The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates

Antioxidants abrogate embelin induced oxidative stress.

<p>(<b>A</b>) A549 cells were pretreated with or without FeTMPyP (10 µM) or NAC (10 mM) for 1h followed by embelin (15 µM) for 4h and ROS generation was detected by DCF staining as described in the “Materials and Methods” section. Cellular fluorescence was captured using an Olympus–IX71 inverted fluorescence microscope equipped with FITC filter settings. (<b>B</b>) Mean fluorescence intensity from three different fields of view were obtained using ImageJ software. * indicates p<0.05 as compared with control and # indicates p<0.05 as compared with embelin treated cells.</p

Embelin induced changes in MAP kinase phosphorylation does not involve cross-talk between MAP kinases.

<p>A549 cells were pre-treated with or without U0126 (5 µM), PD169316 (5 µM), SP600125 (5 µM) for 1h followed by embelin (15 µM) for 4h. Total and phosphorylated levels of ERK 1/2, p38, JNK 1/2 and tubulin (loading control) were detected by Western blotting as described in the “Materials and Methods” section.</p

Effect of embelin and SMAC-N7-Ant peptide on cellular apoptosis.

<p>(<b>A</b>) A549 cells were treated with 15 µM embelin for different time intervals. Following the termination of treatments, caspase-3 activity was measured as indicated in the “Materials and Methods” section. (<b>B</b>) A549 cells were treated with 15 µM embelin for 4h and stained with Annexin-V/FITC and propidium iodide as described in the “Materials and Methods” section. Fluorescence images were captured using an Olympus–IX71 inverted fluorescence microscope equipped with FITC and rhodamine filter settings. Representative images from three different fields of view are shown. (<b>C</b>) Cells were treated with an XIAP inhibitor, SMAC-N7-Ant peptide (100 µM) for 8h. Later, caspase-3 and -9- activities were measured using the tetra-peptide substrates as described under “Materials and Methods” section. For both (<b>A</b>) and (<b>C</b>) data presented are the mean ± SD of three separate experiments. **indicates p<0.01 and * indicates p<0.05 as compared with controls.</p

Embelin induced oxidative stress regulates MAPK mediated apoptosis.

<p>A549 cells were pre-treated with or without the antioxidant FeTMPyP (10 µM) for 1h followed by embelin (15 µM) treatment for 4h. (<b>A</b>) Cellular levels of total and phosphorylated ERK 1/2, p38, JNK 1/2 and tubulin were detected by Western blot followed by chemiluminescence detection as described under “Materials and Methods” section. (<b>B</b>) Under similar experimental conditions as (<b>A</b>), cellular caspase-3 activity was measured as described under “Materials and Methods” section. Data presented are the mean ± SD of three separate experiments. <b>*</b> indicates p<0.01 as compared to control and <b>#</b> indicates p<0.05 as compared to embelin treated cells.</p

Cytotoxicity of embelin in cancer and normal cell lines.

<p>(<b>A</b>) Structure of Embelin (<b>B</b>) Cells were treated with embelin for 48h and following the termination of incubation, cell viability was measured by sulphorhodamine B assay and IC₅₀ values were calculated as mentioned in the “Materials and Methods” section. Data shown are mean ± SD of three separate experiments. * indicates p<0.01as compared with controls.</p

Alterations in pathway based gene expression profile induced by embelin.

<p>A549 cells were treated with embelin (15 µM) for 4h. Microarray analysis was performed as described in “Materials and Methods” section. Genes that showed differential regulation by at least 2-fold with p<0.05 were classified based on functional category and pathways using GeneSpring GX and Genotypic Biointerpreter-Biological Analysis Software. Pathways that predominantly showed differential expression were (<b>A</b>) MAP Kinase pathway, (<b>B</b>) Cytokine-cytokine receptor interaction and (<b>C</b>) p53 pathway. The data has been submitted to GEO database with accession number GSE50545.</p

1-Methyl-4-phenylpyridinium (MPP+)-induced apoptosis and mitochondrial oxidant generation: role of transferrin-receptor-dependent iron and hydrogen peroxide.

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin used in cellular models of Parkinson's Disease. Although intracellular iron plays a crucial role in MPP(+)-induced apoptosis, the molecular signalling mechanisms linking iron, reactive oxygen species (ROS) and apoptosis are still unknown. We investigated these aspects using cerebellar granule neurons (CGNs) and human SH-SY5Y neuroblastoma cells. MPP(+) enhanced caspase 3 activity after 24 h with significant increases as early as 12 h after treatment of cells. Pre-treatment of CGNs and neuroblastoma cells with the metalloporphyrin antioxidant enzyme mimic, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), completely prevented the MPP(+)-induced caspase 3 activity as did overexpression of glutathione peroxidase (GPx1) and pre-treatment with a lipophilic, cell-permeable iron chelator [N, N '-bis-(2-hydroxybenzyl)ethylenediamine-N, N '-diacetic acid, HBED]. MPP(+) treatment increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling)-positive cells which was completely blocked by pre-treatment with FeTBAP. MPP(+) treatment significantly decreased the aconitase and mitochondrial complex I activities; pre-treatment with FeTBAP, HBED and GPx1 overexpression reversed this effect. MPP(+) treatment increased the intracellular oxidative stress by 2-3-fold, as determined by oxidation of dichlorodihydrofluorescein and dihydroethidium (hydroethidine). These effects were reversed by pre-treatment of cells with FeTBAP and HBED and by GPx1 overexpression. MPP(+)-treatment enhanced the cell-surface transferrin receptor (TfR) expression, suggesting a role for TfR-induced iron uptake in MPP(+) toxicity. Treatment of cells with anti-TfR antibody (IgA class) inhibited MPP(+)-induced caspase activation. Inhibition of nitric oxide synthase activity did not affect caspase 3 activity, apoptotic cell death or ROS generation by MPP(+). Overall, these results suggest that MPP(+)-induced cell death in CGNs and neuroblastoma cells proceeds via apoptosis and involves mitochondrial release of ROS and TfR-dependent iron

Effect of WT-syn on time dependent fibrillation of 112-syn.

<p>(<b>A</b>) 112-syn (0.5 mg/mL) was incubated in the presence or absence of different ratios of WT-syn at 37°C in 20 mM Tris buffer, pH 7.5 for 12 h. The increase in ThT fluorescence emission spectrum (470–600 nm) was recorded following excitation at 450 nm. (<b>B</b>) Fold change in the ThT fluorescence of 112-syn, incubated for 12 h in the presence or absence of different ratios of WT-syn. Data presented are the mean ± SD of three separate experiments. *p<0.01 as compared to 0 h reading for 112-syn; #p<0.01 as compared to112-syn alone.</p