ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly,
the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions
(UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies
is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbplx/−) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle
weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate
in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre–Tardbplx/− mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning
of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for
neurodegeneration in ALS with TDP-43 proteinopathies.
"These findings, coupled with the observation that many TDP-43 mutations occur around the prion-like motif, suggest that ALS and FTLD-TDP may be caused by aggregation of TDP-43 which inhibits its normal cellular activities. This loss of TDP-43 function may ultimately be instrumental in disease pathogenesis, and additionally the TDP-43 aggregates may sequester other essential RNAs and proteins . Furthermore, the tremendous variety of reported TDP-43 inclusions suggests distinct mechanisms that may result from the misregulation of disease-specific and potentially overlapping signaling pathways that ultimately lead to TDP-43 accumulation and the subsequent functional deficits characteristic of all diseases with TDP-43 inclusions , . "
[Show abstract][Hide abstract] ABSTRACT: 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 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.
The 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.
These 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.
PLoS ONE 03/2014; 9(3):e90452. DOI:10.1371/journal.pone.0090452 · 3.23 Impact Factor
"Loss of TDP-43 also appears to be deleterious to cell health; knockdown in vitro confers toxicity and knockout results in lethality in vivo, both in utero in constitutive Tardbp−/− mice and in conditional knockout animals in which deletion is postponed until adulthood , , , . Loss of TDP-43 specifically in motor neurons results in cell death and an ALS-like phenotype in mice  and reduced TDP-43 expression in zebrafish and drosophila results in motor deficits , . "
[Show abstract][Hide abstract] ABSTRACT: The majority of cases of frontotemporal lobar degeneration and amyotrophic lateral sclerosis are pathologically defined by the cleavage, cytoplasmic redistribution and aggregation of TAR DNA binding protein of 43 kDa (TDP-43). To examine the contribution of these potentially toxic mechanisms in vivo, we generated transgenic mice expressing human TDP-43 containing the familial amyotrophic lateral sclerosis-linked M337V mutation and identified two lines that developed neurological phenotypes of differing severity and progression. The first developed a rapid cortical neurodegenerative phenotype in the early postnatal period, characterized by fragmentation of TDP-43 and loss of endogenous murine Tdp-43, but entirely lacking aggregates of ubiquitin or TDP-43. A second, low expressing line was aged to 25 months without a severe neurodegenerative phenotype, despite a 30% loss of mouse Tdp-43 and accumulation of lower molecular weight TDP-43 species. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal, but rather were derived from a central portion of human TDP-43. Thus we find that aggregation is not required for cell loss, loss of murine Tdp-43 is not necessarily sufficient in order to develop a severe neurodegenerative phenotype and lower molecular weight TDP-43 positive species in mouse models should not be inherently assumed to be representative of human disease. Our findings are significant for the interpretation of other transgenic studies of TDP-43 proteinopathy.
PLoS ONE 01/2014; 9(1):e86513. DOI:10.1371/journal.pone.0086513 · 3.23 Impact Factor
"Hindlimb clasping is often observed in rodent models of neuromuscular disease including ALS and has been associated with lower motor neuron degeneration, although it can also be caused by muscular dystrophy  and loss of cortical neurons . It has also been noted following motor neuron-specific depletion of TDP43 . Pathological analysis of brains from Tardbp+/Q101X mice did not reveal overt signs of degeneration and in vivo assessment of hindlimb neuromuscular function did not suggest lower motor neuron degeneration at 18 months of age. "
[Show abstract][Hide abstract] ABSTRACT: Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised Tardbp(Q101X) mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the Tardbp(Q101X) mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp(+/Q101X) ) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp(+/Q101X) mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp(+/Q101X) mice were crossed with the SOD1(G93Adl) transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the Tardbp(Q101X) mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community.
PLoS ONE 01/2014; 9(1):e85962. DOI:10.1371/journal.pone.0085962 · 3.23 Impact Factor
Gorana Mandic-Stojmenovic, Elka Stefanova, Valerija Dobricic, Ivana Novakovic, Tanja Stojkovic, Aleksandar Jesic, Vladimir Kostic
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