TARDBP/TDP-43 regulates autophagy in both MTORC1-dependent and MTORC1-independent manners

In a recent paper we addressed the mechanism by which defective autophagy contributes to TARDBP/TDP-43-mediated neurodegenerative disorders. We demonstrated that TARDBP regulates MTORC1-TFEB signaling by targeting RPTOR/raptor, a key component and an adaptor protein of MTORC1. Loss of TARDBP decreased the mRNA stability of RPTOR and this regulation in turn enhanced autophagosomal and lysosomal biogenesis in an MTORC1-dependent manner. Meanwhile, loss of TARDBP could also impair autophagosome-lysosome fusion in an MTORC1-independent manner. Importantly, we found that modulation of MTOR activity by treatment with rapamycin and phosphatidic acid had strong effects on the neurodegenerative phenotypes of TBPH (Drosophila TARDBP)-depleted flies. Taken together, our data reveal that multiple dysfunctions in the autophagic process contribute to TARDBP-linked neurodegeneration and may help to identify potential therapeutic targets in the future.     

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.     

TDP-43在神经退行性疾病中的功能和作用

核蛋白TAR DNA/RNA结合蛋43(TDP-43)目前被认为是肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)、额颞叶变性(frontotemporal lobar degeneration,FTLD)等神经退行性疾病的病理学标记蛋白。在中枢神经系统中,TDP-43作为必要的转录调控因子,参与mRNA前体的剪接,维持RNA稳态和运输。在突变和过表达TDP-43的转基因啮齿类动物模型中,受损伤的神经元呈现出胞核和胞质中TDP-43泛素化、磷酸化聚集,以及细胞周期进程的改变。在此,着重阐述基于TDP-43突变或过表达建立神经退行性疾病动物模型的研究进展,探讨其发病机制、病理学改变及治疗方法。     

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.     

Endogenous TDP-43 localized to stress granules can subsequently form protein aggregates

TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 and accumulation of the protein in the cytosol as ubiquitinated protein aggregates. These protein aggregates may have an important role in subsequent neuronal degeneration in motor neuron disease, frontotemporal dementia and potentially other neurodegenerative diseases. Although the cellular mechanisms driving the abnormal accumulation of TDP-43 are not understood, recent studies have shown that an early change to TDP-43 metabolism in disease may be accumulation in cytosolic RNA stress granules (SGs). However, it is unclear whether the TDP-43 in these SGs progresses to become irreversible protein aggregates as observed in patients. We have shown recently that paraquat-treated cells are a useful model for examining TDP-43 SG localization. In this study, we used the paraquat model to examine if endogenous TDP-43 in SGs can progress to more stable protein aggregates. We found that after treatment of HeLa cells overnight with paraquat, TDP-43 co-localized to SGs together with the ubiquitous SG marker, human antigen R (HuR). However, after a further incubation in paraquat-free, conditioned medium for 6h, HuR-positive SGs were rarely detected yet TDP-43 positive aggregates remained present. The majority of these TDP-43 aggregates were positive for ubiquitin. Further evidence for persistence of TDP-43 aggregates was obtained by treating cultures with cycloheximide after paraquat treatment. Cycloheximide abolished nearly all cytosolic HuR aggregation (SGs) but large TDP-43-positive aggregates remained. Finally, we showed that addition of ERK and JNK inhibitors together with paraquat blocked TDP-43-positive SG formation, while treatment with inhibitors after 24h paraquat exposure failed to reverse the TDP-43 accumulation. This failure was most likely due to the addition of inhibitors after maximal activation of the kinases at 4h post-paraquat treatment. These findings provide strong evidence that once endogenous TDP-43 accumulates in SGs, it has the potential to progress to stable protein aggregates as observed in neurons in TDP-43 proteinopathies. This may provide a therapeutic opportunity to inhibit the transition of TDP-43 from SG protein to aggregate.     

