Heyu Chen

NAD+ metabolism and NAD(+)-dependent enzymes: promising therapeutic targets for neurological diseases.

Numerous studies have indicated that four interacting factors, including oxidative stress, mitochondrial alterations, calcium dyshomeostasis and inflammation, play crucial pathological roles in multiple major neurological diseases, including stroke, Alzheimers disease (AD) and Parkinsons disease (PD). Increasing evidence has also indicated that NAD(+) plays important roles in not only mitochondrial functions and energy metabolism, but also calcium homeostasis and inflammation. The key NAD(+)-consuming enzyme--poly(ADP-ribose) polymerase-1 (PARP-1) and sirtuins--have also been shown to play important roles in cell death and aging, which are two key factors in the pathology of multiple major age-dependent neurological diseases: PARP-1 plays critical roles in both inflammation and oxidative stress-induced cell death; and sirtuins also mediate the process of aging, cell death and inflammation. Thus, it is conceivable that increasing evidence has suggested that NAD(+) metabolism and NAD(+)-dependent enzymes are promising targets for treating a number of neurological illnesses. For examples, the key NAD(+)-dependent enzymes SIRT1 and SIRT2 have been indicated to strongly affect the pathological changes of PD and AD; PARP-1 inhibition can profoundly reduce the brain injury in the animal models of multiple neurological diseases; and administration of either NAD(+) or nicotinamide can also decrease ischemic brain damage. Future studies are necessary to further investigate the roles of NAD+ metabolism and NAD⁺-dependent enzymes in neurological diseases, which may expose novel targets for treating the debilitating illnesses.

SIRT2 is required for lipopolysaccharide-induced activation of BV2 microglia.

It has been reported that inhibition of sirtuin 2 (SIRT2), a sirtuin family protein, can decrease cellular and tissue injuries in models of Parkinson’s disease (PD) and Huntington’s disease (HD); however, the mechanisms underlying these observations have remained unclear. Because inflammation plays key pathological roles in multiple major neurological disorders including PD and HD, in our current study we tested our hypothesis that SIRT2 plays an important role in microglial activation. We found that treatment of BV2 microglia with lipopolysaccharides led to significant increases in NO and inducible nitric oxide synthase mRNA levels, as well as increases in the levels of tumor necrosis factor-&agr; and interleukin 6 mRNA, which indicated microglial activation. These increases were significantly decreased in the cells with SIRT2 silencing-produced decreases in the SIRT2 level. These observations suggest that SIRT2 is required for lipopolysaccharide-induced microglial activation. The findings also suggest that SIRT2 may be a therapeutic target for inhibiting the inflammatory responses in neurological disorders such as PD and HD.

SIRT2 mediates oxidative stress-induced apoptosis of differentiated PC12 cells.

Sirtuin 2 (SIRT2) is a member of the sirtuin family. Previous studies have suggested that SIRT2 mediates the cell death in models of Parkinson’s disease and Huntington’s disease. However, the role of SIRT2 in oxidative stress-induced cell death has remained unclear. In this study, we investigated the roles of SIRT2 in oxidative stress-induced cell death using differentiated PC12 cells as a cell model. We found that H2O2 induced a significant increase in the SIRT2 level in the cells. Both SIRT2 silencing and the SIRT2 inhibitor AGK2 significantly decreased H2O2-induced apoptosis, partially by inhibiting caspase-3 activation. We further found that silencing of SIRT2 led to decreased reactive oxygen species levels in the H2O2-treated cells. Collectively, our observations have suggested that SIRT2 plays a significant role in oxidative stress-induced cell death.

NAD+-Carrying Mesoporous Silica Nanoparticles Can Prevent Oxidative Stress-Induced Energy Failures of Both Rodent Astrocytes and PC12 Cells

