membrane-bound enzyme that transfers electrons from extracellular NADH to extracellular oxygen, making extracellular superoxide.50 NADH oxidase is the major source of superoxide in endothelial cell preparations.50 Amyloidb has NADH oxidase activity and forms extracellular oxygen radicals from extracellular NADH.58 Therefore, NADH generated in the blood of aging patients may enter into a vicious cycle with the generation of oxygen radicals that may damage the vasculature and neurons.

Nicotinamide (Nam) has significant pharmacological activity that is distinct from niacin.59 Nam is a powerful neuroprotective agent that has been in use in clinics to save the lives of thousands of patients with pellagra-induced dementia and brain damage since the 1930s.59 Nam is a precursor for NAD and an inhibitor of poly(ADP-ribose) polymerase (PARP). NAD is primarily involved in the repair of DNA damage in the nucleus since it is a substrate for PARP.59 Of course, NAD and NADH are involved in cellular energy metabolism, especially in mitochondria. The pharmacology of NMN is poorly described. Several compounds similar to NMN, such as cyclic ADP-ribose, ADP-ribose and NaADP (nicotinic acid ADP), are calcium-mobilizing agents.60 NMN can inhibit the enzymatic degradation of NAD by glycohydrolase.61

Visfatin is secreted by visceral adipocytes and macrophages.62 Therefore, damage to the blood-brain barrier by ceramide may attract monocytes/ macrophages that locally produce visfatin. Visfatin induces the formation of TNFa, IL-6 and other cytokines in monocytes and other cells.63 TNFa

stimulates the secretion of adhesion molecules that cause the chemotaxis of white blood cells, such as more monocytes.64–66 Il-6 stimulates the activation of monocytes.64–66 Visfatin then establishes a redox cycle that depends on

extracellular NADH and results in much more damage to the blood-brain barrier. It may be that ceramide and visfatin work together to damage the blood-brain barrier and attract macrophages.67

10.3Oxygen Radicals, Hydrogen Peroxide and Cell Death

Ceramide and visfatin appear to induce the formation of oxygen radicals including peroxynitrite and hydrogen peroxide. Peroxynitrite is charged and does not normally cross cell membranes. It is a powerful protein-nitrating agent that nitrates protein phosphatase type 2A.68 This nitration inhibits the normal

DNA repair enzymes.69 PARP uses NAD as an energy source and as a substrate to poly(ADP-ribosylate) itself and several nuclear enzymes. This poly(ADP-ribosylation) alters the activities of many enzymes.

Neuronal DNA is much more than just an archive of genetic information. It is very actively involved in producing proteins for neurotransmitter synthesis, release, reuptake and other neuronal functions. Damage to neuronal DNA very extensively a ects the ability of neurons to function normally, including maintaining neurotransmitters.70

PARP may ADP-ribosylate various transcription factors that regulate gene transcription.71,72 PARP is a component of positive cofactor 1 activity that regulates class II gene transcription.70 When DNA is damaged, PARP is activated, which inhibits class II gene transcription regulated by RNA polymerase II.73 PARP is also important in the action of p53, the tumor suppressor protein. Both PARP and p53 bind to DNA breaks. PARP can form complexes with p53 that may alter the activity of p53.74 Interestingly, p53 is involved in the inhibition of RNA polymerase III dependent gene transcription.75

It is important to recognize that the PARP referred to above is PARP-1. There are several enzymes with PARP activity.76 It is not known if the other enzymes, PARP-2, PARP-3, tankyrase and V-PARP, can fill in for PARP-1 when PARP inhibitors are used. Clearly, in PARP-1 knockout mice, the other PARP enzymes are still functional and can protect DNA and synthesize poly(ADP-ribose). It is also not known if PARP inhibitors are specific for PARP-1 or can inhibit all forms of PARP.

The energetic consequences of DNA damage and PARP activation are

enormous (Figure 10.2). PARP activation rapidly depletes NAD and ATP levels in the cell, leaving the cell depleted of energy sources.69,77 NADPH

depletion also occurs.77 Glutathione oxidizes. Glycolysis, the pentose phosphate pathway and mitochondrial energetics are a ected.

Recent research indicates that the secondary brain injury associated with stroke is induced by inflammatory processes. Nicotinamide can decrease the recruitment of neutrophils to potential sites of inflammation by inhibiting PARP in neutrophils and other cells.78 In fact, nicotinamide has been recommended for the treatment of arthritic patients since the 1940s. It was found in pilot trails that nicotinamide improved joint mobility and decreased the need for anti-inflammatory medication in arthritic cases.79 In the process of inflammation, the genes for intercellular adhesion molecule 1 and collagenase in neutrophils are activated. Neutrophils are recruited to sites of inflammation. Nitric oxide synthase is activated and oxygen radicals, hydrogen peroxide and nitric oxide are released. These reactive species can damage cellular DNA in the area of inflammation, resulting in apoptosis, necrosis and more serious inflammation.80 PARP has a number of functions in inflammation due to its ability to regulate gene expression.80 Inhibition by nicotinamide of PARP leads to decreased expression of these genes and decreases the extent and severity of inflammation. Nicotinamide also decreases the induction of iNOS, thereby decreasing damage to the blood-brain barrier.81

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Figure 10.2 The energetic consequences of DNA damage caused by oxidative stress. PARP is poly(ADP-ribose) polymerase. NAM is nicotinamide. NMN is nicotinamide mononucleotide. Na is niacin. Na-MN is niacin mononucleotide. Na-AD is niacin adenine dinucleotide. 1 is nicotinamide phosphoribosyl transferase, which requires ATP. 2 is NMN adenyl transferase, which requires ATP. PPP is the pentose phosphate pathway. G-6-P is glucose-6-phosphate. R-5-P is ribose-5-phosphate. G-3-P is 3-phosphoglycerate.

DNA damage leads to cell death. A large amount of DNA fragmentation causes rapid necrosis, with cell swelling and rupture, nuclear swelling and rupture and cytoplasmic vacuole formation. A smaller amount of DNA fragmentation causes apoptosis with cell shrinkage, nuclear condensation and fragmentation, large vacuole formation in the cytoplasm and cell fragmentation forming apoptotic bodies.82 Necrosis is a rapid process that does not require energy. Apoptosis is a delayed process that requires ATP. The center of a brain infarction is typically made up of necrotic cell debris.83 The limit area surrounding the core contains necrotic, apoptotic and normal cells.83