Dopaminergic Cell Line SH-SY5Y Research Articles

Curcumin-loaded a-lactalbumin nanoparticles neuroprotective effects against permethrine-induced neurotoxicity

Curcumin, as is well known, plays the role of an antioxidant, anti-inflammatory and neuroprotective ingredient. Permethrine is a synthetic type I pyrethroidal neurotoxic pesticide that has been involved in causing neurotoxicity in dopaminergic cells and is currently the subject of extensive study in the fight against Parkinson's disease (PD). In the current study, curcumin loaded α-lactalbumin (α-LA) nanoparticles prepared by desolvation technique were used to protect dopaminergic cell line SH-SY5Y against permethrine induced neurotoxicity. These curcumin-loaded nanoparticles, according to transmission electron microscopy, and dynamic light scattering analysis, have the diameters of 100–150 nm, respectively. The encapsulation efficiency was 60.23 % ±3.1 %.The cellular uptake of curcumin was demonstrated to be mediated by these nanoparticles by spectrofluorimetric and Confocal imaging investigations. The α-lactalbumin nanoparticles loaded with curcumin exhibited greater intracellular drug absorption, extended retention, and neuroprotection compared to their corresponding curcumin concentrations. Neuroprotective effect was established by viability experiments and ROS level measurement. Furthermore, in permethrine-induced PD-like features, a-synuclein expression rose and tyrosine hydroxylase expression fell. To further shield dopaminergic neurons from neurotoxicity, curcumin-loaded lactalbumin nanoparticles would be a powerful therapeutic delivery vehicle.

Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer's Disease.

Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer's disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson's disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration.

Peroxiredoxin 5 overexpression decreases oxidative stress and dopaminergic cell death mediated by paraquat.

Peroxiredoxins (Prdxs) are thiol-dependent enzymes that scavenge peroxides. Previously, we found that Prdxs were hyperoxidized in a Parkinson's disease model induced by paraquat (PQ), which led to their inactivation, perpetuating reactive oxygen species (ROS) formation. Herein, we evaluated the redox state of the typical 2-Cys-Prx subgroup. We found that PQ induces ROS compartmentalization in different organelles, reflected by the 2-Cys-Prdx hyperoxidation pattern detected by redox eastern blotting. 2-Cys Prdxs are most vulnerable to hyperoxidation, while atypical 2-Cys Peroxiredoxin 5 (Prdx5) is resistant and is expressed in multiple organelles, such as mitochondria, peroxisomes, and cytoplasm. Therefore, we overexpressed human Prdx5 in the dopaminergic SHSY-5Y cell line using the adenoviral vector Ad-hPrdx5. Prdx5 overexpression was confirmed by western blotting and immunofluorescence (IF) and effectively decreased PQ-mediated mitochondrial and cytoplasmic ROS assessed with a mitochondrial superoxide indicator and DHE through IF or flow cytometry. Decreased ROS mediated by Prdx5 in the main subcellular compartments led to overall cell protection against PQ-induced cell death, which was demonstrated by flow cytometry using Annexin V labeling and 7-AAD. Therefore, Prdx5 is an attractive therapeutic target for PD, as its overexpression protects dopaminergic cells from ROS and death, which warrants further experimental animal studies for its subsequent application in clinical trials.

Insights into the Pharmacokinetics and In Vitro Cell-Based Studies of the Imidazoline I2 Receptor Ligand B06.

The impact of neurodegenerative diseases (ND) is becoming unbearable for humankind due to their vast prevalence and the lack of efficacious treatments. In this scenario, we focused on imidazoline I2 receptors (I2-IR) that are widely distributed in the brain and are altered in patients with brain disorders. We took the challenge of modulating I2-IR by developing structurally new molecules, in particular, a family of bicyclic α-iminophosphonates, endowed with high affinity and selectivity to these receptors. Treatment of two murine models, one for age-related cognitive decline and the other for Alzheimer’s disease (AD), with representative compound B06 ameliorated their cognitive impairment and improved their behavioural condition. Furthermore, B06 revealed beneficial in vitro ADME-Tox properties. The pharmacokinetics (PK) and metabolic profile are reported to de-risk B06 for progressing in the preclinical development. To further characterize the pharmacological properties of B06, we assessed its neuroprotective properties and beneficial effect in an in vitro model of Parkinson’s disease (PD). B06 rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine (6-OHDA) and showed a crucial anti-inflammatory effect in a cellular model of neuroinflammation. This research reveals B06 as a putative candidate for advancing in the difficult path of drug discovery and supports the modulation of I2-IR as a fresh approach for the therapy of ND.

