p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum

In vivo administration of the mitochondrial inhibitor 3-nitropropionic acid (3-NP) produces striatal pathology mimicking Huntington disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. The present study investigated contributions of p53 signaling pathway to autophagy activation and cell death induced by 3-NP. Rat striatum was intoxicated with 3-NP by stereotaxic injection. Morphological and biochemical analyses demonstrated activation of autophagy in striatal cells as evidenced by increased the formation of autophagosomes, the expression of active lysosomal cathepsin B and D, microtubule associate protein light chain 3 (LC3) and conversion of LC3-I to LC3-II. 3-NP upregulated the expression of tumor suppressor protein 53 (p53) and its target genes including Bax, p53-upregulated modulator of apoptosis (PUMA) and damage-regulated autophagy modulator (DRAM). 3-NP-induced elevations in pro-apoptotic proteins Bax and PUMA, autophagic proteins LC3-II and DRAM were significantly reduced by the p53 specific inhibitor pifithrin-α (PFT). PFT also significantly inhibited 3-NP-induced striatal damage. Similarly, 3-NP-induced DNA fragmentation and striatal cell death were robustly attenuated by the autophagy inhibitor 3-methyladenine (3-MA) and bafilomycin A1 (BFA). These results suggest that p53 plays roles in signaling both autophagy and apoptosis. Autophagy, at least partially, contributes to neurodegeneration induced by mitochondria dysfunction.     

Neuroprotective effect of naringin, a dietary flavonoid against 3-nitropropionic acid-induced neuronal apoptosis

The aim of this study was to investigate the protective effect of naringin, a flavonoid on 3-Nitropropionic acid (3-NP)-induced neurodegeneration through the modulation of intrinsic apoptotic cascade in Wistar rats. 3-NP is an irreversible inhibitor of complex II in the mitochondria. 3-NP-induced neurodegeneration has been widely used as an animal model of Huntington’s disease (HD). Increased oxidative stress is one of the major deleterious events in 3-NP-induced neuronal apoptosis. Rats administered with 3-NP showed increase in the levels of lipid peroxidation and protein carbonyl, which was significantly decreased upon naringin treatment (80 mg/kg body weight). 3-NP-induced rats showed decrease in the activities of enzymic antioxidants and reduced levels of non-enzymic antioxidants. Naringin treatment ameliorated the antioxidant status by increasing the activities of enzymic antioxidants and the levels of non-enzymatic antioxidants. 3-NP-induced rats showed decrease in the activities of ATPases in striatum, which was restored to normal level upon naringin treatment. Histopathological observation of the striatal tissue showed protective role of naringin in 3-NP-induced rats. Naringin also reduced the 3-NP-induced apoptosis via decrease in the cytochrome c release from mitochondria and caspase 3 activation as revealed by Western blot. Naringin treatment also decreased the expressions of pro-apoptotic markers like Bad and Bax. Further, naringin antagonized 3-NP-induced decrease in Bcl-2 mRNA expression. The results of this study show evidence on the neuroprotective effect of naringin against 3-NP-induced neuronal apoptosis through its antioxidant and anti-apoptotic effects.     

Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition

Ursodeoxycholic acid (UDCA) has been shown to be a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. 3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, appears to cause apoptotic neuronal cell death in the striatum, reminiscent of the neurochemical and anatomical changes associated with Huntington's disease (HD). This study was undertaken (a) to characterize further the mechanism by which 3-NP induces apoptosis in rat neuronal RN33B cells and (b) to determine if and how the taurine-conjugated UDCA, tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3-NP. Our results indicate that coincubation of cells with TUDCA and 3-NP was associated with an approximately 80% reduction in apoptosis (p < 0.001), whereas neither taurine nor cyclosporin A, a potent inhibitor of the mitochondrial permeability transition (MPT), inhibited cell death. Moreover, TUDCA, as well as UDCA and its glycine-conjugated form, glycoursodeoxycholic acid, prevented mitochondrial release of cytochrome c (p < 0.001), which probably accounts for the observed inhibition of DEVD-specific caspase activity and poly(ADP-ribose) polymerase cleavage. 3-NP decreased mitochondrial transmembrane potential (p < 0.001) and increased mitochondrial-associated Bax protein levels (p < 0.001). Coincubation with TUDCA was associated with significant inhibition of these mitochondrial membrane alterations (p < 0.01). The results suggest that TUDCA inhibits 3-NP-induced apoptosis via direct inhibition of mitochondrial depolarization and outer membrane disruption, together with modulation of Bax translocation from cytosol to mitochondria. In addition, cell death by 3-NP apparently occurs through pathways that are independent of the MPT.     

Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.     

Puerarin Ameliorates 3-Nitropropionic Acid-Induced Neurotoxicity in Rats: Possible Neuromodulation and Antioxidant Mechanisms

Puerarin (daidzein-8-C-glucoside), a major isoflavone glycoside purified from Pueraria lobata, is well reported to have a neuroprotective effect primarily by the antioxidant mechanisms. This investigation was designed to evaluate the efficacy of Puerarin (Pur) to offset 3-nitropropionic acid (3-NP) induced neurotoxicity. Male Wistar strain rats were given 3-NP (20 mg/kg, s.c.) over five consecutive days, whereas Pur (200 mg/kg, i.p.) was administrated 30 min before 3-NP. Rats treated with 3-NP exhibited significant weight loss, reduction of the prepulse inhibition, locomotor hypoactivity and hypothermia. The striata, hippocampi and cortices of the 3-NP treated rats showed abnormal levels of neurotransmitters, oxidative damage and characteristic histopathological lesions. Treatment with Pur ahead of 3-NP, significantly prevented weight loss, PPI deficit, locomotor hypoactivity and hypothermia. Pur treatment blocked the 3-NP-induced neurotransmitters abnormalities (GABA, DA, 5-HT and NE), and normalized the oxidative stress biomarkers (lipid peroxidation, reduced glutathione, glutathione peroxidase). Histopathological examination further affirmed Pur’s neuroprotective effect against 3-NP-induced neurotoxicity. In conclusion, Pur protected the brain tissues from 3-NP induced neurotoxicity primarily by its neuromodulation and antioxidant effect.     

Probucol Increases Striatal Glutathione Peroxidase Activity and Protects against 3-Nitropropionic Acid-Induced Pro-Oxidative Damage in Rats

Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD involve oxidative stress and mitochondrial dysfunction. Administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, in rodents has been proposed as a useful experimental model of HD. This study evaluated the effects of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, on the biochemical parameters related to oxidative stress, as well as on the behavioral parameters related to motor function in an in vivo HD model based on 3-NP intoxication in rats. Animals were treated with 3.5 mg/kg of probucol in drinking water daily for 2 months and, subsequently, received 3-NP (25 mg/kg i.p.) once a day for 6 days. At the end of the treatments, 3-NP-treated animals showed a significant decrease in body weight, which corresponded with impairment on motor ability, inhibition of mitochondrial complex II activity and oxidative stress in the striatum. Probucol, which did not rescue complex II inhibition, protected against behavioral and striatal biochemical changes induced by 3-NP, attenuating 3-NP-induced motor impairments and striatal oxidative stress. Importantly, probucol was able to increase activity of glutathione peroxidase (GPx), an enzyme important in mediating the detoxification of peroxides in the central nervous system. The major finding of this study was that probucol protected against 3-NP-induced behavioral and striatal biochemical changes without affecting 3-NP-induced mitochondrial complex II inhibition, indicating that long-term probucol treatment resulted in an increased resistance against neurotoxic events (i.e., increased oxidative damage) secondary to mitochondrial dysfunction. These data appeared to be of great relevance when extrapolated to human neurodegenerative processes involving mitochondrial dysfunction and indicates that GPx is an important molecular target involved in the beneficial effects of probucol.     

Metabonomic Characterization of the 3-Nitropropionic Acid Rat Model of Huntington’s Disease

3-Nitropropionic acid (3-NP)-induced neurotoxicity can be used as a model for the genetic neurodegenerative disorder Huntington’s disease (HD). A metabolic profiling strategy was adopted to explore the biochemical consequences of 3-NP administered to rats in specific brain regions. ¹H NMR spectroscopy was used to characterize the metabolite composition of several brain regions following 3-NP-intoxication. Dose-dependent increases in succinate levels were observed in all neuroanatomical regions, resulting from the 3-NP-induced inhibition of succinate dehydrogenase. Global decreases in taurine and GABA were observed in the majority of brain regions, whereas altered lipid profiles were observed only in the globus pallidus and dorsal striatum. Depleted phosphatidylcholine and elevated glycerol levels, which are indicative of apoptosis, were also observed in the frontal cortex of the 3-NP model. Many of the metabolic anomalies are consistent with those reported in HD. The 3-NP-induced model of HD provides a means of monitoring potential mechanisms of pathology and therapeutic response for drug interventions, which can be efficiently assessed using metabolic profiling strategies.     

Tolfenamic Acid Attenuates 3-Nitropropionic Acid-Induced Biochemical Alteration in Mice

Tolfenamic acid (TA), a nonsteroidal anti-inflammatory drug, shows neuroprotective effects and alleviates cognitive deficits in transgenic mouse models of Alzheimer’s disease. However, whether TA can prevent the biochemical alterations induced by intraperitoneal injection of 3-nitropropionic acid (3-NP) in mice is still unknown. In this study, the striatal lesion area was measured by 2,3,5-triphenyltetrazolium chloride staining. Glutamate, SDH and ATP levels were tested using colorimetric assay kits. The neuroinflammatory cytokine levels were tested by ELISA kits. The expression of synaptic proteins and the subtypes of the NMDA receptor were tested by western blotting. TA was orally administered 10 days before 3-NP injection (pretreatment) or on the same day as 3-NP injection (co-treatment). TA pretreatment showed the strongest neuroprotective effects: pretreatment significantly attenuated the 3-NP-induced muscular weakness in the forelimb and alterations in glutamate level, mitochondrial function, and pro-inflammatory cytokine release in the brains of mice. These results suggest that TA has preventive and protective effects on 3-NP-induced neurotoxicity.     

FK506 ameliorates cell death features in Huntington's disease striatal cell models

Huntington’s disease (HD) is a genetic neurodegenerative disorder characterized by striatal neurodegeneration, involving apoptosis. FK506, an inhibitor of calcineurin (or protein phosphatase 3, formerly known as protein phosphatase 2B), has shown neuroprotective effects in several cellular and animal models of HD. In the present study, we show the protective effects of FK506 in two striatal HD models, primary rat striatal neurons treated with 3-nitropropionic acid (3-NP) and immortalized striatal STHdh cells derived from HD knock-in mice expressing normal (STHdh^7/7) or full-length mutant huntingtin (FL-mHtt) with 111 glutamines (STHdh^111/111), under basal conditions and after exposure to 3-NP or staurosporine (STS). In rat striatal neurons, FK506 abolished 3-NP-induced increase in caspase-3 activation, DNA fragmentation/condensation and necrosis. Nevertheless, in STHdh^111/111 cells under basal conditions, FK506 did not prevent, in a significant manner, the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, or alter Bax/Bcl-2 ratio, but significantly reverted caspase-3 activation. In STHdh^111/111 cells treated with 0.3 mM 3-NP or 25 nM STS, linked to high necrosis, exposure to FK506 exerted no significant effects on caspase-3 activation. However, treatment of STHdh^111/111 cells exposed to 10 nM STS with FK506 effectively prevented cell death by apoptosis and moderate necrosis. The results suggest that FK506 may be neuroprotective against apoptosis and necrosis under mild cell death stimulus in the presence of FLmHtt.     

Alleviation of autophagy by knockdown of Beclin-1 enhances susceptibility of hippocampal neurons to proapoptotic signals induced by amino acid starvation

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of Beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of Beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of Beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.     

Impaired mitochondrial function results in increased tissue transglutaminase activity in situ

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.     

The effect of Ginkgo biloba extract on 3-nitropropionic acid-induced neurotoxicity in rats

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase enzyme (SDH), induces neurodegeneration similar to that observed in Huntington’s disease (HD). Reduction of prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity, bilateral striatal lesions as well as brain oxidative stress are major features of HD. The present study was designed to investigate neuroprotective effect of Ginkgo biloba extract (EGb 761) on 3-NP induced neurobehavioral changes and striatal lesions.Rats administered 3-NP (20 mg/kg, s.c.) for five consecutive days exhibited PPI deficits and locomotor hypoactivity whereas, pretreatment of animals with EGb 761 (100 mg/kg, i.p. for 15 days) ahead of and during the induction of HD by 3-NP (20 mg/kg for 5 days starting at day 8) ameliorated 3-NP-induced neurobehavioral deficits. Administration of 3-NP increased the level of striatal malondialdehyde (MDA). This effect was prevented in animals pre-treated with EGb 761. Changes in the level of apoptotic regulatory gene expressions, following 3-NP treatment, were demonstrated as both an up-regulation and a down-regulation of the expression levels of striatal Bax and Bcl-xl genes, respectively. In addition, an up-regulation of the expression level of striatal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed. Pre-treatment with EGb 761 caused a down-regulation in striatal GAPDH and Bax together with an up-regulation of striatal Bcl-xl expression level as compared to the 3-NP treated group. Histochemical examination of striatal tissue showed that EGb 761 significantly prevented 3-NP induced inhibition of SDH activity. Histopathological examination further affirmed the neuroprotective effect of EGb 761 against 3-NP toxicity.Taken together, these results suggest that EGb 761 has a neuroprotective role in the current HD paradigm, which may be related to improvement of energy metabolism, antioxidant properties and antiapoptotic effects.     

Mitochondrial NAD+-linked State 3 respiration and complex-I activity are compromised in the cerebral cortex of 3-nitropropionic acid-induced rat model of Huntington's disease

Mitochondrial complex-I dysfunction has been observed in patients of Huntington's disease (HD). We assessed whether such a defect is present in the 3-nitropropionic acid (3-NP) model of HD. Rats treated with 3-NP (10–20 mg/kg i.p., for 4 days) exhibited weight loss, gait abnormalities, and striatal lesions with increased glial fibrillary acidic protein immunostaining on fifth and ninth days, while increase in striatal dopamine and loss of tyrosine hydroxylase immunoreactivity were observed on fifth day following treatment. We report for the first time a dose-dependent reduction in complex-I activity in the cerebral cortex when analyzed spectrophotometrically and by blue native-polyacrylamide gel electrophoresis following 3-NP treatment. The citrate synthase normalized activities of mitochondrial complex-I, -II, -(I + III) and -IV were decreased in the cortex of 3-NP treated rats. In addition, succinate driven State 3 respiration was also significantly inhibited in vivo and in the isolated mitochondria. These findings taken together with the observation of a significant decrease in vivo but not in vitro of State 3 respiration with NAD⁺-linked substrates, suggest complex-I dysfunction in addition to irreversible inhibition of complex-II and succinate dehydrogenase activity as a contributing factor in 3-NP-induced cortico-striatal lesion.     

Effect of exogenous and endogenous antioxidants on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions: insights into Huntington's disease

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of complex II in the mitochondria. 3-NP toxicity has gained acceptance as an animal model of Huntington's disease (HD). In the present study, we confirmed that rats injected with 3-NP (20 mg/kg, i.p., daily for 4 days) exhibit increased oxidative stress in both striatum and cortical synaptosomes as well as lesions in the striatum. Synaptosomal membrane proteins from rats injected with 3-NP exhibited a decrease in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation, and western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal protein oxidation. Treatment of rats with the brain-accessible free radical spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO; 30 mg/kg, i.p., daily 2 h before 3-NP injection) or with N-acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3-NP injection), a known glutathione precursor, before 3-NP treatments protects against oxidative damage induced by 3-NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3-NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3-NP neurotoxicity and perhaps against HD as well.     

