Dopaminergic Neuronal Cell Line Research Articles

GV-971 attenuates α-Synuclein aggregation and related pathology.

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), share a distinct pathological feature, that is, a widespread accumulation of α-synuclein (α-syn) in the brain. There is a significant clinical unmet need for disease-modifying treatments for synucleinopathies. Recently, a seaweed-derived mixture of oligosaccharides sodium oligomannate, GV-971, was approved for Phase 2 clinical trials for PD. This study aimed to further evaluate the therapeutic effects of GV-971 on synucleinopathies using cellular and animal models and explore its associated molecular mechanisms. α-Syn aggregation was assessed, in vitro and ex vivo, by ThT assay. A dopaminergic neuron cell line, Prnp-SNCAA53T mice, and brain slices from PD and DLB patients were used to determine the efficacy of GV-971 in ameliorating α-syn pathology. Measurements of motor functions, including pole, cylinder, and rotarod tests, were conducted on Prnp-SNCAA53T mice 4 weeks after intragastric administration of GV-971 (200 mg day-1 kg-1 ). GV-971 effectively prevented α-syn aggregation and even disassembled pre-aggregated α-syn fibrils, in vitro and ex vivo. In addition, GV-971 was able to rescue α-syn-induced neuronal damage and reduced release of extracellular vesicles (EVs), likely via modulating Alix expression. In the Prnp-SNCAA53T mouse model, when treated at the age of 5 months, GV-971 significantly decreased α-syn deposition in the cortex, midbrain, and cerebellum regions, along with ameliorating the motor dysfunctions. Our results indicate that GV-971, when administered at a relatively early stage of the disease process, significantly reduced α-syn accumulation and aggregation in Prnp-SNCAA53T mice. Furthermore, GV-971 corrected α-syn-induced inhibition of EVs release in neurons, contributing to neuronal protection. Future studies are needed to further assess GV-971 as a promising disease-modifying therapy for PD and other synucleinopathies.

JMJD3 and SNAI2 synergistically protect against Parkinson's disease by mediating the YAP/HIF1α signaling pathway in a mouse model.

This study aimed to characterize the functional relevance and mechanistic basis of the histone demethylase JMJD3 in preserving dopaminergic neuron survival in PD. Mice with MPTP-induced lesions and MN9D dopaminergic neuronal cell lines exposed to 6-OHDA, respectively, were used to simulate in vivo and in vitro PD-like environments. PD-related genes with differential expressions were identified using RNA sequencing of hippocampal tissues collected from MPTP-lesioned mice. A specific lentiviral shRNA vector was used to investigate the effects of JMJD3 on neuron activities in vitro and PD-like phenotypes in vivo. JMJD3 was found to up-regulate the expression of SNAI2 through the inhibition of H3K27me3 enrichment in the SNAI2 promoter region. As a result, the viability of 6-OHDA-exposed MN9D cells was stimulated, and cell apoptosis was diminished. Knockdown of SNAI2 decreased the expression of YAP and HIF1α while also reducing the viability of 6-OHDA-exposed MN9D cells and increasing cell apoptosis. The in vivo experiments demonstrated that JMJD3 activated the SNAI2/YAP/HIF1α signaling pathway, inhibiting PD-like phenotypes in MPTP-lesioned mice. Thus, the findings provide evidence that JMJD3 inhibits the enrichment of H3K27me3 at the SNAI2 promoter, leading to the upregulation of SNAI2 expression and activation of the YAP/HIF1α signaling pathway, ultimately exerting a protective effect on PD mice. This finding suggests that targeting the JMJD3-SNAI2 pathway could be a promising therapeutic strategy for Parkinson's disease. Further in-depth studies are needed to elucidate the underlying mechanisms and identify potential downstream targets of this pathway.

Synthesis, In Vitro Biological Evaluation of Antiproliferative and Neuroprotective Effects and In Silico Studies of Novel 16E-Arylidene-5α,6α-epoxyepiandrosterone Derivatives

Steroids constitute an important class of pharmacologically active molecules, playing key roles in human physiology. Within this group, 16E-arylideneandrostane derivatives have been reported as potent anti-cancer agents for the treatment of leukemia, breast and prostate cancers, and brain tumors. Additionally, 5α,6α-epoxycholesterol is an oxysterol with several biological activities, including regulation of cell proliferation and cholesterol homeostasis. Interestingly, pregnenolone derivatives combining these two modifications were described as potential neuroprotective agents. In this research, novel 16E-arylidene-5α,6α-epoxyepiandrosterone derivatives were synthesized from dehydroepiandrosterone by aldol condensation with different aldehydes followed by a diastereoselective 5α,6α-epoxidation. Their cytotoxicity was evaluated on tumoral and non-tumoral cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, the assessment of the neuroprotective activity of these derivatives was performed in a dopaminergic neuronal cell line (N27), at basal conditions, and in the presence of the neurotoxin 6-hydroxydopamine (6-OHDA). Interestingly, some of these steroids had selective cytotoxic effects in tumoral cell lines, with an IC50 of 3.47 µM for the 2,3-dichlorophenyl derivative in the breast cancer cell line (MCF-7). The effects of this functionalized epoxide on cell proliferation (Ki67 staining), cell necrosis (propidium iodide staining), as well as the analysis of the nuclear area and near neighbor distance in MCF-7 cells, were analyzed. From this set of biological studies, strong evidence of the activation of apoptosis was found. In contrast, no significant neuroprotection against 6-OHDA-induced neurotoxicity was observed for the less cytotoxic steroids in N27 cells. Lastly, molecular docking simulations were achieved to verify the potential affinity of these compounds against important targets of steroidal drugs (androgen receptor, estrogen receptor α, and 5α-reductase type 2, 17α-hydroxylase-17,20-lyase and aromatase enzymes). This in silico study predicted a strong affinity between most novel steroidal derivatives and 5α-reductase and 17α-hydroxylase-17,20-lyase enzymes.

Regulation of Αlpha-Synuclein Gene (SNCA) by Epigenetic Modifier TET1 in Parkinson Disease.

Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

Alpha-synuclein negatively controls cell proliferation in dopaminergic neurons

As researchers grapple with the mechanisms and implications of alpha-synuclein (α-syn) in neuropathology, it is often forgotten that the function(s) of α-syn in healthy cells remain largely elusive. Previous work has relied on observing α-syn localization in the cell or using knockout mouse models. Here, we address the specific role of α-syn in human dopaminergic neurons by disrupting its gene (SNCA) in the human dopaminergic neuron cell line, LUHMES. SNCA-null cells were able to differentiate grossly normally and showed modest effects on gene expression. The effects on gene expression were monodirectional, resulting primarily in the significant decrease of expression for 401 genes, implicating them as direct, or indirect positive targets of α-syn. Gene ontological analysis of these genes showed enrichment in terms associated with proliferation, differentiation, and synapse activity. These results add to the tapestry of α-syn biological functions. Significance statementThe normal functions of α-syn have remained controversial, despite its clear importance in Parkinson's Disease pathology, where it accumulates in Lewy bodies and contributes to neurodegeneration. Its name implies synaptic and nuclear functions, but how it participates at these locations has not been resolved. Via knock-out experiments in dopaminergic neurons, we implicate α-syn as a functional participant in synapse activity and in proliferation/differentiation, the latter being novel and provide insight into α-syn's role in neuronal development.

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.

Camptothecin Regulates Microglia Polarization and Exerts Neuroprotective Effects via Activating AKT/Nrf2/HO-1 and Inhibiting NF-κB Pathways In Vivo and In Vitro.