Identification by proximity labeling of novel lipidic and proteinaceous potential partners of the dopamine transporter

Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain.

Platelet-derived growth factor (PDGF)-BB protects dopaminergic neurons via activation of Akt/ERK/CREB pathways to upregulate tyrosine hydroxylase.

AimsThe neurotropic growth factor PDGF‐BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF‐BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF‐BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study.MethodMPTP‐lesion mice received PDGF‐BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT‐PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF‐BB‐mediated TH upregulation were examined by RT‐PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry.ResultsWe validated a reversal expression of TH in MPTP‐lesion mice upon i.c.v administration of PDGF‐BB for seven days. Similar effects of PDGF‐BB‐mediated TH upregulation were also observed in MPP+‐treated primary neuronal culture and dopaminergic neuronal cell line SH‐SY5Y cells. We next demonstrated that PDGF‐BB rapidly activated the pro‐survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH‐SY5Y cells. We further confirmed the significant induction of p‐CREB in PDGF‐BB‐treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF‐BB‐mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF‐BB‐treated SH‐SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF‐BB treatment.ConclusionOur data demonstrated that PDGF‐BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF‐BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future.

Astaxanthin prevents mitochondrial impairment in the dopaminergic SH-SY5Y cell line exposed to glutamate-mediated excitotoxicity: Role for the Nrf2/HO-1/CO-BR axis

Mitochondrial dysfunction has been viewed in several diseases, including neurological disorders. In the glutamate (GLU)-mediated excitotoxicity, it has been described mitochondrial impairment, disrupted redox environment, and increased rates of cell death in the affected brain areas. Astaxanthin (AST) is a potent antioxidant and anti-inflammatory xanthophyll that also promotes beneficial mitochondria-related effects in brain cells. However, it is not completely clear how AST would be able to promote mitochondrial protection in those cell types. Thus, we investigated here how AST would protect mitochondria in the dopaminergic SH-SY5Y cell line exposed to GLU. AST was administrated to the cells at 1–40 μM for 24 h prior to the exposure to GLU at 80 mM for additional 24 h. AST prevented the GLU-induced impairment in the activity of the Complexes I and V, the loss in mitochondrial membrane potential (MMP), and the decline in the synthesis of ATP. AST also induced an antioxidant effect in the membranes of mitochondria obtained from the GLU-treated SH-SY5Y cells. Inhibition of the enzyme heme oxygenase-1 (HO-1) or silencing of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) suppressed the AST-promoted cellular and mitochondrial protection. Either tricarbonyldichlororuthenium(II) dimer (CORM-2, a source of carbon monoxide - CO) or bilirubin (BR), that are products of the HO-1-biliverdin reductase (BVR) axis, blocked some of the effects caused by GLU in the SH-SY5Y cells. Overall, our data demonstrate that AST prevented mitochondrial dysfunction by a mechanism related to the Nrf2/HO-1 axis in GLU-challenged cells.

Inhibition of γH2AX, COX-2 and regulation of antioxidant enzymes in MPP+-exposed SH-SY5Y cells pre-treated with rutin.

Many plant-derived bioactive compounds such as rutin are reportedly effective in attenuating neuronal death in most neurodegenerative disorders. Parkinson's disease (PD) is characterized by the gradual degeneration of dopaminergic neurons in the substantia nigra of the midbrain, and has previously been modelled in-vitro through the specific neurotoxic activity of 1-methyl-4-phenylpyridinium (MPP+) on dopaminergic neurons. Rutin is a bioflavonoid with multiple pharmacological effects, and this study investigated the neuroprotective effects of rutin in the human dopaminergic SH-SY5Y cell line using the neurotoxin MPP+. SH-SY5Y cells pretreated with rutin, were exposed to MPP+ and evaluated for cell viability, nitric oxide (NO), reactive oxygen species (ROS) and antioxidant enzymes activities. In addition, western blot techniques were used to determine the protein expression levels of γH2AX and COX-2. Rutin significantly attenuated MPP+-induced loss of cell viability, mitigated ROS and NO production and inhibited the disruption of antioxidant enzymes activity. It was also observed that rutin significantly reduced protein expression levels of γH2AX and COX-2 in SH-SY5Y cells treated with MPP+. Taken together, findings from this study tend to suggest that rutin is a promising neuroprotective compound for the treatment of PD through its effects on some of the mechanisms that characterize this neurodegenerative disease.

