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.     

TLR3 regulates mycobacterial RNA-induced IL-10 production through the PI3K/AKT signaling pathway

Cytokine induction in response to Mycobacterium tuberculosis (Mtb) infection is critical for pathogen control, by (i) mediating innate immune effector functions and (ii) instructing specific adaptive immunity. IL-10 is an important anti-inflammatory cytokine involved in pathogenesis of tuberculosis (TB). Here, we show that TLR3, a sensor of extracellular viral or host RNA with stable stem structures derived from infected or damaged cells, is essential for Mtb-induced IL-10 production. Upon Mycobacterium bovis Bacillus Calmette–Guérin (BCG) infection, TLR3^−/− macrophages expressed lower IL-10 but higher IL-12p40 production, accompanied by reduced phosphorylation of AKT at Ser473. BCG-infected TLR3^−/− mice exhibited reduced IL-10 but elevated IL-12 expression compared to controls. Moreover, higher numbers of splenic Th1 cells and reduced pulmonary bacterial burden and tissue damage were observed in BCG-infected TLR3^−/− mice. Finally, BCG RNA induced IL-10 in macrophages via TLR3-mediated activation of PI3K/AKT. Our findings demonstrate a critical role of TLR3-mediated regulation in the pathogenesis of mycobacterial infection involving mycobacterial RNA, which induces IL-10 through the PI3K/AKT signaling pathway.     

Antagonistic Interplay between MicroRNA-155 and IL-10 during Lyme Carditis and Arthritis

MicroRNA-155 has been shown to play a role in immune activation and inflammation, and is suppressed by IL-10, an important anti-inflammatory cytokine. The established involvement of IL-10 in the murine model of Borrelia burgdorferi-induced Lyme arthritis and carditis allowed us to assess the interplay between IL-10 and miR-155 in vivo. As reported previously, Mir155 was highly upregulated in joints from infected severely arthritic B6 Il10^-/- mice, but not in mildly arthritic B6 mice. In infected hearts, Mir155 was upregulated in both strains, suggesting a role of miR-155 in Lyme carditis. Using B. burgdorferi-infected B6, Mir155^-/-, Il10^-/-, and Mir155^-/- Il10^-/- double-knockout (DKO) mice, we found that anti-inflammatory IL-10 and pro-inflammatory miR-155 have opposite and somewhat compensatory effects on myeloid cell activity, cytokine production, and antibody response. Both IL-10 and miR-155 were required for suppression of Lyme carditis. Infected Mir155^-/- mice developed moderate/severe carditis, had higher B. burgdorferi numbers, and had reduced Th1 cytokine expression in hearts. In contrast, while Il10^-/- and DKO mice also developed severe carditis, hearts had reduced bacterial numbers and elevated Th1 and innate cytokine expression. Surprisingly, miR-155 had little effect on Lyme arthritis. These results show that antagonistic interplay between IL-10 and miR-155 is required to balance host defense and immune activation in vivo, and this balance is particularly important for suppression of Lyme carditis. These results also highlight tissue-specific differences in Lyme arthritis and carditis pathogenesis, and reveal the importance of IL-10-mediated regulation of miR-155 in maintaining healthy immunity.     

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.     

TDP-1/TDP-43 potentiates human α-Synuclein (HASN) neurodegeneration in Caenorhabditis elegans

TAR DNA binding protein (TDP-43) is a DNA/RNA binding protein whose pathological role in amyotrophic lateral sclerosis (ALS) and frontal temporal lobe dementia (FTLD) via formation of protein aggregates is well established. In contrast, knowledge concerning its interactions with other neuropathological aggregating proteins is poorly understood. Human α-synuclein (HASN) elicits dopaminergic neuron degeneration via protein aggregation in Parkinson's disease. HASN protein aggregates are also found in TDP-43 lesions and colocalize in Lewy Body Dementia (LBD). To better understand the interactions of TDP-43 and HASN, we investigated the effects of genetic deletion of tdp-1, the Caenorhabditis elegans ortholog of human TDP-43, as well as overexpression of TDP-43, in transgenic models overexpressing HASN^WT and HASN^A53T. Tdp-1 deletion improved the posture, movement, and developmental delay observed in transgenic animals pan-neuronally overexpressing HASN^A53T, and attenuated the loss and impairment of dopaminergic neurons caused by HASN^A53T or HASN^WT overexpression. Tdp-1 deletion also led to a decrease in protein level, mRNA level and aggregate formation of HASN in living animals. RNA-seq studies suggested that tdp-1 supports expression of lysosomal genes and decreases expression of genes involved in heat shock. RNAi demonstrated that heat shock proteins can mediate HASN neuropathology. Co-overexpression of both human TDP-43 and HASN^WT resulted in locomotion deficits, shorter lifespan, and more severe dopaminergic neuron impairments compared to single transgenes. Our results suggest TDP-1/TDP-43 potentiates HASN mediated neurodegeneration in C. elegans. This study indicates a multifunctional role for TDP-1/TDP-43 in neurodegeneration involving HASN.     

UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3^C145S was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43^K263E ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43^K263E ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.     

The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.     

Partial IL-10 inhibition of the cell-mediated immune response in chronic beryllium disease.

Chronic beryllium disease (CBD) provides a human disorder in which to study the delayed type IV hypersensitivity response to persistent Ag that leads to noncaseating pulmonary granuloma formation. We hypothesized that, in CBD, failure of IL-10 to modulate the beryllium-specific, cell-mediated immune response would result in persistent, maximal cytokine production and T lymphocyte proliferation, thus contributing to the development of granulomatous lung disease. To test this hypothesis, we used bronchoalveolar lavage cells from control and CBD subjects to evaluate the beryllium salt-specific production of endogenous IL-10 and the effects of exogenous human rIL-10 (rhIL-10) on HLA expression, on the production of IL-2, IFN-gamma, and TNF-alpha, and on T lymphocyte proliferation. Our data demonstrate that beryllium-stimulated bronchoalveolar lavage cells produce IL-10, and the neutralization of endogenous IL-10 does not increase significantly cytokine production, HLA expression, or T lymphocyte proliferation. Second, the addition of excess exogenous rhIL-10 partially inhibited the beryllium-stimulated production of IL-2, IFN-gamma, and TNF-alpha; however, we measured no change in T lymphocyte proliferation or in the percentage of alveolar macrophages expressing HLA-DP. Interestingly, beryllium salts interfered with an IL-10-stimulated decrease in the percentage of alveolar macrophages expressing HLA-DR. We conclude that, in the CBD-derived, beryllium-stimulated cell-mediated immune response, low levels of endogenous IL-10 have no appreciable effect; exogenous rhIL-10 has a limited effect on cytokine production and no effect on T lymphocyte proliferation or HLA expression.     

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.