An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis.

The dynamic and reversible process of ubiquitin modification controls various cellular activities. Ubiquitin exists as monomers, unanchored chains, or protein-conjugated forms, but the regulation of these interconversions remains largely unknown. Here, we identified a protein designated Rfu1 (regulator of free ubiquitin chains 1), which regulates intracellular concentrations of monomeric ubiquitins and free ubiquitin chains in Saccharomyces cerevisiae. Rfu1 functions as an inhibitor of Doa4, a deubiquitinating enzyme. Rapid loss of free ubiquitin chains upon heat shock, a condition in which more proteins require ubiquitin conjugation, was mediated in part by Doa4 and Rfu1. Thus, regulation of ubiquitin homeostasis is controlled by a balance between a deubiquitinating enzyme and its inhibitor. We propose that free ubiquitin chains function as a ubiquitin reservoir that allows maintenance of monomeric ubiquitins at adequate levels under normal conditions and rapid supply for substrate conjugation under stress conditions.

A novel VCP modulator KUS121 exerts renoprotective effects in ischemia-reperfusion injury with retaining ATP and restoring ERAD-processing capacity.

Acute kidney injury (AKI) is a life-threatening condition and often progresses to chronic kidney disease or the development of other organ dysfunction even after recovery. Despite the increased recognition and high prevalence of AKI worldwide, there has been no established treatment so far. The aim of this study was to investigate the renoprotective effect of Kyoto University substance 121 (KUS121), a novel valosin-containing protein modulator, on AKI. In in vitro experiments, we evaluated cell viability and ATP levels of proximal tubular cells with or without KUS121 under endoplasmic reticulum (ER) stress conditions. In in vivo experiments, the effects of KUS121 were examined in mice with AKI caused by ischemia-reperfusion injury. ER-associated degradation (ERAD)-processing capacity was evaluated by quantification of the ERAD substrate CD3delta-YFP. KUS121 protected proximal tubular cells from cell death under ER stress. The apoptotic response was mitigated as indicated by the suppression of C/EBP homologous protein expression and caspase-3 cleavage, with maintained intracellular ATP levels by KUS121 administration. KUS121 treatment suppressed the elevation of serum creatinine and neutrophil gelatinase-associated lipocalin levels and attenuated renal tubular damage after ischemia-reperfusion. The expression of inflammatory cytokines in the kidney was also suppressed in the KUS121-treated group. Valosin-containing protein expression levels were not altered by KUS121 both in vitro and in vivo. KUS121 treatment restored ERAD-processing capacity associated with potentiation of its upstream pathway, phosphorylated inositol-requiring enzyme-1α, and spliced X box-binding protein-1. In conclusion, these findings indicate that KUS121 can protect renal tubular cells from ER stress-induced injury, suggesting that KUS121 could be a novel and promising therapeutic compound for ischemia-associated AKI.NEW & NOTEWORTHY Novel findings of this study are as follows: 1) Kyoto University substance 121 (KUS121), a novel valosin-containing protein (VCP) modulator, can reduce ATP consumption of VCP; 2) KUS121 reduced endoplasmic reticulum (ER) stress and improved cell viability in proximal tubular cells; 3) KUS121 exerted renoprotective effects against ischemia-reperfusion injury; and 4) KUS121 may prevent ischemic acute kidney injury with ATP retention and restoring ER-associated degradation capacity.

VCP relocalization limits mitochondrial activity, GSH depletion and ferroptosis during starvation in PC3 prostate cancer cells.

