Inhibition of phosphodiesterase 4 by FCPR16 protects SH-SY5Y cells against MPP^+ -induced decline of mitochondrial membrane potential and oxidative stress

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.

S3A-4 Inhibition of Phosphodiesterase 4 by FCPR16 Protects SH-SY5Y Cells against MPP^+-Induced Cell Death through Activating cAMP/PKA/CREB and Epac/Akt Signaling Pathways

Background:Phosphodiesterase 4(PDE4)is a promising target for the treatment of Parkinson’s disease(PD).However,the underlying mechanism has not yet been well elucidated.Additionally,most of current PDE4 inhibitors produce severe nausea and vomiting response in patients,which limit their clinical application.FCPR16 is a novel PDE4 inhibitor with little emetic potential.In the present study,the neuroprotective effect and underlying mechanism of FCPR16 against cellular apoptosis induced by 1-methyl-4-phenylpyridinium(MPP+)were examined in SH-SY5Y cells and primary cultured neurons.Methods:CCK-8 assay,Hoechst staining,lactate dehydrogenase release and flow cytometry were used to study the protective effect of FCPR16 against cell damage caused by MPP+.Mitochondrial membrane potential(Δψm)was measured by JC-1 staining.The extent of oxidation was evaluated using Cell ROXs Deep Red Reagent and malonaldehyde(MDA)kit.Pretreatments with various pathway inhibitors were used to investigate the possible pathways involved in the protection of FCPR16.The phosphorylated and total levels of various proteins were analyzed by Western blot.Results:FCPR16(12.5-50μmol·L-1)dose-dependently reduced MPP+-induced loss 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(25μmol·L-1)significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline ofΔψm and attenuated the expression of MDA level.Further studies disclosed that FCPR16 enhanced the levels of cAMP and the exchange protein directly activated by cAMP(Epac)in SH-SY5Y cells.Western blotting analysis revealed that FCPR16 increased the phosphorylation of cAMP response element-binding protein(CREB)and protein kinase B(Akt)down-regulated by MPP+in SH-SY5Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS andΔψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.We also found that MPP+induced a dose-dependent apoptosis in cultured neurons,and 500μmol·L-1 MPP+caused an approximately 50%loss of cortical neurons,while treatment with FCPR16 reversed the toxic effect of MPP+and enhanced the cell viability in a dose-dependent manner.Conclusion:These results suggest that FCPR16 attenuates MPP+-induced dopaminergic degeneration via lowering ROS and preventing the loss ofΔψm in SH-SY5Y cells.Mechanistically,cAMP/PKA/CREB and Epac/Akt signaling pathways are involved in these processes.

A novel mechanism underlying the protective effect of PDE4 inhibitor against cognitive impairment:inhibiting neuroinflammation through inducing autophagy in microglial cells

OBJECTIVE Inhibition of phosphodiesterase 4(PDE4) improves the learning and memory abilities in Alzheimer disease animal models. The cognition-enhancing effects of PDE4 inhibition involve reduced inflammatory responses in the brain. However,the underlying mechanisms are illunderstood. cA MP induces autophagy,and deficiency of autophagy leads to elevated inflammatory factors.In the present study,we aimed to investigate the contribution of autophagy to the anti-inflammatory effect of PDE4 inhibitor ROF. METHODS Acidic vesicles were traced by Lysotracker(LYT) red and acridine orange(AO) staining. Autophagosomes in BV-2 cells was observed by immunofluorescence staining of microtubule-associated protein 1 light chain 3(LC3). Aβ_(25-35) or lipopolysaccharide(LPS) with ATP were used to activate microglial cells and inflammasome. Cytokine levels were measured by ELISA method. The levels of pro-inflammatory factors and essential proteins involved in the formation of autophagosome were detected by Western blotting. RESULTS ROF increased the level of LC3-Ⅱ,while the level of p62 was decreased. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by AO and LYT red staining. In addition,immunofluorescence indicated a significant increase in punctate LC3. Both LPS plus ATP and Aβ_(25-35) enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1β. Interestingly,these effects were blocked by the treatment of ROF. Moreover,ROF decreased the apoptosis of neuronal N2 a cells in conditioned media from BV-2 microglia. These effects were reversed by inhibition of microglial autophagy.Treatment with ROF also showedenhanced autophagy in mcie treated with LPS. CONCLUSION PDE4 inhibitor ROF inhibits inflammasome activities and reduces the release of IL-1β by inducing autophagy.

FCPR16 induces AMPK-dependent autophagy and provides neuroprotection in SH-SY5Y cells and neurons exposed to MPP^+-induced oxidative damage

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.