Disease-modifying effects of metabolic perturbations in ALS/FTLD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative disorders with considerable clinical, pathological and genetic overlap. Both disorders are characterized by the accumulation of pathological protein aggregates that contain a number of proteins, most notably TAR DNA binding protein 43?kDa (TDP-43). Surprisingly, recent clinical studies suggest that dyslipidemia, high body mass index, and type 2 diabetes mellitus are associated with better clinical outcomes in ALS. Moreover, ALS and FTLD patients have a significantly lower incidence of cardiovascular disease, supporting the idea that an unfavorable metabolic profile may be beneficial in ALS and FTLD. The two most widely studied ALS/FTLD models, super-oxide dismutase 1 (SOD1) and TAR DNA binding protein of 43 kDA (TDP-43), reveal metabolic dysfunction and a positive effect of metabolic strategies on disease onset and/or progression. In addition, molecular studies reveal a role for ALS/FTLD-associated proteins in the regulation of cellular and whole-body metabolism. Here, we systematically evaluate these observations and discuss how changes in cellular glucose/lipid metabolism may result in abnormal protein aggregations in ALS and FTLD, which may have important implications for new treatment strategies for ALS/FTLD and possibly other neurodegenerative conditions.     

TDP-43 proteinopathies: neurodegenerative protein misfolding diseases without amyloidosis

In this review, we summarize recent advances in understanding frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS) and related neurodegenerative disorders that are collectively known as TDP-43 proteinopathies, since transactive response DNA-binding protein 43 (TDP-43) was recently shown to be the major component of the ubiquitinated inclusions that are their pathological hallmarks. TDP-43 proteinopathies are distinct from most other neurodegenerative disorders because TDP-43 inclusions are not amyloid deposits. Besides TDP-43-positive inclusions, both sporadic and familial forms of FTLD and ALS have the pathologic TDP-43 signature of abnormal hyperphosphorylation, ubiquitination and C-terminal fragments in affected brain and spinal cord, suggesting that they share a common mechanism of pathogenesis. Thus, these findings support the concept that FTLD and ALS represent a clinicopathologic spectrum of one disease, that is, TDP-43 proteinopathy.     

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.     

The role of mutant TAR DNA-binding protein 43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

TDP-43 (TAR DNA-binding protein 43) has been identified as a key protein of ubiquitinated inclusions in brains of patients with ALS (amyotrophic lateral sclerosis) or FTLD (frontotemporal lobar degeneration), defining a new pathological disease spectrum. Recently, coding mutations have been identified in the TDP-43 gene (TARDBP), which further confirmed the pathogenic nature of the protein. Today, several animal models have been generated to gain more insight into the disease-causing pathways of the FTLD/ALS spectrum. This mini-review summarizes the current status of TDP-43 models, with a focus on mutant TDP-43.     

Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.     

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.     

Overexpression of ALS-Associated p.M337V Human TDP-43 in Mice Worsens Disease Features Compared to Wild-type Human TDP-43 Mice

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis (ALS), while wild-type TDP-43 is a pathological hallmark of patients with sporadic ALS and frontotemporal lobar degeneration (FTLD). Various in vitro and in vivo studies have also demonstrated toxicity of both mutant and wild-type TDP-43 to neuronal cells. To study the potential additional toxicity incurred by mutant TDP-43 in vivo, we generated mutant human TDP-43 (p.M337V) transgenic mouse lines driven by the Thy-1.2 promoter (Mt-TAR) and compared them in the same experimental setting to the disease phenotype observed in wild-type TDP-43 transgenic lines (Wt-TAR) expressing comparable TDP-43 levels. Overexpression of mutant TDP-43 leads to a worsened dose-dependent disease phenotype in terms of motor dysfunction, neurodegeneration, gliosis, and development of ubiquitin and phosphorylated TDP-43 pathology. Furthermore, we show that cellular aggregate formation or accumulation of TDP-43 C-terminal fragments (CTFs) are not primarily responsible for development of the observed disease phenotype in both mutant and wild-type TDP-43 mice.     

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.     

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.     

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component of the intracellular inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that both monoclonal (60019-2-Ig) and polyclonal (10782-2-AP) anti-TDP-43 antibodies recognize amino acids 203-209 of human TDP-43. The monoclonal antibody labeled human TDP-43 by recognizing Glu204, Asp205 and Arg208, but failed to react with mouse TDP-43. The antibodies stained the abnormally phosphorylated C-terminal fragments of 24-26 kDa in addition to normal TDP-43 in ALS and FTLD brains. Immunoblot analysis after protease treatment demonstrated that the epitope of the antibodies (residues 203-209) constitutes part of the protease-resistant domain of TDP-43 aggregates which determine a common characteristic of the pathological TDP-43 in both ALS and FTLD-TDP. The antibodies and methods used in this study will be useful for the characterization of abnormal TDP-43 in human materials, as well as in vitro and animal models for TDP-43 proteinopathies.     

TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo.     

神经退行性疾病中的TDP-43蛋白聚集和相变

随着全球老龄化人口的急剧增加,神经退行性变已经成为危害公共健康的主要疾病.在神经退行性疾病(肌萎缩侧索硬化症(ALS)、额颞叶变性病(FTLD)和阿尔茨海默病(AD)等)患者脑组织中均能观察到蛋白质聚集形成的包涵体,其中TAR DNA结合蛋白43 (TDP-43)是主要成分之一.目前已发现多个TDP-43基因突变与家族...     

Cytoplasmic accumulation and aggregation of TDP-43 upon proteasome inhibition in cultured neurons

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by intraneuronal deposition of the nuclear TAR DNA-binding protein 43 (TDP-43) caused by unknown mechanisms. Here, we studied TDP-43 in primary neurons under different stress conditions and found that only proteasome inhibition by MG-132 or lactacystin could induce significant cytoplasmic accumulation of TDP-43, a histopathological hallmark in disease. This cytoplasmic accumulation was accompanied by phosphorylation, ubiquitination and aggregation of TDP-43, recapitulating major features of disease. Proteasome inhibition produced similar effects in both hippocampal and cortical neurons, as well as in immortalized motor neurons. To determine the contribution of TDP-43 to cell death, we reduced TDP-43 expression using small interfering RNA (siRNA), and found that reduced levels of TDP-43 dose-dependently rendered neurons more vulnerable to MG-132. Taken together, our data suggests a role for the proteasome in subcellular localization of TDP-43, and possibly in disease.     

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。     

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.     

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.     

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