Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells

One of the functions mediated by sirtuin 1 (SIRT1), the NAD⁺-dependent protein deacetylase, has been suggested to be neuroprotective since resveratrol, a SIRT1 activator, inhibits 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity. In this study, we show that SIRT1 siRNA transfection blocks MPP⁺-induced apoptosis in SH-SY5Y cells. The ratio of potential pro-apoptotic BNIP2 to antiapoptotic BCL-xL was attenuated in SIRT1-deficient cells following MPP⁺ treatment. In addition, BNIP2 shRNA-transfected cells showed reduced cleavage of PARP-1, while BNIP2 overexpression intensified the cleavage in MPP⁺-treated SH-SY5Y cells, suggesting that BNIP2 participates in the MPP⁺-induced apoptosis. Overall, these data imply that SIRT1 may mediate MPP⁺-induced cytotoxicity, possibly through the regulation of BNIP2.     

Upregulated lncRNA small nucleolar RNA host gene 1 promotes 1-methyl-4-phenylpyridinium ion-induced cytotoxicity and reactive oxygen species production through miR-15b-5p/GSK3β axis in human dopaminergic SH-SY5Y cells

Long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) has been demonstrated to be upregulated and play a crucial role in the pathology of Parkinson's disease (PD). However, the exact role of SNHG1 and its underlying mechanisms in PD remains elusive. In this study, we found that SNHG1 and glycogen synthase kinase 3 beta (GSK3β) were upregulated, but miR-15b-5p was downregulated in 1-methyl-4-phenylpyridinium ion (MPP⁺)-treated SH-SY5Y cells. The upregulation of SNHG1 enhanced MPP⁺-induced cellular toxicity in SH-SY5Y cells, as shown by decreased cell viability, increased ROS production, and increased number of TdT-mediated dUTP Nick-End labeling-positive cells, accompanied with the upregulation of cleaved caspase 3 and elevation of cytochrome C release. Meanwhile, SNHG1 knockdown presented the converse effects. SNHG1 was demonstrated to interact with miR-15b-5p. Moreover, SNHG1 could attenuate the inhibitory effects of miR-15b-5p on MPP⁺-induced cytotoxicity and production of ROS. Besides, GSK3β was identified as a direct target of miR-15b-5p. The inhibitory effects of SNHG1 knockdown or miR-15b-5p overexpression on MPP⁺-induced cytotoxicity and reactive oxygen species (ROS) production were abrogated by upregulation of GSK3β. Taken together, these results demonstrate that upregulated lncRNA SNHG1 promotes MPP⁺-induced cytotoxicity and ROS production through the miR-15b-5p/GSK3β axis in human dopaminergic SH-SY5Y cells, suggesting that SNHG1 may act as a potential therapeutic target for PD treatment in the future.     

Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP+-induced oxidative stress and cell death in SH-SY5Y cells

Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP⁺-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP⁺-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP⁺-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling.     

AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP⁺) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP⁺ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.     

LncRNA MALAT1 targeting miR-124-3p regulates DAPK1 expression contributes to cell apoptosis in Parkinson's Disease

Death associated protein kinase 1 (DAPK1) was initially discovered in the progress of gamma-interferon induced programmed cell death, it is a key factor in the central nervous system, including Parkinson's disease (PD). However, the underlying mechanisms of DAPK1 in PD remain unclear and this research work aims to explore the potential mechanisms of DAPK1 in PD. In the study, we exposed SH-SY5Y cells to MPP⁺ and treated mice with MPTP to investigate the roles of DAPK1 in PD and the underlying mechanisms. The results indicated that the expression of DAPK1 is significantly upregulated and negatively correlated with miR-124-3p levels in SH-SY5Y cells treated by MPP⁺, and miR-124-3p mimics could effectively inhibit DAPK1 expressions and alleviate MPP⁺-induced cell apoptosis. In addition, knockdown MALAT1 reduces the levels of DAPK1 and the ratio of SH-SY5Y cell apoptosis, which is reversed via miR-124-3p inhibitor in vitro. Similarly, knockdown MALAT1 could improve behavioral changes and reduce apoptosis by miR-124-3p upregulation and DAPK1 downregulation in MPTP induced PD mice. Taken together, our data showed that lncRNA MALAT1 positively regulates DAPK1 expression by targeting miR-124-3p, and mediates cell apoptosis and motor disorders in PD. In summary, these results suggest that MALAT1/miR-124-3p /DAPK1 signaling cascade mediates cell apoptosis in vitro and in vivo, which may provide experimental evidence of developing potential therapeutic strategies for PD.     

