The Role Of CBS-H₂S Axis In The Pathogenesis Of Parkinson Disease

Objective:In recent years,hydrogen sulfide(H2S)and its donor such as sodium hydrosulfide(NaHS)have been demonstrated to exert neuroprotective effects in different rodent models of Parkinson disease(PD),acting by resolving inflammation and oxidative stress.However,the role of endogenous H2S in the pathologenesis of PD remains elusive.Therefore,this study aims to explore the effect and mechanism of H2S and its synthase in a PD mouse model induced by 1-methy-4-pheny 1-1,2,3,6-tetrahydropyridine(MPTP).Methods:Male C57BL/6J mice(8-10 weeks old,23-28 g weight)were used in this study.PD mouse model was established by intraperitoneally(i.p.)injecting the mice with 14 mg/kg MPTP four times at 2-h intervals.Saline was given as control.To explore the role of(cystathionine β-synthase,CBS)overexpression in MPTP-induced mouse model,the mouse striatum was bilaterally injected with recombinant adeno-associated-virus encoding Cbs(rAAV-Cbs)or its vector(rAAV-Vector)using the stereotaxic apparatus,followed by MPTP or saline administration.The sulfide level in the plasma and striatum was determined by methylene blue method.The protein expression of H2S synthases including CBS,(cystathionine y-lyase,CSE)and(3-mercaptopyruvate sulfurtransferase,3-MST)was evaluated by western blot.The motor balance and coordination ability of the mice was assessed by rotarod,grid walking and balance beam test.The loss of tyrosine hydroxylase(TH)positive neurons/fibers,and activation of astrocytes and microglia in the substantia nigra and striatum were studied by immunohistochemistry and western blotting.The nitrite content which is indicative of nitric oxide(NO)production in the striatum was determined by Griess reagent.The protein expressions of nNOS,3-nitrotyrosine and nitro-a-synuclein in the striatum were studied by western blotting,and iNOS mRNA level was examined by quantitative PCR.In in vitro study,a variety of cells such as primary microglia,astrocytes,midbrain neuron culture,and related cell lines were used and subjected to MPP+ treatment.CBS protein expression was tested using western blotting,and the sulfide production was evaluated by methylene blue method.To establish CBS overexpression in glia culture,mixed glial cells were infected with Lenti-Cbs or Lenti-Vector.To evaluate the effect of glia and glia-derived inflammatory mediators on neuron survival,glia-conditioned medium was harvested and transferred into midbrain neuron culture at 24h after MPP+treatment.The morphology of dopaminergic neurons was then assessed by immunofluorescent staining with the antibody against TH.The midbrain neuron culture was pretreated with 200 μM NaHS or PBS for 10 min,followed by addition with 0.5 mM MPP+ or vehicle.The morphology and neurite length of dopaminergic neurons were examined.MES23.5 and PC12 cells were pretreated with 200μM NaHS or PBS for 10 min,followed by addition with 0.5 mM MPP+ or vehicle.The cell viability of MES23.5 was measured by MTT assay,the expression of nNOS was detected by western blot.Results:1.The decreases of CBS expression and hydrogen sulfide generation in MPTP/MPP+ induced in vivo and in vitro PD models:1)MPTP caused a 25%decrease of sulfide in the striatum at two weeks after administration compared to saline treatment.Unexpectedly,we also detected a 50%decline of plasma sulfide in MPTP-treated mice.2)CBS expression in the striatum was markedly decreased at one week after MPTP injection and remained to be downregulated at two weeks later,while CSE and 3-MST expression remained almost unaltered.However,we did not observe any obvious change in CBS or other two H2S generators in the cortex following MPTP injection.3)CBS expression in astrocytes and primary microglia culture,but not in SH-SY5Y cells or midbrain neuron culture,was significantly decreased.2.CBS overexpression attenuated MPTP-induced neurotoxicity in vivo:1)Western blot analysis showed that striatal CBS expression increased early at 3 days,reached its peak at 7 days.and remained to be markedly elevated at 14 days following rAAV-Cbs injection.Moreover.rAAV-Cbs injection resulted in a significant elevation of sulfide level in the striatum of MPTP-intoxicated mice.This implies that rAAV-Cbs injection successfully induced CBS overexpression and H2S generation in the striatum.2)A significant deficiency in motor coordination and balance was observed in rAAV-Vector(vector)overexpressing mice at two weeks after MPTP injection as compared to saline-treated group.CBS overexpression ameliorated the motor deficits in MPTP-treated mice.3)MPTP caused a dramatic loss of TH+ neurons in both the SN pars compacta and striatum in rAAV-Vector injected mice.and this effect was attenuated in rAAV-Cbs injected mice that overexpress CBS.Western blot analysis of the striatal homogenates also showed the decline of TH protein level in MPTP-treated mice injected with rAAV-Vector(vector),which was obviously alleviated in mice injected with rAAV-Cbs.4)Immunohistochemistry and western blot analyses were conducted to examine the expression of Ibal(a marker of microglia activation)and GFAP(a marker of astrocytes activation)in mouse brains.Our data showed that the numbers of reactive microglia and astrocytes in the substantia nigra and striatum of MPTP-treated rAAV-Cbs mice were significantly lower than those of the vector counterparts.A lower level of Iba-1 protein expression was also detected in the sustantia nigra of CBS overexpressing mice as compared to vector group following MPTP treatment.5)The protein and mRNA analyses showed that the MPTP-induced upregulation of nNOS and iNOS was inhibited in the mice injected with rAAV-Cbs.Here we also observed a significant elevation of nitrotyrosine(an indicator of tyrosine nitration in protein)in the striatum of MPTP-treated vector group,indicating the presence of nitrative stress in MPTP-induced PD model.This increase of 3-nitrotyrosine was obviously inhibited in mice overexpressing CBS.Immunoblotting analysis of striatal nitro-a-Syn level exhibited similar changes.3.Glial CBS overexpression protected dopaminergic neurons against MPP+-induced damage in vitro:1)Transduction with Lenti-Cbs resulted in a marked elevation of CBS expression and H2S generation in glia,although to a less extent in MPP+-treated glia as compared to vehicle group;NO generation and NOS activity was inhibited by CBS expression.2)CBS overexpression in glia relieved the prominent shortening of dopaminergic neurites induced by MPP+-treated glia conditioned medium,implying glial CBS overexpression produced an indirect protection on dopaminergic neurons.3)NaHS pre-treatment alleviated the prominent shortening of dopaminergic neurites induced by MPP+-treatment,NaHS pre-treatment inhibited the decrease of cell viability in MES23.5 and upregulation of nNOS in PC12 cells,stating H2S produced an direct protection on dopaminergic neurons.Conclusion:The sulfide level in the striatum and plasma of MPTP-induced PD model was decreased,accompanied with CBS downregulation in the striatum and glial cells.MPTP injection induced movement impairment in mice,associated with the loss of dopaminergic neurons and activation of glia in the nigro-striatal pathway.Striatal CBS overexpression was able to ameliorate MPTP-induced dopaminergic neurotoxicity in the midbrain,which may be related to the inhibition of glial activation and nitrative stress,and thereby the reduction of a-syn nitration elicited by CBS-H2S axis.