Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for...Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.展开更多
OBJECTIVE To examine whether long-form phosphodiesterase-4(PDE4)knockdown by lentiviral RNA construct containing a speci fi c micro RNA/mi RNAmir hairpin structure reversed depression-like symptoms caused by chronic u...OBJECTIVE To examine whether long-form phosphodiesterase-4(PDE4)knockdown by lentiviral RNA construct containing a speci fi c micro RNA/mi RNAmir hairpin structure reversed depression-like symptoms caused by chronic unpredictable mild stress(CUMS)in mice.METHODS In this research,the study was performed on adult male C57 mice,weighing(25±5)g,kept in a controlled environment.CUMS animal model was used recapitulate a multiple of behavioral characteristics and biochemical states of depression in human.The forced swimming test(FST)and the tail suspension test(TST)were used to detectthe state of depression.Western blotting analysis was used to assess protein levels of c AMP response element binding protein(CREB,unphosphorylated and phosphorylated[p CREB])to explore the neurochemical mechanisms.RESULTS CUMS decreased c AMP levels(P<0.01)and produced depression-like symptoms in FST(P<0.01)and TST(P<0.01).Microinfusions of lentiviruses reversed CUMS-induced c AMP decline(P<0.05)and depression-like symptoms.Moreover,CUMS caused a significant reduction in protein kinase A and CREB phosphorylation,and brain-derived neurotrophic factor transcription,both of which were partially attenuated by lentivirus-mediated knockdown of PDE4D.Also,the phosphorylation of extracellular signal-regulated kinase 1/2 was reduced in CUMS-exposed mice,which was reversed by 4Dmi RNA treatment.Taken together,this study demonstrated that PDE4Dmi RNA improved the CUMS-induced depressionlike symptoms that might be related to the increase in hippocampal c AMP and p CREB expression.CONCLUSION Hence,PDE4D inhibitors can serve as potential antidepressants,and their antidepressant activity is partially mediated by the activation of c AMP signaling pathway in the hippocampus.In other words,long-form PDE4D knockdown may offer a promising treatment for major depression disorder.展开更多
OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxida...OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxidant damage is a reasonable strategy to slow down the progress of PD. FCPR16 is a novel phosphodiesterase4 inhibitor with little emetic potential. Our previous studies showed that FCPR16 was an effective compound for blocking 1-methyl-4-phenylpyridine(MPP+)-induced oxidative damage in SH-SY5Y cells and neurons. However, the detailed mechanisms underlying its protective effect have not been investigated. The level of oxidative stress in neurons is closely related to the balance of mitochondria mass, while autophagy strongly regulates mitochondrial activity in neurons. Our previous study indicated that inhibition of PDE4 or PDE4 knockdown enhanced the activation of autophagy in microglial cells. While whether PDE4 inhibition mediates autophagy in neurons is largely unknown. As described above, autophagy plays a pivotal role in maintaining redox and mitochondrial homeostasis. We were interested in exploring the impact of PDE4 inhibition on autophagy in neurons. METHODS SH-SY5Y cells and neurons was induced with MPP+to mimic PD cell injury in vitro, and MTT assay was used to investigate the viability effects of FCPR16 (50μmol·L-1) with or without different autophagy inhibitors on MPP+-injured SH-SY5Y cells. Detection of apoptosis was performed by PI staining fluorescence. Lysosomes are essential in autophagy, so LYT Red Stain was used to detect lysosomes in SY5Y cells and neurons. Cells were exposed to Cell ROX Deep Red Reagent to detect intracellular reactive oxygen species (ROS). Mitochondrial membrane potential (Δψm)measurement was executed by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl-carbocyanineiodide (JC-1).To better detect intracellular autophagy, we used the CYTO-ID Autophagy detection kit to detect the autophagic vacuoles and monitor autophagic flux. The expression of autophagy related proteins and other related signal molecules were demonstrated by Western blot. As relevant indicators of oxidative stress, 