A brief outline of strong oxidant production is given below. For a more detailed review see [10, 16] where all the appropriate references are also given. The major cellular sources of free radicals under normal physiological conditions are the mitochondria where ~3% of oxygen is incompletely reduced to the superoxide anion. The CNS which comprises about 2% of the body’s mass consumes, when the body is at rest, about 20% of all the oxygen. Hence, there is a large production of superoxide anion by CNS tissue; furthermore, superoxide production by brain increases as one ages [26]. Although relatively innocuous, superoxide can give rise to strong oxidants such as singlet oxygen and interact with other relatively innocuous compounds such as nitric oxide radical or hydrogen peroxide to give rise to the hydroxyl radical, nitrogen dioxide as outlined in Figure 1.

Fig. 1: Strong Oxidants Produced From Superoxide Anion.

These strong oxidants can oxidize proteins, especially thiol proteins, ribonucleic acids as well as polyunsaturated fatty acids. Hence, an important free radical scavenging action is that mediated by superoxide dismutase (SOD), where superoxide is converted to hydrogen peroxide and molecular oxygen as indicated in the following reaction indicated by Figure 2.

Fig. 2: Superoxide Dismutase Action.

There are three SOD isoforms: i) an extracellular Cu,Zn-SOD, a cytosolic Cu,Zn-SOD and a mitochondrial Mn-SOD [9]. The hydrogen peroxide produced is not innocuous since it can be converted to the strong oxidant, the hydroxyl radical, by transition metal ions as indicated in Figure 3.

Fig. 3: Transition Metal Ions And Production Of Strong Oxidants.

Note that superoxide and hydrogen peroxide are involved in the redox cycling of iron and copper, respectively. The hydroxyl radical is a powerful oxidant and can extract an electron from another molecule or can hydroxylate another molecule. Two major means by which the hydroxyl radical can cause cell damage is by causing changes in DNA leading to mutations [3] and by the initiation of a chain of peroxidations of polyunsaturated fatty acids [10]. One should keep in mind that transition metal cations tend to be localized to anionic structures such as the phosphate groups of phospholipids and DNA, a consequence of which is that the generation of hydroxyl radicals tends to be in regions where they can cause the most damage. Figure 4 illustrates how lipid peroxidation chains can be initiated and how they can be prevented. LH represents a polyunsaturated fatty acid, LOOH a lipid hydroperoxide, LOO a lipid peroxyl radical, LO a lipid alkoxyl radical, TOH vitamin E and AscH ascorbic acid.

Fig. 4: Production and Scavenging of Lipid Peroxides.

Note in equations ‘a’ to ‘c’ the initiation of a chain reaction of lipid peroxidations that can only be stopped by either two lipid radicals interacting or by the inactivation of a lipid radical by vitamin E. Inactivation of a lipid peroxyl radical results in the formation of a lipid peroxide and a vitamin E radical. The lipid peroxides can interact with either iron-II or iron-III and give rise to alkoxyl and peroxyl radicals, respectively, each of which can initiate new chains of lipid peroxidation. Peroxidized plasmalemmal, endoplasmic reticular, mitochondrial membrane lipids greatly interfere with cell function. It is vital for the cell to scavenge the peroxides produced. It does this principally using the selenoenzyme glutathione peroxidase (GPx) which in turn uses reduced-glutathione (GSH) as the electron donor as indicated in equation ‘g’ [7]. There are several members of the GPx family: i) a plasma form whose function is unclear since there is very little GSH in plasma [29], ii) a cytosolic and mitochondrial form (GPx1) [4, 7], iii) a membrane-associated phospholipid hydroperoxide glutathione peroxidase (GPx4) [32], and a gastrointestinal glutathione peroxidase (GPx-GI) [5]. Of significance is that GPx activities in brain decreases with age [34]. Oxidized-glutathione (GSSG) is reduced by glutathione reductase using NADPH as the source of electrons. GPx can scavenge both hydrogen peroxide as well as organic peroxides and has a high affinity for such peroxides. Catalase, on the other hand, can scavenge only hydrogen peroxide and has a low affinity for hydrogen peroxide. The efficiency by which GPx can scavenge peroxides increases with increasing GSH concentration [6, 25, 32] (note Figure 5). In other words, relatively small increases in GSH concentration has a marked effect on the ability of GPx to scavenge peroxides. Indeed, increasing intracellular GSH has been demonstrated to increase the ability of cells to scavenge strong oxidants [30, 31], note Figure 5. Conversely decreasing intracellular GSH results in greater damage following oxidative stress [11, 13, 19, 22]. GSH is also important in the regeneration of ascorbate which has been used to reduce the vitamin E radical back to vitamin E. Hence, GSH plays a very central role in the ability of cells to manage oxidative stress [14-16].

