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Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD., Mutations in CHCHD10 can cause amyotrophic lateral sclerosis and frontotemporal dementia. The authors generate mutant Drosophila and HeLa cells to study the underlying mechanisms of CHCHD10S59L -induced degeneration and show it is mediated by TDP-43 and PINK1 pathways.

Prostaglandin (PG) A2 reportedly stimulated expression of heme oxygenase(HO)-1 at the level of transcription via the activation of p38MAPK. Details of the mechanism, however, have not been provided, and this includes identification of the transcription factors responsible for PGA2-induced HO-1 expression. Herein is described an analysis of the role of nuclear factor erythroid 2 related factor 2 (Nrf2) and how PGA2 increases the activity of Nrf2 during PGA2-induced HO-1 expression. Expressions of HO-1 and Nrf2 were analyzed at the levels of both mRNA and protein. Nrf2 siRNA, SB203580, an inhibitor of p38MAPK, and scavengers of reactive oxygen species (ROS) were used to identify the effects of Nrf2, p38MAPK and ROS on PGA2-induced HO-1 expression. Although SB203580 suppressed PGA2-induced HO-1 expression, genetic activation of p38MAPK could not stimulate the transcription of HO-1. Cycloheximide (CHX), an inhibitor of protein translation, almost completely prevented PGA2-induced increase of HO-1 transcription, but it did not prevent the phosphorylation of p38MAPK, which suggests that both de novo protein synthesis and p38MAPK activity are required to induce the transcription of HO-1 in response to PGA2 treatment. In addition, PGA2 increased the level of both Nrf2 mRNA and protein in a dose-dependent manner. Knockdown of Nrf2 using small interfering RNA (siRNA) suppressed PGA2-induced HO-1 expression. The PGA2-induced transcription of Nrf2 was prevented by ROS scavengers such as n-acetyl-l-cysteine and tempol but not CHX. Furthermore, siRNA against p38MAPK did not change the level of nuclear Nrf2 protein. These findings suggest that PGA2 induces HO-1 transcription via an increase in Nrf2 protein, the transcription of which is initiated by an accumulation of ROS that is independent of the p38MAPK activation pathway.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) are a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS-FTD). To elucidate how mutations in CHCHD10 induce disease, we generated a Drosophila melanogaster model of CHCHD10-mediated ALS-FTD. Expression of CHCHD10S59L in Drosophila caused gain-of-function toxicity in eyes, motor neurons, and muscles, in addition to mitochondrial defects in flies and HeLa cells. TDP-43 and PINK1 formed two axes, driving the mutant-dependent phenotypes. CHCHD10S59L expression increased TDP-43 insolubility and mitochondrial translocation. Blocking mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. PINK1 knockdown rescued CHCHD10S59L-mediated phenotypes in Drosophila and HeLa cells. The two PINK1 substrates mitofusin and mitofilin were genetic modifiers of this phenotype. Mitofusin agonists reversed the CHCHD10S59L-induced phenotypes in Drosophila and HeLa cells and increased ATP production in Drosophila expressing C9orf72 with expanded GGGGCC repeats. Two peptides inhibitors of PINK1 mitigated the mitochondrial defects introduced by CHCHD10S59L expression. These findings indicate that TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways by CHCHD10S59L generate dominant toxicity. Therefore, inhibiting PINK1 activity may provide a therapeutic strategy for CHCHD10-associated disease.One Sentence SummaryInhibition of TDP-43 mitochondrial translocation or PINK1 kinase activity mitigates CHCHD10S59L-mediated mitochondrial toxicity.

Prostaglandin (PG) A2, a cyclopentenone PG, arrested the growth of U2OS cells in the G2M phase. While inducing G2M arrest, PGA2 increased the expression of heme oxygenase-1 (HO-1) at the level of transcription along with the accumulation of ROS and the activation of MAPKs including JNK, p38MAPK, and ERK1/2. Among the MAPKs, the inhibition of p38MAPK by a specific chemical inhibitor SB203580, or by RNA interference, but not JNK or ERK1/2, attenuated the PGA2-induced transcription of HO-1. N-acetylcysteine (NAC), a ROS scavenger, prevented PGA2-induced G2M arrest, p38MAPK activation and transcriptional induction of HO-1. PGA2 also stimulated GADD45α expression at the level of transcription, and the knockdown of GADD45α repressed PGA2-induced G2M arrest. Finally, the knockdown of the HO-1 protein elevated PGA2-induced GADD45α expression as well as G2M arrest. Collectively, these results suggest that PGA2 causes an increase in ROS accumulation which initiates both HO-1 transcription via p38MAPK, and G2M arrest via GADD45α transcription, and HO-1 attenuates G2M arrest by modulating the expression of GADD45α.

Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.

A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expression, nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, nutlin‑3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-μ, an inhibitor of the mitochondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-μ reduced HO-1 expression and the phosphorylation of JNK induced by nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented nutlin-3-induced apoptosis. Collectively, these results suggest that nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.

Tight regulation of autophagy is critical for the fate of pancreatic β cells. The autophagy protein ATG5 is essential for the formation of autophagosomes by promoting the lipidation of microtubule-associated protein LC3 (light chain 3). However, little is known about the mechanisms that regulate ATG5 expression levels. In this study, we investigated the regulation of ATG5 expression by HuD. The association of HuD with ATG5 mRNA was analyzed by ribonucleoprotein complex immunoprecipitation and biotin pulldown assays. HuD expression levels in pancreatic β cells were knocked down via siRNA, elevated by overexpression of a HuD-expressing plasmid. The expression levels of HuD, ATG5, LC3, and β-actin were determined by Western blot and quantitative RT-PCR analysis. Autophagosome formation was assessed by fluorescence microscopy in GFP-LC3-expressing cells and in pancreatic tissues from WT and HuD-null mice. We identified ATG5 mRNA as a post-transcriptional target of the mammalian RNA-binding protein HuD in pancreatic β cells. HuD associated with the 3′-UTR of the ATG5 mRNA. Modulating HuD abundance did not alter ATG5 mRNA levels, but HuD silencing decreased ATG5 mRNA translation, and, conversely, HuD overexpression enhanced ATG5 mRNA translation. Through its effect on ATG5, HuD contributed to the lipidation of LC3 and the formation of LC3-positive autophagosomes. In keeping with this regulatory paradigm, HuD-null mice displayed lower ATG5 and LC3 levels in pancreatic β cells. Our results reveal HuD to be an inducer of ATG5 expression and hence a critical regulator of autophagosome formation in pancreatic β cells.

Anthocyanins (ATCs) have been reported to induce apoptosis in various types of cancer cells, stimulating the development of ATCs as a cancer chemotherapeutic or chemopreventive agent. It was recently reported that ATCs can induce autophagy, however, the mechanism for this remains unclear. In the present report, we carried out mechanistic studies of the mechanism involved in ATC-induced autophagy using ATCs extracted from black soybeans (cv. Cheongja 3, Glycine max L.). ATCs clearly induced hallmarks of autophagy, including LC3 puncta formation and the conversion of LC3-I to LC3-II in U2OS human osteosarcoma cells. The induction of autophagy was accompanied by the phosphorylation of multiple protein kinases including extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), protein kinase B (AKT) and adenosyl mono-phosphate-dependent protein kinase (AMPK). While chemical inhibitors against ERK1/2, p38 MAPK, JNK and AKT failed to inhibit ATC-induced autophagy, the suppression of AMPK by compound C (CC) as well as siRNA against AMPK reduced ATC-induced autophagy. The treatment of ATCs resulted in a decrease in intracellular ATP contents and the activation of AMPK by AICAR treatment also induced autophagy. It is noteworthy that the reduction of autophagy via the inhibition of AMPK resulted in enhanced apoptosis in ATC-treated cells. In addition, siRNA against forkhead box O3A (FOXO3a), a downstream target of AMPK, suppressed ATC-induced autophagy and p27KIP1 siRNA increased apoptosis in ATC-treated cells. Collectively, it can be concluded that ATCs induce autophagy in U2OS cells via activation of the AMPK-FOXO3a pathway and protect cells from ATC-induced apoptosis via the AMPK-p27KIP1 pathway. These results also suggest that autophagy-modulating agents could contribute to the efficient development of ATCs as anticancer therapy.

【3-Deazaadenosine (DZA), a potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential ( ${\Delta}{\Psi}_m$ ). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.】