NAD+ and NADH play roles in many important biological processes such as energy metabolism, mitochondrial function, calcium homeostasis, and gene expression. They have emerged as one of the most influential couples in life. Poly (ADP-ribose) polymerase 1 (PARP1) activation leads to NAD+ depletion, which mediates cell death. Intranasal administration of NAD+ can decrease ischemic brain injury by reducing PARP1-induced cell death and is a potential treatment for diseases involving PARP1.

1. NAD + / NADH 在神经细胞死亡中的作用 殷卫海教授 上海交通大学 Med-X 研究院 上海交通大学医学院附属瑞金医院神经病学研究所

2. NAD + / NADH OLD COUPLE POWERFUL COUPLE

3. I. Based on the above discussion, it appears that the classical paradigm regarding the biological functions of NAD and NADP is too narrow to generalize the growing functions of these molecules. It is tempting to propose that a novel paradigm about the biological functions of NAD and NADP may be emerging. From: Ying W. (2008) Antioxidants & Redox Signaling Two of my major new thoughts about NAD

4. II. NAD, together with ATP and Ca 2+ , may be the most fundamental components in life which mediate nearly all of the key biological processes. The close interactions among these components may constitute a ‘ Central Regulatory Network ’ in life. From: Ying W. (2008) Antioxidants & Redox Signaling

6. NADPH NADP + NAD + NADH NAADP Antioxidation Oxidative Stress Reductive biosynthesis Calcium homeostasis Mitochondrial function Energy metabolism Oxidative stress Calcium homeostasis Gene expression Mitochondrial function Energy metabolism Calcium homeostasis Gene expression Cell death Aging Dehydrogenases PARPs Sirtuins ARCs ARTs NADK Dehydrogenases/Oxidases GRx NADPH oxidase G6PDH 6GPDH IDP MEP TDH de novo pathway Salvage pathway L-Trp NMN/NaMN ARCs ETC Oxidases From: Ying W. (2007) Antioxidants & Redox Signaling

7. 1. Roles of NAD+ and NADH in cellular functions 1.1. NAD+ and NADH in energy metabolism (a) Glycolysis (GAPDH); (b) pyruvate / lactate conversion; (c) TCA cycle; (d) electron transport chain; and (e) energy metabolism affected by NAD-dependent SIR2 / PARPs.

9. 1.3. NAD+ and NADH in calcium homeostasis From: Ying W. (2008) Antioxidants & Redox Signaling

10. Ying W. (2007) Antioxidants & Redox Signaling

11. 1.5. NAD + and NADH in aging From: Ying W. (2007) Antioxidants & Redox Signaling

12. Summary NAD + and NADH have emerged as one of the most influential couples in nearly all of the major biological processes in life, including calcium homeostasis, mitochondrial functions, energy metabolism, gene expression, immunological functions, aging and cell death.

14. NAD + Dehydrogenases PARP Poly(ADP-ribosyl)ated proteins + Nam ARTs cADPR + Nam NAD + kinase NADP + sirtuins Deacylated proteins + Nam + O-acetyl-ADP-ribose (ADP-ribosyl)ated proteins + Nam ADP-ribosyl cyclases Salvage pathway Nam / NA de novo pathway NaMN L-Trp L-Kyn Qa Energy metabolism / Mitochondrial functions NADH DNA repair Cell death Gene expression Genomic stability Gene silencing Aging Cell death Calcium homeostasis Antioxidation Calcium homeostasis Signal transduction Immunological regulation Ying W. (2006)

19. From: Weihai Ying. (2006) Frontiers in Bioscience 11:3129-3148.

20. Ischemia/Reperfusion Oxidative stress MNNG DNA Damage PARP-1 Activation PARG PAR-Protein Protein ADP-Ribose NAD+ Depletion Glycolysis MPT Mitochondrial Depolarization CyC/AIF Release ATP Cell Death From: Weihai Ying. (2006) Frontiers in Bioscience 11:3129-3148.

21. PARP-1 mediates MNNG- and chemical OGD-induced Neuronal and astrocyte death

22. MNNG induced increased PAR in the nucleus of neurons

23. PARP-1 activation causes not only ATP depletion, but also depletion of the total pool of (ATP + ADP + AMP)

24. PARP-1 produces NAD + depletion in cells

26. Ying W. et al. (2003) BBRC 308:809-813. NAD + treatment can restore the intracellular NAD + levels in astrocytes treated with the PARP activator MNNG

29. AIF Nuclei Overlay Con MNNG MNNG + NAD + NAD + treatment blocked MNNG-induced AIF translocation

31. * We have further found that NAD + treatment can abolish MNNG-induced mitochondrial permeability transition and mitochondrial depolarization (Alano, Ying and Swanson JBC (2004).

35. Can NAD + be used in vivo to decrease brain injury in cerebral ischemia and other PARP-1 related diseases? We used a rat model of transient focal ischemia to test our hypothesis that NAD + administration can decrease ischemic brain damage. 3. Therapeutic potential of NAD +

36. A key problem for treatment of CNS diseases: William M. Pardridge. (2005) The Blood-Brain Barrier: Bottleneck in Brain Drug Development. NeuroRx. 2: 3–14. A key challenge in establishing effective strategies for neuroprotection: Searching for drug delivery approaches that can overcome the limitations of BBB.

37. The Nose May Help the Brain --- Intranasal Drug Delivery for Treating Neurological Diseases Ying W. (Editorial) Future Neurology

38. Intranasal administration, but not intravenous administration, with the PARG inhibitor gallotannin, decreased ischemic brain injury

39. NAD + treatment can increase intracellular NAD + in a brain slice model

40. Ischemia Ischemia + 10 mg / kg NAD + Intranasal administration with 10 mg / kg NAD + at 2 hrs after ischemic onset can profoundly decreased infarct formation. This treatment did not affect multiple major physiological parameters including temperature, blood pressure, pH etc.

42. Intranasal NAD + administration significantly decreased neurological deficits in rats subject to ischemia-reperfusion

43. What are the mechanisms underlying the protective effects of intranasal NAD + administration against ischemic brain injury?

45. Ischemia Ischemia + GT AIF Nucleus Merge Ischemia-reperfusion induced AIF translocation in rat brains

46. Can NAD + be used for treating other PARP-1-associated diseases ? Our latest study: Intranasal NAD + delivery could decrease traumatic brain injury. TBI TBI + NAD +

48. 4. NADH transport across plasma membranes of cells

50. NADH treatment can increase intracellular NADH levels in astrocytes NADH treatment can increase intracellular NAD + levels in astrocytes

52. P2X 7 R  -Actin

53. Transfection of HEK293 cells with P2X7 receptors led to increased NADH transport

55. 5. Roles of Ca 2+ -Mg 2+ -depenent endonuclease in cell death

56. PARG inhibition may decrease genotoxic agent-induced cell death by multiple mechanisms

57. Post-treatment of the astrocytes with the CME inhibitor ATA abolished MNNG-induced chromatin condensation.

58. ATA post-treatment abolished MNNG-induced DNA fragmentation ATA post-treatment, but not ATA pre-treatment, decreased MNNG-induced cell necrosis

60. Both astrocytes and neurons express CME

62. From: Ying W. (2007) Antioxidants & Redox Signaling

63. From: Ying W. (2007) Antioxidants & Redox Signaling