Abstract
BackgroundTrans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades.ResultsThe protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization.ConclusionThese findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies.
Highlights
Trans-activation Response DNA-binding Protein-43 (TDP-43) Protein Trans-activation response DNA-binding protein (TDP-43) is highly conserved among species and ubiquitously expressed in humans [1]
SDS-PAGE analysis of this material with Coomassie brilliant blue staining demonstrated soluble, full-length, untagged TDP-43 can be isolated by taking advantage of its intrinsic propensity to aggregate, as shown in Figure 2B, and this material is over 95% pure based on densitometry calculations of the gels
The novel techniques described here for untagged TDP-43 purification and the unprecedented demonstration that Casein Kinase II (CKII) can drive TDP-43 filament assembly that outpaces its generic aggregation indicates that free TDP-43 deposition may be controlled in a disease specific manner resulting from the confluence of multiple independent mechanisms
Summary
TDP-43 Protein Trans-activation response DNA-binding protein (TDP-43) is highly conserved among species and ubiquitously expressed in humans [1]. TDP-43 contains two RNA recognition motifs that are involved in its function in RNA stabilization and processing, while the carboxy-terminus is believed to drive a toxic gain of function, as the majority of ALS and FTLD-TDP-linked mutations are found in this glycine-rich region of the protein [3,4,5,6]. Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer’s Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades
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