During periods of crisis, cells must compensate to survive. To this end, cells may need to alter the subcellular localization of crucial proteins. Here, we show that during starvation, VCP, the most abundant soluble ATPase, relocalizes and forms aggregate-like structures at perinuclear regions in PC3 prostate cancer cells. This movement is associated with a lowered metabolic state, in which mitochondrial activity and ROS production are reduced. VCP appears to explicitly sense glutamine levels, as removal of glutamine from complete medium triggered VCP relocalization and its addition to starvation media blunted VCP relocalization. Cells cultured in Gln(+) starvation media exhibited uniformly distributed VCP in the cytoplasm (free VCP) and underwent ferroptotic cell death, which was associated with a decrease in GSH levels. Moreover, the addition of a VCP inhibitor, CB-5083, in starvation media prevented VCP relocalization and triggered ferroptotic cell death. Likewise, expression of GFP-fused VCP proteins, irrespective of ATPase activities, displayed free VCP and triggered cell death during starvation. These results indicate that free VCP is essential for the maintenance of mitochondrial function and that PC3 cells employ a strategy of VCP self-aggregation to suppress mitochondrial activity in order to escape cell death during starvation, a novel VCP-mediated survival mechanism.

Emodin, as a mitochondrial uncoupler, induces strong decreases in adenosine triphosphate (ATP) levels and proliferation of B16F10 cells, owing to their poor glycolytic reserve.

Many types of cancer cells show a characteristic increase in glycolysis, which is called the "Warburg effect." By screening plant extracts, we identified one that decreases cellular adenosine triphosphate (ATP) levels and suppresses proliferation of malignant melanoma B16F10 cells, but not of noncancerous MEF cells. We showed that its active ingredient is emodin, which showed strong antiproliferative effects on B16F10 cells both in vitro and in vivo. Moreover, we also found that emodin can function as a mitochondrial uncoupler. Consistently, three known mitochondrial uncouplers also displayed potent antiproliferative effects and preferential cellular ATP reduction in B16F10 cells, but not in MEF cells. These uncouplers provoked comparable mitochondrial uncoupling in both cell types, but they manifested dramatically different cellular effects. Namely in MEF cells, these uncouplers induced three to fivefold increases in glycolysis from the basal state, and this compensatory activation appeared to be responsible for the maintenance of cellular ATP levels. In contrast, B16F10 cells treated with the uncouplers showed less than a twofold enhancement of glycolysis, which was not sufficient to compensate for the decrease of ATP production. Together, these results raise the possibility that uncouplers could be effective therapeutic agents specifically for cancer cells with prominent "Warburg effect."

Ellagic acid, extracted from Sanguisorba officinalis, induces G1 arrest by modulating PTEN activity in B16F10 melanoma cells.

In Chinese medicine, herbal medicine is commonly used to treat individuals suffering from many types of diseases. We thus expected that some herbal medicines would contain promising compounds for cancer chemotherapy. Indeed, we found that Sanguisorba officinalis extracts strongly inhibit the growth of B16F10 melanoma cells, and we identified ellagic acid (EA) as the responsible ingredient. B16F10 cells treated with EA exhibited strong G1 arrest accompanied by accumulation of p53, followed by inactivation of AKT. Addition of a PTEN inhibitor, but not a p53 inhibitor, abrogated the EA-induced AKT inactivation and G1 arrest. The PTEN inhibitor also diminished EA-induced p53 accumulation. Furthermore, EA apparently increased the protein phosphatase activity of PTEN, as demonstrated by the reduced phosphorylation level of FAK, a protein substrate of PTEN. Furthermore, an in vitro PTEN phosphatase assay on PIP3 showed the direct modulation of PTEN activity by EA. These results suggest that EA functions as an allosteric modulator of PTEN, enhancing its protein phosphatase activity while inhibiting its lipid phosphatase activity. It is notable that a combination of EA and cisplatin, a widely used chemotherapy agent, dramatically enhanced cell death in B16F10 cells, suggesting a promising strategy in chemotherapy.

