Towards a TDP-43-Based Biomarker for ALS and FTLD

TDP-43 accumulates in nerve cells of nearly all cases of amyotrophic lateral sclerosis (ALS; the commonest form of motor neuron disease) and in the majority of Tau-negative frontotemporal lobar degeneration (FTLD). There is currently no biochemical test or marker of disease activity for ALS or FTLD, and the clinical diagnosis depends on the opinion of an experienced neurologist. TDP-43 has a key role in the pathogenesis of ALS/FTLD. Measuring TDP-43 in easily accessible biofluids, such as blood or cerebrospinal fluid, might reduce diagnostic delay and offer a readout for use in future drug trials. However, attempts at measuring disease-specific forms of TDP-43 in peripheral biofluids of ALS and FTLD patients have not yielded consistent results, and only some of the pathological biochemical features of TDP-43 found in human brain tissue have been detected in clinical biofluids to date. Reflecting on the molecular pathology of TDP-43, this review provides a critical overview on biofluid studies and future directions to develop a TDP-43-based clinical biomarker for ALS and FTLD.     

Inflammation Induces TDP-43 Mislocalization and Aggregation

TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43^A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.     

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.     

TDP-43-induced death is associated with altered regulation of BIM and Bcl-xL and attenuated by caspase-mediated TDP-43 cleavage

Abnormal aggregates of transactive response DNA-binding protein-43 (TDP-43) and its hyperphosphorylated and N-terminal truncated C-terminal fragments (CTFs) are deposited as major components of ubiquitinated inclusions in most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). The mechanism underlying the contribution of TDP-43 to the pathogenesis of these neurodegenerative diseases remains unknown. In this study, we found that a 2-5-fold increase in TDP-43 expression over the endogenous level induced death of NSC34 motor neuronal cells and primary cortical neurons. TDP-43-induced death is associated with up-regulation of Bim expression and down-regulation of Bcl-xL expression. siRNA-mediated reduction of Bim expression attenuates TDP-43-induced death. Accumulated evidence indicates that caspases are activated in neurons of ALS and FTLD-U patients, and activated caspase-mediated cleavage of TDP-43 generates CTFs of TDP-43. Here, we further found that the ER (endoplasmic reticulum) stress- or staurosporine-mediated activation of caspases leads to cleavage of TDP-43 at Asp(89) and Asp(169), generating CTF35 (TDP-43-(90-414)) and CTF27 (TDP-43-(170-414)) in cultured neuronal cells. In contrast to TDP-43, CTF27 is unable to induce death while it forms aggregates. CTF35 was weaker than full-length TDP-43 in inducing death. A cleavage-resistant mutant of TDP-43 (TDP-43-D89E/D169E) showed stronger death-inducing activity than wild-type TDP-43. These results suggest that disease-related activation of caspases may attenuate TDP-43-induced toxicity by promoting TDP-43 cleavage.     

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.     

ALS-Associated TDP-43 Induces Endoplasmic Reticulum Stress,Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.     

Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43

Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition.     

RNP2 of RNA Recognition Motif 1 Plays a Central Role in the Aberrant Modification of TDP-43

Phosphorylated and truncated TAR DNA-binding protein-43 (TDP-43) is a major component of ubiquitinated cytoplasmic inclusions in neuronal and glial cells of two TDP-43 proteinopathies, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Modifications of TDP-43 are thus considered to play an important role in the pathogenesis of TDP-43 proteinopathies. However, both the initial cause of these abnormal modifications and the TDP-43 region responsible for its aggregation remain uncertain. Here we report that the 32 kDa C-terminal fragment of TDP-43, which lacks the RNP2 motif of RNA binding motif 1 (RRM1), formed aggregates in cultured cells, and that similar phenotypes were obtained when the RNP2 motif was either deleted from or mutated in full-length TDP-43. These aggregations were ubiquitinated, phosphorylated and truncated, and sequestered the 25 kDa C-terminal TDP-43 fragment seen in the neurons of TDP-43 proteinopathy patients. In addition, incubation with RNase decreased the solubility of TDP-43 in cell lysates. These findings suggest that the RNP2 motif of RRM1 plays a substantial role in pathological TDP-43 modifications and that it is possible that disruption of RNA binding may underlie the process of TDP-43 aggregation.     

Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.     

TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides

TAR DNA binding protein 43 (TDP-43) is a key target in amyotrophic lateral sclerosis (ALS) treatment. Here, based on hydrophobic tagging strategy, we designed and synthesized a series of single or double hydrophobic tags conjugated peptides D1-D8. Among them, it was found that D4 displayed strongest ability to induce TDP-43 degradation in cells. D4 could reduce TDP-43 induced cytotoxicity. Besides, D4 could reduce TDP-43 levels in a transgenic drosophila model.     

The Tau Tubulin Kinases TTBK1/2 Promote Accumulation of Pathological TDP-43

Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.     

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.     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.     

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.     

Sustained Expression of TDP-43 and FUS in Motor Neurons in Rodent's Lifetime

TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) are two highly conserved ribonucleoproteins. Pathogenic mutations of the TDP-43 or the FUS gene are all linked to amyotrophic lateral sclerosis (ALS) that is characterized by progressive degeneration of motor neurons. To better understand the correlation of ALS disease genes with the selectivity of chronic motor neuron degeneration, we examined the longitudinal expression of the TDP-43 and the FUS genes in C57BL6 mice and in Sprague-Dawley rats. TDP-43 and FUS were robustly and ubiquitously expressed in the postnatal mice and rats, but were markedly decreased in the adult rodents. In adulthood, TDP-43 and FUS proteins were even undetectable in peripheral organs including skeletal muscles, liver, and kidney, but were constantly expressed at substantial levels in the central nervous system. Motor neurons expressed the TDP-43 and the FUS genes at robust levels throughout rodent''s lifetime. Moreover, TDP-43 and FUS were accumulated in the cytoplasm of motor neurons in aged animals. Our findings suggest that TDP-43 and FUS play an important role in development and that constant and robust expression of the genes in motor neurons may render the neurons vulnerable to pathogenic mutation of the TDP-43 or the FUS gene. To faithfully model the pathology of TDP-43- or FUS gene mutations in rodents, we must replicate the expression patterns of the TDP-43 and the FUS gene in animals.