Full-length TAR DNA-binding Protein-43 Research Articles

Multiscale simulations of TDP-43 full-length uncover molecular determinants of its phase separation beyond N/C-terminal oligomerization.

TAR DNA-binding protein 43 (TDP-43) is a multidomain protein involved in the regulation of RNA processing, and its aggregates have been observed in neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 is composed of a well-folded N-terminal domain (NTD), two RNA recognition motifs (RRM1/2), an intrinsically disordered C-terminal domain (CTD), and short flexible linkers between folded domains. Recently, TDP-43 has been shown to undergo liquid-liquid phase separation (LLPS) to form membraneless compartments referred to as biomolecular condensates. While NTD oligomerization was shown to promote the phase separation of full-length TDP-43 strongly, CTD can also undergo LLPS on its own. However, the complete picture of domain-level intermolecular interactions of full-length TDP-43 within the biological condensates has remained unclear due to the challenges associated with its purification. Here, we utilize a multiscale simulation approach to get molecular-level insights into the role of interdomain interactions in TDP-43 phase separation. Atomistic, single-chain simulations show that interdomain contacts are mediated by electrostatic interactions. Our coarse-grained, condensed phase simulations also reveal that electrostatic interactions involving RRM/NTD-linker modulate the phase separation of TDP-43, in addition to the NTD/CTD-oligomerization. These findings should pave the way for a greater understanding of molecular determinants of TDP-43 phase separation.

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-β-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology. Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions. Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-β structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature. Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies. KEY MESSAGES Cytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-β structure, and do not show a fibrillar morphology. TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

Biophysical characterization of full-length TAR DNA-binding protein (TDP-43) phase separation.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are associated with deposition of cytosolic inclusion bodies of TAR DNA-binding protein 43 (TDP-43) in brain and motor neurons. We induced phase separation of purified full-length TDP-43 devoid of large tags using a solution-jump method, and monitored it with an array of biophysical techniques. The tetramethylrhodamine-5-maleimide- or Alexa488-labeled protein formed rapidly (<1 min) apparently round, homogeneous and 0.5-1.0μm wide assemblies, when imaged using confocal fluorescence, bright-field, and stimulated emission depletion microscopy. The assemblies, however, had limited internal diffusion, as assessed with fluorescence recovery after photobleaching, and did not coalesce, but rather clustered into irregular bunches, unlike those formed by the C-terminal domain. They were enriched with α-helical structure, with minor contributions of β-sheet/random structure, had a red-shifted tryptophan fluorescence and did not bind thioflavin T. By monitoring with turbidimetry both the formation of the spherical species and their further clustering under different experimental conditions, we carried out a multiparametric analysis of the two phenomena. In particular, both processes were found to be promoted by high protein concentrations, salts, crowding agents, weakly by reducing agents, as the pH approached a value of 6.0 from either side (corresponding to the TDP-43 isoionic point), and as the temperature approached a value of 31°C from either side. Important differences were found with respect to the TDP-43 C-terminal domain. Our multiparametric results also provide explanations to some of the solubility data obtained on full-length TDP-43 that were difficult to explain following the multiparametric analysis acquired on the C-terminal domain.

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

Phosphorylated TAR DNA-binding protein-43: Aggregation and antibody-based inhibition

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of β-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 μg/mL: 45 μg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.

Distinct brain-derived TDP-43 strains from FTLD-TDP subtypes induce diverse morphological TDP-43 aggregates and spreading patterns in vitro and in vivo.

The heterogeneity in the distribution and morphological features of TAR DNA-binding protein-43 (TDP-43) pathology in the brains of frontotemporal lobar degeneration (FTLD-TDP) patients and their different clinical manifestations suggest that distinct pathological TDP-43 strains could play a role in this heterogeneity between different FTLD-TDP subtypes (A-E). Our aim was to evaluate the existence of distinct TDP-43 strains in the brains of different FTLD-TDP subtypes and characterise their specific seeding properties in vitro and in vivo. We used an inducible stable cell line expressing a mutant cytoplasmic TDP-43 (iGFP-NLSm) to evaluate the seeding properties of distinct pathological TDP-43 strains. Brain-derived TDP-43 protein extracts from FTLD-TDP types A (n=6) and B (n=3) cases induced the formation of round/spherical phosphorylated TDP-43 aggregates that morphologically differed from the linear and wavy wisps and bigger heterogeneous filamentous (skein-like) aggregates induced by type E (n=3) cases. These morphological differences correlated with distinct biochemical banding patterns of sarkosyl-insoluble TDP-43 protein recovered from the transduced cells. Moreover, brain-derived TDP-43 extracts from type E cases showed higher susceptibility to PK digestion of full-length TDP-43 and the most abundant C-terminal fragments that characterise type E extracts. Finally, we showed that intracerebral injections of different TDP-43 strains induced a distinctive morphological and subcellular distribution of TDP-43 pathology and different spreading patterns in the brains of CamKIIa-hTDP-43NLSm Tg mice. We show the existence of distinct TDP-43 strains in the brain of different FTLD-TDP subtypes with distinctive seeding and spreading properties in the brains of experimental animal models.

