Expression of mutated human TDP-43-encoding cDNAs in worm neurons results in a disease model recapitulating key features of human TDP-43 proteinopathy disorders including motor and behavioral abnormalities, reduced lifespan, accumulation of detergent insoluble, ubiquitinated, and phosphorylated abnormal TDP-43 protein species, and neurodegenerative changes (see Table S2
for a summary of phenotypes). Expression of neither low nor high levels of normal human TDP-43 in transgenic C. elegans
results in significant neurodegeneration, but does lead to moderate and progressive motor dysfunction. However, familial ALS mutant versions of TDP-43 (G290A, M337V, or A315T) expressed at similar levels cause a severe and progressive motor phenotype indicative of significant motor neuron dysfunction () demonstrating the neurotoxic effect of the TDP-43 mutations. In ALS and FTLD-U disease phenotypes worsen over time. Likewise, in this model expression of TDP-43 leads to progressive paralysis () and reduced lifespan. Interestingly, in worms the correlation between average lifespan and severity of motor dysfunction is limited (Lakowski and Hekimi, 1998
; Ailion et al., 1999
; Munoz and Riddle, 2003
). The disconnect between lifespan and motor function may be due to the opposing effects of paralysis-induced caloric restriction and rapid aging due to motor dysfunction. The WT-low expressing line has a shorter lifespan and slightly worse locomotion than the WT-high, despite lower levels of TDP-43. However, the WT-low line has low levels of detectable S409/410 phosphorylation present only in its detergent insoluble fraction () while WT-high does not have S409/410 phosphorylation in any fraction. The relatively worse phenotype of WT-low as compared to WT-high is consistent with the hypothesis linking S409/410 phosphorylation to TDP-43 toxicity.
TDP-43 is predicted to contain both nuclear localization and nuclear export signals (Ayala et al., 2008
; Winton et al., 2008
), indicative of a cellular function that involves shuttling between the nucleus and cytoplasm. While TDP-43 is normally found in the nucleus, it can relocate to the cytoplasm in response to neuronal injury (Moisse et al., 2009
). Abnormal TDP-43 forms pathologic inclusions in both compartments with varying frequency in different TDP-43 proteinopathy disorders. For instance, cytoplasmic neuronal and glial inclusions are common in ALS, but vary widely in subtypes of FTLD-U. Nuclear inclusions of TDP-43 are common in some subtypes of FTLD-U, such as those caused by progranulin mutations, but rare in others (Forman et al., 2007
). Nuclear neuronal inclusions containing TDP-43 also predominate in inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD). Likewise, in this model of TDP-43 proteinopathy, aggregated TDP-43 localizes predominantly to the nucleus of neurons for both normal and mutated TDP-43 transgenic lines (). While some studies demonstrate cytoplasmic localization of TDP-43 in response to ALS causing mutations (Barmada et al., 2010
), in others nuclear localization of aggregates predominates and drives neurodegeneration (Wegorzewska et al., 2009
). The lack of correlation between TDP-43 cellular localization and neurotoxicity may indicate that localization alone is not responsible for TDP-43 driven neurodegeneration.
Loss of motor neurons is a key finding in post mortem analysis of the nervous systems of ALS patients. Given the prominent motor phenotype in TDP-43 transgenic animals, we examined the effects of wild type and familial ALS mutant forms of TDP-43 on neurons in living animals. Interestingly, worms expressing wild type TDP-43 did not exhibit GABAergic neurodegeneration, despite obvious motor deficits. However, we observe progressive and severe degeneration of the VD and DD classes of GABAergic motor neurons in animals expressing familial ALS mutant forms of TDP-43 (). We also observed degeneration of ADE and CEP but not PDE classes of dopaminergic neurons in mutant but not wild type TDP-43 lines (data not shown). Neurodegeneration in mutant but not wild type TDP-43 expressing animals likely underlies the differences in severity of motor dysfunction in mutant versus wild type TDP-43 expressing animals, and may underlie additional sensory or behavioral problems yet to be explored in this model. It is likely that mutant TDP-43 neurodegeneration is limited to specific subsets of neurons in C. elegans, since worms expressing mutant TDP-43 are viable and have grossly normal development and adult morphology. Whether TDP-43 destroys any additional populations of neurons and, if so, analyzing what features they share with GABAergic and dopaminergic neurons will be important future work. Human TDP-43 proteinopathies feature degeneration of specific subtypes of neurons, and worms provide a tractable system to explore the specificity of TDP-43 neurodegeneration.
