Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell-based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl-2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase-3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy-related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase-independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases.

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Hypothalamic tuberoinfundibular dopamine (TIDA) neurons remain unaffected in Parkinson disease (PD) while there is significant degeneration of midbrain nigrostriatal dopamine (NSDA) neurons. A similar pattern of susceptibility is observed in acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse and rotenone rat models of degeneration. It is not known if the resistance of TIDA neurons is a constitutive or induced cell-autonomous phenotype for this unique subset of DA neurons. In the present study, treatment with a single injection of MPTP (20 mg/kg; s.c.) was employed to examine the response of TIDA versus NSDA neurons to acute injury. An acute single dose of MPTP caused an initial loss of DA from axon terminals of both TIDA and NSDA neurons, with recovery occurring solely in TIDA neurons by 16 h post-treatment. Initial loss of DA from axon terminals was dependent on a functional dopamine transporter (DAT) in NSDA neurons but DAT-independent in TIDA neurons. The active metabolite of MPTP, 1-methyl, 4-phenylpyradinium (MPP+), reached higher concentration and was eliminated slower in TIDA compared to NSDA neurons, which indicates that impaired toxicant bioactivation or distribution is an unlikely explanation for the observed resistance of TIDA neurons to MPTP exposure. Inhibition of protein synthesis prevented TIDA neuron recovery, suggesting that the ability to recover from injury was dependent on an induced, rather than a constitutive cellular mechanism. Further, there were no changes in total tyrosine hydroxylase (TH) expression following MPTP, indicating that up-regulation of the rate-limiting enzyme in DA synthesis does not account for TIDA neuronal recovery. Differential candidate gene expression analysis revealed a time-dependent increase in parkin and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) expression (mRNA and protein) in TIDA neurons during recovery from injury. Parkin expression was also found to increase with incremental doses of MPTP. The increase in parkin expression occurred specifically within TIDA neurons, suggesting that these neurons have an intrinsic ability to up-regulate parkin in response to MPTP-induced injury. These data suggest that TIDA neurons have a compensatory mechanism to deal with toxicant exposure and increased oxidative stress, and this unique TIDA neuron phenotype provides a platform for dissecting the mechanisms involved in the natural resistance of central DA neurons following toxic insult.

Traditional herbal medicines have been processed to enhance their therapeutic effects, remove or reduce toxicity and side effects, and facilitate preparation and storage. In addition, the increased biological activities have been thought to be closely related to the compounds which are modified through processing.1-3) Therefore, there has been a growing interest in the bioactive constituents of herbal drugs modified by processing. [4][5][6] The root of ginseng, Panax ginseng C.A. MEYER (Araliaceae), has been heat processed to make white ginseng (WG, roots dried after peeling) and red ginseng (RG, steamed at 98-100°C and dried ginseng roots without peeling) for consumption. Especially, RG is more common as a functional food than WG in Asian countries, because steaming induces a change in the chemical constituents and enhances the biological activities of ginseng. [7][8][9][10] Recently, a method to increase the RG-specific ginsenosides by steaming WG at a higher temperature than RG was developed.2,11) This heat processed ginseng is termed sun ginseng (SG), and we have been investigating its enhanced free radical scavenging activity compared to conventional ginsengs and its active constituents.Phenolic compounds and maltol were responsible for the increased free radical scavenging activities of SG. In addition, Maillard reaction products (MRPs) were also thought to be related to the increased antioxidant activity of ginseng by heat processing, 5,6) because the Maillard reaction has been thought to be the major source correlated with increased efficacy by heat processing in various crude drugs or foods. 12,13) On the other hand, ginsenoside is one of the easily changeable components of ginseng by heat processing, 2,14) but heat processing-induced chemical and activity changes of ginsenosides considering the Maillard reaction have not yet been fully elucidated. In addition, the contents of less-polar ginsenosides Rg 3 , Rk 1 , Rg 5 , and maltol are known to increase by heat processing, and these compounds are supposed to be produced by the deglycosylation of diol-type ginsenosides and the Maillard reaction in ginseng by steaming. 6,14) Therefore, there is a need to investigate the structure and activity changes of isolated ginsenosides by heat processing with amino acids.In this study, we investigated the hydroxyl radical (· OH) scavenging activity changes of ginsengs and ginsenoside-Rb 2 (Rb 2 ) by heat processing using an electron spin resonance spectrometer (ESR). No report has compared the · OH scavenging activities of WG, RG, and SG using ESR, and ESR is thought as a versatile tool to detect free radicals even with slightly insoluble ginsenosides in suspension. In addition, Rb 2 , a well known diol-type triterpene glycoside that exists abundantly in Panax ginseng, was steamed with glycine, a frequently used amino acid in the Maillard reaction model system, 15) and the chemical and · OH scavenging activity changes were analyzed and compared. Shillim-Dong, Kwanak-gu, Seoul 151-742, Korea. Received October 7, 20...