3-nitrotyrosine (3-NT) and highly toxic peroxide4-hydroxynonenal (4-HNE) were detected with 3-NT and 4-HNE ELISA kits. RESULTS FCPR16 could significantly decrease the expression of p62, an autophagy substrate, at 6 and 12 h, while FCPR16 enhanced the level of LC3-Ⅱ. Similarly, FCPR16 increased the lysosomes fluorescence and CYTO-ID signal in cells and neurons, while it could be blockby 3-methyladenine (3-MA) and hydroxychloroquine sulfate (HCQ). Simultaneously, Treatment of SH-SY5Y cells with FCPR16 prevented MPP+-induced production of ROS and the decline ofΔψm. Importantly, we also found that FCPR16 phosphorylated and thus activated AMPK in SH-SY5Y cells treated with MPP+. In contrast, blockade of the AMPK pathway with compound C blocked the role of FCPR16 in autophagy enhancement. MPP+-induced a significant increase in PI-positive cells, while FCPR16 decreased the ratio of PI positive cells and 3-MA and compound C could block the protective effect. Additionally, FCPR16 reduced MPP+-induced decline of cell viability, and 3-MA and compound C could block the protective effect. CONCLUSION Deficits in autophagy have been proven to participate the pathology of PD and targeting autophagic function has been viewed as a potential therapeutic strategy for the clearance of toxic proteins (such asα-synuclein) and of impaired mitochondria. this is the first time that PDE4 inhibition has been shown to induce autophagic enhancement both in SH-SY5Y cells and in primary cultured neurons. In addition, our findings indicate that inhibition of PDE4 by FCPR16 protects against MPP+-induced oxidative stress and cellular injury in SH-SY5Y cells and neurons through the activation of AMPK-dependent autophagy. Taken together, these results show that PDE4 is a promising target for developing novel drugs against neuronal apoptosis and FCPR16 may be a potential compound for the prevention and treatment of PD.展开更多
基金NationalNatural Science Foundation of China (81773698)Funding from Guangzhou Science and Technology Department (2015B020211007,201604020112).
文摘Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.
基金The project supported by National Natural Science Foundation of China(81301099,81373384)Natural Science Foundation of Guangdong Province(S2013040014202)+1 种基金China Postdoctoral Science Foundation(2013M542192)National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2012ZX09J1211003C)
文摘OBJECTIVE To examine whether long-form phosphodiesterase-4(PDE4)knockdown by lentiviral RNA construct containing a speci fi c micro RNA/mi RNAmir hairpin structure reversed depression-like symptoms caused by chronic unpredictable mild stress(CUMS)in mice.METHODS In this research,the study was performed on adult male C57 mice,weighing(25±5)g,kept in a controlled environment.CUMS animal model was used recapitulate a multiple of behavioral characteristics and biochemical states of depression in human.The forced swimming test(FST)and the tail suspension test(TST)were used to detectthe state of depression.Western blotting analysis was used to assess protein levels of c AMP response element binding protein(CREB,unphosphorylated and phosphorylated[p CREB])to explore the neurochemical mechanisms.RESULTS CUMS decreased c AMP levels(P<0.01)and produced depression-like symptoms in FST(P<0.01)and TST(P<0.01).Microinfusions of lentiviruses reversed CUMS-induced c AMP decline(P<0.05)and depression-like symptoms.Moreover,CUMS caused a significant reduction in protein kinase A and CREB phosphorylation,and brain-derived neurotrophic factor transcription,both of which were partially attenuated by lentivirus-mediated knockdown of PDE4D.Also,the phosphorylation of extracellular signal-regulated kinase 1/2 was reduced in CUMS-exposed mice,which was reversed by 4Dmi RNA treatment.Taken together,this study demonstrated that PDE4Dmi RNA improved the CUMS-induced depressionlike symptoms that might be related to the increase in hippocampal c AMP and p CREB expression.CONCLUSION Hence,PDE4D inhibitors can serve as potential antidepressants,and their antidepressant activity is partially mediated by the activation of c AMP signaling pathway in the hippocampus.In other words,long-form PDE4D knockdown may offer a promising treatment for major depression disorder.