cDNA Microarray Analysis of Changes in Gene Expression Associated with MPP+ Toxicity in SH-SY5Y Cells

cDNA microarray analysis of 1-methyl-4-phenyl-pyridinium (MPP⁺) toxicity (1 mM, 72 h) in undifferentiated SH-SY5Y cells identified 48 genes that displayed a signal intensity greater than the mean of all differentially expressed genes and a two-fold or greater difference in normalized expression. RT-PCR analysis of a subset of genes showed that c-Myc and RNA-binding protein 3 (RMB3) expression decreased by 50% after 72 h of exposure to MPP⁺ (1 mM) but did not change after 72 h of exposure to 6-hydroxydopamine (25 M), rotenone (50 nM), and hydrogen peroxide (600 M). Exposure of retinoic acid (RA)-differentiated SH-SY5Y cells to MPP⁺ (1 mM, 72 h) also resulted in a decrease in RMB3 expression and an increase in GADD153 expression. In contrast, c-Myc expression was slightly increased in RA-differentiated cells. Collectively, these data provide new insights into the molecular mechanisms of MPP⁺ toxicity and show that MPP⁺ can elicit distinct patterns of gene expression in undifferentiated and RA-differentiated SH-SY5Y cells.     

Cdk5 suppression blocks SIRT1 degradation via the ubiquitin-proteasome pathway in Parkinson's disease models

The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family, may have a neuroprotective effect in multiple neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS). Many studies have suggested that overexpression-induced or resveratrol-treated activation of SIRT1 could significantly ameliorate several neurodegenerative diseases in mouse models. However, the type of SIRT1, protein expression levels and underlying mechanisms remain unclear, especially in PD. In this study, the results demonstrated that SIRT1 knockout markedly worsened the movement function in MPTP-lesioned animal model of PD. SIRT1 expression was found to be markedly decreased not only in environmental factor PD models, neurotoxin MPP⁺-treated primary culture neurons and MPTP-induced mice but also in genetic factor PD models, overexpressed α-synuclein-A30PA53T SH-SY5Y stable cell line and hm²α-SYN-39 transgenic mouse strain. Importantly, the degradation of SIRT1 during MPP⁺ treatment was mediated by the ubiquitin-proteasome pathway. Furthermore, the results indicated that cyclin-dependent kinase 5 (Cdk5) was also involved in the decrease of SIRT1 expression, which could be efficiently blocked by the inhibition of Cdk5. In conclusion, our findings revealed that the Cdk5-dependent ubiquitin-proteasome pathway mediated degradation of SIRT1 plays a vital role in the progression of PD.     

Integrated insight into the molecular mechanisms of selenium-modulated,MPP+-induced cytotoxicity in a Parkinson’s disease model