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BackgroundNeural stem cells (NSCs) hold great potential for the treatment of neurodegenerative diseases. However, programmed cell death (PCD) provoked by the harsh conditions evident in the diseased brain greatly undermines the potential of NSCs. Currently, the mechanisms of PCD that effect NSCs remain largely unknown.ResultsWe have previously reported that hippocampal neural stem (HCN) cells derived from the adult rat brain undergo autopahgic cell death (ACD) following insulin withdrawal without hallmarks of apoptosis despite their normal apoptotic capabilities. In this study, we demonstrate that glycogen synthase kinase 3β (GSK-3β) induces ACD in insulin-deprived HCN cells. Both pharmacological and genetic inactivation of GSK-3β significantly decreased ACD, while activation of GSK-3β increased autophagic flux and caused more cell death without inducing apoptosis following insulin withdrawal. In contrast, knockdown of GSK-3α barely affected ACD, lending further support to the critical role of GSK-3β.ConclusionCollectively, these data demonstrate that GSK-3β is a key regulator of ACD in HCN cells following insulin withdrawal. The absence of apoptotic indices in GSK-3β-induced cell death in insulin-deprived HCN cells corroborates the notion that HCN cell death following insulin withdrawal represents the genuine model of ACD in apoptosis-intact mammalian cells and identifies GSK-3β as a key negative effector of NSC survival downstream of insulin signaling.

Background and Purpose An urgent need exists to develop therapies for stroke which have high efficacy, long therapeutic time windows and acceptable toxicity. We undertook preclinical investigations of a novel therapeutic approach involving supplementation with carnosine, an endogenous pleiotropic dipeptide. Methods Efficacy and safety of carnosine treatment was evaluated in rat models of permanent or transient middle cerebral artery occlusion. Mechanistic studies used primary neuronal/astrocytic cultures and ex vivo brain homogenates. Results Intravenous treatment with carnosine exhibited robust cerebroprotection in a dose-dependent manner, with long clinically-relevant therapeutic time windows of 6 h and 9 h in transient and permanent models, respectively. Histological outcomes and functional improvements including motor and sensory deficits were sustained at 14 d post-stroke onset. In safety and tolerability assessments, carnosine did not exhibit any evidence of adverse effects or toxicity. Moreover, histological evaluation of organs, complete blood count, coagulation tests and the serum chemistry did not reveal any abnormalities. In primary neuronal cell cultures and ex vivo brain homogenates, carnosine exhibited robust anti-excitotoxic, antioxidant, and mitochondria protecting activity. Conclusion In both permanent and transient ischemic models, carnosine treatment exhibited significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. Carnosine was well tolerated and exhibited no toxicity. Mechanistic data show that it influences multiple deleterious processes. Taken together, our data suggest that this endogenous pleiotropic dipeptide is a strong candidate for further development as a stroke treatment.