Genetic screening for potassium channel mutations in Japanese autosomal dominant spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease characterized by cerebellar ataxia. Many causative genes have been identified to date, the most common etiology being the abnormal expansion of repeat sequences, and the mutation of ion channel genes also play an important role in the development of SCA. Some of them encode calcium and potassium channels. However, due to limited reports about potassium genes in SCA, we screened 192 Japanese individuals with dominantly inherited SCA who had no abnormal repeat expansions of causative genes for potassium channel mutations (KCNC3 for SCA13 and KCND3 for SCA19/SCA22) by target sequencing. As a result, two variants were identified from two patients: c.1973G>A, p.R658Q and c.1018G>A, p.V340M for KCNC3, and no pathogenic variant was identified for KCND3. The newly identified p.V340M exists in the extracellular domain, and p.R658Q exists in the intracellular domain on the C-terminal side, although most of the reported KCNC3 mutations are present at the transmembrane site. Adult-onset and slowly progressive cerebellar ataxia are the main clinical features of SCA13 and SCA19 caused by potassium channel mutations, which was similar in our cases. SCA13 caused by KCNC3 mutations may present with deep sensory loss and cognitive impairment in addition to cerebellar ataxia. In this study, mild deep sensory loss was observed in one case. SCA caused by potassium channel gene mutations is extremely rare, and more cases should be accumulated in the future to elucidate its pathogenesis due to channel dysfunction.

Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

Glucose-stimulated single pancreatic islets sustain increased cytosolic ATP levels during initial Ca2+ influx and subsequent Ca2+ oscillations.

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca(2+) levels throughout the islets during high glucose conditions. This Ca(2+) elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K(+) channels, leading to the depolarization of membranes, which in turn induces Ca(2+) elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca(2+) levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca(2+) dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca(2+) levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca(2+) increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca(2+) influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca(2+) level in the subsequent oscillation phases. Importantly, Ca(2+) oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.

Different dynamic movements of wild-type and pathogenic VCPs and their cofactors to damaged mitochondria in a Parkin-mediated mitochondrial quality control system.

VCP/p97 is a hexameric ring-shaped AAA(+) ATPase that participates in various ubiquitin-associated cellular functions. Mis-sense mutations in VCP gene are associated with the pathogenesis of two inherited diseases: inclusion body myopathy associated with Paget's disease of the bone and front-temporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). These pathogenic VCPs have higher affinities for several cofactors, including Npl4, Ufd1 and p47. In Parkin-dependent mitochondrial quality control systems, VCP migrates to damaged mitochondria (e.g., those treated with uncouplers) to aid in the degradation of mitochondrial outer membrane proteins and to eliminate mitochondria. We showed that endogenous Npl4 and p47 also migrate to mitochondria after uncoupler treatment, and Npl4, Ufd1 or p47 silencing causes defective mitochondria clearance after uncoupler treatment. Moreover, pathogenic VCPs show impaired migration to mitochondria, and the exogenous pathogenic VCP expression partially inhibits Npl4 and p47 localization to mitochondria. These results suggest that the increased affinities of pathogenic VCPs for these cofactors cause the impaired movement of pathogenic VCPs. In adult flies, exogenous expression of wild-type VCP, but not pathogenic VCPs, reduces the number of abnormal mitochondria in muscles. Failure of pathogenic VCPs to function on damaged mitochondria may be related to the pathogenesis of IBMPFD and ALS.

KUS121, a VCP modulator, has an ameliorating effect on acute and chronic heart failure without calcium loading via maintenance of intracellular ATP levels.

AIMS: As heart failure (HF) progresses, ATP levels in myocardial cells decrease, and myocardial contractility also decreases. Inotropic drugs improve myocardial contractility but increase ATP consumption, leading to poor prognosis. Kyoto University Substance 121 (KUS121) is known to selectively inhibit the ATPase activity of valosin-containing protein, maintain cellular ATP levels, and manifest cytoprotective effects in several pathological conditions. The aim of this study is to determine the therapeutic effect of KUS121 on HF models. METHODS AND RESULTS: Cultured cell, mouse, and canine models of HF were used to examine the therapeutic effects of KUS121. The mechanism of action of KUS121 was also examined. Administration of KUS121 to a transverse aortic constriction (TAC)-induced mouse model of HF rapidly improved the left ventricular ejection fraction and improved the creatine phosphate/ATP ratio. In a canine model of high frequency-paced HF, administration of KUS121 also improved left ventricular contractility and decreased left ventricular end-diastolic pressure without increasing the heart rate. Long-term administration of KUS121 to a TAC-induced mouse model of HF suppressed cardiac hypertrophy and fibrosis. In H9C2 cells, KUS121 reduced ER stress. Finally, in experiments using primary cultured cardiomyocytes, KUS121 improved contractility and diastolic capacity without changing peak Ca2+ levels or contraction time. These effects were not accompanied by an increase in cyclic adenosine monophosphate or phosphorylation of phospholamban and ryanodine receptors. CONCLUSIONS: KUS121 ameliorated HF by a mechanism totally different from that of conventional catecholamines. We propose that KUS121 is a promising new option for the treatment of HF.

Prostaglandin E receptor EP1 forms a complex with dopamine D1 receptor and directs D1-induced cAMP production to adenylyl cyclase 7 through mobilizing G(ßγ) subunits in human embryonic kidney 293T cells.

The mechanism underlying the crosstalk between multiple G protein-coupled receptors remains poorly understood. We previously reported that prostaglandin E receptor EP1 facilitates dopamine D1 receptor signaling in striatal slices and promotes behavioral responses induced by D1 receptor agonists. Here, using human embryonic kidney (HEK)-293T cells expressing D1 and EP1, we have analyzed the mechanism underlying EP1-mediated facilitation of D1 receptor signaling. Fluorescent immunostaining showed that EP1 and D1 receptors are partly colocalized in the cells, and coprecipitation experiments revealed a molecular complex of EP1 and D1 receptors. Treatment of the cells with 17S,17,20-dimethyl-2,5-ethano-6-oxo-PGE1 (ONO-DI-004), an EP1-selective agonist, enhanced cAMP production induced by D1 agonists (±)-6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide (SKF-81297) and 6-chloro-2,3,4,5-tetrahydro-1-(3-methylphenyl)-3-(2-propenyl)-1H-3-benzazepine-7,8-diol hydrobromide (SKF-83822). Although this facilitative effect of EP1 stimulation was not affected by pharmacologic blockade of EP1-induced Ca²âº increase, it was blocked by overexpression of G(tα) as a G(ßγ) scavenger. Consistently, depletion of adenylyl cyclase (AC) 7, a G(ßγ)-sensitive AC isoform, abolished the facilitative action of EP1 on D1-induced cAMP production. Notably, neither G(tα) overexpression nor AC7 depletion affected cAMP production induced by D1 stimulation alone. In contrast, depletion of AC6, another AC isoform, reduced cAMP production induced by D1 stimulation alone, but spared its facilitation by EP1 stimulation. Collectively, these data suggest that, through complex formation with D1, EP1 signaling directs the D1 receptor through G(ßγ) to be coupled to AC7, an AC isoform distinct from those used by the D1 receptor alone, in HEK-293T cells.

In vivo fluorescent adenosine 5'-triphosphate (ATP) imaging of Drosophila melanogaster and Caenorhabditis elegans by using a genetically encoded fluorescent ATP biosensor optimized for low temperatures.

Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important functions, the spatiotemporal dynamics of ATP levels inside living multicellular organisms is unclear. In this study, we modified the genetically encoded Förster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 °C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.

Survival and process outgrowth of human iPSC-derived cells expressing Purkinje cell markers in a mouse model for spinocerebellar degenerative disease.