Pathological lesions containing detergent insoluble, phosphorylated, and aggregated TDP-43 are a key feature of TDP-43 proteinopathies like FTLD-U and ALS and can be found in both the nucleus and cytoplasm of neurons (reviewed in (Gendron et al., 2010
; Geser et al., 2010
)). The vast majority of TDP-43 proteinopathy cases exhibit abnormal TDP-43 aggregates in the absence of TDP-43 mutations. Similarly, in this model we see TDP-43 detergent insoluble aggregates independent of ALS causing TDP-43 mutations (). However, the severity of neuronal dysfunction or degeneration does not correlate with protein aggregation. This observation suggests aggregation of TDP-43 does not drive neurotoxicity, because while mutant TDP-43 is decidedly more toxic than wild type TDP-43, we observe considerably more aggregated wild type TDP-43 protein than mutant TDP-43 protein. Thus, we conclude that mutant and wild type TDP-43 cause neuronal dysfunction by distinct mechanisms or aggregation does not play a causal role in neurotoxicity, but rather may be a cellular consequence of TDP-43 dysfunction.
Caspase cleavage of TDP-43 has been suggested to be important in the pathogenic mechanism of ALS and FTLD-U (Zhang et al., 2009
), and caspases are known to mediate apoptotic cell death. We observe truncated species in all TDP-43 transgenic lines roughly in proportion to the level of full length TDP-43 protein expressed (). Thus, we explored the requirements for ced-3
in TDP-43 mediated motor dysfunction and neurotoxicity. In C. elegans, ced-
3 is a caspase gene essential for apoptosis, while ced-4
is a caspase activator gene required for normal apoptosis. Loss of ced-3
function does not prevent wild type or mutant TDP-43 induced movement defects or neurodegeneration () demonstrating that caspase-dependent cleavage and activation of conventional apoptosis pathways are not required in the pathology and neurotoxicity of TDP-43. Furthermore, truncated TDP-43 products persist in the absence of the CED-3 caspase () (Nishimoto et al., 2010
). Likewise, we see little relationship between the level of truncated species and the degree of neurodegeneration, since TDP-43 WT-high has the highest level of truncated species and TDP-43 WT-low has the lowest level of truncated species (), yet neither has significant neurodegenerative changes. These findings are consistent with observations in cultured mammalian cells, where TDP-43 fragments persist in the absence of caspase function (Nishimoto et al., 2010
). It is possible other mechanisms of cell death such as necrosis or cellular atrophy are contributing to TDP-43 driven neurodegeneration (Bodansky et al., 2010
). Future work will address more precisely what mechanism is involved in neuronal death in this TDP-43 model.
The theme of aberrant phosphorylation of misfolded or aggregated proteins has been noted in the pathological characterization of several neurodegenerative diseases besides ALS and FTLD-U. For instance hyperphosphorylation of microtubule binding protein tau is a diagnostic hallmark of Alzheimer’s disease (reviewed in (Buee et al., 2000
)), while hyperphosphorylation of α-synuclein occurs in Parkinson’s disease (reviewed in (Thomas and Beal, 2007
; Kahle, 2008
)). Whether phosphorylation is a cause or consequence of protein aggregation and neurotoxicity remains a debated point for both disorders. Interestingly, we see marked enrichment of pS409/410 species in ALS mutant TDP-43 expressing lines ( Non-ionic detergent and ionic detergent fractions), correlating TDP-43 phosphorylation with the severity of motor dysfunction and neurodegeneration.
In ALS mutant TDP-43 lines, phosphorylated TDP-43 is only observed in detergent soluble and insoluble protein fractions, indicating the toxic phosphorylated species may be misfolded. This may also hold true for the differences observed in the WT-low versus WT-high expressing lines. The more toxic WT-low line has low level, but detectable phosphorylated TDP-43 while WT-high has none (). The abundance of phosphorylation of ALS mutated TDP-43 as compared to low or undetectable phospho TDP-43 in wild type lines highlight potentially different mechanisms for toxicity of wild type and ALS mutant TDP-43. Furthermore, we demonstrate that ALS mutant TDP-43 is no more toxic than wild type TDP-43 when phosphorylation of the S409/410 site is prevented (); however, this same removal of phosphorylation has no effect on the toxicity of otherwise wild type TDP-43 (Fig S7B
). Likewise, pseudo-phosphorylation on its own does not cause any dramatic increase in TDP-43 toxicity (Fig S7
), suggesting either that phosphorylated TDP-43 is only toxic in combination with ALS causing mutations, or that pseudo-phosphorylation at positions 409/410 does not accurately recapitulate the structural changes of authentic phosphorylation of TDP-43 at S409/410.
