The Novel Neuroprotective Compound KMS99220 Has an Early Anti-neuroinflammatory Effect via AMPK and HO-1, Independent of Nrf2

We have previously reported a novel synthetic compound KMS99220 that prevented degeneration of the nigral dopaminergic neurons and the associated motor deficits, suggesting a neuroprotective therapeutic utility for Parkinson's disease. Microglia are closely associated with neuroinflammation, which plays a key role in the pathogenesis of neurodegenerative diseases. In this study, we investigated the effects of KMS99220 on the signaling involving AMP-activated protein kinase (AMPK) and heme oxygenase-1 (HO-1), the enzymes thought to regulate inflammation. KMS99220 was shown to elevate the enzyme activity of purified AMPK, and phosphorylation of cellular AMPK in BV2 microglia. It increased the level of HO-1, and this was attenuated by AMPK inhibitors. KMS99220 lowered phosphorylation of IκB, nuclear translocation of NFκB, induction of inducible nitric oxide synthase, and generation of nitric oxide in BV2 cells that had been challenged with lipopolysaccharide. This anti-inflammatory response involved HO-1, because both its pharmacological inhibition and knockdown of its expression abolished the response. The AMPK inhibitors also reversed the anti-inflammatory effects of KMS99220. The induction of HO-1 by KMS99220 occurred within 1 h, and this appeared not to involve the transcription factor Nrf2, because Nrf2 knockdown did not affect the compound's HO-1 inducing- and anti-inflammatory effects in this time window. These findings indicated that KMS99220 leads to AMPK-induced HO-1 expression in microglia, which in turn plays an important role in early anti-inflammatory signaling. Together with its neuroprotective property, KMS99220 may serve as a feasible therapeutic agent against neuroinflammation and neurodegeneration.

Enhanced Efficacy of Human Brain-Derived Neural Stem Cells by Transplantation of Cell Aggregates in a Rat Model of Parkinson's Disease

OBJECTIVE: Neural tissue transplantation has been a promising strategy for the treatment of Parkinson's disease (PD). However, transplantation has the disadvantages of low-cell survival and/or development of dyskinesia. Transplantation of cell aggregates has the potential to overcome these problems, because the cells can extend their axons into the host brain and establish synaptic connections with host neurons. In this present study, aggregates of human brain-derived neural stem cells (HB-NSC) were transplanted into a PD animal model and compared to previous report on transplantation of single-cell suspensions. METHODS: Rats received an injection of 6-OHDA into the right medial forebrain bundle to generate the PD model and followed by injections of PBS only, or HB-NSC aggregates in PBS into the ipsilateral striatum. Behavioral tests, multitracer (2-deoxy-2-[18F]-fluoro-D-glucose ([18F]-FDG) and [18F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([18F]-FP-CIT) microPET scans, as well as immunohistochemical (IHC) and immunofluorescent (IF) staining were conducted to evaluate the results. RESULTS: The stepping test showed significant improvement of contralateral forelimb control in the HB-NSC group from 6-10 weeks compared to the control group (p<0.05). [18F]-FP-CIT microPET at 10 weeks posttransplantation demonstrated a significant increase in uptake in the HB-NSC group compared to pretransplantation (p<0.05). In IHC and IF staining, tyrosine hydroxylase and human beta2 microglobulin (a human cell marker) positive cells were visualized at the transplant site. CONCLUSION: These results suggest that the HB-NSC aggregates can survive in the striatum and exert therapeutic effects in a PD model by secreting dopamine.

Role of Oxidative Stress in Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder associated with a selective loss of the dopamine(DA)rgic neurons in the substantia nigra pars compacta and the degeneration of projecting nerve fibers in the striatum. Because there is currently no therapy that delays the neurodegenerative process, modification of the disease course by neuroprotective therapy is an important unmet clinical need. Toward this end, understanding cellular mechanisms that render the nigral neurons particularly vulnerable have been a subject of intensive research. Increasing evidence suggests that oxidative stress plays a major role. The metabolism of DA itself contributes to oxidative stress, resulting in modification of intracellular macromolecules whose functions are important for cell survival. Mitochondrial dysfunction and the consequent increase in reactive oxygen species also trigger a sequence of events that leads to cell demise. In addition, activated microglia produce nitric oxide and superoxide during neuroinflammatory responses, and this is aggravated by the molecules released by damaged DAergic neurons such as alpha-synuclein, neuromelanin and matrix metalloproteinase-3. Ways to reduce oxidative stress therefore can provide a therapeutic strategy. NAD(P)H:quinone reductase (NQO1) and other antioxidant enzymes, whose gene expression are commonly under the regulation of the transcription factor Nrf2, can serve as target proteins utilized toward development of disease-modifying therapy for PD.

Inactivation of Aconitase by Tetrahydrobiopterin in DArgic Cells: Relevance to PD

Oxidative damage is thought to be a major cause of the progression of dopamine (DA)rgic neurodegeneration as in Parkinson's disease. We have previously reported that tetrahydrobiopterin (BH4), an endogenous molecule required for DA synthesis, exerts oxidative stress to DA-producing cells and facilitates the production of DA quinone. It is known that aconitase, present in both mitochondrial and cytosolic forms, act as an reactive oxygen species (ROS) sensor, and that their inactivation leads to further generation of ROS. In the present study we investigated whether the BH4-associated vulnerability of DA cells might involve aconitase. In DArgic cell line CATH.a, BH4 treatment caused reduction of activity of both mitochondrial and cytosolic aconitases, and this appeared to be due to direct inactivation of the pre-existing enzyme molecules. Although most of the activity reduced by BH4 was increased upon reactivation reaction under a reducing condition, the restoration was not complete, suggesting that irreversible and covalent modification has occurred. The aconitase inactivation was exacerbated in the presence of DA and attenuated in the presence of tyrosine hydroxylase inhibitor a-methyl-p-tyrosine, suggesting the involvement of DA. The degree of inactivation increased when the cells were treated with the quinone reductase inhibitor dicoumarol and decreased in the presence of quinone reductase inducer sulforaphane. Taken together, BH4 appeared to lead to both reversible and irreversible inactivation of aconitase and that this is facilitated by the presence of DA and accumulation of DA quinone.