基金National Natural Science Foundation of China(81773698)Guangzhou Science and Technology Department(2015B020211007 and 201604020112)
文摘OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxidant damage is a reasonable strategy to slow down the progress of PD. FCPR16 is a novel phosphodiesterase4 inhibitor with little emetic potential. Our previous studies showed that FCPR16 was an effective compound for blocking 1-methyl-4-phenylpyridine(MPP+)-induced oxidative damage in SH-SY5Y cells and neurons. However, the detailed mechanisms underlying its protective effect have not been investigated. The level of oxidative stress in neurons is closely related to the balance of mitochondria mass, while autophagy strongly regulates mitochondrial activity in neurons. Our previous study indicated that inhibition of PDE4 or PDE4 knockdown enhanced the activation of autophagy in microglial cells. While whether PDE4 inhibition mediates autophagy in neurons is largely unknown. As described above, autophagy plays a pivotal role in maintaining redox and mitochondrial homeostasis. We were interested in exploring the impact of PDE4 inhibition on autophagy in neurons. METHODS SH-SY5Y cells and neurons was induced with MPP+to mimic PD cell injury in vitro, and MTT assay was used to investigate the viability effects of FCPR16 (50μmol·L-1) with or without different autophagy inhibitors on MPP+-injured SH-SY5Y cells. Detection of apoptosis was performed by PI staining fluorescence. Lysosomes are essential in autophagy, so LYT Red Stain was used to detect lysosomes in SY5Y cells and neurons. Cells were exposed to Cell ROX Deep Red Reagent to detect intracellular reactive oxygen species (ROS). Mitochondrial membrane potential (Δψm)measurement was executed by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl-carbocyanineiodide (JC-1).To better detect intracellular autophagy, we used the CYTO-ID Autophagy detection kit to detect the autophagic vacuoles and monitor autophagic flux. The expression of autophagy related proteins and other related signal molecules were demonstrated by Western blot. As relevant indicators of oxidative stress, 3-nitrotyrosine (3-NT) and highly toxic peroxide4-hydroxynonenal (4-HNE) were detected with 3-NT and 4-HNE ELISA kits. RESULTS FCPR16 could significantly decrease the expression of p62, an autophagy substrate, at 6 and 12 h, while FCPR16 enhanced the level of LC3-Ⅱ. Similarly, FCPR16 increased the lysosomes fluorescence and CYTO-ID signal in cells and neurons, while it could be blockby 3-methyladenine (3-MA) and hydroxychloroquine sulfate (HCQ). Simultaneously, Treatment of SH-SY5Y cells with FCPR16 prevented MPP+-induced production of ROS and the decline ofΔψm. Importantly, we also found that FCPR16 phosphorylated and thus activated AMPK in SH-SY5Y cells treated with MPP+. In contrast, blockade of the AMPK pathway with compound C blocked the role of FCPR16 in autophagy enhancement. MPP+-induced a significant increase in PI-positive cells, while FCPR16 decreased the ratio of PI positive cells and 3-MA and compound C could block the protective effect. Additionally, FCPR16 reduced MPP+-induced decline of cell viability, and 3-MA and compound C could block the protective effect. CONCLUSION Deficits in autophagy have been proven to participate the pathology of PD and targeting autophagic function has been viewed as a potential therapeutic strategy for the clearance of toxic proteins (such asα-synuclein) and of impaired mitochondria. this is the first time that PDE4 inhibition has been shown to induce autophagic enhancement both in SH-SY5Y cells and in primary cultured neurons. In addition, our findings indicate that inhibition of PDE4 by FCPR16 protects against MPP+-induced oxidative stress and cellular injury in SH-SY5Y cells and neurons through the activation of AMPK-dependent autophagy. Taken together, these results show that PDE4 is a promising target for developing novel drugs against neuronal apoptosis and FCPR16 may be a potential compound for the prevention and treatment of PD.