Microglia, the main immune cells in the brain, participate in the innate immune response in the central nervous system (CNS). Studies have shown that microglia can be polarized into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. Accumulated evidence suggests that over-activated M1 microglia release pro-inflammatory mediators that damage neurons and lead to Parkinson’s disease (PD). In contrast, M2 microglia release neuroprotective factors and exert the effects of neuroprotection. Camptothecin (CPT), an extract of the plant Camptotheca acuminate, has been reported to have anti-inflammation and antitumor effects. However, the effect of CPT on microglia polarization and microglia-mediated inflammation responses has not been reported. In our study we found that CPT improved motor performance of mice and reduced the loss of neurons in the substantia nigra (SN) of the midbrain in LPS-injected mice. In the mechanism study, we found that CPT inhibited M1 polarization of microglia and promotes M2 polarization via the AKT/Nrf2/HO-1 and NF-κB signals. Furthermore, CPT protected the neuroblastoma cell line SH-SY5Y and dopaminergic neuron cell line MN9D from damage mediated by microglia activation. In conclusion, our results demonstrate that CPT regulates the microglia polarization phenotype via activating AKT/Nrf2/HO-1 and inhibiting NF-κB pathways, inhibits neuro-inflammatory responses, and exerts neuroprotective effects in vivo and in vitro.

Alpha-lipoic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced cell injury by inhibiting autophagy and apoptosis

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and BclxL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

Chloride intracellular channel 4 participate in the protective effect of Ginkgolide B in MPP+ injured MN9D cells: insight from proteomic analysis

BackgroundGinkgolide B (GB), the extract of G. biloba leaves, has been shown to be protective against many neurological disorders, including Parkinson’s disease (PD). Efforts have been made to synthesized ginkgolides analogs and derivatives with more targeted and smaller molecular weight. In the present study, four GB derivatives (GBHC-1-GBHC-4) were synthesized, and their protective roles in N-methyl-4-phenylpyridinium (MPP +) injured MN9D dopaminergic neuronal cell line were evaluated. Also, cell response mechanisms upon these GB derivatives treatment were analyzed by iTRAQ proteomics.MethodsMN9D cells were treated with MPP + to induce in vitro cell models of PD. Four GB derivatives (GBHC-1-GBHC-4) were synthesized, and their protective roles on cell viability and apoptosis in in vitro PD model cells were evaluated by CCK8 assay, fluorescence-activated cell sorting and DAPI staining, respectively. The proteomic profiles of MPP+ injured MN9D cells pretreated with or without GB and GB derivatives were detected using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique.ResultsPretreatment with GBHC-1-GBHC-4 noticeably increased cell viability and attenuated cell apoptosis in MPP+ -injured MN9D cells. Using proteomic analysis, we identified differentially expressed proteins upon GB and GB derivatives treatment. Chloride intracellular channel 4 (CLIC4) and “protein processing in endoplasmic reticulum” pathways participated in the protective roles of GB and GBHC-4. GB and GBHC-4 pretreatment could significantly reverse MPP+ -induced CLIC4 expression and translocation from cytoplasm to nucleus of MN9D cells.ConclusionsQuantitative comparative proteomic analysis identified differentially expressed proteins associated with GB and GB derivatives. We further verified the expression of CLIC4 by western blotting and immunocytochemistry assay. This bio-information on the identified pathways and differentially expressed proteins such as CLIC4 provide more targeted directions for the synthesis of more effective and targeted GB derivatives for the treatment of neurological disorders.

Pulmonary function and functional capacity in patients with mucopolysaccharidosis

