Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis.

Inhalation of molecular hydrogen (H2) gas ameliorates oxidative stress-induced acute injuries in the brain. Consumption of water nearly saturated with H2 also prevents chronic neurodegenerative diseases including Parkinson's disease in animal and clinical studies. However, the molecular mechanisms underlying the remarkable effect of a small amount of H2 remain unclear. Here, we investigated the effect of H2 on mitochondria in cultured human neuroblastoma SH-SY5Y cells. H2 increased the mitochondrial membrane potential and the cellular ATP level, which were accompanied by a decrease in the reduced glutathione level and an increase in the superoxide level. Pretreatment with H2 suppressed H2O2-induced cell death, whereas post-treatment did not. Increases in the expression of anti-oxidative enzymes underlying the Nrf2 pathway in H2-treated cells indicated that mild stress caused by H2 induced increased resistance to exacerbated oxidative stress. We propose that H2 functions both as a radical scavenger and a mitohormetic effector against oxidative stress in cells.

Cytoprotective role of mitochondrial amyloid beta peptide-binding alcohol dehydrogenase against a cytotoxic aldehyde.

Recent reports on amyloid beta peptide (A beta) binding-alcohol dehydrogenase (ABAD) have revealed the link of A beta with oxidative stress derived from mitochondria in the pathogenesis of Alzheimer's disease (AD). As a novel function of ABAD, we speculate that ABAD may detoxify aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). To verify this speculation, we transfected cDNA encoding ABAD into cultured cells (HeLa and SH-SY5Y), where ABAD was localized to mitochondria. ABAD-transfectants decreased the levels of externally added 4-HNE in cultured medium as detected by TLC and became resistant against external 4-HNE. Moreover, ABAD suppressed the cytotoxic effects caused by cellular 4-HNE, which were produced through excess reactive oxygen species (ROS) by treatment with an inhibitor of mitochondrial respiration, antimycin A or by adding H(2)O(2). Catabolism of 4-HNE by ABAD was inhibited by A beta, resulting in the abolishment of the cytoprotective function by ABAD against ROS. These results propose an additional role of ABAD in neural cell death in AD: ABAD detoxifies aldehydes, such as 4-HNE derived from lipid peroxides in healthy brains, and inhibited by A beta in the development of AD.

Cell-type specific occurrence of apoptosis in taste buds of the rat circumvallate papilla.

The present study employed immunohistochemistry for single-stranded DNA (ssDNA) to detect apoptotic cells in taste buds of the rat circumvallate papilla. Double-labeling of ssDNA and markers for each cell type - phospholipase C beta2 (PLCbeta2) and alpha-gustducin for type II cells, neural cell adhesion molecule (NCAM) for type III cells, and Jacalin for type IV cells - was also performed to reveal which types of cells die by apoptosis. We detected approximately 16.8% and 14.0% of ssDNA-immunoreactive nuclei among PLCbeta2-immunoreactive and alpha-gustducinimmunoreactive cells, respectively, but rarely found ssDNA-immunoreactive cells among NCAM-immunoreactive or Jacalin-labeled cells, indicating that type II cells die by apoptosis. We also applied double labeling of ssDNA and human blood group antigen H (AbH) - which mostly labels type I cells as well as other cell types - and found that approximately 78% of ssDNA-immunoreactive cells were labeled with AbH, indicating that apoptosis also occurs in type I cells. The present results revealed that apoptosis occurs in both type I cells (dark cells) and type II cells (light cells), suggesting that there are two major cell lineages (dark cell and light cell lineages) for the differentiation of taste bud cells. In summury, type IV cells differentiate into dark and light cells and type III cells differentiate to type II cells within the light cell line.

Age-dependent neurodegeneration accompanying memory loss in transgenic mice defective in mitochondrial aldehyde dehydrogenase 2 activity.

Oxidative stress may underlie age-dependent memory loss and cognitive decline. Toxic aldehydes, including 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxides, are known to accumulate in the brain in neurodegenerative disease. We have previously shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies HNE by oxidizing its aldehyde group. To investigate the role of such toxic aldehydes, we produced transgenic mice, which expressed a dominant-negative form of ALDH2 in the brain. The mice had decreased ability to detoxify HNE in their cortical neurons and accelerated accumulation of HNE in the brain. Consequently, their lifespan was shortened and age-dependent neurodegeneration and hyperphosphorylation of tau were observed. Object recognition and Morris water maze tests revealed that the onset of cognitive impairment correlated with the degeneration, which was further accelerated by APOE (apolipoprotein E) knock-out; therefore, the accumulation of toxic aldehydes is by itself critical in the progression of neurodegenerative disease, which could be suppressed by ALDH2.

Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2.

Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.

Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis.

We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells.