The Anxiolytic Drug Buspirone Prevents Rotenone-Induced Toxicity in a Mouse Model of Parkinson’s Disease

A pharmacological and genetic blockade of the dopamine D3 receptor (D3R) has shown to be neuroprotective in models of Parkinson’s disease (PD). The anxiolytic drug buspirone, a serotonin receptor 1A agonist, also functions as a potent D3R antagonist. To test if buspirone elicited neuroprotective activities, C57BL/6 mice were subjected to rotenone treatment (10mg/kg i.p for 21 days) to induce PD-like pathology and were co-treated with increasing dosages of buspirone (1, 3, or 10 mg/kg i.p.) to determine if the drug could prevent rotenone-induced damage to the central nervous system (CNS). We found that high dosages of buspirone prevented the behavioural deficits caused by rotenone in the open field test. Molecular and histological analyses confirmed that 10 mg/kg of buspirone prevented the degeneration of TH-positive neurons. Buspirone attenuated the induction of interleukin-1β and interleukin-6 expression by rotenone, and this was paralleled by the upregulation of arginase-1, brain-derived neurotrophic factor (BDNF), and activity-dependent neuroprotective protein (ADNP) in the midbrain, striatum, prefrontal cortex, amygdala, and hippocampus. Buspirone treatment also improved mitochondrial function and antioxidant activities. Lastly, the drug prevented the disruptions in the expression of two neuroprotective peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). These results pinpoint the neuroprotective efficacy of buspirone against rotenone toxicity, suggesting its potential use as a therapeutic agent in neurodegenerative and neuroinflammatory diseases, such as PD.     

Nadh: Ubiquinone oxidoreductase in obligate aerobic yeasts

The strictly aerobic yeasts Candida pinus, Cryptococcus albidus, Rhodotorula minuta, Rhodotorula mucilaginosa and Trichosporon beigelii possess mitochondrial NADH dehydrogenases with significant features of the NADH:ubiquinone oxidoreductase (complex I). These species show in all growth phases and under standard cultivation conditions, NADH dehydrogenases of approximately 700 kDa, which are sensitive to rotenone, a specific inhibitor of this complex. Identical results were obtained with the weakly fermenting C. pinus. The facultatively fermenting yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus do not possess the 700 kDa-complex and are insensitive to rotenone. In S. cerevisiae, a rotenone-insensitive NADH dehydrogenase of about 500–600 kDa is detected only in stationary phase cells. As in Neurospora crassa, upon incubation of the obligately aerobic yeast R. mucilaginosa with chloramphenicol, an intermediate NADH dehydrogenase of approximately 350 kDa was formed, which was insensitive to rotenone.     

Mitochondrial Dysfunction and Oxidative Stress Promote Apoptotic Cell Death in the Striatum via Cytochrome c/Caspase-3 Signaling Cascade Following Chronic Rotenone Intoxication in Rats

Parkinson's disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration. Evidence suggests that mitochondrial dysfunction may be linked to PD through a variety of different pathways, including free-radical generation and dysfunction of the mitochondrial Complex I activity. In Lewis rats, chronic systemic administration of a specific mitochondrial Complex I inhibitor, rotenone (3 mg/kg/day) produced parkinsonism-like symptoms. Increased oxidized proteins and peroxynitrite, and mitochondrial or cytosol translocation of Bim, Bax or cytochrome c in the striatum was observed after 2-4 weeks of rotenone infusion. After 28 days of systemic rotenone exposure, imunohistochemical staining for tyrosine hydroxylase indicated nigrostriatal dopaminergic neuronal cell degeneration. Characteristic histochemical (TUNEL or activated caspase-3 staining) or ultrastructural (electron microscopy) features of apoptotic cell death were present in the striatal neuronal cell after chronic rotenone intoxication. We conclude that chronic rotenone intoxication may enhance oxidative and nitrosative stress that induces mitochondrial dysfunction and ultrastructural damage, resulting in translocation of Bim and Bax from cytosol to mitochondria that contributes to apoptotic cell death in the striatum via cytochrome c/caspase-3 signaling cascade.     

天麻钩藤饮抑制铁死亡-脂质代谢保护鱼藤酮诱导的PC12细胞损伤

目的：研究天麻钩藤饮（TGY）对鱼藤酮（Rot）诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞损伤的影响及可能的机制。方法：采用Rot建立和培养PC12细胞神经损伤模型并利用MTT比色法测定细胞存活率,确定Rot的最佳造模浓度以及TGY的有效干预浓度。流式细胞仪检测细胞的脂质活性氧（ROS）水平并用荧光倒置显微镜观察细胞荧光强度的变化;生化试剂盒检测超氧化物歧化酶（SOD）,谷胱甘肽（GSH）的水平和活力变化以及丙二醛（MDA）含量的变化。透射电子显微镜观察细胞线粒体形态变化,蛋白免疫印迹法检测细胞内谷胱甘肽过氧化物酶4（GPX4）,长链脂酰辅酶A合成酶4（ACSL4）,溶血卵磷脂酰基转移酶3（LPCAT3）蛋白表达。结果：0.6 μmol/L Rot处理细胞24 h后,细胞存活率接近50%（56.7%±9.9%）,TGY预处理12 h后可抑制Rot的损伤程度。同时,减少乳酸脱氢酶（LDH）的漏出率,并呈现一定的量效关系。Rot处理后的细胞内脂质ROS含量升高,而给予TGY预处理后有效降低Rot损伤的细胞内脂质ROS的含量,降低Rot处理后的MDA水平,并提高SOD活力和GSH水平。通过透射电镜观察到与正常组相比,0.6 μmol/L Rot处理后细胞的线粒体皱缩,TGY预保护处理后,PC12细胞的线粒体形态有一定程度的改善。Western blot的结果呈现出TGY预处理后可以在一定程度提高Rot损伤后GPX4的表达,下降ACSL4、LPCAT3的表达。 结论：TGY可以上调GPX4蛋白的表达,下调ACSL4、LPCAT3蛋白的表达,抑制不饱和脂肪酸的氧化,降低脂质过氧化水平和ROS的含量,从而改善神经损伤。     

NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

Cholesterol plays a crucial role in the brain, where its metabolism is particularly regulated by astrocytic activity. Indeed, adult neurons suppress their own cholesterol biosynthesis and import this sterol through ApoE-rich particles secreted from astrocytes. Recent evidence suggests that nerve growth factor (NGF) may exert neurotrophic activity by influencing cell metabolism. Nevertheless, the effect of NGF on glial cholesterol homeostasis has still not been elucidated. Thus, the aim of this project is to assess whether NGF could influence cholesterol metabolism in glial cells. To reach this objective, the U373 astrocyte-derived cell line was used as an experimental model. Immunoblot and ELISA analysis showed that proteins and enzymes belonging to the cholesterol metabolism network were increased upon NGF treatment in glial cells. Furthermore, NGF significantly increased ApoE secretion and the amount of extracellular cholesterol in the culture medium. Co-culture and U373-conditioned medium experiments demonstrated that NGF treatment efficiently counteracted rotenone-mediated cytotoxicity in N1E-115 neuronal cells. Conversely, neuroprotection mediated by NGF treatment was suppressed when N1E-115 were co-cultured with ApoE-silenced U373 cells. Taken together, these data suggest that NGF controls cholesterol homeostasis in glial cells. More importantly, NGF exerts neuroprotection against oxidative stress, which is likely associated with the induction of glial ApoE secretion.     

鱼藤酮的高效液相色谱分析方法研究

介绍一种鱼藤酮的高效液相色谱定量分析方法。采用hypersil C18色谱柱,在以v(乙腈)∶v(水)=60∶40为流动相,流速为1.0 mL/m in,检测波长为294 nm的条件下,用外标法对鱼藤酮原药中的有效成分进行定量分析。方法的标准差为0.28,变异系数为0.39%,线性相关系数为0.9999,平均回收率为99.54%。     

Neural-Induced Human Adipose Tissue-Derived Stem Cells Conditioned Medium Ameliorates Rotenone-Induced Toxicity in SH-SY5Y Cells

Parkinson’s disease (PD) is an age-related neurodegenerative disease (NDD) characterized by the degenerative loss of dopaminergic neurons in the substantia nigra along with aggregation of α-synuclein (α-syn). Neurogenic differentiation of human adipose-derived stem cells (NI-hADSCs) by supplementary factors for 14 days activates different biological signaling pathways. In this study, we evaluated the therapeutic role of NI-hADSC-conditioned medium (NI-hADSC-CM) in rotenone (ROT)-induced toxicity in SH-SY5Y cells. Increasing concentrations of ROT led to decreased cell survival at 24 and 48 h in a dose- and time-dependent manner. Treatment of NI-hADSC-CM (50% dilution in DMEM) against ROT (0.5 μM) significantly increased the cell survival. ROT toxicity decreased the expression of tyrosine hydroxylase (TH). Western blot analysis of the Triton X-100-soluble fraction revealed that ROT significantly decreased the oligomeric, dimeric, and monomeric phosphorylated Serine129 (p-S129) α-syn, as well as the total monomeric α-syn expression levels. ROT toxicity increased the oligomeric, but decreased the dimeric and monomeric p-S129 α-syn expression levels. Total α-syn expression (in all forms) was increased in the Triton X-100-insoluble fraction, compared to the control. NI-hADSC-CM treatment enhanced the TH expression, stabilized α-syn monomers, reduced the levels of toxic insoluble p-S129 α-syn, improved the expression of neuronal functional proteins, regulated the Bax/Bcl-2 ratio, and upregulated the expression of pro-caspases, along with PARP-1 inactivation. Moreover, hADSC-CM treatment decreased the cell numbers and have no effect against ROT toxicity on SH-SY5Y cells. The therapeutic effects of NI-hADSC-CM was higher than the beneficial effects of hADSC-CM on cellular signaling. From these results, we conclude that NI-hADSC-CM exerts neuroregenerative effects on ROT-induced PD-like impairments in SH-SY5Y cells.     

1,5-Anhydro-D-fructose Protects against Rotenone-Induced Neuronal Damage In Vitro through Mitochondrial Biogenesis

Mitochondrial functional abnormalities or quantitative decreases are considered to be one of the most plausible pathogenic mechanisms of Parkinson’s disease (PD). Thus, mitochondrial complex inhibitors are often used for the development of experimental PD. In this study, we used rotenone to create in vitro cell models of PD, then used these models to investigate the effects of 1,5-anhydro-D-fructose (1,5-AF), a monosaccharide with protective effects against a range of cytotoxic substances. Subsequently, we investigated the possible mechanisms of these protective effects in PC12 cells. The protection of 1,5-AF against rotenone-induced cytotoxicity was confirmed by increased cell viability and longer dendritic lengths in PC12 and primary neuronal cells. Furthermore, in rotenone-treated PC12 cells, 1,5-AF upregulated peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression and enhanced its deacetylation, while increasing AMP-activated protein kinase (AMPK) phosphorylation. 1,5-AF treatment also increased mitochondrial activity in these cells. Moreover, PGC-1α silencing inhibited the cytoprotective and mitochondrial biogenic effects of 1,5-AF in PC12 cells. Therefore, 1,5-AF may activate PGC-1α through AMPK activation, thus leading to mitochondrial biogenic and cytoprotective effects. Together, our results suggest that 1,5-AF has therapeutic potential for development as a treatment for PD.