Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Tributyltin induces epigenetic changes and decreases the expression of nuclear respiratory factor-1.

Tributyltin (TBT), a common organotin environmental pollutant, has been widely used as a component of marine antifouling paints. We previously reported that exposure to TBT inhibits the expression and DNA binding of nuclear respiratory factor-1 (NRF-1) and causes neurotoxicity. In the present study, we focused on the epigenetic effects of TBT and investigated whether TBT decreases NRF-1 expression via epigenetic modifications in SH-SY5Y human neuroblastoma cells. First, we found that exposure to 300 nM TBT decreases NRF-1 expression. We examined epigenetic changes induced by TBT, and showed that TBT causes hypermethylation of the NRF-1 promoter region, increases the amount of methyl-CpG-binding protein 2 (MeCP2) bound to the NRF-1 promoter, and alters the expression of DNA methyltransferases and ten-eleven translocation (TET) demethylation enzymes. These results suggest that epigenetic changes play an important role in regulation of NRF-1 expression. Next, we investigated effect of NRF-1 expression decrease on cells, and TBT reduces mitochondrial membrane potential and overexpression of NRF-1 rescued this reduction in membrane potential. Thus, we suggested that NRF-1 is important for maintaining mitochondrial membrane potential. Our study indicates that TBT causes epigenetic changes such as hypermethylation, which increases recruitment of MeCP2 to the NRF-1 promoter and probably lead to decreased of NRF-1 expression and mitochondrial membrane potential. Therefore, this research provides new evidence of the epigenetic action caused by organotin.

Mild MPP+ exposure-induced glucose starvation enhances autophagosome synthesis and impairs its degradation.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, mainly characterised by the progressive loss of dopaminergic neurons. MPP+ has been widely used as a PD-related neurotoxin, and their reports suggested the several hypotheses for neuronal cell death. However, most of these hypotheses come from the studies about the acute MPP+ exposure. We previously revealed that mild MPP+ exposure (10 and 200 µM), which induces gradual cell death, impairs autophagosome degradation at 48 h. In the present study, we further investigated the specific events of mild MPP+ exposure and revealed that mild MPP+ exposure causes the cell death through glucose starvation, but not acute toxic model (2.5 and 5 mM). At 36 h after mild MPP+ exposure, autophagosome synthesis was enhanced owing to glucose starvation and continued to enhance until 48 h, despite impaired autophagosome degradation. Inhibition of autophagosome synthesis reduced mild MPP+-induced cell death. In conclusion, we clarified that glucose starvation-enhanced autophagosome synthesis occurs at an earlier stage than impaired autophagosome degradation and is important in mild MPP+ toxicity.

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease-associated ubiquitin C-terminal hydrolase L1 and alters its structure and function.

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ) is an endogenous parkinsonism-inducing dopamine derivative. Here, we investigated the interaction between 3',4'DHBnTIQ and UCH-L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3',4'DHBnTIQ binds to UCH-L1 specifically at Cys152 in vitro. In addition, 3',4'DHBnTIQ treatment increased the amount of UCH-L1 in the insoluble fraction of SH-SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH-SY5Y cells. Catechol-modified UCH-L1 as well as insoluble UCH-L1 were detected in the midbrain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated PD model mice. Structurally as well as functionally altered UCH-L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH-L1 by neurotoxic endogenous compounds such as 3',4'DHBnTIQ might play a key role in onset and progression of idiopathic PD. We investigated the interaction between ubiquitin C-terminal hydrolase L1 (UCH-L1) and the brain endogenous parkinsonism inducer 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ). Our results indicate that 3',4'DHBnTIQ binds to UCH-L1 specifically at cysteine 152 and induces its aggregation. 3',4'DHBnTIQ also inhibits the hydrolase activity of UCH-L1. Catechol-modified as well as insoluble UCH-L1 were detected in the midbrains of MPTP-treated Parkinson's disease (PD) model mice. Conjugation of UCH-L1 by neurotoxic endogenous compounds like 3',4'DHBnTIQ might play a key role in onset and progression of PD.