Mechanistic and Structural Characterization of Full‐Length, Aggregation‐Prone, TDP‐43 Protein

TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) [1]. TDP-43 is a protein that functions in transcriptional repression, the modulation of RNA metabolism, and stress granule formation within cells. As in the case with many other proteins that are partially destructed, characterization of full-length recombinant TDP-43 has proved difficult. This protein is commonly modified post-translationally (PTMs) into aggregated and mutated structures in the cytoplasm and nucleus, including phosphorylation, acetylation, and C-terminal cleavages [2]. Cytoplasmic mislocalization and post-translational modifications of TDP-43 have been linked to aggregate formation, proposedly important for TDP-43-toxicity mechanisms [3, 4]. Since pertinent research has only recently been established on the protein, the pathogenicity and functionality of TDP-43 in ALS has yet to be determined, thus providing us with a critical knowledge gap in TDP-43's functions. Thus, systematic in vitro studies were conducted in our lab to characterize the basic aggregation mechanisms of full-length, native TDP-43 and establish a working model for the in vitro analyses of the protein. Current research on the full-length protein have discovered notable findings regarding the intermolecular properties of TDP-43, providing critical insight into the hydrophobicity of its structure. Along with these intermolecular characteristics and its poor solubility in vitro, the full structure of TDP-43 has not been fully defined. Herein, the in vitro aggregation of TDP-43 protein was achieved and characterized using various spectroscopic methods and electron microscopy. Data indicate that TDP-43 protein is an inherently aggregation-prone protein from the application of several in vitro conditions to induce misfolding of the protein, followed by turbidity and Thioflavin T (ThT) fluorescence assays to monitor β-sheet formation. Transmission electron microscopy (TEM) allowed for visualization of the structural morphologies of these TDP-43 aggregates. As a result, data indicate that full-length TDP-43 protein aggregated under specific environmental conditions and resulted in a high turbidity and ThT intensity. The in vitro analysis of TDP-43 aggregation is a valuable model for future protein studies and provides a preliminary platform for screening therapies towards a viable treatment for ALS.

Granulins modulate liquid–liquid phase separation and aggregation of the prion-like C-terminal domain of the neurodegeneration-associated protein TDP-43

TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hallmarks of both FTLD and ALS are the toxic cytoplasmic inclusions of the prion-like C-terminal fragments of TDP-43 CTD (TDP-43 C-terminal domain), formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. Both full-length TDP-43 and its CTD are also known to form stress granules by coacervating with RNA in the cytoplasm during stress and may be involved in these pathologies. Furthermore, mutations in the PGRN gene, leading to haploinsufficiency and diminished function of progranulin (PGRN) protein, are strongly linked to FTLD and ALS. Recent reports have indicated that proteolytic processing of PGRN to smaller protein modules called granulins (GRNs) contributes to FTLD and ALS progression, with specific GRNs exacerbating TDP-43-induced cytotoxicity. Here we investigated the interactions between the proteolytic products of both TDP-43 and PGRN. Based on structural disorder and charge distributions, we hypothesized that GRN-3 and GRN-5 could interact with the TDP-43 CTD. We show that, under both reducing and oxidizing conditions, GRN-3 and GRN-5 interact with and differentially modulate TDP-43 CTD aggregation and/or liquid-liquid phase separation in vitro GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid-liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS.

Differential toxicity of TAR DNA-binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.

The N-Terminal Domain of ALS-Linked TDP-43 Assembles without Misfolding.

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.