Fig. 5: Oxidative Stress Is Inversely Related to Intracellular GSH.

In Figure 5, taken from [30], the amount of strong oxidant (such as the hydroxyl radical) being formed is measured by the oxidation of the non-fluorescent DCFH to DCF under a perturbation that causes increased hydrogen peroxide formation. In this experiment hydrogen peroxide was caused to form by exposing oligodendrocyte precursors to blue light (480 nm at 140 mW/m2): this causes excitation of compounds such as riboflavin which in turn donates an electron to molecular oxygen resulting in formation of superoxide that in turn is dismutated to hydrogen peroxide. Intracellular GSH was manipulated in these cells from the ~5 nmoles/mg protein normally found in these cells to ~22 nmoles/mg protein, the level that is normally found in astrocytes. Note that a relatively modest increase in GSH from 5 nmoles/mg protein to 7 nmoles/mg protein can halve the oxidative stress experienced by these cells.

GSH is synthesized according to the following two reactions in Figure 6.

Fig. 6: GSH Synthesis.

Glutamyl-cysteine synthase (GCS: reaction ‘i’) is the rate-limiting enzyme in regulating GSH levels [21]. It is a heterodimer comprised of a 73 kD heavy subunit and a 27.7 kD light subunit. The activity of glutamyl-cysteine synthase, the key enzyme in GSH synthesis drops with age [12]. Another important enzyme regulating the intracellular levels of GSH is glutathione reductase, which uses NADPH as the electron donor to reduce oxidized-glutathione to reduced-glutathione. Cerebral GSH levels has been demonstrated to decrease while GSSG levels rise with age [12]. GSH is not only important for the scavenging of peroxides but also for the reduction of oxidized-ascorbic acid which is necessary for the regeneration of vitamin E. GSH also plays an important role in determining how readily proinflammatory genes can be activated since GSH acts as the intracellular redox buffer. High GSH:GSSG ratios tend to inhibit the activation of the redox-sensitive transcription factor NF-kappaB [8]. NF-kappaB is the key transcription factor involved in activating transcription of pro-inflammatory genes such as cyclo-oxygenase-2, inducible nitric oxide synthase, intercellular adhesion molecules and proinflammatory cytokines [1].

Most neurodegenerative diseases become more common as we age. There are a number of known reasons for this, including increased inefficiencies in mitochondrial function leading to increased superoxide production, decreased abilities to produce GSH and to reduce oxidized-glutathione to GSH, decreased activities of GPx, etc. It is my thesis that raising GSH in neural and endothelial cells will inhibit the formation of strong oxidants and thus delay the onset of a variety of neurodegenerative diseases. Increases in tissue GSH can be accomplished either by administering compounds that increase intracellular levels of the rate-limiting amino acid, cysteine, for GSH synthesis or by promoting the activity of the rate-limiting enzyme, glutamyl-cysteine synthase. Administration of the cysteine pro-drug, N-acetylcysteine has been demonstrated to result in better survival of motoneurons in a mouse model of motoneuron disease (see paper by Henderson and Roder in this Symposium). One of the research directions in which my laboratory is interested is raising intracellular GSH by promoting the activity of glutamyl-cysteine synthase.

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