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.     

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.     

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

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

Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein-RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.     

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 is a culprit in human neurodegeneration, and not just an innocent bystander

In 2006 the protein TDP-43 was identified as the major ubiquitinated component deposited in the inclusion bodies found in two human neurodegenerative diseases, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenesis of both disorders is unclear, although they are related by having some overlap of symptoms and now by the shared histopathology of TDP-43 deposition. Now, in 2008, several papers have been published in quick succession describing mutations in the TDP-43 gene, showing they can be a primary cause of amyotrophic lateral sclerosis. There are many precedents in neurodegenerative disease in which rare single-gene mutations have given great insight into understanding disease processes, which is why the TDP-43 mutations are potentially very important.     

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.     

Circ-CL5A1受TDP-43调控并抑制肝细胞癌转移的研究

目的:探索Circ-COL5A1的生物学功能、调控机制和作用机制,进而为HCC转移的干预提供候选分子并进一步了解HCC转移。方法:通过前期工作基础选定目标分子Circ-COL5A1。通过慢病毒转染在HCC细胞系中过表达Circ-COL5A1,进而通过划痕愈合实验、transwell实验观察Circ-COL5A1的生物学功能。通过生物信息学分析、表达干扰实验和RNA免疫共沉淀(RIP)实验探究目标分子的调控机制。通过western blot技术、实时定量PCR(qRT-PCR)技术对目标分子的下游作用机制进行初步探索。结果:Circ-COL5A1在肝癌干细胞中表达下调,而且Circ-COL5A1过表达的HCC细胞系侵袭和迁移能力减弱。在Circ-COL5A1生物学合成过程中,RNA结合蛋白TDP-43可以富集其线性前体,并在环化结构形成后解离。Circ-COL5A1还可以降低其亲本基因V型胶原蛋白α1链(COL5A1)的蛋白质表达水平,这可能会影响多个信号通路进而干预HCC的转移过程。结论:内源性的Circ-COL5A1可以抑制HCC的转移能力,可以为阻断HCC转移提供候选分子。TDP-43的促进环状RNA形成提示RNA结合蛋白是环状RNA生物学合成过程中的重要调控因子。Circ-COL5A1可以通过转录后调控抑制其亲本基因COL5A1的表达。     

Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent.

Annexin I (also called lipocortin 1) is a 37-kDa member of the annexin family of proteins. It has been proposed to be involved in the regulation of cell growth and differentiation, apoptosis, and inflammation. Previously, we have reported that annexin I displays a chaperone-like function (Kim, G.Y., Lee, H.B., Lee, S.O., Rhee, H.J. & Na, D.S. (1997) Biochem. Mol. Biol. Int. 43, 521-528). To determine the possibility that annexin I is a stress protein, we examined whether expression of annexin I and annexin I mRNA increases in response to stresses in A549 and HeLa cells. Treatments of cells with heat, hydrogen peroxide or sodium arsenite resulted in (a) an increase in annexin I and annexin I mRNA and (b) translocation of annexin I from the cytoplasm to the nucleus and perinuclear region. The annexin I gene promoter region, cloned upstream of a reporter gene, was inducible in response to heat, hydrogen peroxide, and sodium arsenite. These results indicate that annexin I serves as a stress protein and annexins may constitute a new class of stress proteins.     

Vgl1, a multi-KH domain protein,is a novel component of the fission yeast stress granules required for cell survival under thermal stress

Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.     

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.     

Isoprostanes, a novel markers of oxidative stress in schizophrenic disorders

It is a well known fact that 3H-panthenol (PL) has a high bioavailability, so we studied its biotransformation and its protective action against lipoperoxide activation in homogenates and mitochondrial-synaptosomal fraction (11 000 g) of rat brain. The lipoperoxidation was initialized by Fe²⁺-ascorbate complex (Fe²⁺-Asc). In experiments in vivo , after 30 min, we demonstrated accumulation of intermediate products of CoA biosynthesis – pantothenic acid (PA), phospho-PA, and phosphopantetheine – in postmitochondrial fraction of brain, by using a HPLC technique. Addition of the PL (10 m m ) to brain hemispheres homogenates or mitochondrial-synaptosomal fraction caused a remarkable reduction of malondialdehyde production. However, 30 min preincubation with the PL, but not with PA, was ineffective. The data obtained may be a reason for a high neuroprotective activity of PL in curing brain diseases with vessel or alcohol-induced damages.