An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson's Disease1.
Ontology highlight
ABSTRACT:
SUBMITTER: Vasquez V
PROVIDER: S-EPMC6221916 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
Publications
An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson's Disease1.
Vasquez Velmarini V Mitra Joy J Perry George G Rao K S KS Hegde Muralidhar L ML
Journal of Alzheimer's disease : JAD 20180101 2
Altered expression of α-synuclein is linked to Parkinson's disease (PD). A major challenge to explore how the increased α-synuclein affect neurotoxicity is the lack of a suitable human neuronal cell model that mimics this scenario. Its expression in neural precursors affects their differentiation process, in addition to the neuronal adaptability and variability in maintaining a constant level of expression across passages. Here, we describe an SH-SY5Y line harboring Tet-ON SNCA cDNA cassette tha ...[more]
Similar Datasets
Project description:Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (αSyn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study αSyn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different αSyn constructs: αSyn, αSyn coupled to the fluorescence protein Venus (αSyn-Venus), and αSyn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of αSyn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly αSyn-overexpressing cell lines holding high potential for the screening for modulators of αSyn aggregation and αSyn-mediated toxicity.
| S-EPMC6591385 | biostudies-literature
Project description:BackgroundADP/ATP translocase 1 (ANT1) is involved in the exchange of cytosolic ADP and mitochondrial ATP, and its defection plays an important role in mitochondrial pathogenesis. To reveal an etiological implication of ANT1 for Parkinson's disease (PD), a neurodegenerative disorder, a mouse model treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neuroblastoma cell model induced by 1-methyl-4-pehny1-pyridine were utilized in this study.ResultsThe tissue-specific abundance in ANT1 in mouse brains was accessed using the analysis of Western blot and immunohistochemistry. Down-regulated soluble ANT1 was found to be correlated with PD, and ANT1 was associated with PD pathogenesis via forming protein aggregates with α-synuclein. This finding was confirmed at cellular level using neuroblastoma cell models. ANT1 supplement in neuronal cells revealed the protective roles of ANT1 against cytotoxicity caused by MPP+. Protein interaction assay, coupled with the analysis of LC-MS/MS, silver-stained SDS-PAGE and Western blot against anti-ANT1 antibody respectively, illustrated the interaction of ANT1 with α-synuclein using the expressed α-synuclein as a bite. Additionally, a significant increasing ROSs was detected in the MPP+-treated cells.ConclusionsThis study indicated that ANT1 was a potentially causative factor of PD, and led to neuropathogenic injury via promoting the formation of protein aggregates with α-synuclein. This investigation potentially promotes an innovative understanding of ANT1 on the etiology of PD and provides valuable information on developing potential drug targets in PD treatment or reliable biomarkers in PD prognostication.
| S-EPMC8272299 | biostudies-literature
Project description:BackgroundOverexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models.ResultsHere we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary.ConclusionsSince curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.
| S-EPMC2879277 | biostudies-literature
Project description:Parkinson's disease (PD) is one of the most common neurodegenerative disorders. However, its cellular and molecular mechanisms still wrap in the mist. This is partially caused by the absence of appropriate animal models mimicking sporadic PD that constitutes the majority of cases. Previously, we reported that a cysteine protease, asparagine endopeptidase (AEP), is activated in an age-dependent manner, and cleaves α-synuclein in the brain of sporadic PD patients. The AEP-derived α-synuclein 1-103 fragment is required for the pathogenesis of PD. Thus, we designed and characterized a novel transgenic mouse line expressing α-synuclein 1-103 (designated N103 mice). This model shows an abundant accumulation of pathological α-synuclein in the central nervous system, loss of dopaminergic neurons in the substantia nigra, and progressive striatal synaptic degeneration. The N103 mice also manifest age-dependent PD-like behavioral impairments. Notably, the mice show weight loss and constipation, which are the common non-motor symptoms in PD. The RNA-sequencing analysis found that the transcriptomics pattern was extensively altered in N103 mice. In conclusion, the N103 mouse line, as a brand-new tool, might provide new insights into PD research.
| S-EPMC8569470 | biostudies-literature
Project description:Altogether results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein through its co-localization with cytochrome c or on it's own, affects several biological pathways which contribute to increased neuronal death in Maneb and paraquat (MP)-induced model of Parkinson's disease.
2016-11-29 | GSE89562 | GEO
Project description:BackgroundCell-to-cell propagation of α-synuclein (α-syn) aggregates is thought to contribute to the pathogenesis of Parkinson's disease (PD) and underlie the spread of α-syn neuropathology. Increased pro-inflammatory cytokine levels and activated microglia are present in PD and activated microglia can promote α-syn aggregation. However, it is unclear how microglia influence α-syn cell-to-cell transfer.MethodsWe developed a clinically relevant mouse model to monitor α-syn prion-like propagation between cells; we transplanted wild-type mouse embryonic midbrain neurons into a mouse striatum overexpressing human α-syn (huα-syn) following adeno-associated viral injection into the substantia nigra. In this system, we depleted or activated microglial cells and determined the effects on the transfer of huα-syn from host nigrostriatal neurons into the implanted dopaminergic neurons, using the presence of huα-syn within the grafted cells as a readout.ResultsFirst, we compared α-syn cell-to-cell transfer between host mice with a normal number of microglia to mice in which we had pharmacologically ablated 80% of the microglia from the grafted striatum. With fewer host microglia, we observed increased accumulation of huα-syn in grafted dopaminergic neurons. Second, we assessed the transfer of α-syn into grafted neurons in the context of microglia activated by one of two stimuli, lipopolysaccharide (LPS) or interleukin-4 (IL-4). LPS exposure led to a strong activation of microglial cells (as determined by microglia morphology, cytokine production and an upregulation in genes involved in the inflammatory response in the LPS-injected mice by RNA sequencing analysis). LPS-injected mice had significantly higher amounts of huα-syn in grafted neurons. In contrast, injection of IL-4 did not change the proportion of grafted dopamine neurons that contained huα-syn relative to controls. As expected, RNA sequencing analysis on striatal tissue revealed differential gene expression between LPS and IL-4-injected mice; with the genes upregulated in tissue from mice injected with LPS including several of those involved in an inflammatory response.ConclusionsThe absence or the hyperstimulation of microglia affected α-syn transfer in the brain. Our results suggest that under resting, non-inflammatory conditions, microglia modulate the transfer of α-syn. Pharmacological regulation of neuroinflammation could represent a future avenue for limiting the spread of PD neuropathology.
