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

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis

死にゆく細胞のATP濃度変化を詳細に可視化することに成功 --積極的にATP濃度を下げる因子を明らかに--. 京都大学プレスリリース. 2020-10-22.ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a ‘programmed’ process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP

A VCP modulator, KUS121, as a promising therapeutic agent for post-traumatic osteoarthritis

京大開発の薬剤「KUS121」の変形性膝関節症への効果を確認 --外傷性変形性関節症の治療薬として臨床応用へ--. 京都大学プレスリリース. 2020-12-17.Post-traumatic osteoarthritis (PTOA) is a major cause which hinders patients from the recovery after intra-articular injuries or surgeries. Currently, no effective treatment is available. In this study, we showed that inhibition of the acute stage chondrocyte death is a promising strategy to mitigate the development of PTOA. Namely, we examined efficacies of Kyoto University Substance (KUS) 121, a valosin-containing protein modulator, for PTOA as well as its therapeutic mechanisms. In vivo, in a rat PTOA model by cyclic compressive loading, intra-articular treatments of KUS121 significantly improved the modified Mankin scores and reduced damaged-cartilage volumes, as compared to vehicle treatment. Moreover, KUS121 markedly reduced the numbers of TUNEL-, CHOP-, MMP-13-, and ADAMTS-5-positive chondrocytes in the damaged knees. In vitro, KUS121 rescued human articular chondrocytes from tunicamycin-induced cell death, in both monolayer culture and cartilage explants. It also significantly downregulated the protein or gene expression of ER stress markers, proinflammatory cytokines, and extracellular-matrix-degrading enzymes induced by tunicamycin or IL-1β. Collectively, these results demonstrated that KUS121 protected chondrocytes from cell death through the inhibition of excessive ER stress. Therefore, KUS121 would be a new, promising therapeutic agent with a protective effect on the progression of PTOA

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

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα 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–RARα 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 RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα 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 RARα 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

Real-Time Imaging of Rabbit Retina with Retinal Degeneration by Using Spectral-Domain Optical Coherence Tomography

Background: Recently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT). Methodology/Principal Findings: Wild type (WT) and RP rabbits (aged 4–20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch’s membrane (ELM–BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors

Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5

A Trace Amount of Galactose, a Major Component of Milk Sugar, Allows Maturation of Glycoproteins during Sugar Starvation

Milk sugar is composed of glucose and galactose. Galactose is less suitable as an energy source than glucose. Thus, it has been a puzzle as to why mammals utilize galactose as a major component of milk sugar. Here we show that in hypoglycemic conditions, the presence of a trace amount of galactose, but not glucose, is able to maintain the production of mature glycoproteins and to abolish cell-death-inducing endoplasmic reticulum stress. In severely sugar-limited conditions, both glucose and galactose enter into the glycolytic pathway, but galactose is not able to raise the phosphofructokinase 1 activity, leading to the accumulation of fructose-6-phosphate, which in turn is utilized for the maturation of glycoproteins (e.g., growth factor receptors) and allows the activation of their intracellular signaling and prevents cell death from hypoglycemic conditions. Thus trace amounts of galactose may play unexpectedly important roles in the growth of infants and their protection during starvation

BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells

ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed "BTeam". BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the Îμ subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7-4.1 mM) and mitochondrial matrix (2.4-2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations