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.     

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.     

Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43

Intracellular aggregates of phosphorylated TDP-43 are a major component of ubiquitin-positive inclusions in the brains of patients with frontotemporal lobar degeneration and ALS and are considered a pathological hallmark. Here, to gain insight into the mechanism of intracellular TDP-43 accumulation, we examined the relationship between phosphorylation and aggregation of TDP-43. We found that expression of a hyperactive form of casein kinase 1 δ (CK1δ1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 (ubiquitin- and p62-positive) in cultured neuroblastoma SH-SY5Y cells. Insoluble phosphorylated TDP-43 prepared from cells co-expressing TDP-43 and CK1δ1-317 functioned as seeds for TDP-43 aggregation in cultured cells, indicating that CK1δ1-317-induced aggregated TDP-43 has prion-like properties. A striking toxicity and alterations of TDP-43 were also observed in yeast expressing TDP-43 and CK1δ1-317. Therefore, abnormal activation of CK1δ causes phosphorylation of TDP-43, leading to the formation of cytoplasmic TDP-43 aggregates, which, in turn, may trigger neurodegeneration.     

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 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.     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells

The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)(n) consensus motif in 80% of 3'-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3'-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.     

ALS skin fibroblasts reveal oxidative stress and ERK1/2-mediated cytoplasmic localization of TDP-43

The main hallmark of many forms of familiar and sporadic amyotrophic lateral sclerosis (ALS) is a reduction in nuclear TDP-43 protein and its inclusion in cytoplasmic aggregates in motor neurons. In order to understand which cellular and molecular mechanisms underlie the mislocalization of TDP-43, we examined human skin fibroblasts from two individuals with familial ALS, both with mutations in TDP-43, and two individuals with sporadic ALS, both without TDP-43 mutations or mutations in other ALS related genes. We found that all ALS fibroblasts had a partially cytoplasmic localization of TDP-43 and had reduced cell metabolism as compared to fibroblasts from apparently healthy individuals. ALS fibroblasts showed an increase in global protein synthesis and an increase in 4E-BP1 and rpS6 phosphorylation, which is indicative of mTORC1 activity. We also observed a decrease in glutathione (GSH), which suggests that oxidative stress is elevated in ALS. ERK1/2 activity regulated the extent of oxidative stress and the localization of TDP-43 in the cytoplasm in all ALS fibroblasts. Lastly, ALS fibroblasts showed reduced stress granule formation in response to H₂O₂ stress. In conclusion, these findings identify specific cellular and molecular defects in ALS fibroblasts, thus providing insight into potential mechanisms that may also occur in degenerating motor neurons.     

TDP-43 regulates its mRNA levels through a negative feedback loop

TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.     

Charge and redox states modulate granulin—TDP-43 coacervation toward phase separation or aggregation

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1–7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN’s absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.     

TDP-43: gumming up neurons through protein-protein and protein-RNA interactions

Since the discovery that 43 kDa TAR DNA binding protein (TDP-43) is involved in neurodegeneration, studies of this protein have focused on the global effects of TDP-43 expression modulation on cell metabolism and survival. The major difficulty with these global searches, which can yield hundreds to thousands of variations in gene expression level and/or mRNA isoforms, is our limited ability to separate specific TDP-43 effects from secondary dysregulations occurring at the gene expression and various mRNA processing steps. In this review, we focus on two biochemical properties of TDP-43: its ability to bind RNA and its protein-protein interactions. In particular, we overview how these two properties may affect potentially very important processes for the pathology, from the autoregulation of TDP-43 to aggregation in the cytoplasmic/nuclear compartments.     

Gene Targeting of Mouse Tardbp Negatively Affects Masp2 Expression

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp.     

C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

Background

TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing.

Results

We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs.

Conclusions

Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.     

Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm

Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.     

Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells

We report phosphorylated and ubiquitinated aggregates of TAR DNA binding protein of 43 kDa (TDP-43) in SH-SY5Y cells similar to those in brains of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). Two candidate sequences for the nuclear localization signal were examined. Deletion of residues 78-84 resulted in cytoplasmic localization of TDP-43, whereas the mutant lacking residues 187-192 localized in nuclei, forming unique dot-like structures. Proteasome inhibition caused these to assemble into phosphorylated and ubiquitinated TDP-43 aggregates. The deletion mutants lacked the exon skipping activity of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Our results suggest that intracellular localization of TDP-43 and proteasomal function may be involved in inclusion formation and neurodegeneration in TDP-43 proteinopathies.