Aim To test the hypothesis that NAD+-carrying mesoporous silica nanoparticles (M-MSNs@NAD+) can effectively deliver NAD+ into cells to produce cytoprotective effects. Methods & Materials NAD+ was incorporated into M-MSNs. Primary rat astrocyte cultures and PC12 cells were treated with H2O2, followed by post-treatment with M-MSNs@NAD+. After various durations of the post-treatment, intracellular NAD+ levels, intracellular ATP levels and lactate dehydrogenase (LDH) release were determined. Results & Discussion M-MSNs can be effectively loaded with NAD+. The M-MSNs@NAD+ can significantly attenuate H2O2-induced NAD+ and ATP decreases in both astrocyte cultures and PC12 cells. M-MSNs@NAD+ can also partially prevent the H2O2-induced LDH release from both astrocyte cultures and PC12 cells. In contrast, the NAD+ that is spontaneously released from the M-MSNs@NAD+ is insufficient to prevent the H2O2-induced damage. Conclusions Our study has suggested the first approach that can effectively deliver NAD+ into cells, which provides an important basis both for elucidating the roles of intracellular NAD+ in biological functions and for therapeutic applications of NAD+. Our study has also provided the first direct evidence demonstrating a key role of NAD+ depletion in oxidative stress-induced ATP decreases.

NAD + /NADH Metabolism and NAD + -Dependent Enzymes in Cell Death and Ischemic Brain Injury: Current Advances and Therapeutic Implications

NAD(+) and NADH play crucial roles in a variety of biological processes including energy metabolism, mitochondrial functions, and gene expression. Multiple studies have indicated that NAD(+) administration can profoundly decrease oxidative cell death as well as ischemic and traumatic brain injury, suggesting NAD(+) metabolism as a promising therapeutic target for cerebral ischemia and head injury. Cumulating evidence has suggested that NAD(+) can produce its protective effects by multiple mechanisms, including preventing mitochondrial alterations, enhancing energy metabolism, preventing virtually all forms of cell death including apoptosis, necrosis and autophagy, inhibiting inflammation, directly increasing antioxidation capacity of cells and tissues, and activating SIRT1. Increasing evidence has also suggested that NADH metabolism is a potential therapeutic target for treating several neurological disorders. A number of studies have further indicated that multiple NAD(+)-dependent enzymes such as sirtuins, polymerase(ADP-ribose) polymerases (PARPs) and CD38 mediate cell death and multiple biological processes. In this article, an overview of the recent findings regarding the roles of NAD(+)/NADH and NAD(+)-dependent enzymes in cell death and ischemic brain injury is provided. These findings have collectively indicated that NAD(+)/NADH and NAD(+)-dependent enzymes play fundamental roles in oxidative stress-induced cell death and ischemic brain injury, which may become promising therapeutic targets for brain ischemia and multiple other neurological disorders.

SIRT2 mediates NADH‐induced increases in Nrf2, GCL, and glutathione by modulating Akt phosphorylation in PC12 cells

SIRT2 plays important roles in multiple biological processes. It is unclear whether SIRT2 affects antioxidant capacity by modulating Nrf2, a key transcription factor for multiple antioxidant genes. By studying NADH‐treated differentiated PC12 cells, we found that NADH induced a significant increase in the nuclear Nrf2, which was prevented by both SIRT2 siRNA and SIRT2 inhibitor, AGK2. SIRT2 siRNA also blocked the NADH‐induced increases in glutamate cysteine ligase (GCL) and glutathione. Moreover, SIRT2 siRNA and AGK2 blocked NADH‐induced Akt phosphorylation, and inhibition of Akt phosphorylation prevented NADH‐induced increases in the nuclear Nrf2 and glutathione. Collectively, our study shows that SIRT2 regulates nuclear Nrf2 levels by modulating Akt phosphorylation, thus modulating the levels of GCL and total glutathione.

Malate-aspartate shuttle inhibitor aminooxyacetic acid leads to decreased intracellular ATP levels and altered cell cycle of C6 glioma cells by inhibiting glycolysis

NADH shuttles, including malate-aspartate shuttle (MAS) and glycerol-3-phosphate shuttle, can shuttle the reducing equivalents of cytosolic NADH into mitochondria. It is widely accepted that the major function of NADH shuttles is to increase mitochondrial energy production. Our study tested the hypothesis that the novel major function of NADH shuttles in cancer cells is to maintain glycolysis by decreasing cytosolic NADH/NAD(+) ratios. We found that AOAA, a widely used MAS inhibitor, led to decreased intracellular ATP levels, altered cell cycle and increased apoptosis and necrosis of C6 glioma cells, without affecting the survival of primary astrocyte cultures. AOAA also decreased the glycolytic rate and the levels of extracellular lactate and pyruvate, without affecting the mitochondrial membrane potential of C6 cells. Moreover, the toxic effects of AOAA were completely prevented by pyruvate treatment. Collectively, our study has suggested that AOAA may be used to selectively decrease glioma cell survival, and the major function of MAS in cancer cells may be profoundly different from its major function in normal cells: The major function of MAS in cancer cells is to maintain glycolysis, instead of increasing mitochondrial energy metabolism.

