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.     

Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.     

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-Associated TDP-43 Induces Endoplasmic Reticulum Stress,Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.     

HuR-positive stress granules: Potential targets for age-related osteoporosis

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target β-catenin by binding and recruiting β-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of β-catenin into HuR-positive SGs, facilitated β-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.     

An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.     

Inhibition of autophagy,lysosome and VCP function impairs stress granule assembly

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.Cells respond to stresses, like heat shock or oxidative agents, which lead to protein aggregation, by activating the protein quality control (PQC) and attenuating translation.¹ The PQC consists of molecular chaperones and degradation systems and is an essential player of the proteotoxic stress response. To minimize protein aggregation, chaperones assist protein folding; when this is not effective, chaperones assist in targeting damaged substrates for clearance by the ubiquitin–proteasome system (UPS) and the lysosome-based degradation systems.^{2, 3} In parallel, polysomes disassemble, releasing ribosomes, mRNAs, defective ribosomal products (DRIPs) and newly synthesized proteins, which, due to the stress, are prone to aggregation and are subjected to PQC and degradation.⁴The mRNAs encoding ‘housekeeping'' proteins released from disassembling polysomes are sequestered into stress granules (SGs), non-membranous cytoplasmic foci where mRNAs are stored during stress.⁵ SGs have heterogeneous compositions and contain translationally silent mRNAs, early initiation factors, small, but not large, ribosomal subunits, mRNA-binding proteins, kinases and signaling molecules.⁵ Selective sequestration of these components within SGs occurs in a challenging subcellular environment where aggregate-prone substrates (released by polysomes) tend to accumulate. SG assembly is also triggered by the self-aggregation of RNA-binding proteins that contain prion-like domains, including T-cell-restricted intracellular antigen-1 (TIA-1).⁶ Unlike prionogenic fibrillar aggregates, SGs are dynamic structures, which disassemble within few hours after their formation.Due to the heterogeneous composition of SGs and to the crowded molecular environment, SGs may, indirectly, require PQC assistance for proper assembly and disassembly. A number of SG components have a role in PQC, including ubiquitin and E3 ubiquitin ligases (TNF receptor-associated factor 2 and Roquin),^{7, 8, 9, 10} while proteasome inhibition induces SGs.¹¹ Histone deacetylase 6 (HDAC6), another SG component,⁸ facilitates the clearance of misfolded ubiquitinated proteins and participates in their targeting to the aggresome, a perinuclear structure that forms in response to an overload of un/misfolded proteins and enhances the degradation of toxic proteins.¹² Moreover, HDAC6 binds to another SG component, Ras-GTPase-activating protein SH3 domain-binding protein (G3BP), which modulates the de-ubiquitinating enzyme ubiquitin specific peptidase 10 (USP10), which is also required for SG formation.^{13, 14} Although the exact role of these PQC components in SG dynamics is only partly understood, these findings suggest that PQC and SGs are interconnected systems. SGs are degraded via macroautophagy (which we call autophagy) via a mechanism requiring the ubiquitin-selective chaperone valosin-containing protein (VCP).¹⁵ VCP modulates the ubiquitin-dependent proteolysis of selective clients by proteasome, ER-associated degradation and/or autophagosomes;^{16, 17, 18} this underscores the link between SGs and proteostasis. Here, we investigated whether impairment of PQC, autophagy and lysosomes affects SG assembly. We demonstrate that inhibition of VCP, autophagy or lysosomes affects SG formation, morphology and composition.     

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.     

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

Background

The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.

Methods

We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.

Results

Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.

Conclusions

Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

     

The chemotherapeutic agent bortezomib induces the formation of stress granules

Background

Cytoplasmic stress granules (SGs) are specialized storage sites of untranslated mRNAs whose formation occurs under different stress conditions and is often associated with cell survival. SGs-inducing stresses include radiations, hypoxia, viral infections, and chemical inhibitors of specific translation initiation factors. The FDA-approved drug bortezomib (Velcade^®) is a peptide boronate inhibitor of the 26S proteasome that is very efficient for the treatment of myelomas and other hematological tumors. Solid tumors are largely refractory to bortezomib. In the present study, we investigated the formation of SGs following bortezomib treatment.

Results

We show that bortezomib efficiently induces the formation of SGs in cancer cells. This process involves the phosphorylation of translation initiation factor eIF2α by heme-regulated inhibitor kinase (HRI). Depletion of HRI prevents bortezomib-induced formation of SGs and promotes apoptosis.

Conclusions

This is the first study describing the formation of SGs by a chemotherapeutic compound. We speculate that the activation of HRI and the formation of SGs might constitute a mechanism by which cancer cells resist bortezomib-mediated apoptosis.

     

Roles of YB-1 under arsenite-induced stress: Translational activation of HSP70 mRNA and control of the number of stress granules

Background

When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.

Method

Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.

Results

Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.

Conclusion

In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.

General significance

Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.     

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.     

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.     

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.     

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.     

RhoA/ROCK1 signaling regulates stress granule formation and apoptosis

Cells form stress granules (SGs), in response to unfavorable environments, to avoid apoptosis, but it is unclear whether and how SG formation and cellular apoptosis are coordinately regulated. In this study we detected the small GTPase, Ras homolog gene family member A (RhoA), and its downstream kinase, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), in SG, and found that their stress-induced activities were important for SG formation and subsequent global translational repression. Importantly, only activated RhoA and ROCK1 were sequestered into SG. Sequestration of activated ROCK1 into SG prevented ROCK1 from interacting with JNK-interacting protein 3 (JIP-3) and its activation of c-Jun N-terminal kinase (JNK), a pathway triggering apoptosis, thereby protecting cells from apoptosis. This study identifies a specific signaling pathway, mediated by RhoA and ROCK1, which determines cell fate by promoting SG formation or initiating apoptosis during stress.