S-(+)-Pentedrone and R-(+)-methylone as the most oxidative and cytotoxic enantiomers to dopaminergic SH-SY5Y cells: Role of MRP1 and P-gp in cathinones enantioselectivity

Cathinone derivatives are the most representative group within new drugs market, which have been described as neurotoxic. Since cathinones, as pentedrone and methylone, are available as racemates, it is our aim to study the neuronal cytotoxicity induced by each enantiomer. Therefore, a dopaminergic SH-SY5Y cell line was used to evaluate the hypothesis of enantioselectivity of pentedrone and methylone enantiomers on cytotoxicity, oxidative stress, and membrane efflux transport (confirmed by in silico studies). Our study demonstrated enantioselectivity of these cathinones, being the S-(+)-pentedrone and R-(+)-methylone the most oxidative enantiomers and also the most cytotoxic, suggesting the oxidative stress as main cytotoxic mechanism, as previously described in in vitro studies. Additionally, the efflux transporter multidrug resistance associated protein 1 (MRP1) seems to play, together with GSH, a selective protective role against the cytotoxicity induced by R-(−)-pentedrone enantiomer. It was also observed an enantioselectivity in the binding to P-glycoprotein (P-gp), another efflux protein, being the R-(−)-pentedrone and S-(−)-methylone the most transported enantiomeric compounds. These results were confirmed, in silico, by docking studies, revealing that R-(−)-pentedrone is the enantiomer with highest affinity to MRP1 and S-(−)-methylone and R-(−)-pentedrone are the enantiomers with highest affinity to P-gp. In conclusion, our data demonstrated that pentedrone and methylone present enantioselectivity in their cytotoxicity, which seems to involve different oxidative reactivity as well as different affinity to the P-gp and MRP1 that together with GSH play a protective role.

Mitochondrial UQCRC1 mutations cause autosomal dominant parkinsonism with polyneuropathy

Parkinson's disease is a neurodegenerative disorder with a multifactorial aetiology. Nevertheless, the genetic predisposition in many families with multi-incidence disease remains unknown. This study aimed to identify novel genes that cause familial Parkinson's disease. Whole exome sequencing was performed in three affected members of the index family with a late-onset autosomal-dominant parkinsonism and polyneuropathy. We identified a novel heterozygous substitution c.941A>C (p.Tyr314Ser) in the mitochondrial ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) gene, which co-segregates with disease within the family. Additional analysis of 699 unrelated Parkinson's disease probands with autosomal-dominant Parkinson's disease and 1934 patients with sporadic Parkinson's disease revealed another two variants in UQCRC1 in the probands with familial Parkinson's disease, c.931A>C (p.Ile311Leu) and an allele with concomitant splicing mutation (c.70-1G>A) and a frameshift insertion (c.73_74insG, p.Ala25Glyfs*27). All substitutions were absent in 1077 controls and the Taiwan Biobank exome database from healthy participants (n = 1517 exomes). We then assayed the pathogenicity of the identified rare variants using CRISPR/Cas9-based knock-in human dopaminergic SH-SY5Y cell lines, Drosophila and mouse models. Mutant UQCRC1 expression leads to neurite degeneration and mitochondrial respiratory chain dysfunction in SH-SY5Y cells. UQCRC1 p.Tyr314Ser knock-in Drosophila and mouse models exhibit age-dependent locomotor defects, dopaminergic neuronal loss, peripheral neuropathy, impaired respiratory chain complex III activity and aberrant mitochondrial ultrastructures in nigral neurons. Furthermore, intraperitoneal injection of levodopa could significantly improve the motor dysfunction in UQCRC1 p.Tyr314Ser mutant knock-in mice. Taken together, our in vitro and in vivo studies support the functional pathogenicity of rare UQCRC1 variants in familial parkinsonism. Our findings expand an additional link of mitochondrial complex III dysfunction in Parkinson's disease.

Protective effects of valproic acid on 6-hydroxydopamine-induced neuroinjury.

Oxidative stress may play critically important roles in the etiology of Parkinson's disease (PD). 6-Hydroxydopamine (6-OHDA) is a physiological neurotoxin reported to induce oxidative-induced apoptosis of dopaminergic neurons in PD mice models. Valproic acid (VPA), a clinical mood stabilizer, is a HDAC inhibitor with neuroprotective capacities. In the study, we aim at examining the feasibility of VPA as a protector for dopaminergic neurons against damage from 6-OHDA, and the intracellular mechanisms. The 6-OHDA-induced neurotoxicity to the human dopaminergic cell line SH-SY5Y was applied for examining VPA protective effects. Pretreatment with VPA was able to improve cell viability and reduce 6-OHDA-induced reactive oxygen species. Furthermore, a significant suppression of apoptotic caspases including cleaved caspase-3, caspase-7, and caspase-9 was observed. The results also revealed VPA decreased the 6-OHDA-induced Bax/Bcl2 ratio, as measured at protein level. These novel findings indicate that VPA may be capable of protecting the SH-SY5Y dopaminergic neuronal cells from 6-OHDA-induced toxicity via the deceasing of apoptotic caspases (cleaved caspase-3, caspase-7, and caspase-9) and reducing of the Bax/Bcl2 ratio. Very possibly, VPA could serve as not only a mood stabilizer but also a potential antidote for PD prevention.

Phosphodiesterase 7 Regulation in Cellular and Rodent Models of Parkinson's Disease.

Parkinson's disease is characterized by a loss of dopaminergic neurons in the ventral midbrain. This disease is diagnosed when around 50% of these neurons have already died; consequently, therapeutic treatments start too late. Therefore, an urgent need exists to find new targets involved in the onset and progression of the disease. Phosphodiesterase 7 (PDE7) is a key enzyme involved in the degradation of intracellular levels of cyclic adenosine 3', 5'-monophosphate in different cell types; however, little is known regarding its role in neurodegenerative diseases, and specifically in Parkinson's disease. We have previously shown that chemical as well as genetic inhibition of this enzyme results in neuroprotection and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson's disease. Here, we have used in vitro and in vivo models of Parkinson's disease to study the regulation of PDE7 protein levels. Our results show that PDE7 is upregulated after an injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures and after lipopolysaccharide or 6-hidroxydopamine injection in the Substantia nigra pars compacta of adult mice. PDE7 increase takes place mainly in degenerating dopaminergic neurons and in microglia cells. This enhanced expression appears to be direct since 6-hydroxydopamine and lipopolysaccharide increase the expression of a 962-bp fragment of its promoter. Taking together, these results reveal an essential function for PDE7 in the pathways leading to neurodegeneration and inflammatory-mediated brain damage and suggest novel roles for PDE7 in neurodegenerative diseases, specifically in PD, opening the door for new therapeutic interventions.

Protective effect of gastrodin against methamphetamine-induced autophagy in human dopaminergic neuroblastoma SH-SY5Y cells via the AKT/mTOR signaling pathway

Methamphetamine (METH) has been shown to induce neuropathological dysfunction and irreversible brain cell damage. Prior studies indicated the involvement of autophagy in METH-induced neurotoxicity. However, the underlying mechanism by which autophagy contributes to METH-induced neurotoxicity remains elusive. Gastrodin, a primary bioactive constituent of Gastrodia elata—an orchid used in traditional Chinese medicine—is used widely to treat stroke, dementia, and headache. This study investigates whether METH induces autophagy in the human dopaminergic neuroblastoma cell line SH-SY5Y, then examines the neuroprotective effects of gastrodin against autophagy in METH-treated SH-SY5Y cells. The effects of METH on the protein expressions of autophagy-related genes (LC3B and Beclin-1) were evaluated with and without gastrodin. The presence of autophagosomes in the METH-induced treatment with and without gastrodin is revealed through transmission electron microscopy. Pharmacological intervention was employed to study the role of the AKT/mTOR signaling pathway in the gastrodin-mediated neuroprotection against METH-induced autophagy. The present results indicate that METH exposure elevates the protein expression levels of LC3B and Beclin-1 in a dose- and time-dependent manner. Gastrodin is observed to block the METH-induced upregulation of LC3B and Beclin-1 protein expression significantly. Gastrodin is found to exhibit an anti-autophagic effect on the inhibition of the METH-induced Beclin-1 protein expression, partly via the AKT/mTOR These findings may aid the development of a gastrodin-based therapeutic strategy for treating METH-induced neurotoxicity.

