Real-Time Characterization of Cell Membrane Disruption by α-Synuclein Oligomers in Live SH-SY5Y Neuroblastoma Cells.
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ABSTRACT:
SUBMITTER: Parres-Gold J
PROVIDER: S-EPMC7484231 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alpha-Synuclein (aSyn) is a chameleon-like protein. Its overexpression and intracellular deposition defines neurodegenerative α-synucleinopathies including Parkinson's disease. Whether aSyn up-regulation is the cause or the protective reaction to α-synucleinopathies remains unresolved. Remarkably, the accumulation of aSyn is involved in cancer. Here, the neuroblastoma SH-SY5Y cell line was genetically engineered to overexpress aSyn at low and at high levels. aSyn cytotoxicity was assessed by the MTT and vital-dye exclusion methods, observed at the beginning of the sub-culture of low-aSyn overexpressing neurons when cells can barely proliferate exponentially. Conversely, high-aSyn overexpressing cultures grew at high rates while showing enhanced colony formation compared to low-aSyn neurons. Cytotoxicity of aSyn overexpression was indirectly revealed by the addition of pro-oxidant rotenone. Pretreatment with partially reduced graphene oxide, an apoptotic agent, increased toxicity of rotenone in low-aSyn neurons, but, it did not in high-aSyn neurons. Consistent with their enhanced proliferation, high-aSyn neurons showed elevated levels of SMP30, a senescence-marker protein, and the mitosis Ki-67 marker. High-aSyn overexpression conferred to the carcinogenic neurons heightened tumorigenicity and resistance to senescence compared to low-aSyn cells, thus pointing to an inadequate level of aSyn stimulation, rather than the aSyn overload itself, as one of the factors contributing to α-synucleinopathy.
| S-EPMC7016025 | biostudies-literature
Project description:Aggregation of α-synuclein (α-syn) is closely linked to Parkinson's disease (PD) and the related synucleinopathies. Aggregates spread through the brain during the progression of PD, but the mechanism by which this occurs is still not known. One possibility is a self-propagating, templated-seeding mechanism, but this cannot be established without quantitative information about the efficiencies and rates of the key steps in the cellular process. To address this issue, we imaged the uptake and seeding of unlabeled exogenous α-syn fibrils by SH-SY5Y cells and the resulting secreted aggregates, using super-resolution microscopy. Externally-applied fibrils very inefficiently induced self-assembly of endogenous α-syn in a process accelerated by the proteasome. Seeding resulted in the increased secretion of nanoscopic aggregates (mean 35 nm diameter), of both α-syn and Aβ. Our results suggest that cells respond to seed-induced disruption of protein homeostasis predominantly by secreting nanoscopic aggregates; this mechanism may therefore be an important protective response by cells to protein aggregation.
| S-EPMC8139990 | biostudies-literature
Project description:Genistein, a potent antioxidant compound, protects dopaminergic neurons in a mouse model of Parkinson's disease. However, the mechanism underlying this action remains unknown. This study investigated human SH-SY5Y cells overexpressing the A53T mutant of α-synuclein. Four groups of cells were assayed: a control group (without any treatment), a genistein group (incubated with 20 μM genistein), a rotenone group (treated with 50 μM rotenone), and a rotenone + genistein group (incubated with 20 μM genistein and then treated with 50 μM rotenone). A lactate dehydrogenase release test confirmed the protective effect of genistein, and genistein remarkably reversed mitochondrial oxidative injury caused by rotenone. Western blot assays showed that BCL-2 and Beclin 1 levels were markedly higher in the genistein group than in the rotenone group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed that genistein inhibited rotenone-induced apoptosis in SH-SY5Y cells. Compared with the control group, the expression of NFE2L2 and HMOX1 was significantly increased in the genistein + rotenone group. However, after treatment with estrogen receptor and NFE2L2 channel blockers (ICI-182780 and ML385, respectively), genistein could not elevate NFE2L2 and HMOX1 expression. ICI-182780 effectively prevented genistein-mediated phosphorylation of NFE2L2 and remarkably suppressed phosphorylation of AKT, a protein downstream of the estrogen receptor. These findings confirm that genistein has neuroprotective effects in a cell model of Parkinson's disease. Genistein can reduce oxidative stress damage and cell apoptosis by activating estrogen receptors and NFE2L2 channels.
| S-EPMC6108222 | biostudies-literature
Project description:Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
| S-EPMC9124689 | biostudies-literature
Project description:Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO is challenged by its low abundance and structural and morphological complexity. Here, we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. First, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 7 best leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus, a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.
| S-EPMC10016340 | biostudies-literature
Project description:Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.