Abstract WP97: Neuroprotective Effect of Progranulin Against Focal Cerebral Ischemia via Inhibition of Proteolysis of TDP-43 by Caspase-3

Introduction: We previously demonstrated that progranulin (PGRN), a glycoprotein growth factor, may be a novel therapeutic target that provides vascular protection and anti-neuroinflammation properties related in part to vascular endothelial growth factor and interleukin 10, respectively. We also found that PGRN could provide neuroprotection in part by inhibition of cytoplasmic redistribution of TAR DNA-binding protein-43 (TDP-43), although its exact mechanism remains poorly understood. The purpose of this study is to determine the mechanism of neuroprotection by PGRN against ischemic neuronal injury. Methods: The middle cerebral artery of adult PGRN knock-out (KO) mice (C57BL/6 background) and wild-type (WT) mice was occluded for 90 minutes. Immunohistochemical analysis using an antibody against TDP-43 was performed to investigate the subcellular localization of TDP-43 after ischemia. We also performed Western blot analysis to investigate the expression levels of TDP-43 and activated caspase-3 using cerebral cortex tissues from a rat autologous thromboembolic model with delayed tissue plasminogen activator (tPA) treatment (4 hours after ischemia). Results: Twenty-four hours after reperfusion, neuronal cells showing cytoplasmic redistribution of TDP-43 were more frequently observed in PGRN KO mice than in WT mice (P&lt;0.01). In a rat autologous thromboembolic model with delayed tPA treatment, the expression level of full-length of TDP-43 decreased 24 hours after ischemia via proteolytic degradation. However, intravenous administration of recombinant PGRN with delayed tPA treatment inhibited the decrease in the expression level of full-length TDP-43 as well as the increase in the expression level of activated caspase-3 compared with that of the control protein. Conclusion: This study demonstrated that PGRN might protect neuronal cells against focal cerebral ischemia via inhibition of proteolysis and abnormal cytoplasmic redistribution of TDP-43 by caspase-3.

Phosphorylated TDP-43 becomes resistant to cleavage by calpain: A regulatory role for phosphorylation in TDP-43 pathology of ALS/FTLD

TAR DNA-binding protein-43 (TDP-43) pathology, which includes the presence of abnormal TDP-43-containing inclusions with a loss of nuclear TDP-43 in affected neurons, is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobar degeneration (FTLD). TDP-43 in the pathological brains and spinal cords of ALS/FTLD patients is abnormally fragmented and phosphorylated. It is believed that the generation of aggregation-prone TDP-43 fragments initiates TDP-43 pathology, and we previously reported that calpain has an important role in the generation of such aggregation-prone TDP-43 fragments. However, the role of phosphorylation in TDP-43 pathology has not been largely elucidated, despite previous observations that several kinases and their kinases are involved in TDP-43 phosphorylation. Here, we investigated the role of TDP-43 phosphorylation in the calpain-dependent cleavage of TDP-43 and found that phosphorylated, full-length TDP-43 and calpain-dependent TDP-43 fragments were more resistant to cleavage by calpain than endogenous full-length TDP-43 was. These results suggest that both phosphorylated and calpain-cleaved TDP-43 fragments persist intracellularly for a length of time that is sufficient for self-aggregation, thereby serving as seeds for inclusions.

Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death.

The presence of lower molecular weight species comprising the C-terminal region of TAR DNA-binding protein 43 (TDP-43) is a characteristic of TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we have identified a novel splice variant of TDP-43 that is upregulated in ALS and generates a 35-kDa N-terminally truncated species through use of an alternate translation initiation codon (ATGMet85), denoted here as Met85-TDP-35. Met85-TDP-35 expressed ectopically in human neuroblastoma cells exhibited reduced solubility, cytoplasmic distribution, and aggregation. Furthermore, Met85-TDP-35 sequestered full-length TDP-43 from the nucleus to form cytoplasmic aggregates. Expression of Met85-TDP-35 in primary motor neurons resulted in the formation of Met85-TDP-35-positive cytoplasmic aggregates and motor neuron death. A neo-epitope antibody specific for Met85-TDP-35 labeled the 35-kDa lower molecular weight species on immunoblots of urea-soluble extracts from ALS-FTLD disease-affected tissues and co-labeled TDP-43-positive inclusions in ALS spinal cord sections, confirming the physiological relevance of this species. These results show that the 35-kDa low molecular weight species in ALS-FTLD can be generated from an abnormal splicing event and use of a downstream initiation codon and may represent a mechanism by which TDP-43 elicits its pathogenicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1412-5) contains supplementary material, which is available to authorized users.

Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations

TAR DNA-binding protein 43 (TDP-43) is a major component of the pathologic inclusions observed in the motor neurons of amyotrophic lateral sclerosis (ALS) patients. We examined TDP-43 expression in primary fibroblasts cultures from 22 ALS patients, including cases with SOD1 (n = 4), TARDBP (n = 4), FUS (n = 2), and C9ORF72 (n = 3) mutations and 9 patients without genetic defect. By using a phosphorylation-independent antibody, 15 patients showed notable alterations of TDP-43 level in the nuclear or cytoplasmic compartments. In particular, a marked accumulation of TDP-43 was observed in the cytoplasm of all cases with C9ORF72 and TARDBP mutations, 1 patient with FUS mutation and 3 patients without genetic defect. Patients with SOD1 mutations revealed a significant reduction of TDP-43 in the nuclei without cytoplasmic mislocalization. These changes were associated with the presence of truncated and phosphorylated TDP-43 species. Our results show that fibroblasts recapitulate some of hallmark TDP-43 abnormalities observed in neuronal cells. The reduction of full-length TDP-43 level in mutant SOD1 cells indicates that at least some SOD1 mutations alter TDP-43 metabolism.

A Nonsense Mutation in Mouse Tardbp Affects TDP43 Alternative Splicing Activity and Causes Limb-Clasping and Body Tone Defects

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 TardbpQ101X mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the TardbpQ101X 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 SOD1G93Adl 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 TardbpQ101X 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.

The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation

TAR DNA-binding protein-43 (TDP-43) is the principal component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and the most common pathological subtype of frontotemporal dementia—frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). To date, the C-terminus of TDP-43, which is aggregation-prone and contains almost all ALS-associated mutations, has garnered much attention while the functions of the N-terminus of TDP-43 remain largely unknown. To bridge this gap in our knowledge, we utilized novel cell culture and computer-assisted models to evaluate which region(s) of TDP-43 regulate its folding, self-interaction, biological activity and aggregation. We determined that the extreme N-terminus of TDP-43, specifically the first 10 residues, regulates folding of TDP-43 monomers necessary for proper homodimerization and TDP-43-regulated splicing. Despite such beneficial functions, we discovered an interesting dichotomy: full-length TDP-43 aggregation, which is believed to be a pathogenic process, also requires the extreme N-terminus of TDP-43. As such, we provide new insight into the structural basis for TDP-43 function and aggregation, and we suggest that stabilization of TDP-43 homodimers, the physiologically active form of TDP-43, may be a promising therapeutic strategy for ALS and FTLD-TDP.

Nucleus Accumbens 1, a Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad protein binds to TAR DNA-binding protein 43 and has a potential role in Amyotrophic Lateral Sclerosis

Protein degradation is a critical component of cellular maintenance. The intracellular translocation and targeting of the Ubiquitin Proteasome System (UPS) differentially coordinates a protein’s half-life and thereby its function. Nucleus Accumbens 1 (NAC1), a member of the Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex (POZ/BTB) family of proteins, participates in the coordinated proteolysis of synaptic proteins by mediating recruitment of the UPS to dendritic spines. Here we report a novel interaction between NAC1 and TAR DNA-binding protein 43 (TDP-43), a protein identified as the primary component of ubiquitinated protein aggregates found in patients with Amyotrophic Lateral Sclerosis (ALS). In vitro translated full-length TDP-43 associated with both the POZ/BTB domain and the non-POZ/BTB domain of NAC1 in GST pulldown assays. Other POZ/BTB proteins (including zinc finger POZ/BTB proteins and atypical POZ/BTB proteins) showed weak interactions with TDP-43. In addition, NAC1 and TDP-43 were present in the same immunocomplexes in different regions of mouse brain and spinal cord. In primary spinal cord cultures, TDP-43 expression was mainly nuclear, whereas NAC1 was both nuclear and cytoplasmic. In order to mimic ALS-like toxicity in the spinal cord culture system, we elevated extracellular glutamate levels resulting in the selective loss of motor neurons. Using this model, it was found that glutamate toxicity elicited a dose-dependent translocation of TDP-43 out of the nucleus of cholinergic neurons and increased the co-localization of NAC1 and TDP-43. These findings suggest that NAC1 may function to link TDP-43 to the proteasome; thereby, facilitating the post-translational modifications of TDP-43 that lead to the development of ALS.

