GALNT9 enrichment attenuates MPP+-induced cytotoxicity by ameliorating protein aggregations containing α-synuclein and mitochondrial dysfunction.

BACKGROUND: GALNTs (UDP-GalNAc; polypeptide N-acetylgalactosaminyltransferases) initiate mucin-type O-GalNAc glycosylation by adding N-GalNAc to protein serine/threonine residues. Abnormalities in O-GalNAc glycosylation are involved in various disorders such as Parkinson's disease (PD), a neurodegenerative disorder. GALNT9 is potentially downregulated in PD patients. METHODS: To determine whether GALNT9 enrichment ameliorates cytotoxicity related to PD-like variations, a pcDNA3.1-GALNT9 plasmid was constructed and transfected into SH-SY5Y cells to establish a GALNT9-overexpressing cell model. RESULTS: Downregulation of GALNT9 and O-GalNAc glycosylation was confirmed in our animal and cellular models of PD-like variations. GALNT9 supplementation greatly attenuated cytotoxicity induced by MPP+ (1-Methyl-4-phenylpyridinium iodide) since it led to increased levels of tyrosine hydroxylase and dopamine, reduced rates of apoptosis, and significantly ameliorated MPP+-induced mitochondrial dysfunction by alleviating abnormal levels of mitochondrial membrane potential and reactive oxygen species. A long-lasting mPTP (mitochondrial permeability transition pores) opening and calcium efflux resulted in significantly lower activity in the cytochrome C-associated apoptotic pathway and mitophagy process, signifying that GALNT9 supplementation maintained neuronal cell health under MPP+ exposure. Additionally, it was found that glycans linked to proteins influenced the formation of protein aggregates containing α-synuclein, and GALNT9 supplement dramatically reduced such insoluble protein aggregations under MPP+ treatment. Glial GALNT9 predominantly appears under pathological conditions like PD-like variations. CONCLUSIONS: GALNT9 enrichment improved cell survival, and glial GALNT9 potentially represents a pathogenic index for PD patients. This study provides insights into the development of therapeutic strategies for the treatment of PD.

Sodium butyrate activates the KATP channels to regulate the mechanism of Parkinson's disease microglia model inflammation.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder. Microglia-mediated neuroinflammation has emerged as an involving mechanism at the initiation and development of PD. Activation of adenosine triphosphate (ATP)-sensitive potassium (KATP ) channels can protect dopaminergic neurons from damage. Sodium butyrate (NaB) shows anti-inflammatory and neuroprotective effects in some animal models of brain injury and regulates the KATP channels in islet ß cells. In this study, we aimed to verify the anti-inflammatory effect of NaB on PD and further explored potential molecular mechanisms. METHODS: We established an in vitro PD model in BV2 cells using 1-methyl-4-phenylpyridinium (MPP+ ). The effects of MPP+ and NaB on BV2 cell viability were detected by cell counting kit-8 assays. The morphology of BV2 cells with or without MPP+ treatment was imaged via an optical microscope. The expression of Iba-1 was examined by the immunofluorescence staining. The intracellular ATP content was estimated through the colorimetric method, and Griess assay was conducted to measure the nitric oxide production. The expression levels of pro-inflammatory cytokines and KATP channel subunits were evaluated by reverse transcription-quantitative polymerase chain reaction and western blot analysis. RESULTS: NaB (5 mM) activated the KATP channels through elevating Kir6.1 and Kir6.1 expression in MPP+ -challenged BV2 cells. Both NaB and pinacidil (a KATP opener) suppressed the MPP+ -induced activation of BV2 cells and reduced the production of nitrite and pro-inflammatory cytokines in MPP+ -challenged BV2 cells. CONCLUSION: NaB treatment alleviates the MPP+ -induced inflammatory responses in microglia via activation of KATP channels.

CircHIVEP2 alleviates Parkinson's nerve damage and inflammatory response by targeting miR-485-3p.