Biochemical profile and in vitro neuroprotective properties of Carpobrotus edulis L., a medicinal and edible halophyte native to the coast of South Africa

This work reports the nutritional profile and in vitro neuroprotective properties of leaves of Carpobrotus edulis L., a medicinal and edible succulent species native to the coast of South Africa. Biomass was evaluated for proximate composition and for contents in carotenoids, liposoluble pigments and minerals. Hexane, dichloromethane, ethyl acetate and methanol extracts were prepared by Soxhlet extraction from dried biomass and evaluated for in vitro inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), capacity to attenuate hydrogen peroxide (H2O2)-induced injury in the human dopaminergic cell line SH-SY5Y and for anti-neuroinflammatory potential on lipopolysaccharide (LPS)-stimulated microglia cells. Extracts were evaluated for antioxidant activity by four complementary methods, total content of phenolics, tannins and flavonoids. Finally the profile of the main phenolic compounds was determined by high performance liquid chromatography with diode array detection (HPLC-DAD). C. edulis has a high moisture content, high levels of crude protein, fibre, ash, carotenoids, calcium and iron and a low fat level. The extracts were able to efficiently scavenge the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), reduce iron and chelate copper and iron ions, and exhibited different levels of phenolic compounds in the order ethyl acetate>methanol>dichloromethane>hexane. The main compounds detected were gallic and salicylic acids and quercetin, all in the ethyl acetate extract. The extracts allowed a dual and potent inhibition of AChE and BuChE. The dichloromethane and methanol extracts had the strongest capacity to prevent cell death induced by H2O2, and the methanol extract had anti-neuronflammatory properties. All together our results suggest that consumption of leaves of C. edulis can contribute for a balanced diet, and that they may add to the improvement of cognitive functions. It also suggests possible novel biotechnological applications of C. edulis such as source of molecules and/or products for the food and/or pharmaceutical industries. Studies aiming to the isolation and identification of the bioactive compounds are already in progress.

Clathrin-Dependent Uptake of Paraquat into SH-SY5Y Cells and Its Internalization into Different Subcellular Compartments.

The herbicide paraquat (PQ) is an exogenous toxin that allows the selective activation of dopaminergic neurons in the mesencephalon to induce injury and also causes its apoptosis in vitro. However, uptake mechanisms between PQ and neurons remain elusive. To address this issue, we undertook a study of PQ endocytosis in a dopaminergic SH-SY5Y cell line as well as explored the subsequent subcellular location and potential functional analysis of PQ. The PQ was found to bind the SH-SY5Y cell membrane and then became internalized via a clathrin-dependent pathway. PQ was internalized by many subcellular organelles in a time- and dose-dependent manner. Interestingly, the taken up PQ and secretogranin III (SCG3), which became dysregulated with PQ treatment that induced SH-SY5Y apoptosis in our previous study, colocalized in cytoplasmic vesicles. Taken together, our findings indicate that PQ is endocytosed by SH-SY5Y cells and that its multiple, subcellular localizations indicate PQ may potentially be involved in subcellular-level functions. More importantly, PQ distributing preferentially into SCG3-positive vesicles demonstrates its selective targeting which may affect SCG3 and cargoes carried by SCG3-positive vesicles. Therefore, it is reasonable to infer that PQ toxic insults may potentially interfere with neurotransmitter storage and transport associated with secretory granules.

TCF11/Nrf1-Mediated Induction of Proteasome Expression Prevents Cytotoxicity by Rotenone.

Precise regulation of cellular protein degradation is essential for maintaining protein and redox homeostasis. The ubiquitin proteasome system (UPS) represents one of the major degradation machineries, and UPS disturbances are strongly associated with neurodegeneration. We have previously shown that the transcription factor TCF11/Nrf1 induces antioxidant response element-mediated upregulation of UPS components in response to proteotoxic stress. Knockout of TCF11/Nrf1 is embryonically lethal, and therefore, the present investigation describes the role of oxidative stress in regulating TCF11/Nrf1-dependent proteasome expression in a model system relevant to Parkinson's disease. Using the human dopaminergic neuroblastoma cell line SH-SY5Y and mouse nigrostriatal organotypic slice cultures, gene and protein expression analysis and functional assays revealed oxidative stress is induced by the proteasome inhibitor epoxomicin or the mitochondrial complex I inhibitor rotenone and promotes the upregulation of proteasome expression and function mediated by TCF11/Nrf1 activation. In addition, we show that these stress conditions induce the unfolded protein response. TCF11/Nrf1, thus, has a cytoprotective function in response to oxidative and proteotoxic stress. Innovation and Conclusion: We here demonstrate that adaption of the proteasome system in response to oxidative stress is dependent on TCF11/Nrf1 in this model system. We conclude that TCF11/Nrf1, therefore, plays a vital role in maintaining redox and protein homeostasis. This work provides a vital insight into the molecular mechanisms of neurodegeneration due to oxidative stress by rotenone, and further studies investigating the role of TCF11/Nrf1 in the human condition would be of considerable interest. Antioxid. Redox Signal. 25, 870-885.

