A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity

Abstract

Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.

Fig. 1.

Yeast TDP-43 proteinopathy model. (A) TDP-43 or TDP-43-GFP expression in yeast cells was detected by immunoblotting with a mouse polyclonal TDP-43 antibody. Phosphoglycerate kinase 1 (PGK1) was used as a loading control. (B) Fluorescence microscopy was used to visualize the subcellular localization of C-terminally GFP-tagged TDP-43 fusion proteins. Cells were stained with DAPI to visualize nuclei. Whereas GFP alone was distributed between the cytoplasm and nucleus (Top), one integrated copy of TDP-43-GFP strongly localized to the nucleus (Middle) with the occasional formation of intranuclear foci. Expressing TDP-43-GFP from a 2-μm (2 μ) high-copy plasmid profoundly altered its localization because the majority of TDP-43 was now found in multiple cytoplasmic foci (Bottom).

Fig. 2.

TDP-43 is toxic to yeast cells. Yeast cells were transformed with a galactose-inducible GFP construct, untagged TDP-43, or a TDP-43-GFP fusion. Serial dilutions of transformants were spotted on glucose- or galactose-containing agar plates, and growth was assessed after 2 days. Whereas the transformants grew equally well on the control glucose plates, TDP-43 expression profoundly affected growth.

Fig. 3.

TDP-43 inclusions are distinct from polyglutamine aggregates. (A–H) Yeast cells expressing CFP-fusions of htt25Q, htt103Q, or TDP-43 were visualized by fluorescence microscopy. htt25Q-CFP was diffusely distributed (A and D), whereas htt103Q-CFP and TDP-43-CFP formed cytoplasmic aggregates in wild-type cells (strain BY4741) (B and C). (D–G) Deletion of Hsp104 (in cells isogenic to the wild-type BY4741 strain) eliminated the aggregation of htt103Q (E) but not that of TDP-43 (F). Similarly, Hsp104 deletion abolished htt103Q toxicity but not TDP-43 toxicity (G). (H) Filter retardation assay demonstrates SDS-insoluble htt103Q inclusions, unable to pass through cellulose acetate membrane, whereas TDP-43 are fully soluble by this assay. (I) SDD-AGE gel electrophoresis was used to compare TDP-43 aggregates to those of the yeast prion determinant Rnq1. Monomeric and high-molecular-mass amyloid-like forms are detected in yeast lysates expressing Rnq1-YFP, whereas only the monomeric form is detected for TDP-43. Protein samples were incubated in the presence of increasing SDS concentrations (0.1%, 1%, 2%) and detected by using immunoblotting with an anti-GFP antibody (cross-reacts with YFP).

Fig. 4.

Dissecting the molecular determinants of TDP-43 aggregation and toxicity. (A) A diagram illustrating the domain structure of TDP-43 along with various truncation constructs used in this study (a–l). (B) Structure/function analysis testing the effects of truncations on TDP-43-GFP localization by fluorescence microscopy and toxicity by spotting assays (C). The C-terminal region is required for aggregation and toxicity (compare constructs a and d), but by itself is not sufficient (construct j). A construct harboring the C-terminal region along with RRM2 recapitulates the complete aggregation and toxicity of full-length TDP-43 (construct g). RRM, RNA recognition motif; C-term, C-terminal region; NLS, nuclear localization signal.