Neuronal adenosine A<Subscript>2A</Subscript> receptor overexpression is neuroprotective towards 3-nitropropionic acid-induced striatal toxicity: a rat model of Huntington’s disease

The A_2A adenosine receptor (A_2AR) is widely distributed on different cellular types in the brain, where it exerts a broad spectrum of pathophysiological functions, and for which a role in different neurodegenerative diseases has been hypothesized or demonstrated. To investigate the role of neuronal A_2ARs in neurodegeneration, we evaluated in vitro and in vivo the effect of the neurotoxin 3-nitropropionic acid (3-NP) in a transgenic rat strain overexpressing A_2ARs under the control of the neural-specific enolase promoter (NSEA_2A rats). We recorded extracellular field potentials (FP) in corticostriatal slice and found that the synaptotoxic effect of 3-NP was significantly reduced in NSEA_2A rats compared with wild-type animals (WT). In addition, after exposing corticostriatal slices to 3-NP 10 mM for 2 h, we found that striatal cell viability was significantly higher in NSEA_2A rats compared to control rats. These in vitro results were confirmed by in vivo experiments: daily treatment of female rats with 3-NP 10 mg/kg for 8 days induced a selective bilateral lesion in the striatum, which was significantly reduced in NSEA_2A compared to WT rats. These results demonstrate that the overexpression of the A_2AR selectively at the neuronal level reduced 3-NP-induced neurodegeneration, and suggest an important function of the neuronal A_2AR in the modulation of neurodegeneration.     

三硝基丙酸动物模型和亨廷顿氏病

亨廷顿氏病（Huntington’s disease,HD）是一类神经系统退行性疾病,主要病理改变累及基底节神经元以及纹状体的固有神经元,患者主要临床特征为进行性恶化的运动、认知及精神障碍三联征。3-硝基丙酸（3-NP）是线粒体毒性药物,主要作用于氧化呼吸链并抑制机体的三羧酸循环。本文将就HD的发病机制及病理学特征以及3-NP介导的动物模型的优缺点做一综述。     

Protein phosphorylation and oocyte maturation : I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, α-naphthylphosphate

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. α-Naphthylphosphate (α-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation at 3.5 mM; 100% above 6 mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, α-NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of α-NP-induced maturation (35–45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18–20 min). The addition of α-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to α-NP, only α-NP was found capable of inducing maturation when microinjected into oocytes. α-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although α-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that α-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, α-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of α-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation.     

Combination therapy with Coenzyme Q10 and creatine produces additive neuroprotective effects in models of Parkinson's and Huntington's Diseases

Coenzyme Q₁₀ (CoQ₁₀) and creatine are promising agents for neuroprotection in neurodegenerative diseases via their effects on improving mitochondrial function and cellular bioenergetics and their properties as antioxidants. We examined whether a combination of CoQ₁₀ with creatine can exert additive neuroprotective effects in a MPTP mouse model of Parkinson's disease, a 3-NP rat model of Huntington's disease (HD) and the R6/2 transgenic mouse model of HD. The combination of the two agents produced additive neuroprotective effects against dopamine depletion in the striatum and loss of tyrosine hydroxylase neurons in the substantia nigra pars compacta (SNpc) following chronic subcutaneous administration of MPTP. The combination treatment resulted in significant reduction in lipid peroxidation and pathologic α-synuclein accumulation in the SNpc neurons of the MPTP-treated mice. We also observed additive neuroprotective effects in reducing striatal lesion volumes produced by chronic subcutaneous administration of 3-NP to rats. The combination treatment showed significant effects on blocking 3-NP-induced impairment of glutathione homeostasis and reducing lipid peroxidation and DNA oxidative damage in the striatum. Lastly, the combination of CoQ₁₀ and creatine produced additive neuroprotective effects on improving motor performance and extending survival in the transgenic R6/2 HD mice. These findings suggest that combination therapy using CoQ₁₀ and creatine may be useful in the treatment of neurodegenerative diseases such as Parkinson's disease and HD.     

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.