Poly(ADP-ribose) polymerase activation mediates synchrotron radiation X-ray-induced damage of rodent testes by exacerbating DNA damage and apoptotic changes

Abstract Purposes: Synchrotron radiation (SR) X-ray has great potential for cancer treatment and medical imaging. It is of significance to investigate the mechanisms underlying the effects of SR X-ray irradiation on biological tissues, and search for the strategies for preventing the damaging effects of SR X-ray irradiation on normal tissues. The major aim of our current study is to test our hypothesis that poly(ADP-ribose) polymerase (PARP) plays a significant role in SR X-ray-induced tissue damage. Methods and materials: The testes of rodents were pre-treated with PARP inhibitor 3-aminobenzamide (3-AB) or antioxidant N-acetyl-acetylcysteine (NAC), followed by SR X-ray irradiation. PARP activation, double-strand DNA breaks (DSB), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) signals, caspase-3 activity and weight of the testes were determined. Results: SR X-ray irradiation produced dose-dependent increases in poly(ADP-ribose) (PAR) formation – an index of PARP activation, which can be prevented by NAC administration. Administration of 10 or 20 mg/kg 3-AB attenuated a variety of tissue injury induced by SR X-ray, including caspase-3 activation, increases in TUNEL signals and loss of testical weight. The PARP inhibitor also significantly decreased SR X-ray-induced γ-H2AX signal – a marker of DSB. Conclusions: Our study has provided the first evidence suggesting that SR X-ray can induce PARP activation by generating oxidative stress, which leads to various tissue injuries at least partially by exacerbating DNA damage and apoptotic changes.

Malate-Aspartate Shuttle Inhibitor Aminooxyacetate Acid Induces Apoptosis and Impairs Energy Metabolism of Both Resting Microglia and LPS-Activated Microglia.

Abstract NADH shuttles mediate the transfer of the reducing equivalents of cytosolic NADH into mitochondria. Cumulating evidence has suggested that malate-aspartate shuttle (MAS), one of the two types of NADH shuttles, plays significant roles in such biological processes as glutamate synthesis in neurons. However, there has been no information regarding the roles of NADH shuttle in the survival and energy metabolism of microglia. In current study, using microglial BV2 cells as a cellular model, we determined the roles of MAS in the survival and energy metabolism of microglia by using aminooxyacetate acid (AOAA)—a widely used MAS inhibitor. Our study has suggested that AOAA can effectively inhibit the MAS activity of the cells. We also found that AOAA can induce both early- and late-stage apoptosis of resting microglia and lipopolysaccharides (LPS)-activated microglia. AOAA also induced mitochondrial depolarization, increases in the cytosolic Ca2+ concentrations, and decreases in the intracellular ATP levels. Moreover, our study has excluded the possibility that the major nonspecific effect of AOAA—inhibition of GABA transaminase—is involved in theses effects of AOAA. Collectively, our study has provided first information suggesting significant roles of MAS in the survival and energy metabolism in both resting microglia and LPS-activated microglia.

Therapeutic Potential of Intranasal Delivery of Drugs and Cells for Stroke and Other Neurological Diseases

Although numerous studies have suggested the pathological mechanisms underlying stroke-induced brain damage, most clinical trials on the drug treatment of ischemic stroke have been unsuccessful. One of the key obstacles for establishing effective therapies for stroke and other neurological diseases is the blockage of entrance of drugs and therapeutic cells into the brain by the blood-brain barriers (BBB). A number of studies have suggested that intranasal drug delivery is a promising approach for effectively delivering drugs into the brain by bypassing the BBB. There may be at least one intracellular transport-mediated route and two extracellular transport-mediated routes for the nose-to-brain delivery. Recent studies have further suggested that intranasal delivery may also deliver therapeutic cells into the brain more effectively and less invasively compared to traditional approaches. However, multiple key questions regarding intranasal drug and cell delivery for treating neurological disorders remain unanswered. Future studies on intranasal delivery in humans as well as the mechanisms underlying the intranasal delivery may suggest novel biological mechanisms and markedly enhance our capacity of treating stroke and other neurological diseases.