translation when cells are under duress [90]. TDP43 and FUS are incorporated into cytoplasmic stress granules upon exposure to oxidative, heat, or endoplasmic reticulum stress, but return to their normal (predominantly nuclear) localization when the stress is removed [41,53,55,56,91,92]. The reversible aggregation of TDP43 and FUS, and the resulting sequestration of any bound RNAs, may be essential to their physiological functions [87]; however, disrupting the precarious balance between aggregate formation and dissolution may also contribute to the irreversible development of inclusions and the pathogenic deposition of RNA binding proteins in ALS [93] (Fig.…”

“…Prolonged or repeated insults over time may erode the ability of the cell to dissociate stress granules efficiently, altering the kinetics of the reaction and increasing stress granule stability [94,95]. ALS-linked mutations in TARDBP and FUS disrupt stress granule dynamics, favoring larger and less soluble granules [53,56]. In addition, valosin-containing protein (VCP) deficiency or chemical inhibition of VCP blocks stress granule disassembly in human cells [96].…”

“…Deletion of the RNA-binding domains in TDP43 and FUS both prevents their inclusion in stress granules and reduces their toxicity [34,[39][40][41]. Moreover, components of stress granules have been detected in TDP43-rich cytoplasmic deposits in spinal neurons of patients with ALS [56,99,101], but are less common in cortical neurons [102]. These results may be explained by the preferential accumulation of fulllength TDP43 in spinal neurons of patients with ALS, in contrast to the deposition of carboxy-terminal fragments of TDP43 in cortical neurons [103].…”