Neurodegenerative Diseases - Leonard Petrucelli

Cell and Animal Models of TDP-43 Proteinopathies

TDP-43 is a principal component of ubiquitin-positive inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U). TDP-43 pathology is also observed, to varying degrees, in other neurodegenerative disorders, including Alzheimer's disease, hippocampal sclerosis, Lewy body disease, parkinsonism-dementia complex of Guam, corticobasal degeneration, Pick's disease and Perry syndrome. In order to elucidate both the normal functions of TDP-43 as well as how TDP-43 dysfunction causes neuronal death, we have developed cell culture, Caenorhabditis elegans (C. elegans) and mouse models of TDP-43 proteinopathies.

Modeling TDP-43 proteinopathies in cells

We and others have shown that TDP-43 is cleaved by caspases to produce fragments of approximately 35 and 25 kDa [1-4]. We have also shown that the overexpression and aggregation of the 25 kDa C-terminal fragment generated by caspase cleavage of TDP-43 (GFP-TDP_220-414) is detrimental to neuroblastoma cells [5]. The aggregation of GFP-TDP_220-414 is associated with increased cytotoxicity which likely results from a toxic gain of function since aggregate formation neither alters the nuclear distribution or function of endogenous full-length TDP-43 [5]. We thus believe that compounds that decrease the aggregation of TDP-43 will provide neuroprotection to patients suffering from TDP-43 proteinopathies. To identify such compounds, we have generated a human neuroblastoma cell line (M17D3 cells) that inducibly expresses GFP-TDP_220-414 in the absence of doxycycline (Dox). Removal of Dox from the culture media leads to the time-dependent expression of GFP-TDP_220-414 and the formation of cytoplasmic GFP-TDP_220-414 inclusions (Fig.1). We are currently using this model to screen a library of ~58,000 small-molecules with increased probability of oral bioavailability and blood brain barrier penetration to identify compounds that diminish both TDP-43 aggregation and toxicity. Compounds of interest will be validated using:

1. in vitro aggregation models of recombinant TDP-43;
2. secondary cell model systems (for example, primary neurons expressing aggregation-prone TDP-43 products);
3. our transgenic mouse models of TDP-43 proteinopathies (see below)

Fig.1: Time-dependent expression and aggregation of GFP-TDP_220-414 in M17D3 neuroblastoma cells. To determine if GFP-TDP_220-414 forms cytoplasmic inclusions in a time-dependent manner, M17D3 cells were grown in the absence or presence of 1 µg/ml doxycycline (Dox), which controls the induction of GFP-TDP_220-414 expression. Cells were fixed and GFP fluorescence was examined by confocal microscopy on days 2, 4 and 6. No GFP signal is present in cells treated with Dox, confirming that GFP-TDP_220-414 expression is tightly regulated. In contrast, GFP-TDP_220-414 inclusions are observed after only 2 days in Dox-free media and they markedly increase in number by day 4. At later time-points (days 5-7), larger, but fewer, inclusions are present per cell, suggesting that the larger inclusions are formed by the aggregation of small inclusions. Scale bar, 20 µM.

Modeling TDP-43 proteinopathies in the nematode C. elegans.

To model function and neurotoxicity of TDP-43 in vivo, we have engineered C. elegans with neuronal expression of either wild-type or mutant human TDP-43 (Fig.2). Transgenic worms with neuronal expression of human TDP-43 exhibit "uncoordinated" movement and have abnormal motor neuron synapses. Just as overexpression of human TDP-43 results in the worms having an uncoordinated phenotype, so does the neuronal overexpression of TDP-1, the C.elegans ortholog of TDP-43. By using the uncoordinated phenotype as an indicator of TDP-43-induced neurotoxicity, we have investigated the contribution of specific TDP-43 domains and its subcellular localization in promoting this phenotype. Furthermore, we have shown that the orthologous C. elegans TDP-1 is functionally conserved in cell culture-based splicing assays of a CFTR minigene. By investigating the conserved functions between TDP-43 and TDP-1, we intend to determine the roles of TDP-43 in RNA homeostasis in neurons under basal or stressful conditions.

Fig.2: Pan-neuronal expression of eGFP::TDP-43 (green) or the ALS evoking mutants eGFP::TDP-43.N345K and eGFP::TDP-43.M337V in the model organism C.elegans. Mutant GFP::TDP-43 has altered sub-nuclear distribution in C.elegans neurons. Merged image composed of GFP channel and Nomarski DIC images.

Mouse models of TDP-43 proteinopathies

We are in the process of generating and characterizing transgenic mouse models of TDP-43 proteinopathies to allow us to determine the behavioral, biochemical, and neuropathological impact of wild-type and mutant TDP-43 expression. Given that the majority of TDP-43 proteinopathies are not associated with mutations in the gene encoding TDP-43, it is essential to develop model systems that can be used to elucidate the normal function of wild-type TDP-43 in the central nervous system and to determine if wild-type TDP-43 can directly cause neurodegeneration. Therefore, we have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion protein (Prp) promoter. To understand how ALS-specific mutations contribute to disease, we are also generating a conditional mouse model expressing M337V TDP-43, as well as a control line conditionally expressing wild-type TDP-43. The ability to control the expression TDP-43 will allow us to determine if TDP-43 pathology can be prevented, halted or reversed by suppression of TDP-43 expression. Overall, these transgenic TDP-43 mice will be valuable tools in understanding the normal role of TDP-43 and will provide an essential resource to dissect the pathogenic mechanisms between wild-type and mutant TDP-43.

References cited on this page:

1. Zhang, Y.J., et al., Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci, 2007. 27(39): p. 10530-4.
2. Dix, M.M., G.M. Simon, and B.F. Cravatt, Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell, 2008. 134(4): p. 679-91.
3. Dormann, D., et al., Proteolytic processing of TAR DNA binding protein-43 by caspases produces C-terminal fragments with disease defining properties independent of progranulin. J Neurochem, 2009. 9: p. 9.
4. Nishimoto, Y., et al., Characterization of alternative isoforms and inclusion body of the TAR DNA binding protein-43. J Biol Chem, 2009. 3: p. 3.
5. Zhang, Y.J., et al., Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci U S A, 2009. 21: p. 7607-7612.