Cytoplasmic mislocalization and mitochondrial colocalization of TDP-43 are common features between normal aged and young mice

Transactive response DNA binding protein 43 (TDP-43) pathologies have been well recognized in various neurodegenerative disorders including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). However, there have been limited studies on whether there are any TDP-43 alterations in normal aging. We investigated TDP-43 distribution in different brain regions in normal aged (n = 3 for 26- or 36-month-old) compared to young (n = 3 for 6- or 12-month-old) mice. In both normal aged and young mice, TDP-43 and phosphorylated TDP-43 (pTDP-43) demonstrated a unique pattern of distribution in neurons in some specific brain regions including the pontine nuclei, thalamus, CA3 region of the hippocampus, and orbital cortex. This pattern was demonstrated on higher magnification of high-resolution double fluorescence images and confocal microscopy as mislocalization of TDP-43 and pTDP-43, characterized by neuronal nuclear depletion and cytoplasmic accumulation in these brain regions, as well as colocalization between TDP-43 or pTDP-43 and mitochondria, similar to what has been described previously in neurodegenerative disorders. All these findings were identical in both normal aged and young mice. In summary, TDP-43 and pTDP-43 mislocalization from nucleus to cytoplasm and their colocalization with mitochondria in the specific brain regions are present not only in aging, but also in young healthy states. Our findings provide a new insight for the role of TDP-43 proteinopathy in health and diseases, and that aging may not be a critical factor for the development of TDP-43 proteinopathy in subpopulations of neurons.Impact statementDespite increasing evidence implicating the important role of TDP-43 in the pathogenesis of a wide range of age-related neurodegenerative diseases, there is limited study of TDP-43 proteinopathy and its association with mitochondria during normal aging. Our findings of cytoplasmic accumulation of TDP-43 that is highly colocalized with mitochondria in neurons in selective brain regions in young animals in the absence of neuronal loss provide a novel insight into the development of TDP-43 proteinopathy and its contribution to neuronal loss.     

Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior

Pathological modifications in the highly conserved and ubiquitously expressed heterogeneous ribonucleoprotein TDP-43 were recently associated to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a late-onset disorder that affects predominantly motoneurons [Neumann, M. et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133, Sreedharan, J. et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668-1672, Kabashi, E. et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40, 572-574]. However, the function of TDP-43 in vivo is unknown and a possible direct role in neurodegeneration remains speculative. Here, we report that flies lacking Drosophila TDP-43 appeared externally normal but presented deficient locomotive behaviors, reduced life span and anatomical defects at the neuromuscular junctions. These phenotypes were rescued by expression of the human protein in a restricted group of neurons including motoneurons. Our results demonstrate the role of this protein in vivo and suggest an alternative explanation to ALS pathogenesis that may be more due to the lack of TDP 43 function than to the toxicity of the aggregates.     

Characterization of a series of 4-aminoquinolines that stimulate caspase-7 mediated cleavage of TDP-43 and inhibit its function

Dysfunction of the heterogeneous ribonucleoprotein TAR DNA binding protein 43 (TDP-43) is associated with neurodegeneration in diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we examine the effects of a series of 4-aminoquinolines with affinity for TDP-43 upon caspase-7-induced cleavage of TDP-43 and TDP-43 cellular function. These compounds were mixed inhibitors of biotinylated TG6 binding to TDP-43, binding to both free and occupied TDP-43. Incubation of TDP-43 and caspase-7 in the presence of these compounds stimulated caspase-7 mediated cleavage of TDP-43. This effect was antagonized by the oligonucleotide TG12, prevented by denaturing TDP-43, and exhibited a similar relation of structure to function as for the displacement of bt-TG6 binding to TDP-43. In addition, the compounds did not affect caspase-7 enzyme activity. In human neuroglioma H4 cells, these compounds lowered levels of TDP-43 and increased TDP-43 C-terminal fragments via a caspase-dependent mechanism. Subsequent experiments demonstrated that this was due to induction of caspases 3 and 7 leading to increased PARP cleavage in H4 cells with similar rank order of the potency among the compounds tests for displacement of bt-TG6 binding. Exposure to these compounds also reduced HDAC-6, ATG-7, and increased LC3B, consistent with the effects of TDP-43 siRNA described by other investigators. These data suggest that such compounds may be useful biochemical probes to further understand both the normal and pathological functions of TDP-43, and its cleavage and metabolism promoted by caspases.     

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.     

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.     

