Fig. 1

Hcy-induced neuronal toxicity is associated with mitochondrial dysfunction and apoptosis. A Simplified diagram showing Hcy as an intermediate of Met and Cys metabolism. B Bar plot showing percentage of apoptotic cell death in SK-N-SH (human neuroblastoma), B35 (rat neuroblastoma) and rat primary cortical neurons upon Hcy (5 mM) treatment for 24 h. C Loss of mitochondrial membrane potential (ΔΨm) in Hcy treated SK-N-SH cells as measured by potentiometric TMRE probe (500 nM). D Representative confocal images showing the release of Cytochrome c (Cyto c) from mitochondria in cyto c-GFP expressing primary cortical neurons treated with Hcy. Scale bar 20 µm. Bar graph represents the percentage of neurons showing Cyto c release upon Hcy treatment. E Representative immunoblot showing Cyto c level in isolated mitochondrial and cytosolic fraction of SK-N-SH cells post Hcy treatment. F Confocal images showing high level of mitochondrial fragmentation in mito-DsRed expressing SK-N-SH cells treated with Hcy. Insets showing zoomed portions of the images and bar graph showing the average length distribution of different mitochondrial populations. Scale bar 10 µm. G Representative transmission electron micrograph showing distorted mitochondrial structures with disrupted cristae in SK-N-SH cells treated with Hcy. Arrowheads represent mitochondria. Scale bar 0.5 µm. Bar graph showing the quantitation of the TEM images representing the percentage of damaged mitochondrial population per cell. H Representative immunoblot showing activated caspase-3 in primary neurons upon Hcy treatment. β-actin was used as a loading control. I Bars showing caspase-3 activation by measuring its enzymatic activity using AcDEVD substrate in Hcy treated SK-N-SH cells. J Pan caspase inhibitor zVAD-FMK (5 µM) rescued Hcy-induced toxicity in both primary cortical neurons and SK-N-SH cells. Concentration of Hcy was 5 mM for cell lines and 0.75 mM for primary neurons. Data are shown as Mean ± SEM with n ≥ 3. **P < 0.01. ****P < 0.0001