ObjectiveParkinson’s disease (PD) is a neurodegenerative disease that is associated with oxidative stress. Due to the anti-inflammatory and antioxidant functions of Selenium (Se), this molecule may have neuroprotective functions in PD; however, the involvement of Se in such a protective function is unclear.Methods1-methyl-4-phenylpyridinium (MPP⁺), which inhibits mitochondrial respiration, is generally used to produce a reliable cellular model of PD. In this study, a MPP⁺-induced PD model was used to test if Se could modulate cytotoxicity, and we further capture gene expression profiles following PC12 cell treatment with MPP⁺ with or without Se by genome wide high-throughput sequencing.ResultsWe identified 351 differentially expressed genes (DEGs) and 14 differentially expressed long non-coding RNAs (DELs) in MPP⁺-treated cells when compared to controls. We further document 244 DEGs and 27 DELs in cells treated with MPP⁺ and Se vs. cells treated with MPP⁺ only. Functional annotation analysis of DEGs and DELs revealed that these groups were enriched in genes that respond to reactive oxygen species (ROS), metabolic processes, and mitochondrial control of apoptosis. Thioredoxin reductase 1 (Txnrd1) was also identified as a biomarker of Se treatment.ConclusionsOur data suggests that the DEGs Txnrd1, Siglec1 and Klf2, and the DEL AABR07044454.1 which we hypothesize to function in cis on the target gene Cdkn1a, may modulate the underlying neurodegenerative process, and act a protective function in the PC12 cell PD model. This study further systematically demonstrated that mRNAs and lncRNAs induced by Se are involved in neuroprotection in PD, and provides novel insight into how Se modulates cytotoxicity in the MPP⁺-induced PD model.     

Protective effect of urocortin on 1-methyl-4-phenylpyridinium-induced dopaminergic neuronal death

Recent studies have indicated that the corticotropin releasing hormone (CRF)-related peptide, urocortin, restores key indicators of damage in animal models for Parkinson’s disease (PD). However, the molecular mechanism for the neuroprotective effect of urocortin is unknown. 1-Methy-4-phenylpyridinium (MPP⁺) induces dopaminergic neuronal death. In the present study, MPP⁺-induced neuroblastoma SH-SY5Y cell death was significantly attenuated by urocortin in a concentration-dependent manner. The protective effect of urocortin involved the activation of CRF receptor type 1, resulting in the increase of cyclic AMP (cAMP) levels. Various cAMP-enhancing reagents mimicked the effect of urocortin, while inhibitors for protein kinase A (PKA) blocked the effect of urocortin, strongly implicating the involvement of cAMP-PKA pathway in the neuroprotective effect of urocortin on MPP⁺-induced cell death. As the downstream of this signal pathway, urocortin promoted phosphorylation of both glycogen synthase kinase 3β and extracellular signal-regulated kinases, which are known to promote cell survival. These neuroprotective signaling pathways of urocortin may serve as potential therapeutic targets for PD.     

Miltirone Attenuates Reactive Oxygen Species-Dependent Neuronal Apoptosis in MPP+-Induced Cell Model of Parkinson’s Disease Through Regulating the PI3K/Akt Pathway

Miltirone is a phenanthrene-quinone derived from Salvia miltiorrhiza Bunge with anti-inflammatory and anti-oxidant effects. Our study aimed to explore the protective effect of miltirone on 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of Parkinson’s disease (PD). PharmMapper database was employed to predict the targets of miltirone. PD-related genes were identified using GeneCards database. The overlapping genes between miltirone and PD were screened out using Venn diagram. KEGG analysis was performed using DAVID and KOBAS databases. Cell viability, reactive oxygen species (ROS) generation, apoptosis, and caspase-3 activity were detected by CCK-8 assay, a ROS assay kit, TUNEL, and caspase-3 activity assay, respectively. Effect of miltirone on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was explored by western blot analysis. A total of 214 targets of miltirone and 372 targets related to PD were attained, including 29 overlapping targets. KEGG analysis demonstrated that the 29 overlapping targets were both significantly enriched in the PI3K/Akt pathway. MPP⁺ stimulation reduced the cell viability in SH-SY5Y cells and neuronal primary cultures derived from human brain. Miltirone or N-acetylcysteine (NAC) attenuated MPP⁺-induced reduction in cell viability, ROS production, SOD activity reduction, apoptosis, and increase of caspase-3 activity. Additionally, miltirone recuperated MPP⁺-induced inactivation of the PI3K/Akt pathway. Moreover, treatment with LY294002, an inhibitor of the PI3K/Akt pathway, reversed the inhibitory effect of miltirone on MPP⁺-induced ROS generation and apoptosis in SH-SY5Y cells and neuronal primary cultures. In conclusion, miltirone attenuated ROS-dependent apoptosis in MPP⁺-induced cellular model of PD through activating the PI3K/Akt pathway.