Dysfunction of protein handling has been implicated in many neurodegenerative diseases and inhibition of the ubiquitin-proteasome system (UPS) has been linked to the formation of protein aggregates and proteinopathies in such diseases. While proteasomal inhibition could trigger an array of downstream protein handling changes including up-regulation of heat shock proteins (HSPs), induction of molecular chaperones, activation of the ER stress/unfolded protein response (UPR), autophagy and aggresome formation, little is known of the relationship of proteasomal inhibition to the sequence of activation of these diverse protein handling systems. In this study we utilized the reversible proteasome inhibitor MG132 and examined the activity of several major protein handling systems in the immortalized dopaminergic neuronal N27 cell line. In the early phase (up to 6 h after proteasomal inhibition), MG132 induced time-dependent proteasomal inhibition which resulted in stimulation of the UPR, increased autophagic flux and stimulated heat shock protein response as determined by increased levels of phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), C/EBP homologous protein (CHOP)/GADD153, turnover of autophagy related microtubule-associated protein 1 light chain 3 (LC3) and increased levels of Hsp70 respectively. After prolonged proteasomal inhibition induced by MG132, we observed the formation of vimentin-caged aggresome-like inclusion bodies. A recovery study after MG132-induced proteasomal inhibition indicated that the autophagy-lysosomal pathway participated in the clearance of aggresomes. Our data characterizes the relationship between proteasome inhibition and activation of other protein handling systems. These data also indicated that the induction of alternate protein handling systems and their temporal relationships may be important factors that determine the extent of accumulation of misfolded proteins in cells as a result of proteasome inhibition.

Polydatin protects SH-SY5Y in models of Parkinson's disease by promoting Atg5-mediated but parkin-independent autophagy

Parkinson's disease (PD), the second most common chronic neurodegenerative disorder, broadly remains incurable. Both genetic susceptibility and exposure to deleterious environmental stimuli contribute to dopaminergic neuron degeneration in the substantia nigra. Hence, reagents that can ameliorate the phenotypes rendered by genetic or environmental factors should be considered in PD therapy. In this study, we found that polydatin (Pol), a natural compound extracted from grapes and red wines, significantly attenuated rotenone- (Rot) or Parkin deficiency-induced mitochondrial dysfunction and cell death in SH-SY5Y, a human dopaminergic neuronal cell line. We showed that Pol significantly attenuated the Rot-induced decrease in cell viability, mitochondrial membrane potential (MMP), and Sirt 1 expression and increase in cell death, reactive oxygen species (ROS) and DJ1 expression. Rot resulted in a decrease in mTOR/Ulk-involved autophagy and an increase in PGC1β/mfn2-involved mitochondrial fusion, which was inhibited by Pol. We further demonstrated that the protective effects of Pol are partially blocked when autophagy-related gene 5 (Atg5) is genetically inactivated, suggesting that Pol-mediated neuroprotection requires Atg5. Moreover, Pol rescued Parkin knockdown-induced oxidative stress, mitochondrial dysfunction, autophagy impairment, and mitochondrial fusion enhancement. Interestingly, Pol treatment could also rescue the mitochondrial morphological abnormality and motorial dysfunction of a Drosophila PD model induced by Parkin deficiency. Thus, Pol could represent a useful therapeutic strategy as a disease-modifier in PD by decreasing oxidative stress and regulating autophagic processes and mitochondrial fusion.

Mycoplasma infection of cultured cells induces oxidative stress and attenuates cellular base excision repair activity

Mycoplasma contamination is a major concern for in vitro cell culture models as its resistance to most antibiotics, which makes the prevention and treatment of infection challenging. Furthermore, numerous studies show that Mycoplasma infection alters a variety of cellular processes, in a wide range of cell lines. However, there is a lack of information pertaining to the effects of Mycoplasma infection on genomic stability. In this study, a dopaminergic neuronal cell line (BE-M17), a popular in vitro model for Parkinson’s disease, was used to evaluate the effect of Mycoplasma infection on genomic instability, and base excision repair (BER) activity, using single cell gel electrophoresis (the comet assay). The results showed that Mycoplasma infection induced oxidative stress in the absence of an inflammatory response, with markedly increased levels of DNA damage [strand breaks/alkali-labile sites (SB/ALS), and oxidised purines], compared to uninfected cells. The source of the oxidative stress may have been increased ROS generation, or attenuation of cellular antioxidant capacity (or a combination of both). Uninfected cells initially repaired SB/ALS more rapidly than infected cells, although SB/ALS were fully repaired in both uninfected and infected cells 2 h after H2O2 challenge. However, while uninfected cells showed complete repair of oxidised purines within 24 h, for the infected cells, these were not fully repaired even after 30 h. In conclusion, this study showed that not only does Mycoplasma infection induce oxidative stress and DNA damage, but it also decreases the efficiency of the main pathway responsible for the repair of oxidatively damaged DNA i.e. BER. In this in vitro model, there is no mechanism for infection-induced inflammation, which could be a source of increased ROS production. Therefore, further studies are needed to evaluate how Mycoplasma infection causes oxidatively damaged DNA, and how it modulates cellular DNA repair.