Biochemical and histopathological alterations in TAR DNA‐binding protein‐43 after acute ischemic stroke in rats

Nuclear factor TAR DNA-binding protein-43 (TDP-43) is considered to play roles in pathogenesis of human neurodegenerative diseases, so-called TDP-43 proteinopathy, via its proteolytic cleavage, abnormal phosphorylation, subcellular redistribution, and insolubilization generating TDP-43-positive neuronal intracellular inclusions. The purpose of this study was to elucidate biochemical and histopathological alternations in TDP-43 specific to acute ischemic stroke. Adult male rats were subjected to a 90-min middle cerebral artery occlusion. We examined the proteolytic cleavage, phosphorylation, subcellular localization, and solubility of TDP-43 by immunoblottings and histopathological examinations using the ischemic and sham-operated cortex. The level of full-length TDP-43 (43 kDa) decreased and that of the 25-kDa C-terminal fragment increased after acute ischemic stroke, which can be explained by proteolytic cleavage of TDP-43. Cytoplasmic redistribution and altered nuclear distribution of TDP-43 was observed after acute ischemic stroke, whereas abnormal phosphorylation and insolubilization of TDP-43 as well as formation of intracellular inclusions were not observed. Ischemic neurons with the cytoplasmic redistribution of TDP-43 expressed ubiquitin and activated caspase 3 and were terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling-positive. In conclusion, biochemical and histopathological alterations in TDP-43 were identified in rats after acute ischemic stroke, although there was very less similarity between TDP-43 alterations observed in acute ischemic stroke and those observed in TDP-43 proteinopathy.

TDP-43 neurotoxicity and protein aggregation modulated by heat shock factor and insulin/IGF-1 signaling

TAR DNA-binding protein 43 (TDP-43) plays a key role in the neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The nature of the TDP-43-mediated neurotoxicity associated with these diseases is not yet understood. Here, we have established transgenic Caenorhabditis elegans models that express human TDP-43 variants in the nervous system, including the full-length wild-type (WT) and mutant proteins and a pathologic C-terminal fragment. The C. elegans models developed severe locomotor defects associated with the aggregation of TDP-43 in neurons. In comparison to parallel Cu/Zn superoxide dismutase worm models, transgenic full-length TDP-43, including the WT protein, was highly neurotoxic. In addition, TDP-43 demonstrated an unusually high tendency to aggregate, a property intrinsic to the WT protein. The C-terminal 25 kDa fragment of TDP-43 was unstable but remarkably aggregation-prone. Distinct disulfide-linked TDP-43 dimers and oligomers were detected. In C. elegans, the neurotoxicity and the protein aggregation of TDP-43 were regulated by environmental temperature and heat shock transcriptional factor 1, indicating that a deficiency in protein quality control is a risk factor for TDP-43 proteinopathy. Furthermore, the neurotoxicity and the protein aggregation of TDP-43 can be significantly attenuated by a deficiency in the insulin/insulin-like growth factor 1 (IGF-1) signaling in C. elegans and mammalian cells. These results suggest that protein misfolding underlies the aging-dependent neurodegeneration associated with TDP-43 and that the insulin/IGF-1 signaling may be a target for therapies.

Both cytoplasmic and nuclear accumulations of the protein are neurotoxic in Drosophila models of TDP-43 proteinopathies

Recently, the TAR DNA-binding protein-43 (TDP-43) has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patient with amyotrophic lateral sclerosis or frontotemporal lobar degeneration. Pathological proteins are abnormally hyperphosphorylated and partially cleaved to generate C-terminal fragments. In this issue, we addressed the mechanism underlying TDP-43 toxicity in vivo, using Drosophila as an experimental model. We developed new Drosophila transgenic models expressing different variants of full-length human TDP-43 proteins presenting different subcellular localizations: a wild-type form of hTDP-43 and two mutants forms of the protein, hTDP-43mutNLS and hTDP43mutNES, which lack nuclear localization signals (NLS) and nuclear export signals (NES), respectively. Using an inducible GAL4 system, we found that both nuclear and cytoplasmic accumulations of TDP-43 in adult neurons lead to reduction of lifespan in Drosophila, the gradient of toxicity being hTDP-43>hTDP-43mutNLS>hTDP43mutNES. This toxicity occurs regardless of inclusions formation. In the other hand, in retina, muscle and glial cells, only the accumulation of cytoplasmic species of TDP-43 was toxic. Biochemical data showed that human TDP-43 proteins expressed in adult fly neurons are abnormally phosphorylated on the disease-specific Ser409/Ser410 site and processed. In conclusion, our data show that TDP-43 expression in flies recapitulates several biochemical key features of human TDP-43 proteinopathies, including abnormal phosphorylation on a disease-specific site and processing of the protein. Moreover, our TDP-43 Drosophila models indicate that distinct pathways of TDP-43 toxicity might operate depending on the cell type.