OBJECTIVE: Dysregulation of covalently closed circular RNAs (circRNAs) has been associated with neurological disorders, the role of circHIVP2 in Parkinson's disease (PD) and its molecular mechanism is not well understood. METHODS: 127 patients with PD and 85 healthy people were enrolled. RT-qPCR was employed to examine the levels of circHIVEP2. ROC curve to explore the diagnostic. Mpp+ induced the SH-SY5Y to construct an in vitro PD cell model. Cell viability, apoptosis, and secretion levels of inflammatory factors were analyzed by CCK-8, flow cytometry, and ELISA assay. CircHIVEP2 targets miRNA predicted by bioinformatics database and validated by the dual luciferase reporter and RIP assays. RESULTS: CircHIVEP2 was typically lower in PD patients than in controls. CircHIVEP2 has certain specificity and sensitivity to recognize PD patients from healthy individuals. miR-485-3p, a target miRNA of circHIVEP2, was significantly elevated in PD patients. Additionally, MPP+ induction reduced cell viability and promoted apoptosis and inflammatory factor overproduction. However, overexpression of circHIVEP2 significantly inhibited the effects of MPP+, but this inhibition was significantly attenuated by elevated miR-485-3p. CONCLUSION: circHIVEP2 is a potential diagnostic biomarker for PD, and its upregulation mitigated MPP+-induced nerve damage and inflammation and this may be through targeted by the miR-485-3p.

Downregulation of TRPM7, TRPM8, and TRPV1 channels modulate apoptotic parameters and neurodegenerative markers: Focus on neuronal differentiation and Parkinson's disease model.

The transient receptor potential channel (TRP) channels are expressed in neuronal tissues and involved in neurological diseases such as pain, epilepsy, neuronal apoptosis, and neurodegenerative diseases. Formerly, we have investigated how neuronal differentiation changes TRP channels expression profile and how Parkinson's disease model is related with this expression levels. We have found that transient receptor potential channel melastatin subtype 7 (TRPM7), transient receptor potential channel melastatin subtype 8 and transient receptor potential channel vanilloid subtype 1 (TRPV1) channels have pivotal effects on differentiation and 1-Methyl-4-phenylpyridinium (MPP+ )-induced Parkinson's disease model in SH-SY5Y cells. In this study, we have investigated that downregulation of the TRP channels to evaluate how differentiation status changes to Parkinson's disease pathological hallmarks. We have also performed to other analyses to elucidate these TRP channels' function in MPP+ -induced neurotoxicity related apoptosis, cell viability, caspase 3 and 9 enzyme activities, intracellular reactive oxygen species production, mitochondrial depolarization levels, Ca2+ signaling, Alpha-synuclein and Dopamine levels, mono amino oxidase A and B enzymatic activities, both in differentiated and undifferentiated neuronal cells. Herein we have concluded that especially TRPM7 and TRPV1 channels have distinct role in Parkinson's disease pathology via their activity changings in pathological state, and downregulation of these channels or specific antagonists can be useful for the possible treatment strategy for Parkinson's disease and related markers.

LincRNA-p21 Promotes Cellular Senescence by Down-regulating the Wnt/ß-catenin Pathway in MPP+-treated SH-SY5Y Cells.

AIM AND OBJECTIVE: Long intergenic non-coding RNA-p21 (lincRNA-p21) plays a critical role in various senescence-associated physiological and pathological conditions. We aimed to explore the senescence-associated effects of lincRNA-p21 in 1-methyl-4-phenylpyridinium (MPP+) treated neuroblastoma SH-SY5Y cell line as a therapeutic target. MATERIALS AND METHODS: The RNA expression levels of lincRNA-p21, p53, p16, and telomere length were examined with reverse transcription-quantitative polymerase chain reaction (RTqPCR). The Telo TAGGG™ Telomerase PCR ELISA PLUS Kit was used to determine telomerase activity. Cellular viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay. Western blot was performed to analyze ß-catenin protein expression. Besides, oxidative stress was evaluated by Jaggregate- forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolocarbocyanine++ + iodide (JC­1) stain, fluorescence spectrophotometry, colorimetric assay, and malondialdehyde (MDA) formation. RESULTS: This research demonstrated that MPP+ caused a distinct increase in the expression of LincRNA- p21 in SH-SY5Y cells. MPP+ induced cellular senescence with decreasing cellular proliferation and viability, increasing expression levels of senescence-associated makers such as genes p53 and p16, accompanied by significantly decreasing telomere length and telomerase activity. At the same time, these effects were abolished by silencing lincRNA-p21 with small interfering RNA (siRNA). On the contrary, ß-catenin silencing contributes to reversing anti-senescent effects caused by lincRNA-p21 silencing. Moreover, modifying lincRNA-p21 exerted an anti-senescent influence depending on decreasing oxidant stress. CONCLUSION: Our study showed that in the treatment of MPP+, lincRNA-p21 might serve a role in the SH-SY5Y cell senescence by modulating the Wnt/ß-catenin pathway, as well as increasing oxidant stress. Thus, trying to target lincRNA-p21 may have important therapeutic and practical implications for PD.