CRISPR/Cas9 Knockout‐Based Genome Wide Screening for Pathways Associated with Organochlorine Pesticide Toxicity in Human Dopaminergic Neurons

Environmental exposure to organochlorine pesticides (OCP) has been implicated as a risk factor for Parkinson's disease (PD), a devastating movement disorder characterized by massive loss of dopamine (DA) producing neurons in the substantia nigra and clinical symptoms including bradykinesia, rigidity and tremor. PD is the second most prevalent neurodegenerative disease and >95% of PD cases are usually not clinically diagnosed until after the age of sixty. OCPs are highly persistent in the environment and are extremely bio‐accumulative. Altered iron distribution in the brain and accumulation in the substania nigra is also observed in PD and may play a role in PD pathogenesis. To gain insight into the role of OCP exposure and iron dysregulation in PD pathogenesis, we carried out a genome wide (Brunello library, 19,114 genes, 76,411 sgRNA) CRISPR/Cas9 screen in SH‐SY5Y human dopaminergic neuronal cell line exposed to sub toxic doses of dieldrin or iron over one month to identify cellular components which modulate their toxicity. NGS sequencing of sensitive and resistant clones identified multiple genes, which when mutated significantly altered sensitivity to dieldrin or iron. Pathway analysis of the dieldrin data set implicated the protein degradation pathways previously implicated in inherited forms of PD centered on Parkin2 and SCNA. These results suggest a potentially common pathogenesis of idiopathic and inherited forms of PD. Additionally, the dieldrin results also suggested a potential role for the mTOR pathway, which is a signaling pathway that integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival, is significantly involved in dieldrin associated toxicity. Fe toxicity on the other hand was shown to significantly alter dopamine receptor 2 (D2) signaling pathways. Together, these studies provide potentially novel insight into the dopaminergic toxicity associated with PD. Currently, we are conducting targeted validation of candidate genes identified in this study. Our work represents one of the first to apply genome wide CRISPR screens in a neuronal cell line and may reveal how an environmental toxicant and iron dysregulation in the brain contribute to PD. Additionally, this work may allow us to identify molecular mediators of neurodegeneration as potential therapeutic targets and/or diagnostic biomarkers.Support or Funding InformationUniversity of Florida Opportunity Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The “ATCC” cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while “UKN” cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) β-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties.Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content.We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Characterization of Parkinson’s disease-related pathogenic TMEM230 mutants

ABSTRACTParkinson’s disease (PD) is the second most common neurodegenerative disease. Although most PD cases are sporadic, 5–10% of them are hereditary and several pathogenic mutations in related genes have been identified. Mutations in TMEM230 were recently identified as a cause of autosomal dominant PD. However, the basic properties of the mutant proteins are not yet known. We examined stability and neurotoxicity, important characteristics of PD pathogenesis-related proteins, of WT TMEM230 and two pathogenic mutants, R78L and PG5ext, in a dopaminergic neuronal cell line. Our study showed that amount of protein expressed in the same vector backbone was R78L > WT > PG5ext. The stabilities of the mutant proteins were similar to each other, but lower than that of the WT. In addition, overexpression of mutants and WT TMEM230 caused similar levels of neurotoxicity upon MPP+ treatment when compared to the cells transfected with an empty vector. Because the proteins encoded by two PD-causing genes, TMEM230 and LRRK2, function in vesicle trafficking, we tested whether they interact. LRRK2 neither interacts with, nor phosphorylates TMEM230. We also investigated the levels of several Rab proteins (Rab1A, 5, 7, 8A and 11) involved in vesicle trafficking after TMEM230 overexpression. However, there was no clear difference of any Rab proteins among cells transfected with an empty vector, TMEM230 WT and mutants-expressing cells, suggesting that TMEM230 does not directly regulate these Rab proteins. Thus, these TMEM230 PG5ext and R78L mutant proteins are not distinctly different from the WT proteins except for their stability.Abbreviations: LRRK2: Leucine-rich repeat kinase 2; PD: Parkinson's disease; AD: Alzheimer's disease; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FACS: fluorescence-activated cell sorting; PBS: phosphate buffered saline; FBS: fetal bovine serum; PI: propidium iodide.