Cx43 Mediates Resistance against MPP⁺-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway.

Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson’s disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP+-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP+-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP+-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.

ChemInform Abstract: 3,4‐seco‐28‐Nor‐oleanane Triterpenes from Camellia japonica Protect from Neurotoxicity in a Rotenone Model of Parkinson′s Disease.

A natural material extract library (Korea Bioactive Natural Material Bank) was screened with respect to their protective effects on neuronal cells, and a 70% ethanol extract from the flowers of Camellia japonica turned out to be a potential hit. Bioassay-guided fractionation of this active extract led to the isolation of six new 3,4-seco-28-nor-oleanane triterpenoids (1–6). The molecular structures of these new triterpenoids were elucidated through extensive spectroscopic analyses, including high-resolution MS and 1D- and 2D-NMR data. In a rotenone model of Parkinson's disease (PD), compounds 3–6 effectively protected against neurotoxicity in the human dopaminergic SH-SY5Y cell line. Among these 3,4-seco-28-nor-oleanane triterpenoids, 4,17β,29-trihydroxy-16-oxo-3,4-seco-28-norolean-12-en-3-oic acid n-butyl ester (5) exerted the strongest neuroprotective effect by suppressing the expression of α-synuclein and the intracellular production of reactive oxygen species (ROS) induced by rotenone treatment. In addition, compound 5 induced microtubule-associated protein 1A/1B-light chain 3 (LC3), which is known as an autophagy biomarker. These results suggest a new class of chemical entities for developing bioactive compounds for PD therapy.

3,4-seco-28-Nor-oleanane triterpenes from Camellia japonica protect from neurotoxicity in a rotenone model of Parkinson's disease

A natural material extract library (Korea Bioactive Natural Material Bank) was screened with respect to their protective effects on neuronal cells, and a 70% ethanol extract from the flowers of Camellia japonica turned out to be a potential hit. Bioassay-guided fractionation of this active extract led to the isolation of six new 3,4-seco-28-nor-oleanane triterpenoids (1–6). The molecular structures of these new triterpenoids were elucidated through extensive spectroscopic analyses, including high-resolution MS and 1D- and 2D-NMR data. In a rotenone model of Parkinson's disease (PD), compounds 3–6 effectively protected against neurotoxicity in the human dopaminergic SH-SY5Y cell line. Among these 3,4-seco-28-nor-oleanane triterpenoids, 4,17β,29-trihydroxy-16-oxo-3,4-seco-28-norolean-12-en-3-oic acid n-butyl ester (5) exerted the strongest neuroprotective effect by suppressing the expression of α-synuclein and the intracellular production of reactive oxygen species (ROS) induced by rotenone treatment. In addition, compound 5 induced microtubule-associated protein 1A/1B-light chain 3 (LC3), which is known as an autophagy biomarker. These results suggest a new class of chemical entities for developing bioactive compounds for PD therapy.

3,4-methylenedioxypyrovalerone (MDPV) Induces Cytotoxic Effects on Human Dopaminergic SH-SY5Y Cells

Background. Synthetic cathinones are a rapidly growing group of psychostimulant drugs usually referred to as bath salts and have been used as an alternative to classic amphetamine-like drugs, with 3,4-methylenedioxypyrovalerone (MDPV) being one of the most prevalent constituents. Consistent with the effects of other psychostimulants, MDPV may induce neurotoxicity by altering monoamine systems in the brain or by inducing neuronal apoptosis. Purpose. The aim of this study was to evaluate the effects of MDPV on the human dopaminergic cell line SH-SY5Y. Experimental design. After 24-hour exposure to MDPV (100µ Mt o 2.5 mM), cytotoxicity, cellular proliferation, and apoptosis were evaluated, whereas reactive oxygen species (ROS) production was evaluated at 2 h, 4 h, 6 h, 22 h, and 24 h. Results. MDPV increased ROS production after 1 h, 4 h, and 6 h of exposure in all but the highest concentration; a moderate increase was observed at 22 h and 24 h. Only high concentrations of the drug decreased cellular proliferation and induced apoptosis and necrosis. Conclusion. MDPV induces dopaminergic toxicity by decreasing cellular proliferation and by increasing apoptosis and necrosis. The production of ROS may play a role in the early response to the drug.