Cannabinoid type 2 receptor activation inhibits MPP+-induced M1 differentiation of microglia through activating PI3K/Akt/Nrf2 signal pathway.

BACKGROUND: Growing evidence indicates that cannabinoid type 2 (CB2) receptor activation inhibits neuroinflammation in the pathogenesis of Parkinson's disease (PD). Nonetheless, the precise mechanisms of CB2 receptor-mediated neuroprotection have not been fully elucidated. The differentiation of microglia from the M1 to M2 phenotype plays a vital role in neuroinflammation. METHODS: In the present study, we investigated the effect of CB2 receptor activation on the M1/M2 phenotypic transformation of microglia treated with 1-methyl-4-phenylpyridinium (MPP+). The M1 phenotype microglia markers, including inducible nitric oxide (iNOS), interleukin 6 (IL-6), and CD86, and the M2 phenotype microglia markers, including arginase-1 (Arg-1), IL-10, and CD206, were detected by western blots and flow cytometry. The levels of phosphoinositide-3-kinase (PI3K)/Akt and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined by Western blots. Subsequent addition of Nrf2 inhibitors initially revealed the specific mechanism by which CB2 receptors affect phenotypic changes in microglia. RESULTS: Our results showed that pretreatment with JWH133 significantly inhibited the MPP+-induced up-regulation of M1 phenotype microglia markers. Meanwhile, JWH133 increased the levels of M2 phenotype microglia markers. JWH133-mediated effects were blocked by co-treatment with AM630. Mechanism studies found that MPP+ treatment downregulated PI3K, Akt phosphorylated proteins, and nuclear Nrf2 protein. JWH133 pretreatment promoted PI3K/Akt activation and facilitated nuclear translocation of Nrf2, which was reversed by the PI3K inhibitor. Further studies showed that Nrf2 inhibitors inverted the effect of JWH133 on microglia polarization. CONCLUSION: The results indicate that CB2 receptor activation promotes MPP+-induced microglia transformation from M1 to M2 phenotype through PI3K/Akt/Nrf2 signaling pathway.

2-Aminoethoxydiphenyl borate ameliorates mitochondrial dysfunctions in MPTP/MPP+ model of Parkinson's disease.

Mitochondrial dysfunction is closely linked with the pathophysiology of several neurodegenerative disorders including Parkinson's disease (PD). Despite several therapeutic advancements related to symptomatic modification of PD pathology, strategies targeting mitochondrial dysfunctions remain largely elusive. Recently, transient receptor potential (TRP) channels have been shown to play a pivotal role in the control of mitochondrial and neuronal functioning in PD. In this study, the effect of 2-aminoethoxydiphenyl borate (2-APB), TRP channel blocker was investigated in the context of mitochondrial dysfunctions in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-administered Sprague Dawley rats. MPP+-treated SH-SY5Y cells exhibited reductions in cell viability, generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential. Co-treatment with 2-APB led to an increase in cell viability, reduction in intracellular and mitochondrial ROS and improvement in mitochondrial membrane potential compared to MPP+-treated SH-SY5Y cells. In addition, intranigral administration of MPTP led to a significant reduction in motor function in the rats. Fourteen days of 2-APB (3 and 10 mg/kg, i.p.) treatment improved behavioural parameters. MPTP-induced decrease in complex I activity and mitochondrial potential were also blocked by 2-APB in the mitochondria isolated from the brain regions i.e. midbrain and striatum. MPTP-induced decrease in tyrosine hydroxylase levels were also restored by 2-APB. Moreover, MPTP-induced reduction in proteins involved in mitochondrial biogenesis, viz. peroxisome proliferator-activated-receptor-gamma coactivator and mitochondrial transcription factor-A were increased after 2-APB treatment in vivo. In summary, 2-APB has a promising neuroprotective role in the MPP+/MPTP models of PD via targeting mitochondrial dysfunctions and biogenesis.

Garcinol protects SH-SY5Y cells against MPP+-induced cell death by activating DJ-1/SIRT1 and PGC-1α mediated antioxidant pathway in sequential stimulation of p-AMPK mediated autophagy.

Parkinson's disease (PD), a chronic and progressive neurodegenerative disease, can reduce the population of dopaminergic neurons in the substantia nigra. The cause of this neuronal death remains unclear. 1-Methyl-4-phenylpyridinium ion (MPP+) is a potent neurotoxin that can destroy dopaminergic (DA) neurons and promote PD. Garcinol, a polyisoprenylated benzophenone derivative, was extracted from Garcinia indica and is an important active compound it has been used as an anticancer, antioxidant, and anti-inflammatory, agent and it can suppress reactive oxygen species (ROS) mediated cell death in a PD model. Human neuroblastoma (SH-SY5Y) cells (1 × 105 cells) were treated with MPP+ (1 mM) for 24 h to induce cellular ROS production. The formation of ROS was suppressed by pretreatment with different concentrations of garcinol (0.5 and 1.0 µM) for 3 h in SH-SY5Y cells. The present study found that MPP+ treatment increased the formation of reactive oxygen species (ROS), and the increased ROS began to promote cell death in SH-SY5Y cells. However, our natural compound garcinol effectively blocked MPP+-mediated ROS formation by activating the DJ-1/SIRT1 and PGC-1α mediated antioxidant pathway. Further findings indicate that the activated SIRT1 can also regulate p-AMPK-mediated autophagy to protect the neurons from the damage it concludes that garcinol sub-sequential regulates intracellular autophagy in this model, and the productive efficacy of garcinol was confirmed by western blot analysis and MitoSOX DCFDA and MTT assays. The results showed garcinol increased protection due to the prevention of MPP+-induced ROS and the promotion of cell survival.

Knockdown of SEMA7A alleviates MPP+ -induced apoptosis and inflammation in BV2 microglia via PPAR-γ activation and MAPK inactivation.

INTRODUCTION: The inflammation mediated by microglial cells plays an important role in the process of neurodegenerative diseases. Recent evidence indicates that semaphorin 7A (SEMA7A) is implicated in various neurodegenerative diseases, but whether it plays a role in Parkinson's disease (PD) remains unclear. METHODS: In this study, 1.0 mmol/L 1-methyl-4-phenylpyridinium (MPP+ )-stimulated mouse microglia (BV2) cells were used as an in vitro model of PD. The expression of SEMA7A was detected by quantitative polymerase chain reaction. Cell Counting Kit-8 and apoptosis kits were used to analyze the viability and apoptosis of BV-2 cells. The content of IL-6, IL-ß, and tumor necrosis factor-α was determined by ELISA (enzyme-linked immunosorbent assay) kit. Western blot was used to detect the protein expression level of the inducible NO synthase and cyclooxygenase-2. RESULTS: Our findings indicated that SEMA7A expression in BV2 cells was upregulated after MPP+ stimulation. Knockdown of SEMA7A promoted cell viability while it inhibited apoptosis and the expression of proinflammatory enzymes and proinflammatory cytokines. Silencing SEMA7A-induced peroxisome proliferator-activated receptor-gamma (PPAR-γ) activation and mitogen-activated protein kinase (MAPK) signaling pathway inactivation. Furthermore, a PPAR-γ inhibitor and an MAPK activator promoted the effect of MPP+ on cell viability, apoptosis, and inflammation of BV2 cells; what is more, the PPAR-γ inhibitor and MAPK activator blocked the inhibitory effect of SEMA7A downregulation on MPP+ -induced injury. CONCLUSION: In general, knockdown of SEMA7A inhibits MPP+ -induced BV2 cell apoptosis and inflammation via PPAR-γ activation and MAPK inactivation, which may provide a new therapy target for PD.

Resilin, an insect-derived elastomeric protein, protects dopaminergic neurons in Parkinson disease models.

Parkinson disease (PD) is a prevalent neurodegenerative disorder that is characterized by motor and behavioral disturbances, including resting tremors, rigidity, bradykinesia, and postural instability. The primary cause of PD is the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region that subsequently reduces the dopamine content in the striatum (ST); this is a promising therapeutic target for PD. Resilin is an elastomeric protein with high strain, low stiffness, and high resilience that is found in insect cuticles. However, scant evidence supports the application of resilin in neurodegenerative diseases, including PD. Herein, we investigated the protective effects of resilin on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in mouse models and explored the mechanisms underlying its action. Resilin significantly and concentration-dependently reduced 1-methyl-4-phenylpyridinium+ (MPP+)-induced apoptotic neurotoxicity in differentiated PC12 and SH-SY5Y cells. Moreover, resilin prevented dopamine depletion in ST, and immunohistochemical findings indicated that resilin protects against dopaminergic neuronal loss induced by MPTP in the SNpc and ST. Behavioral studies using pole and rotarod tests showed significantly improved PD-related motor impairment in mice treated with resilin. We then explored the molecular mechanisms underlying the apoptosis of dopaminergic neurons using protein arrays and discovered that resilin inhibits dopaminergic neuronal death through the apoptosis signaling factors cytochrome c and caspases-9 and -3 in the SNpc. Thus, resilin has potential in treating PD by controlling apoptosis signals.

Long noncoding RNA BACE1-antisense transcript plays a critical role in Parkinson's disease via microRNA-214-3p/Cell death-inducing p53-target protein 1 axis.

This study aimed to analyze the function and latent mechanism of long noncoding RNA BACE1-antisense transcript (lncRNA BACE1-AS) in MPP+-induced SH-SY5Y cells. SH-SY5Y cells were cultivated in 1 mM MPP+ for 24 h to establish Parkinson's disease (PD) model in vitro. TargetScan and luciferase reporter assay were conducted to predict and verify the interaction between microRNA (miR)-214-3p and CDIP1 (Cell death-inducing p53-target protein 1). Cell viability, lactate dehydrogenase (LDH) release, and cell apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT), LDH, and flow cytometer. The secretion of inflammatory factors and representative biomarkers of oxidative stress, including reactive oxygen species (ROS) and superoxide dismutase (SOD) were assessed using enzyme-linked immunosorbent assay (ELISA) and specific assay kits. Results suggested that lncRNA BACE1-AS was over-expressed and miR-214-3p was under-expressed in MPP+-stimulated SH-SY5Y cells. Further analyses revealed that MPP+ inhibited cell viability; enhanced cell apoptosis, Cleaved Caspase-3 expression and Cleaved Caspase-3/GAPDH ratio; induced oxidative stress and inflammation in SH-SY5Y cells were inhibited by lncRNA BACE1-AS-siRNA transfection; and all these inhibitions were reversed by miR-214-3p inhibitor. In addition, we found that CDIP1 was directly targeted by miR-214-3p and up-regulated in MPP+-stimulated SH-SY5Y cells. Further functional assays suggested that CDIP1-plasmid reversed the effects of miR-214-3p mimic on MPP+-stimulated SH-SY5Y cells. In conclusion, lncRNA BACE1-AS regulates SH-SY5Y cell proliferation, apoptosis, inflammatory response, and oxidative stress through direct regulation of miR-214-3p/CDIP1 signaling axis, and could be a potential candidate associated with the diagnosis and treatment of PD.

miR-126-5p Targets SP1 to Inhibit the Progression of Parkinson's Disease.

BACKGROUND: At present, symptomatic treatment may improve the life quality of Parkinson's disease (PD) patients to a certain extent but cannot completely cure PD. Therefore, it is urgent medical problem to be solved for improving the efficacy and safety of PD treatment. METHODS: SH-SY5Y and SK-N-SH cells were treated with 1-methyl-4-phenylpyridinium (MPP+) to establish PD model cells. miR-126-5p and specific protein-1 (SP1) expression levels were detected by quantitative Real-Time PCR (qRT-PCR). Western blot was applied to measure protein levels of SP1, Bax, and Bcl-2. The viabilities and apoptosis rates of treated cells were measured using cell counting kit-8 assay and flow cytometry analysis. Enzyme-linked immunosorbent assay was performed to measure TNF-α and IL-1ß releases. Interaction between miR-126-5p and SP1 was examined by dual-luciferase reporter assay. RESULTS: MPP+ treatment greatly downregulated miR-126-5p expression while upregulated SP1 expression in SH-SY5Y and SK-N-SH cells in a time- and does-dependent manner. Overexpression of miR-126-5p facilitated cell viability, while reduced cell apoptosis and inflammatory responses induced by MPP+ treatment. Moreover, SP1 was a target of miR-126-5p and could be negatively regulated by miR-126-5p. Overexpression of SP1 could reverse the effects of miR-126-5p on MPP+-administrated cells. CONCLUSION: Our results suggested that miR-126-5p attenuated the neurotoxicity induced by MPP+ in vitro through targeting SP1 (Graphical abstract), which further enhanced our understanding of the pathological mechanism of PD.