Transcranial magnetic stimulation promotes the proliferation of dopaminergic neuronal cells in vitro

Transcranial magnetic stimulation (TMS) is a safe and non-invasive treatment for neurological disorders. TMS has been approved as a treatment for major depressive disorders by the US Food and Drug Administration (FDA) in 2008. Due to the phenomenon of electromagnetic induction, a time-varying magnetic field induces an electric field in the conductive tissues in the brain, TMS has the ability to activate neurons in vivo. However, the effects of the magnetic fields on neurons in cell culture have not been investigated adequately. The magnetic fields affect the neurons when the potential across the neuronal membrane exceeds the threshold which in turn causes an action potential. Based on these theories, we investigated the effects of the magnetic fields generated by a monophasic stimulator with a 70 mm double coil on rat dopaminergic neuronal cell lines (N27). The directions of the magnetic fields in each coil of the double coil oppose each other. The effects of changing the direction of the magnetic field on N27 neurons was also investigated. The results of the experiments showed that both of the fields perpendicular to the coil surface promoted the proliferation of N27 dopaminergic neurons. In order to investigate the gene expression and protein expression affected by TMS, quantitative Polymerase Chain Reaction (qPCR) was used. Here we report changes in glial cell line-derived neurotrophic factor (GDNF) in dopaminergic neuronal cells (N27) after TMS treatment.

Breaking down autophagy and the Ubiquitin Proteasome System

Autophagy is an evolutionarily conserved catabolic process that is involved in cellular homeostasis and stress responses. Although basal levels of autophagy are essential for cellular homeostasis, dysregulated autophagy is linked to neurodegeneration. Recent studies using genetic or neurotoxin-based models of Parkinson's disease (PD) detect autophagy. We demonstrate that neurotoxins induce autophagy in dopaminergic neuronal cell line and primary cultured neurons. Based on previous reports, including ones from our laboratory, which show that elevated reactive oxygen species (ROS) and cytosolic calcium are implicated in dopaminergic neurodegeneration, we reasoned that these triggers may play critical roles in determining dysregulated autophagy. Similarly, we have demonstrated that ROS-mediated signals play an essential role in 6-hydroxydopamine (6-OHDA)-induced apoptosis, whereas MPP+ causes elevations in cytosolic calcium and calpain activation. By using these experimental models, we specifically address the question as to whether an increase in ROS or cytosolic calcium governs abnormal flux of autophagy as well as the ubiquitin proteasome system (UPS). So far, our data support a notion that ROS and cytosolic calcium act on a distinct flux of autophagy and the UPS. Our data also raise the possibility of interplay between autophagy and other cell death modes (e.g., caspase- or calpain-dependent cell death) during dopaminergic neurodegeneration.

Systemic Analysis of miRNAs in PD Stress Condition: miR-5701 Modulates Mitochondrial-Lysosomal Cross Talk to Regulate Neuronal Death.