| S-EPMC6697982 | biostudies-literature
Project description:The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.
| S-EPMC2978097 | biostudies-literature
Project description:Parkinson's disease is characterized by a progressive accumulation of alpha-Synuclein (αSyn) neuronal inclusions called Lewy bodies in the nervous system. Lewy bodies can arise from the cell-to-cell propagation of αSyn, which can occur via sequential steps of secretion and uptake. Here, by fusing a removable short signal peptide to the N-terminus of αSyn, we developed a novel mouse model with enhanced αSyn secretion and cell-to-cell transmission. Expression of the secreted αSyn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). This combination of promoter and viral vector induced a robust expression in neurons but not in the glia of injected mice. Biochemical characterization of the secreted αSyn revealed that, in cultured cells, this protein is released to the extracellular milieu via conventional secretion. The released αSyn is then internalized and processed by acceptor cells via the endosome-lysosome pathway indicating that the secreted αSyn is cell-to-cell transmitted. The secreted αSyn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson's disease-like phenotype that includes a robust αSyn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of Parkinson's disease. In summary, a novel animal model of Parkinson's disease based on enhanced cell-to-cell transmission of αSyn was developed. The neuron-produced cell-to-cell transmitted αSyn triggers all phenotypic features of experimental Parkinson's disease in mice.
| S-EPMC10771530 | biostudies-literature
Project description:Idiopathic Parkinson's disease is the second most common neurodegenerative disease and is estimated to be approximately 30% heritable. Genome wide association studies have revealed numerous loci associated with risk of development of Parkinson's disease. The majority of genes identified in these studies are expressed in glia at either similar or greater levels than their expression in neurons, suggesting that glia may play a role in Parkinson's disease pathogenesis. The role of individual glial risk genes in Parkinson's disease development or progression is unknown, however. We hypothesized that some Parkinson's disease risk genes exert their effects through glia. We developed a Drosophila model of α-synucleinopathy in which we can independently manipulate gene expression in neurons and glia. Human wild type α-synuclein is expressed in all neurons, and these flies develop the hallmarks of Parkinson's disease, including motor impairment, death of dopaminergic and other neurons, and α-synuclein aggregation. In these flies, we performed a candidate genetic screen, using RNAi to knockdown 14 well-validated Parkinson's disease risk genes in glia and measuring the effect on locomotion in order to identify glial modifiers of the α-synuclein phenotype. We identified 4 modifiers: aux, Lrrk, Ric, and Vps13, orthologs of the human genes GAK, LRRK2, RIT2, and VPS13C, respectively. Knockdown of each gene exacerbated neurodegeneration as measured by total and dopaminergic neuron loss. Knockdown of each modifier also increased α-synuclein oligomerization. These results suggest that some Parkinson's disease risk genes exert their effects in glia and that glia can influence neuronal α-synuclein proteostasis in a non-cell-autonomous fashion. Further, this study provides proof of concept that our novel Drosophila α-synucleinopathy model can be used to study glial modifier genes, paving the way for future large unbiased screens to identify novel glial risk factors that contribute to PD risk and progression.
| S-EPMC8502212 | biostudies-literature
Project description:BackgroundParkinson's disease (PD) is the second most prevalent movement disorder characterized by up to 80% loss of dopamine (DA) neurons and accumulation of Lewy body deposits composed of α-synuclein (α-syn). Accumulation of α-syn is associated with microglial activation, leading to a pro-inflammatory environment linked with the pathogenesis of PD. Along with microglia, CD4 and CD8 T cells are observed in SNpc. The contribution of T-cells to PD development remains unclear with studies demonstrating that they may mediate neurodegeneration or act in a neuroprotective manner.MethodsHere, we assessed the contribution of T cells to PD neurodegeneration using an adeno-associated virus (AAV) coding human wild-type α-syn or GFP injected into the substantia nigra pars compacta (SNpc) in T cell deficient (athymic nude) and T cell competent (heterozygous) rats. The rats were behaviorally assessed with cylinder test to test paw bias. Following behavior testing, brains were collected and analyzed for markers of dopamine neuron, microglial activation, T cells, and α-syn expression.ResultsInjection of AAV9-α-syn unilaterally into the SN of T cell competent rats resulted in a significant paw bias in comparison to the controls at 60 days post-injection. Conversely, T cell-deficient rats injected with AAV9-α-syn showed no deficit in paw bias. As expected, injected T cell competent rats demonstrated a significant increase in microglial activation (MHCII staining) as well as significant dopaminergic neuron loss. In contrast, the T cell-deficient counterparts did not show a significant increase in microglial activation or significant neuron loss compared to the control animals. We also observed CD4 and CD8 T cells in SNpc following microglial MHCII expression and dopaminergic neuron loss. The time course of T cell entry correlates with upregulation of MHCII and the peak loss of TH+ cells in the SNpc.ConclusionThese data demonstrate that T cell infiltration and microglial upregulation of MHCII are involved in α-synuclein-mediated DA neuron loss in this rat model of PD.
| S-EPMC7429710 | biostudies-literature