LINC00943 acts as miR-338-3p sponge to promote MPP+-induced SK-N-SH cell injury by directly targeting SP1 in Parkinson's disease.

BACKGROUND: Abnormal expression of long non-coding RNA (lncRNA) is associated with the progression of Parkinson's disease (PD). LINC00943 has been proved to play an important role in the development of PD, so its role and mechanism in PD progression are worth further exploration. METHODS: MPTP was used to construct PD mice model, and its active ingredient MPP+ was used to construct PD cell model. Cell proliferation and apoptosis were determined by MTT assay, EdU staining and flow cytometry. The protein levels of Cyclin D1, Bcl-2 and specificity protein 1 (SP1) were tested by western blot analysis. The concentrations of inflammation factors were examined by ELISA assay. The expression levels of LINC00943, microRNA (miR)-338-3p and SP1 were measured using quantitative real-time PCR. The interaction between miR-338-3p and LINC00943 or SP1 was confirmed using dual-luciferase reporter assay and RIP assay. RESULTS: Our data showed that LINC00943 was highly expressed in the brain tissues of MPTP-treated mice and MPP+-induced SK-N-SH cells. Knockdown of LINC00943 could promote the proliferation, while inhibit the apoptosis and inflammation of MPP+-induced SK-N-SH cells to alleviate cell injury. In terms of mechanism, we pointed out that LINC00943 could sponge miR-338-3p, and miR-338-3p could target SP1. The negative regulation of si-LINC00943 on MPP+-induced SK-N-SH cell injury could be reversed by miR-338-3p inhibitor. Moreover, miR-338-3p had a protective effect on SK-N-SH cells from MPP+-induced injury, which could be reversed by SP1 overexpression. Additionally, we confirmed that LINC00943 positively regulated SP1 via sponging miR-338-3p. CONCLUSION: To sum up, our data revealed that knockdown LINC00943 might alleviate PD progression through regulating the miR-338-3p/SP1 axis.

Regulation of nitric oxide/reactive oxygen species redox signaling by nNOS splicing variants.

We previously demonstrated different expression patterns of the neuronal nitric oxide synthase (nNOS) splicing variants, nNOS-µ and nNOS-α, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-µ and nNOS-α and investigated intracellular redox signaling in nNOS-expressing PC12 cells, stimulated with a neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP+), to enhance the nNOS uncoupling reaction. Using in vitro studies, we show that nNOS-µ produced nitric oxide (NO), as did nNOS-α, in the presence of tetrahydrobiopterin (BH4), an important cofactor for the enzymatic activity. However, nNOS-µ generated more NO and less superoxide than nNOS-α in the absence of BH4. MPP + treatment induced more reactive oxygen species (ROS) production in nNOS-α-expressing PC12 cells than in those expressing nNOS-µ, which correlated with the intracellular production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a downstream messenger of nNOS redox signaling, and apoptosis in these cells. Furthermore, post-treatment with 8-nitro-cGMP aggravated MPP+-induced cytotoxicity via activation of the H-Ras/extracellular signal-regulated kinase signaling pathway. In conclusion, our results provide strong evidence that nNOS-µ exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.

LncRNA JHDM1D-AS1 Suppresses MPP + -Induced Neuronal Injury in Parkinson's Disease via miR-134-5p/PIK3R3 Axis.

Parkinson's disease (PD) is a multi-factorial neurodegenerative disease. Long noncoding RNAs (lncRNAs) have been revealed to be involved in the process of PD. Herein, this study aimed to investigate the potential function and mechanism of JHDM1D-AS1 (JHDM1D antisense 1) in PD process. 1-Methyl-4-phenylpyridinium (MPP +)-induced SK-N-SH cells were used to conduct expression and function analyses. Levels of genes and proteins were examined using real-time reverse transcription PCR (RT-qPCR) and Western blot. Cell viability and apoptosis were determined using CCK-8 assay, flow cytometry, and Western blot, respectively. ELISA analysis was performed for the detection of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured using commercial kits. The direct interactions between miR-134-5p and PIK3R3 (Phosphoinositide-3-Kinase Regulatory Subunit 3) or JHDM1D-AS1 were verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. JHDM1D-AS1 expression was decreased by MPP + in SK-N-SH cells in a dose- or time-dependent manner. Functionally, JHDM1D-AS1 overexpression attenuated MPP + -evoked neuronal apoptosis, inflammation, and oxidative stress. Mechanistically, JHDM1D-AS1 competitively bound to miR-134-5p to upregulate the expression of its target PIK3R3. Rescue experiments suggested that miR-134-5p upregulation reversed the inhibitory effects of JHDM1D-AS1 on MPP + -induced neuronal injury. Moreover, inhibition of miR-134-5p protected neurons against MPP + -induced neuronal apoptosis, inflammation, and oxidative stress, which were abolished by PIK3R3 silencing. JHDM1D-AS1 protected against MPP + -induced neuron injury via miR-134-5p/PIK3R3 axis, suggesting the potential involvement of this axis in PD process.

Annexin A2 degradation contributes to dopaminergic cell apoptosis via regulating p53 in neurodegenerative conditions.

BACKGROUND: P53 overexpression has been shown to involve in mitochondria-mediated dapaminergic neuron cell death in Parkinson's disease. However, the exactly molecular mechanisms responsible for the p53-dependent intrinsic cell death in neurodegenerative conditions remain unclearly. Annexin A2 is a multifunctional protein that negatively regulates p53 expression. The purpose of this study was to explore the mechanism of p53 dependent dopaminergic cell death and implication of Annexin A2 in cellular apoptosis in 1-methyl-4-phenylpyridinium (MPP+)-induced PC12 cells. METHODS: The cell viability of neural PC12 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide assay. Flow cytometry was used to evaluate the apoptosis and mitochondrial transmembrane potential of neural PC12 cells. The expression of p53 and Annexin A2 was analyzed by western blot assay. RESULTS: The present study showed that the exposure of PC12 cells to neurotoxin MPP+ increased the expression levels of p53 and the discharge of mitochondrial transmembrane potential. Notably, Annexin A2 degradation was also observed in this cellular model of Parkinson's disease, in a time and dose-dependent manner. This expressing change of Annexin A2 was in direct proportion to the loss of cell viability of PC12 cells, and this expression pattern was in inverse proportion to p53 levels in this cellular model of Parkinson's disease. CONCLUSION: These results indicated that Annexin A2 degradation plays a crucial role the degeneration of dapaminergic cells of Parkinson's disease, and Annexin A2 downregulation-mediated the cell death is closely associated with mitochondrial dysfunction via p53-dependent pathway; thus provide a novel therapeutic target for Parkinson's disease treatment.

UPRmt activation protects against MPP+-induced toxicity in a cell culture model of Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPRmt) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPRmt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPRmt in idiopathic PD is unknown. The present study examined the link between the UPRmt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells. Treatment with MPP + induced activation of the UPRmt, reflected by an increase in the expression of UPRmt-related chaperones, proteases, and transcription mediators. UPRmt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP+ treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPRmt transcription factor) conferred protection against MPP+-induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPRmt activation in MPP + -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD.

circSAMD4A participates in the apoptosis and autophagy of dopaminergic neurons via the miR­29c­3p­mediated AMPK/mTOR pathway in Parkinson's disease.