Parkinson's disease (PD) is complex neurological disorder and is prevalent in the elderly population. This is primarily due to loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) region of the brain. The modulators of the selective loss of dopaminergic neurons in PD are still not well understood. The small non-coding RNAs specifically miRNAs fine-tune the protein levels by post-transcriptional gene regulation. The role of miRNAs in PD pathogenesis is still not well characterized. In the current study, we identified the miRNA expression pattern in 6-OHDA-induced PD stress condition in SH-SY5Y, dopaminergic neuronal cell line. The targets of top 5 miRNAs both up- and down regulated were analyzed by using StarBase. The putative pathways of identified miRNAs included neurotrophin signaling, neuronal processes, mTOR, and cell death. The level of miR-5701 was significantly downregulated in the presence of 6-OHDA. The putative targets of miR-5701 miRNA include genes involved in lysosomal biogenesis and mitochondrial quality control. The transfection of miR-5701 mimic decreased the transcript level of VCP, LAPTM4A, and ATP6V0D1. The expression of miR-5701 mimic induces mitochondrial dysfunction, defect in autophagy flux, and further sensitizes SH-SY5Y cells to 6-OHDA-induced cell death. To our knowledge, the evidence in the current study demonstrated the dysregulation of specific pattern of miRNAs in PD stress conditions. We further characterized the role of miR-5701, a novel miRNA, as a potential regulator of the mitochondrial and lysosomal function determining the fate of neurons which has important implication in the pathogenesis of PD.

Assessment of an Immortalized Rat Dopaminergic Neuronal Cell Line as a Potential Model for Studying Dopamine Transporter Function

Dopamine (DA) is a neurotransmitter which controls several psychological and physiological processes including, but not limited to, reward, motivation, attention, and motor activity. Disruption of DA homeostasis in the brain is implicated in several neurological diseases such as attention‐deficit hyperactivity disorder (ADHD), schizophrenia, depression, and Parkinson disease. The DA transporter (DAT) is an integral plasma membrane protein that actively translocates extracellular DA into the presynaptic neuron. DAT serves as a primary mechanism by which synaptic DA levels and neurotransmission is spatially and temporally modulated. DAT is a major site of action for the psychostimulants cocaine and amphetamine, and for therapeutic drugs such as methylphenidate and bupropion used to treat ADHD and depression. Because DAT function is crucial for DA homeostasis, the protein is posited to play an important role in the etiology of DA imbalance diseases. Thus, a greater understanding of DAT functional and regulatory molecular mechanisms is critical as this may lead to additional effective therapeutic interventions. Currently, many cell models are used in exploring DAT function and regulation. However, it is uncertain if any of these cell lines adequately replicate dopaminergic neuron physiology. Primary dopaminergic neuron cultures from brain tissue appear to provide the most direct and reliable model for studying dopaminergic functions but are removed from their specialized neuronal environment and limited in number, restricting their usefulness for many biochemical assays. In addition, primary cultures are labor intensive and challenging to prepare and maintain. To address these issues, we are currently examining the feasibility of the neuronal cell line, 1RB3AN27 (N27), derived from immortalized rat primary dopaminergic neurons as an improved cell model for studying DAT. We have assessed these cells for expression of key dopaminergic proteins such as tyrosine hydroxylase (TH), syntaxin 1A, and native DAT via immunoblotting, finding modest TH and syntaxin 1A and very low DAT protein levels. Differentiation in the presence of dibutyryl cAMP and dehydroepiandrosterone failed to elevate protein levels of these dopaminergic markers, although more experimental replicates are needed to confirm these observations. We are currently determining the levels of additional proteins involved in DAT regulation and DA biosynthesis such as palmitoyl acyltransferases (PATs), DOPA decarboxylase, and Nurr1. Due to very low DAT levels in both undifferentiated and differentiated cells, we transfected these cells with human (h) or rat (r) DAT, creating lines stably expressing these DAT forms. Both the rDAT and hDAT N27 lines showed robust DA uptake capacity compared to the native N27 line in which little to no DA uptake was detected. In an initial kinetic analysis, the Km DA determined for the hDAT N27 line was lower than that determined using other heterologous cell systems but was similar to that determined using rat striatal synaptosomes. Taken together, preliminary results suggest that these cell lines may serve as suitable neuronal models in investigating DAT function and regulation. However, further studies are required to make this determination.Support or Funding InformationNational Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)