Growth Inhibitory Effects and Mechanisms of Ceramides in Renal Cell Carcinoma Cells / 대한비뇨기과학회지

PURPOSE: Because metastatic renal cell carcinoma responds to various forms of therapy with low remission rates, safe therapeutic agents is urgently needed. Ceramide is a potent and specific suppressor of cell growth and an inducer of apoptosis via an intracellular mediation of the sphingomyelin cycle. The present study was designed to assess the growth inhibitory effects and their mechanisms of C2-ceramide and C6-ceramide in renal cell carcinoma cells. MATERIALS AND METHODS: A standard microculture tetrazolium(MTT) assay was used to measure the cytotoxicity of C2-ceramide and C6-ceramide in renal cell carcinoma cell line A498. Apoptosis was confirmed by DNA fragmentation assay using agarose gel and TdT-mediated biotin-dUTP nicked-end labelling(TUNEL) technique. C2-ceramide and C6-ceramide were injected to the A498 tumor which was formed after A498 cells were implanted subcutaneously in athymic mice. Growth inhibitory effects of ceramides were examined biweekly. RESULTS: The survival fractions of A498 cells were 92.6+/-6.0, 82.8+/-14.0, 66.4+/-11.3, 41.8+/-9.6 and 24.3+/-6.3% for the concentrations of C2-ceramide 2, 4, 6, 8 and 10microM, repectively. IC50 of C2-ceramide was approximately 6.7microM. The survival fractions of A498 cells were 60.9+/-5.0, 23.4+/-3.0, 8.7+/-2.1, 5.0+/-1.2 and 3.3+/-0.6% for the concentrations of C6-ceramide 2, 4, 6, 8 and 10microM, respectively. IC50 of C6-ceramide was about 2.3microM. There were DNA fragmentations in A498 cells treated with C2-ceramide or C6-ceramide on the agarose gel and apoptotic tumor cells were also identified after treatment of C2-ceramide and C6-ceramide in TUNEL method. In in vivo study using athymic mice, the growth of A498 tumors was significantly suppressed by C2-ceramide and C6-ceramide. In vivo tumor suppressive effect was more prominent with C6-ceramide than with C2-ceramide. There`s no toxicity-related death of ceramide-treated athmic mice for 3 months. CONCLUSIONS: C2-ceramide and C6-ceramide have the growth inhibitory effects in human renal cell carcinoma cell line A498 by apoptosis mechanism in vitro and they have the in vivo tumor suppressive effects in athymic mice. C6-ceramide was more effective than C2-ceramide in both in vitro cytotoxicity test and in vivo animal experiment of growth inhibition. Therefore, ceramides may be used to treat metastatic renal cell carcinoma in the future.

Effect of Protein Kinase C Inhibitor (Staurosporine) in Human Invasive Bladder Tumor Cells / 대한비뇨기과학회지

Protein Kinase C which is a Ca++ -activated, phospholipid - dependent enzyme phosphorylates numerous protein substrates and participates in intracellular signaling processes. Protein kinase C is associated with a wide range of biological effects including stimulus-secretion coupling, induction of cellular proliferation and differentiation, activation of nuclear transcription factors and cell surface receptors and tumor promotion. Programmed cell death, referred to apoptosis is an active, energy-dependent process in which the cell participates in its own destruction during apoptosis. There is condensation and fragmentation of nuclear chromatin, accompanied by a marked decline in total cell volume, dilation of the endoplasmic reticulum and general compacting of cellular organelles. Thereafter, there is fragmentation of both nucleus and cytoplasm to give rise to small membrane-bound vesicles known as apoptotic bodies. Protein kinase C may have the regulatory role in apoptosis. Staurosporine is a potent protein kinase C inhibitor. Staurosporine inhibited the growth of human invasive bladder tumor cells, T24 in MTT test. The survival fractions of human invasive bladder tumor cells T24 were 100.0%, 76.0%, 62.5% and 18.1% with staurosporine concentration 0nM, 10nM, 100nM and 1000nM, respectively. From the results we identified that staurosporine inhibited the growth of T24 cells markedly in a dose dependent manner(P<0.05). 12-hour exposure of T24 cells to staurosporine failed to induce DNA fragmentation at the concentrations of 0nM, 10nM and 100nM but promoted fragmentation at the concentration of 1000nM, showing typical ladder pattern on agarose gel electrophoresis. On the examination of cellular morphology, T24 cells showed the features of apoptosis such as cell shrinkage, nuclear condensation and formation of bleb and apoptotic bodies after exposure to staurosporine of 10nM, 100nM and 1000nM concentrations. These results suggest that staurosporine have remarkable cytotoxic effect against human invasive bladder tumor cells T 24 and the mechanism of cytotoxicity may be apoptosis.

A study on catecholamine concentrations in the perilymph and the CSF of normal guinea pig / 대한이비인후과학회지

No abstract available.