Previous pathological analysis of post-mortem ALS and FTLD-U CNS samples clearly demonstrates S409/410 phosphorylation in TDP-43 lesions, but not normal CNS tissue (Inukai et al., 2008
; Neumann et al., 2009
). Taken together, previously published pathological and functional findings (Inukai et al., 2008
; Neumann et al., 2009
) and our data support a critical role for TDP-43 phosphorylation in the mechanism of neurodegeneration in familial ALS disease.
TDP-43 has been the focus of intensive study since its identification as the pathological protein in ALS and FTLD-U ubiquitinated inclusions (Arai et al., 2006
; Neumann et al., 2006
). Efforts to understand the mechanism of disease have led to the development of model systems exhibiting some features of TDP-43 pathology. The first mammalian model was recently described demonstrating viral mediated expression of wild type human TDP-43 in the substantia nigra of rats. These animals show loss of dopaminergic neurons and display behavioral phenotypes. They also mimic some features of TDP-43 pathology including cytoplasmic localization and ubiquitination (Tatom et al., 2009
). A mouse model expressing wild type human TDP-43 in most central nervous system neurons displayed dose-dependent degeneration of motor neurons, spastic quadriplegia, accumulation of truncated, detergent insoluble TDP-43 products and shortened lifespan (Wils et al., 2010
)). Expression of ALS-mutant TDP-43 (M337V) in rats led to widespread neurodegeneration, muscle atrophy, paralysis, and shortened lifespan, phenotypes which were not observed with expression of wild type human TDP-43 (Zhou et al., 2010
). Another recent study describes neuronal expression of ALS-mutant TDP-43 (A315T) in mice, resulting in selective neurodegeneration, progressive gait abnormalities, and shortened lifespan (Wegorzewska et al., 2009
). Our observations that high levels of detergent insoluble TDP-43 can occur without significant neurodegeneration (summarized in Table S2
) are consistent with the mouse and rat models of mutant TDP-43 neurotoxicity, where TDP-43 exhibited little or no aggregation or inclusion formation (Wegorzewska et al., 2009
; Zhou et al., 2010
). This indicates TDP-43 driven neurodegeneration can occur in the absence of protein aggregation.
is a simple system allowing us to model the cellular and molecular changes in neurodegeneration, although it cannot accurately model more complex pathological features associated with mammalian nervous system dynamics. For instance, the selective and variable neuronal vulnerability in FTLD-U (Seeley et al., 2006
) or the focal onset, progression, and spreading of ALS (reviewed in (Ravits and La Spada, 2009
)) require a model with the complexity of the mammalian CNS architecture. These questions can best be addressed in the study of vertebrate animal models of neurodegeneration or in study of patients themselves. However, this C. elegans
model of TDP-43 proteinopathies is highly relevant to understanding TDP-43 related disease mechanisms because of its potential for dissection of the determinants of TDP-43 neurotoxicity at the cellular and molecular levels. For instance, we demonstrate the reversal of the motor dysfunction phenotype of ALS mutant TDP-43 but not wild type TDP-43 with ablation of the serine 409/410 phosphorylation sites and the subsequent loss of phospho-TDP-43 protein. While this model lacks the cytoplasmic mislocalization of TDP-43 commonly seen in ALS, we do demonstrate specific and robust nuclear aggregation of TDP-43 without any detectable cytoplasmic TDP-43 (). This nuclear aggregation of TDP-43 is not typical of ALS, but is commonly seen in some FTLD-U cases. Thus we present this as a generic model of TDP-43 proteinopathy rather than a specific model of a single human disease.
Use of C. elegans
as a model system permits unbiased approaches to genetically manipulate a model of human neurodegenerative diseases (Kraemer et al., 2006
; Kuwahara et al., 2008
; Guthrie et al., 2009
). This TDP-43 proteinopathy model is likewise tractable for genome-wide forward and reverse genetic screens which will allow identification of novel modifiers of TDP-43 activity. Furthermore, this model will facilitate exploration of the mechanism of TDP-43 motor neurotoxicity, including dissection of which molecular features of TDP-43 pathology cause motor neuron dysfunction, and which are secondary effects or markers of motor neuron degeneration. In addition, the worm model has potential as a system to rapidly screen for pharmacological modifiers of TDP-43 pathology, potentially guiding development of therapeutic interventions. For example, identification of compounds or mutations preventing phosphorylation of TDP-43 at S409/410 may be important neuroprotective strategies for TDP-43 proteinopathy disorders.