The Effects Of HDAC Regulation Via Autophagy On The Pathogenesis And Neuroprotection Of Parkinson's Disease

Part 1 Effects of Different Environmental Toxins on Histone Deacetylase Isoforms and PD-related Molecules in Dopaminergic Cell ModelObjective To investigate the effects of different environmental toxins on histone deacetylase isoforms and PD-related molecules in SH-SY5Y dopaminergic cells.Methods SH-SY5Y cells were treated with rotenone or fenpropathrin for different doses and different periods. Western blot and Real-time PCR were used to detect the protein and mRNA levels of HDAC1, HDAC4, HDAC6, a-synuclein, VMAT2 and DAT. Laser Scanning Confocal Microscope (LSCM) was used to observe subcellular localization and protein aggregates of HDAC1, HDAC4, HDAC6, a-synuclein and ubiquitin.Results Western blot analysis indicated that rotenone treatment increased the protein levels of HDAC1, HDAC4, HDAC6 in SH-SY5Y cells in a dose-and time-denpendent manner. In particular, rotenone induced a-synuclein oligomers in a dose-denpendent manner. Real-time PCR indicated that fenpropathrin treatment upregulated the mRNA levels of HDAC1, HDAC4, HDAC6 and a-synuclein in SH-SY5Y in a dose-denpendent manner, while the mRNA levels of VMAT2 and DAT were decreased in a dose-dependent manner. LSCM showed that rotenone enhanced HDAC1 staining in the nucleus in a time-denpendent manner. HDAC4 protein distributed uniformly in the cytoplasm of untreated SH-SY5Y, with a small fraction appeared in the nucleus. The exposure to rotenone led to the enhanced cytoplasmic and nuclear distribution of HDAC4. Moreover, co-localization of HDAC4 and a-synuclein was observed following rotenone treatment. Similarly, HDAC6 co-located with a-synuclein in the cytoplasm and HDAC6 staining was greatly elevated and upon rotenone treatment, whereas this protein distributed uniformly in the cytoplasm of untreated SH-SY5Y.Conclusion Rotenone or fenpropathrin treatment induces changes in expression levels and subcellular localization of histone deacetylase isoforms and PD-related molecules in SH-SY5Y dopaminergic cells.Part 2 Effects of Different Environmental Toxins on Histone Deacetylase Isoforms and PD-related Molecules in Mouse ModelsObjective To investigate the effects of different environmental toxins on histone deacetylase isoforms and PD-related molecules in mouse modelsMethods Male C57BL/6 mice aged 12 months were divided into 2 groups:control group, rotenone treated group (30mg/kg/d, p.o.,5 times per week for 8 weeks). Male C57BL/6 mice aged 4 months were divided into 4 groups:control group, fenpropathrin treated group (10mg/kg/d, i.p.) and fenpropathrin treated group (25mg/kg/d, i.p.,5 times per week for 8 weeks). Laser Scanning Confocal Microscope (LSCM) was used to observe subcellular localization and protein aggregates of HDAC1, HDAC4, HDAC6, a-synuclein and ubiquitin. Real-time PCR was used to detect the mRNA levels of HDAC1, HDAC4, HDAC6, a-synuclein, VMAT2 and DAT.Results LSCM showed that chronic rotenone treatment enhanced HDAC1 staining in the nucleus of dopaminergic neurons in the substantia nigra. HDAC4 protein distributed uniformly in the cytoplasm of normal dopaminergic neurons, with a small fraction appeared in the nucleus. Chronic exposure to rotenone led to the enhanced cytoplasmic and nuclear staining as well as the formation of HDAC4-positive aggregates. Similarly, HDAC6 staining in the cytoplasm and neurites of dopaminergic neurons was greatly elevated upon rotenone treatment, whereas this protein distributed uniformly in the cytoplasm of normal neurons. Additionally, obvious a-synuclein and ubiquitin aggregation were also observed in dopaminergic neurons of rotenone-lesioned mice. Real-time PCR indicated that fenpropathrin treatment downregulated the mRNA levels of HDAC1, HDAC4, HDAC6, VMAT2 and DAT in the substantia nigra in a dose-and time-denpendent manner, whereas the mRNA level of a-synuclein was upregulated upon fenpropathrin exposure. In addition, fenpropathrin treatment also decreased the mRNA levels of VMAT2 and DAT in the striatum in a time-denpendent manner.Conclusion Rotenone or fenpropathrin treatment induces changes in expression levels and subcellular localization of histone deacetylase isoforms and PD-related molecules in mouse models.Part 3 Mechanisms Underlying Neuroprotective Effects of HDAC Inhibitors in Rotenone-induced Dopaminergic Cell ModelObjective To explore the possible mechanisms underlying neuroprotective effects of HDAC inhibitors in rotenone-induced dopaminergic cellsMethods SH-SY5Y/SK-N-SH cells were treated with rotenone, combined with various concentrations of NaB or VPA pretreatment. In addition, SK-N-SH cells were treated with rotenone with NaB/VPA pretreatment under the condition of autophagic flux disruption induced by 3-MA or Chloroquine. Western blot was used to detect the protein levels of a-synuclein oligomers, pSerl29a-synuclein, LC3, P62, beclin 1 and GRP78.Results Western blot showed that NaB or VPA pretreatment was able to increase beclin 1 level and the ratio of LC3-?/LC-? in a dose-dependent manner in rotenone-induced dopaminergic cells. And the protein level of a-synuclein oligomers, pSerl29a-synuclein, P62 and GRP78 were decreased with NaB/VPA pretreatment. Moreover,3-MA or Chloroquine pretreatment could partially block the effect of autophagy induction by NaB or VPA.Conclusion NaB protects against rotenone-induced neurotoxicity in mice by regulating autophagy-related signaling pathways including restoring Beclin-1 expression and reducing mTOR expression.Part 4 Mechanisms Underlying Neuroprotective Effects of HDAC Inhibitors against Rotenone Neurotoxicity in Wild-type, VMAT2 and DAT Heterozygote Knockout MiceObjective To investigate whether NaB or VPA could enhance autophagy and thus exert neuroprotective effects in PD models induced by chronic rotenone administration in wild-type, VMAT2 and DAT heterozygote knockout mice.Methods Male wild-type C57BL/6 mice aged 12 months were divided into 4 groups: control group, rotenone-only treated group (30mg/kg/d, p.o.), group treated with both NaB (0.3g/kg/d, i.p.) and rotenone, group treated with both VPA (50mg/kg/d, i.p.) and rotenone (5 times per week for 8 weeks). Male VMAT2 and DAT heterozygote mice aged 12 months were divided into 3 groups:control group, rotenone-only treated group (30mg/kg/d, p.o.) and group treated with both NaB (0.3g/kg/d, i.p.) and rotenone (5 times per week for 8 weeks). Then, animals were subjected to behavioral evaluations by Rotarod and pole test, neurochemical analysis by HPLC with electrochemical detection, immunohistochemical and immunofluorescence assay, western blot analysis and ultrastructural observation by Transmission Electron Microscope (TEM).Results Administration of NaB or VPA significantly attenuated rotenone-induced motor deficits, depletion of striatal dopamine and loss of TH-positive neurons in the substantia nigra in wild-type, VMAT2 and DAT heterozygote knockout mice. Moreover, NaB or VPA significantly reduced accumulation of a-synuclein and ubiquitin in dopaminergic neurons and hippocampal neurons, which were elevated in rotenone-treated mice. Furthermore, compared with animals treated with rotenone only, NaB or VPA administration significantly reduced P62 accumulation, increased the number of LC3-II-positive autophagosomes. In rotenone-induced wild-type and DAT heterozygote mice, mitochondria in substantia nigra and striatum showed abnormal structure with distorted cristae and swollen shape. In contrast, these changes were distinctly diminished upon NaB treatment showing normal mitochondrial morphology. Moreover, NaB treatment greatly increased the number of autophagic vacuoles (AVs) which include autophagosomes and autolysosomes. In particular, an distinct increase in autolysosomes were observed upon NaB treatment.Conclusion Our study indicates that NaB or VPA attenuates rotenone-induced neurodegeneration via enhancing autophagy and preventing a-synuclein aggregation.