Parkinson's disease (PD) can lead to movement injury and cognitive dysfunction. Although advances have been made in attenuating PD, the effect of inhibiting the development of PD remains disappointing. Therefore, the present study aimed at investigating the etiology of Parkinson's disease and developing an alternative therapeutic strategy for patients with PD. A PD mouse model was established using an intraperitoneal injection of 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine hydrochloride (MPTP­HCl; 30 mg/kg/day for 5 days), and a PD cellular model was established by treating SH­SY5Y cells with different concentrations of 1­methyl­4­phenylpyridinium (MPP+) for 24 h. The expression levels of circular RNA sterile α motif domain containing 4A (circSAMD4A) and microRNA (miR)­29c­3p in both midbrain tissues and SH­SY5Y cells were detected via reverse transcription­quantitative PCR. The interaction between circSAMD4A and miR­29c­3p was verified using a dual­luciferase reporter experiment. Apoptosis­, autophagy­ and 5'AMP­activated protein kinase (AMPK)/mTOR cascade­associated proteins in midbrain tissues and SH­SY5Y cells were detected using western blotting. Furthermore, TUNEL staining and flow cytometry were used to analyze cell apoptosis. It was found that circSAMD4A was upregulated, while miR­29c­3p was downregulated in both PD animal and cellular models. Moreover, circSAMD4A directly targeted and negatively regulated miR­29c­3p. Further studies identified that circSAMD4A knockdown inhibited MPTP­ or MPP+­induced apoptosis and autophagy; however, these effects were abolished by an miR­29c­3p inhibitor. In addition, circSAMD4A knockdown repressed phosphorylated­AMPK expression and increased mTOR expression in MPTP­ or MPP+­induced PD models, the effects of which were reversed by a miR­29c­3p inhibitor. Collectively, these results suggested that circSAMD4A participated in the apoptosis and autophagy of dopaminergic neurons by modulating the AMPK/mTOR cascade via miR­29c­3p in PD.

p53-mediated ferroptosis is required for 1-methyl-4-phenylpyridinium-induced senescence of PC12 cells.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and striatum. Aging is the most important risk factor of PD. Ferroptosis is an iron-dependent form of cell death associated with PD. However, it is not clear whether ferroptosis accelerates PD by promoting cellular senescence. This study investigated the mechanism of 1-methyl-4-phenylpyridinium (MPP+) -induced PC12 cells injury. We found that MPP+ induced cell senescence with increased ß-galactosidase activity and the expression of p53, p21 and p16 activation in cells. In addition, MPP+ treatment showed smaller mitochondria and increased membrane density, downregulation of ferritin heavy chain 1 expression and upregulation of acyl-CoA synthetase long chain family member 4 expression, and enhanced levels of oxidative stress, which were important characteristics of ferroptosis. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, was tested to eliminate MPP+-induced cell senescence. Fer-1 downregulated the expression of p53 and upregulated the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase-4 (GPX4) in MPP+-induced ferroptosis. Inhibition of p53 eliminated cell senescence by upregulation the expression of of SLC7A11 and GPX4. Thus, these results suggest that MPP+ induces senescence in PC12 cells via the p53/ SLC7A11/ GPX4 signaling pathway in the ferroptosis regulation mechanism.

RIPK1 inhibitor ameliorates the MPP+/MPTP-induced Parkinson's disease through the ASK1/JNK signalling pathway.

Receptor-interacting protein kinase 1 (RIPK1) is up-regulated in patients with neurodegenerative diseases. Our study aimed to explore the underlying mechanisms that involved in the neurotoxic function of RIPK1 in Parkinson's disease (PD). MPP+/MPTP-induced PD cellular and mice models were used in this study. The results showed that RIPK1 was high expressed and activated in MPP+-treated SH-SY5Y cells and MPTP-induced PD mice. Overexpression of RIPK1 facilitated cell apoptosis, necrosis, inflammation response, ROS production and mitochondrial dysfunction in MPP+- treated SH-SY5Y cells, while the RIPK1 inhibitor Nec-1s has an opposite effect. In addition, the Apoptosis-signaling kinase-1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated during the overexpression of RIPK1, and inhibiting the ASK1/JNK signal by the ASK1 inhibitor partially reversed the decline of cell viability, the increase of cell apoptosis, necrosis and inflammation induced by RIPK1 overexpression in MPP+-treated SH-SY5Y cells. Further studies suggested that the inhibition of RIPK1 by Nec-1s largely alleviated the behavioural impairment in PD mice. Hence, our study indicated that the RIPK1 inhibitor Nec-1s has neuroprotective effects against PD through inactivating the ASK1/JNK signalling pathway.