| S-EPMC7813998 | biostudies-literature
Project description:Parkinson's disease (PD) is a neurodegenerative disorder, and the hallmarks of this disease include iron deposition and α-synuclein (α-syn) aggregation. Hepcidin could reduce iron in the central and peripheral nervous systems. Here, we hypothesized that hepcidin could further decrease α-syn accumulation via reducing iron. Therefore, rotenone or α-syn was introduced into human neuroblastoma SH-SY5Y cells to imitate the pathological progress of PD in vitro. This study investigated the clearance effects of hepcidin on α-syn induced by a relatively low concentration of rotenone exposure or α-syn overexpression to elucidate the potential clearance pathway involved in this process. We demonstrated that SH-SY5Y cell viability was impaired after rotenone treatment in a dose-dependent manner. α-syn expression and iron content increased under a low concentration rotenone (25 nM for 3 days) treatment in SH-SY5Y cells. Pre-treatment with hepcidin peptide suppressed the abovementioned effects of rotenone. However, hepcidin did not affect treatment with rotenone under high iron conditions. Hepcidin also played a role in reducing α-syn accumulation in rotenone and α-syn overexpression conditions. We identified that the probable clearance effect of hepcidin on α-syn was mediated by the autophagy pathway using pretreatment with autophagy inhibitors (3-MA and CQ) and detection of autophagy protein markers (LC3II/I and p62). In conclusion, hepcidin eliminated α-syn expression via the autophagy pathway in rotenone-treated and α-syn overexpression SH-SY5Y cells. This study highlights that hepcidin may offer a potential therapeutic perspective in α-syn accumulation diseases.
| S-EPMC7596286 | biostudies-literature
Project description:Parkinson's disease (PD) is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies (LBs) in the surviving SNc neurons. LBs formation is caused by the accumulation of α-synuclein (α-syn) or phosphorylated α-syn at serine-129 (pSer129-α-syn), which is implicated in the pathological progression of PD. Salidroside (Sal), the main active ingredient of the root of Rhodiola rosea L., has been reported to have potent neuroprotective properties in our previous investigations. Here, we investigated the effects of Sal on 6-OHDA and overexpresssion of WT/A30P-α-syn-induced pathological α-syn increase and the mechanism behind it in SH-SY5Y cells. We found Sal displays neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. Sal decreased the pSer129-α-syn level mainly by maintaining the normal function of ubiquitin-proteasome system (UPS). Furthermore, Sal promoted the clearance of α-syn and protected the cell viability mainly through recovered the 20S proteasome activity in WT/A30P-α-syn-transfected cells. These data provide new mechanistic insights into the neuroprotective effects of Sal and Sal may be a promising therapy to slow neurodegeneration in PD. Highlights: Sal protects cells and decreases the pSer129-α-syn protein level in 6-OHDA-induced impairmental and dysfunctional SH-SY5Y cells. Sal promotes the clearance of α-syn and protects the cell viability mainly through recovering the 20S proteasome activity in WT/A30P-α-syn plasmids transfected cells. Maintaining the normal function of the UPS may be one of the important mechanisms of Sal in neuroprotective effects.
| S-EPMC5917065 | biostudies-literature
Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to staphylococcal α-hemolysin oligomers (rAHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with 3nM of extracted oligomers (rAHL) for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.
2015-04-01 | E-GEOD-60543 | biostudies-arrayexpress
Project description:(1) Background: Parkinson's disease (PD) is the most common movement disorder. Imbalanced protein homeostasis and α-syn aggregation are involved in PD pathogenesis. Autophagy is related to the occurrence and development of PD and can be regulated by histone deacetylases (HDACs). Various inhibitors of HDACs exert neuroprotective effects within in vitro and in vivo models of PD. HDAC4, a class Ⅱ HDAC, colocalizes with α-synuclein and ubiquitin in Lewy bodies and also accumulates in the nuclei of dopaminergic neurons in PD models. (2) Methods: In the present study, the gene expression profile of HDACs from two previously reported datasets in the GEO database was analyzed, and the RNA levels of HDAC4 in brain tissues were compared between PD patients and healthy controls. In vitro, SH-SY5Y cells transfected with HDAC4 shRNA or pretreated with mc1568 were treated with 1 μM of rotenone for 24 h. Then, the levels of α-syn, LC3, and p62 were detected using Western blot analysis and immunofluorescent staining, and cell viabilities were detected using Cell Counting Kit-8 (CCK-8). (3) Results: HDAC4 was highly expressed in PD substantia nigra and locus coeruleus. Mc1568, an inhibitor of HDAC4, decreased α-synuclein levels in rotenone-treated SH-SY5Y cells in a concentration-dependent manner and activated autophagy, which was impaired by rotenone. The knockdown of HDAC4 reversed rotenone-induced α-syn accumulation in SH-SY5Y cells and protected the neurons by enhancing autophagy. (4) Conclusions: HDAC4 is a potential therapeutic target for PD. The inhibition of HDAC4 by mc1568 or a gene block can reduce α-syn levels by regulating the autophagy process in PD. Mc1568 is a promising therapeutic agent for PD and other disorders related to α-syn accumulation.
| S-EPMC10136981 | biostudies-literature