Purkinje cells are the sole output neurons of the cerebellar cortex and play central roles in the integration of cerebellum-related motor coordination and memory. The loss or dysfunction of Purkinje cells due to cerebellar atrophy leads to severe ataxia. Here we used in vivo transplantation to examine the function of human iPS cell-derived cerebellar progenitors in adult transgenic mice in which Purkinje-specific cell death occurs due to cytotoxicity of polyglutamines. Transplantation using cerebellar organoids (42-48 days in culture), which are rich in neural progenitors, showed a viability of >50% 4 weeks after transplantation. STEM121+ grafted cells extended their processes toward the deep cerebellar nuclei, superior cerebellar peduncle, and vestibulocerebellar nuclei. The transplanted cells were mostly located in the white matter, and they were not found in the Purkinje cell layer. MAP2-positive fibers seen in the molecular layer of cerebellar cortex received VGluT2 inputs from climbing fibers. Transplanted neural progenitors overgrew in the host cerebellum but were suppressed by pretreatment with the γ-secretase inhibitor DAPT. Hyperproliferation was also suppressed by transplantation with more differentiated organoids (86 days in culture) or KIRREL2-positive cells purified by FACS sorting. Transplanted cells expressed Purkinje cell markers, GABA, CALB1 and L7, though they did not show fan-shaped morphology. We attempted to improve neuronal integration of stem cell-derived cerebellar progenitors by transplantation into the adult mouse, but this was not successfully achieved. Our findings in the present study contribute to regenerative medical application for cerebellar degeneration and provide new insights into cerebellar development in future.

Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs.

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.

In vivo analysis of the role of aberrant histone deacetylase recruitment and RAR alpha blockade in the pathogenesis of acute promyelocytic leukemia.

The promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARalpha to inhibit RARalpha function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1-RARalpha fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARalpha mutants, as well as that of PML-RARalpha mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARalpha did act as a bona fide DN RARalpha mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARalpha mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML(-/-) and p53(-/-) compound mutants lends support to a model by which the RARalpha and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

Valosin-containing protein (VCP) in novel feedback machinery between abnormal protein accumulation and transcriptional suppression.

Abnormal protein accumulation is often observed in human neurodegenerative disorders such as polyglutamine diseases and Parkinson disease. Genetic and biochemical analyses indicate that valosin-containing protein (VCP) is a crucial molecule in the pathogenesis of human neurodegenerative disorders. We report here that VCP was specifically modified in neuronal cells with abnormal protein accumulation; this modification caused the translocation of VCP into the nucleus. Modification-mimic forms of VCP induced transcriptional suppression with deacetylation of core histones, leading to cell atrophy and the decrease of de novo protein synthesis. Preventing VCP nuclear translocation in polyglutamine-expressing neuronal cells and Drosophila eyes mitigated neurite retraction and eye degenerations, respectively, concomitant with the recovery of core histone acetylation. This represents a novel feedback mechanism that regulates abnormal protein levels in the cytoplasm during physiological processes, as well as in pathological conditions such as abnormal protein accumulation in neurodegenerations.

Enhanced ATPase activities as a primary defect of mutant valosin-containing proteins that cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia.

Valosin-containing protein (VCP) has been shown to colocalize with abnormal protein aggregates, such as nuclear inclusions of Huntington disease and Machado-Joseph disease, Lewy bodies in Parkinson disease. Several mis-sense mutations in the human VCP gene have been identified in patients suffering inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). Recently, we have shown that VCP possesses both aggregate-forming and aggregate-clearing activities. Here, we showed that in cells treated with proteasome inhibitors VCP first appeared as several small aggregates throughout the cells; and then, these small aggregates gathered together into a single big aggregate. Subcellular localization and ATPase activity of VCP clearly influenced the localization of the aggregates. Furthermore, all tested IBMPFD-causing mutant VCPs, possessed elevated ATPase activities and enhanced aggregate-forming activities in cultured cells. In Drosophila, these mutants and VCP(T761E), a super active VCP, did not appear to spontaneously induce eye degeneration, but worsened the phenotype when co-expressed with polyglutamines. Unexpectedly, these VCPs did not apparently change sizes and the amounts of polyglutamine aggregates in Drosophila eyes. Elevated ATPase activities, thus, may be a hidden primary defect causing IBMPFD pathological phenotypes, which would be revealed when abnormal proteins are accumulated, as typically observed in aging.

KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats.

Currently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

Maintaining ATP levels via the suppression of PERK-mediated rRNA synthesis at ER stress.

Currently, [(3)H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [alpha-(33)P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1alpha or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2alpha. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2alpha knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.

Glucosamine induces autophagy via an mTOR-independent pathway.

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 microM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.