PTK2/FAK regulates UPS impairment via SQSTM1/p62 phosphorylation in TARDBP/TDP-43 proteinopathies

ABSTRACT

TARDBP/TDP-43 (TAR DNA binding protein) proteinopathies are a common feature in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer disease (AD). However, the molecular mechanisms underlying TARDBP-induced neurotoxicity are largely unknown. In this study, we demonstrated that TARDBP proteinopathies induce impairment in the ubiquitin proteasome system (UPS), as evidenced by an accumulation of ubiquitinated proteins and a reduction in proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2/FAK (PTK2 protein tyrosine kinase 2) as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduced ubiquitin aggregates and attenuated TARDBP-induced cytotoxicity in a Drosophila model of TARDBP proteinopathies. We further identified that phosphorylation of SQSTM1/p62 (sequestosome 1) at S403 (p-SQSTM1 [S403]), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TARDBP overexpression and is dependent on the activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of SQSTM1 (SQSTM1^S403A) significantly repressed the accumulation of insoluble poly-ubiquitinated proteins and neurotoxicity induced by TARDBP overexpression in neuronal cells. In addition, TBK1 (TANK binding kinase 1), a kinase that phosphorylates S403 of SQSTM1, was found to be involved in the PTK2-mediated phosphorylation of SQSTM1. Taken together, our data suggest that the PTK2-TBK1-SQSTM1 axis plays a critical role in the pathogenesis of TARDBP by regulating neurotoxicity induced by UPS impairment. Therefore, targeting the PTK2-TBK1-SQSTM1 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TARDBP proteinopathies.Abbreviations: ALP: macroautophagy/autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; ATXN2: ataxin 2; BafA1: bafilomycin A₁; cCASP3: cleaved caspase 3; CSNK2: casein kinase 2; FTLD: frontotemporal lobar degeneration; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; OPTN: optineurin; PTK2/FAK: PTK2 protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.     

基于GNM方法研究人TDP-43-DNA相互作用动力学及关键位点

TARDNA结合蛋白43(transactive response DNA binding protein 43,TDP-43),一种可变剪切因子,可以特异性地结合富含TG序列的DNA,涉及多种神经退行性疾病.分子动力学模拟方法虽然是研究分子间相互作用强有力的工具,但它非常耗时,且难以对有大的构象变化的体系进行充分采样...     

Identification of casein kinase-1 phosphorylation sites on TDP-43

TAR DNA-binding protein of 43 kDa (TDP-43) is deposited as hyperphosphorylated cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions and amyotrophic lateral sclerosis. In this study, we identified 29 phosphorylation sites on recombinant TDP-43 that are phosphorylated by casein kinase-1 (CK1). Interestingly, 18 of them were located in the C-terminal glycine-rich region of TDP-43. Our results indicate that CK1-mediated phosphorylation may play a role in the pathogenesis of these diseases.     

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.     

Neurodegeneration-associated TDP-43 interacts with fragile X mental retardation protein (FMRP)/Staufen (STAU1) and regulates SIRT1 expression in neuronal cells

Despite the identification of the 43 kDa transactive response DNA-binding protein (TDP-43) as a major pathological signatory protein in a wide range of neurodegenerative diseases, the mechanistic role of TDP-43 in neurodegenerative disorders is still poorly understood. Here, we report that TDP-43 is physically associated with fragile X mental retardation protein (FMRP) and Staufen (STAU1) to form a functional complex. Differential microarray analysis revealed that the expression of a collection of functionally important genes including Sirtuin (SIRT1) is regulated by this complex. RNA-immunoprecipitation (RIP) and RNA pull-down assays demonstrated that TDP-43/FMRP/STAU1 specifically binds to the 3'-UTR of SIRT1 mRNA, and that knockdown the expression of any one of these three proteins resulted in the reduction of SIRT1 mRNA and protein. SIRT1 is implicated in double-stranded DNA break repair and is required for cell survival. Indeed, depletion of TDP-43/FMRP/STAU1 sensitizes cells to apoptosis and DNA damages. Collectively, our results revealed a molecular mechanism for the cellular function of TDP-43 and might shed new light on the understanding of the mechanistic role of TDP-43 in neurodegenerative diseases.     

Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. We previously characterized a new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. In the present study, we demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub⁺) TBPH pathology. Although we found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, we herein showed that an augmented soluble cytoplasmic Ub⁺-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. We demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend our knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.