     

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP⁺) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP⁺-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]     

SIRT1 mediates salidroside‐elicited protective effects against MPP+‐induced apoptosis and oxidative stress in SH‐SY5Y cells: involvement in suppressing MAPK pathways

Parkinson's disease (PD) is a progressive neurodegenerative disease, leading to tremor, rigidity, bradykinesia, and gait impairment. Salidroside has been reported to exhibit antioxidative and neuroprotective properties in PD. However, the underlying neuroprotective mechanisms effects of salidroside are poorly understood. Recently, a growing body of evidences suggest that silent information regulator 1 (SIRT1) plays important roles in the pathophysiology of PD. Hence, the present study investigated the roles of SIRT1 in neuroprotective effect of salidroside against N‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced SH‐SY5Y cell injury. Our findings revealed that salidroside attenuates MPP⁺‐induced neurotoxicity as evidenced by the increase in cell viability, and the decreases in the caspase‐3 activity and apoptosis ratio. Simultaneously, salidroside pretreatment remarkably increased SIRT1 activity, SIRT1 mRNA and protein levels in MPP⁺‐treated SH‐SY5Y cell. However, sirtinol, a SIRT1 activation inhibitor, significantly blocked the inhibitory effects of salidroside on MPP⁺‐induced cytotoxicity and apoptosis. In addition, salidroside abolished MPP⁺‐induced the production of reactive oxygen species (ROS), the up‐regulation of NADPH oxidase 2 (NOX2) expression, the down‐regulations of superoxide dismutase (SOD) activity and glutathione (GSH) level in SH‐SY5Y cells, while these effects were also blocked by sirtinol. Finally, we found that the inhibition of salidroside on MPP⁺‐induced phosphorylation of p38, extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) were also reversed by sirtinol in SH‐SY5Y cells. Taken together, these results indicated that SIRT1 contributes to the neuroprotection of salidroside against MPP⁺‐induced apoptosis and oxidative stress, in part through suppressing of mitogen‐activated protein kinase (MAPK) pathways.     

PEP-1-HO-1 prevents MPTP-induced degeneration of dopaminergic neurons in a Parkinson’s disease mouse model

Heme oxygenase-1 (HO-1) degrades heme to carbon dioxide, biliverdin, and Fe²⁺, which play important roles in various biochemical processes. In this study, we examined the protective function of HO-1 against oxidative stress in SH-SY5Y cells and in a Parkinson’s disease mouse model. Western blot and fluorescence microscopy analysis demonstrated that PEP-1-HO-1, fused with a PEP-1 peptide can cross the cellular membranes of human neuroblastoma SH-SY5Y cells. In addition, the transduced PEP-1-HO-1 inhibited generation of reactive oxygen species (ROS) and cell death caused by 1-methyl-4-phenylpyridinium ion (MPP⁺). In contrast, HO-1, which has no ability to transduce into SH-SY5Y cells, failed to reduce MPP⁺-induced cellular toxicity and ROS production. Furthermore, intraperitoneal injected PEP-1-HO-1 crossed the blood-brain barrier in mouse brains. In a PD mouse model, PEP-1-HO-1 significantly protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity and dopaminergic neuronal death. Therefore, PEP-1-HO-1 could be a useful agent in treating oxidative stress induced ailments including PD. [BMB Reports 2014; 47(10): 569-574]