TDP-43 physically interacts with amyotrophic lateral sclerosis-linked mutant CuZn superoxide dismutase

Mutations in the CuZn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43) genes are linked to familial amyotrophic lateral sclerosis, ALS1 and ALS10, respectively. In addition, TDP-43 is a major component protein of the ubiquitinated aggregates observed in sporadic ALS (SALS) patients. However, it remains unclear whether these ALS groups partly have a shared pathogenesis. In the present study, we demonstrate that mutant SOD1, but not wild-type SOD1, interacts with TDP-43 by co-immunoprecipitation assays using cultured cells and G93A mutant SOD1 transgenic mice. The region responsible for this interaction within SOD1 is the dimer interface, namely, the N- and C-terminal regions. Deletion mutants of TDP-43 with or without nuclear localization sequence interacted with mutant SOD1. Cell fractionation assays using cultured cells showed that mutant SOD1 was localized in the cytosolic fraction but not in the nuclear fraction. TDP-43 was detected both in the nuclear and cytosolic fractions, suggesting that mutant SOD1 interacts with TDP-43 in the cytoplasm. Mutant SOD1 overexpression led to an increased amount of mutant SOD1 and, to some extent, its interacting proteins including TDP-43 in the detergent-insoluble fraction. These results indicate that mutant SOD1 could affect the solubility/insolubility of its interacting proteins including TDP-43 through physical interactions. Our findings may contribute to the understanding of links among SALS, ALS1 and ALS10.     

Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation

ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology.     

Using yeast models to probe the molecular basis of amyotrophic lateral sclerosis

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative disease attributable to the death of motor neurons. Associated with ALS are mutations in the genes encoding SOD1 (superoxide dismutase 1), FUS (fused in Sarcoma) protein and TDP-43 (TAR DNA-binding protein-43) each of which leads to aggregation of the respective protein. For example, the ALS-associated mutations in the hSOD1 (human SOD1) gene typically destabilize the native SOD homodimer, leading to misfolding, aggregation and degradation of SOD1. The ALS-associated pathology is not a consequence of the functional inactivation of SOD1 itself, but is rather due to a toxic gain-of-function triggered by mutant SOD1. Recently, the molecular basis of a number of human neurodegenerative diseases resulting from protein misfolding and aggregation, including fALS (familial ALS), was probed by using the baker's yeast, Saccharomyces cerevisiae, as a highly tractable model. Such studies have, for example, identified novel mutant SOD1-specific interactions and demonstrated that mutant SOD1 disrupts mitochondrial homoeostasis. Features of ALS associated with TDP-43 aggregation have also been recapitulated in S. cerevisiae including the identification of modulators of the toxicity of TDP-43. In this paper, we review recent studies of ALS pathogenesis using S. cerevisiae as a model organism and summarize the potential mechanisms involved in ALS progression.     

Disease-Associated Mutations of TDP-43 Promote Turnover of the Protein Through the Proteasomal Pathway

TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy.     

Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.

Methodology/Principal Findings

Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.

Conclusion/Significance

We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.     

TDP-43 Dimerizes in Human Cells in Culture

TAR DNA-binding protein-43 (TDP-43) is a 43-kDa nuclear protein involved in regulation of gene expression. Abnormally, phosphorylated, ubiquitinated, and aggregated TDP-43 constitute a principal component of neuronal and glial cytoplasmic and nuclear inclusions in the brains of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), although the molecular mechanism that triggers aggregate formation remains unknown. By Western blot analysis using anti-TDP-43 antibodies, we identified a band with an apparent molecular mass of 86-kDa in HEK293, HeLa, and SK-N-SH cells in culture. It was labeled with both N-terminal-specific and C-terminal-specific TDP-43 antibodies, enriched in the cytosolic fraction, and the expression levels were reduced by TDP-43 siRNA but unaltered by treatment with MG-132 or by expression of ubiqulin-1 or casein kinase-1. By immunoprecipitation analysis, we found the interaction between the endogenous full-length TDP-43 and the exogenous Flag-tagged TDP-43, and identified the N-terminal half of TDP-43 spanning amino acid residues 3–183 as an intermolecular interaction domain. When the tagged 86-kDa tandemly connected dimer of TDP-43 was overexpressed in HEK293, it was sequestered in the cytoplasm and promoted an accumulation of high-molecular-mass TDP-43-immunoreactive proteins. Furthermore, the 86-kDa band was identified in the immunoblot of human brain tissues, including those of ALS. These results suggest that the 86-kDa band represents dimerized TDP-43 expressed constitutively in normal cells under physiological conditions.     

Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.     

SOD1 and TDP-43 animal models of amyotrophic lateral sclerosis: recent advances in understanding disease toward the development of clinical treatments

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with no cure. Breakthroughs in understanding ALS pathogenesis came with the discovery of dominant mutations in the superoxide dismutase 1 gene (SOD1) and other genes, including the gene encoding transactivating response element DNA binding protein-43 (TDP-43). This has led to the creation of animal models to further our understanding of the disease and identify a number of ALS-causing mechanisms, including mitochondrial dysfunction, protein misfolding and aggregation, oxidative damage, neuronal excitotoxicity, non-cell autonomous effects and neuroinflammation, axonal transport defects, neurotrophin depletion, effects from extracellular mutant SOD1, and aberrant RNA processing. Here we summarise the SOD1 and TDP-43 animal models created to date, report on recent findings supporting the potential mechanisms of ALS pathogenesis, and correlate this understanding with current developments in the clinic.     

Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation

TAR DNA-binding protein 43 (TDP-43) is the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Although normal TDP-43 is a nuclear protein, pathological TDP-43 is redistributed and sequestered as insoluble aggregates in neuronal nuclei, perikarya, and neurites. Here we recapitulate these pathological phenotypes in cultured cells by altering endogenous TDP-43 nuclear trafficking and by expressing mutants with defective nuclear localization (TDP-43-DeltaNLS) or nuclear export signals (TDP-43-DeltaNES). Restricting endogenous cytoplasmic TDP-43 from entering the nucleus or preventing its exit out of the nucleus resulted in TDP-43 aggregate formation. TDP-43-DeltaNLS accumulates as insoluble cytoplasmic aggregates and sequesters endogenous TDP-43, thereby depleting normal nuclear TDP-43, whereas TDP-43-DeltaNES forms insoluble nuclear aggregates with endogenous TDP-43. Mutant forms of TDP-43 also replicate the biochemical profile of pathological TDP-43 in FTLD-U/ALS. Thus, FTLD-U/ALS pathogenesis may be linked mechanistically to deleterious perturbations of nuclear trafficking and solubility of TDP-43.     

Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein-RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.     

TDP-43 functions and pathogenic mechanisms implicated in TDP-43 proteinopathies

Given the critical role for TDP-43 in diverse neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), there has been a recent surge in efforts to understand the normal functions of TDP-43 and the molecular basis of dysregulation that occurs in TDP-43 proteinopathies. Here, we highlight recent findings examining TDP-43 molecular functions with particular emphasis on stress-mediated regulation of TDP-43 localization, putative downstream TDP-43 target genes and RNAs, as well as TDP-43 interacting proteins, all of which represent viable points of therapeutic intervention for ALS, FTLD-TDP and related proteinopathies. Finally, we review current mouse models of TDP-43 and discuss their similarities and potential relevance to human TDP-43 proteinopathies including ALS and FTLD-TDP.     

Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress

Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.     

Methylene Blue Protects against TDP-43 and FUS Neuronal Toxicity in C. elegans and D. rerio

The DNA/RNA-binding proteins TDP-43 and FUS are found in protein aggregates in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and related dementia, but little is known about the neurotoxic mechanisms. We have generated Caenorhabditis elegans and zebrafish animal models expressing mutant human TDP-43 (A315T or G348C) or FUS (S57Δ or R521H) that reflect certain aspects of ALS including motor neuron degeneration, axonal deficits, and progressive paralysis. To explore the potential of our humanized transgenic C. elegans and zebrafish in identifying chemical suppressors of mutant TDP-43 and FUS neuronal toxicity, we tested three compounds with potential neuroprotective properties: lithium chloride, methylene blue and riluzole. We identified methylene blue as a potent suppressor of TDP-43 and FUS toxicity in both our models. Our results indicate that methylene blue can rescue toxic phenotypes associated with mutant TDP-43 and FUS including neuronal dysfunction and oxidative stress.     

Understanding the role of TDP-43 and FUS/TLS in ALS and beyond

Dominant mutations in two DNA/RNA binding proteins, TDP-43 and FUS/TLS, are causes of inherited Amyotrophic Lateral Sclerosis (ALS). TDP-43 and FUS/TLS have striking structural and functional similarities, implicating alterations in RNA processing as central in ALS. TDP-43 has binding sites within a third of all mouse and human mRNAs in brain and this binding influences the levels and splicing patterns of at least 20% of those mRNAs. Disease modeling in rodents of the first known cause of inherited ALS-mutation in the ubiquitously expressed superoxide dismutase (SOD1)-has yielded non-cell autonomous fatal motor neuron disease caused by one or more toxic properties acquired by the mutant proteins. In contrast, initial disease modeling for TDP-43 and FUS/TLS has produced highly varied phenotypes. It remains unsettled whether TDP-43 and FUS/TLS mutants provoke disease from a loss of function or gain of toxicity or both. TDP-43 or FUS/TLS misaccumulation seems central not just to ALS (where it is found in almost all instances of disease), but more broadly in neurodegenerative disease, including frontal temporal lobular dementia (FTLD-U) and many examples of Alzheimer's or Huntington's disease.     

Modeling ALS and FTLD proteinopathies in yeast: An efficient approach for studying protein aggregation and toxicity

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.Key words: amyotrophic lateral sclerosis, ALS, frontotemporal lobar degeneration, FTLD, yeast, amyloid, prion, FUS, TDP-43, SOD1     

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.     

The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.     

TDP-43 identified from a genome wide RNAi screen for SOD1 regulators

Amyotrophic Lateral Sclerosis (ALS) is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1) that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43), a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis.