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Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado (S.A.M., V.V.); and Alcohol Research Center and Department of Pharmacology, University of Colorado Health Sciences Center at Fitzsimmons, School of Medicine, Aurora, Colorado (R.A.D.)
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Abstract |
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-hydroxybutyric aciduria, and pyridoxine-dependent seizures, most of which are characterized by neurological abnormalities. Several pharmaceutical agents and environmental toxins are also known to disrupt or inhibit aldehyde dehydrogenase function. It is, therefore, possible to speculate that reduced detoxification of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde from impaired or deficient aldehyde dehydrogenase function may be a contributing factor in the suggested neurotoxicity of these compounds. This article presents a comprehensive review of what is currently known of both the neurotoxicity and respective metabolism pathways of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde with an emphasis on the role that aldehyde dehydrogenase enzymes play in the detoxification of these two aldehydes. |
I. Introduction |
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; O'Brien et al., 2005). In the central nervous system (CNS1), the catecholamines, dopamine, norepinephrine, and epinephrine, are intraneuronally metabolized to their respective aldehyde metabolite by monoamine oxidase (MAO). Dopamine is deaminated to 3,4-dihydroxyphenylacetaldehyde (DOPAL), and both norepinephrine and epinephrine are deaminated to form 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL). Increasing evidence suggests that these catecholamine-derived aldehydes may in fact be neurotoxins, and their intraneuronal accumulation has been theorized as one mechanism that may be involved in cell death associated with neurodegenerative conditions, including Parkinson's disease (PD) and Alzheimer's disease (AD) (Mattammal et al., 1995; Burke et al., 2003). Aldehydes, including DOPAL and DOPEGAL, are detoxified by various enzyme systems including aldehyde dehydrogenase (ALDH), which is exclusively responsible for their oxidative metabolism (Fig. 1). This article presents a comprehensive review of the role of DOPAL and DOPEGAL in the CNS. First, a general overview of the biological significance and reactivity of aldehydic compounds is given, followed by an introduction to biogenic aldehydes. Second, the intraneuronal formation of DOPAL and DOPEGAL, their possible transport systems, and their identification and quantification in biological samples will be reviewed in detail. Third, the neurotoxicity of DOPAL and DOPEGAL will be examined (including possible mechanisms of toxicity), including their potential role in cell death and neurodegeneration. Finally, the metabolism of DOPAL and DOPEGAL by various enzyme systems will be comprehensively examined with an emphasis on the role of the human ALDH isozymes and the impact of ALDH dysfunction.
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II. Aldehydes |
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Aldehydes are organic compounds containing terminal carbonyl groups. They can be divided into four general carbonyl classes: 1) saturated alkanals, such as formaldehyde, acetaldehyde, and hexanal; 2) unsaturated alkenals, such as acrolein, 4-hydroxy-2-nonenal (4-HNE), and crotonaldehyde; 3) aromatic aldehydes, such as benzaldehyde, DOPAL and DOPEGAL; and 4) dicarbonyls such as glyoxal and malondialdehyde (MDA). These compounds are widespread in nature and ubiquitous in the environment. Various aldehydes, including formaldehyde, acetaldehyde, and acrolein, are produced during combustion and are present in smog and cigarette smoke (Rickert et al., 1980; Destaillats et al., 2002). Motor vehicle exhaust represents a major source of environmental aldehydes in air both through direct emission of aldehydes and through the emission of hydrocarbons, which can give rise to aldehydes. Cigarette smoke is also an important source of aldehydes; interestingly, second-hand smoke can contain significantly higher levels of aldehydes than first-hand smoke. Aldehydes are also used or generated in a wide variety of industrial applications (O'Brien et al., 2005). Formaldehyde is used in the production of resins, polyurethane, and polyester plastics and as a fumigant and a preservative in animal feed. Acetaldehyde also has many industrial uses including use in alkyd resin production. Drugs and environmental agents are also important aldehyde precursors. The hepatotoxins allyl alcohol and ethanol are directly metabolized to their corresponding aldehydes, acrolein, and acetaldehyde, respectively. Moreover, acrolein, ethanol, and other agents, such as carbon tetrachloride, also can induce the formation of lipid peroxidation-derived aldehydes (O'Brien et al., 2005). Many drugs, including the anticancer drugs cyclophosphamide and ifosfamide, are also metabolized to aldehyde intermediates (Maki and Sladek, 1993).
A range of aliphatic and aromatic dietary aldehydes, including citral, benzaldehyde, acetaldehyde, and formaldehyde, exist naturally in various foods, particularly in fruits and vegetables, to which they impart flavor and odor (Lindahl, 1992). Cooking fumes also contain a variety of aldehydes. Similarly, aldehydes including hexenal and cinnamaldehyde are approved by the U.S. Food and Drug Administration for use as flavoring additives and spices. In animals, aldehydes, including acrolein, benzaldehyde, and hexanal, act as communication molecules, having roles in attraction or defense (Schauenstein et al., 1977). Likewise, plant species produce aldehydes, including various hexenals, as part of a natural pesticide system against animals and insects. Interestingly, some insects have evolved to feed on these toxic plants and, therefore, can exploit plant-derived aldehydes for their own use in the chemical defense against predators (Williams et al., 2001).
Aldehydes are also generated as physiologically derived intermediates during the biotransformation of many endogenous compounds, including lipids, amino acids, neurotransmitters, and carbohydrates. For example, more than 200 aldehyde species arise from the oxidative degradation of cellular membrane lipids (lipid peroxidation), including 4-HNE and MDA (Esterbauer, 1993). Amino acid catabolism generates several aldehyde intermediates including glutamate -semialdehyde, produced during proline and arginine metabolism, and malonate semialdehyde, produced during valine catabolism (Vasiliou et al., 2004). Neurotransmitters, such as -aminobutyric acid (GABA), serotonin, norepinephrine, epinephrine, and dopamine, also give rise to aldehyde metabolites (Duncan and Sourkes, 1974; Gibson et al., 1998). Carbohydrate metabolism and ascorbate auto-oxidation generate glycolaldehyde and the dicarbonyl, glyoxal (O'Brien et al., 2005).
Aldehydes play vital roles in normal physiological and therapeutic processes. For example, the aldehyde retinal is essential for vision and its ALDH-dependent oxidation product, retinoic acid, is critical for embryonic development (Siegenthaler et al., 1990). Betaine, the ALDH-oxidation product of betaine aldehyde, is an osmolyte and methyl donor that has been shown to protect cells and organs from osmotic stress-induced toxicity (Horio et al., 2001). Other critical processes involving aldehydes include the biosynthesis of neurotransmitters. The inhibitory neurotransmitter GABA can be formed through the ALDH-dependent oxidation of its aldehyde precursor, -aminobutyraldehyde (Ambroziak and Pietruszko, 1987). In addition, the excitatory neurotransmitter, glutamate, is formed by the ALDH-induced oxidation of glutamate -semialdehyde. In terms of a therapeutic role, aldehyde intermediates can mediate the efficacy of certain drugs. The antineoplastic agent cyclophosphamide, through its aldehyde intermediate aldophosphamide, gives rise to phosphoramide mustard and acrolein, which are responsible for its tumor-cell killing effects (Sladek et al., 1989).
Although some aldehydes are essential for normal biological processes, many are cytotoxic and even carcinogenic (Yokoyama et al., 1996; Feng et al., 2004) (Fig. 1). Aldehydes are strong electrophilic compounds with terminal carbonyl groups, making them highly reactive. In fact, the aldehyde group is the most reactive among the functional groups of biomolecules. In addition to the electrophilic carbonyl carbon, 
and 
-unsaturated aldehydes, considered bifunctional aldehydes, such as 4-HNE and acrolein, contain a second electrophile at the -carbon. Furthermore, 4-hydroxylalkenel aldehydes, including 4-HNE, contain a hydroxyl group that can also participate in reactions. Unlike free radicals, aldehydes are relatively long-lived and, therefore, they not only react with targets in the same vicinity of their formation but also can diffuse or be transported to reach sites that are some distance away (Esterbauer et al., 1991).
Because of their electrophilic nature, aldehydes form adducts with various cellular nucleophiles, resulting in impaired cellular homeostasis, dramatically reduced enzyme activity, and even DNA damage (Sayre et al., 2001; Schaur, 2003). Aldehydes readily form adducts with glutathione (GSH) (Esterbauer et al., 1975), nucleic acids (Basu et al., 1988), and protein amino acids (Nadkarni and Sayre, 1995). Furthermore, the ability of aldehydes to cross-link proteins and DNA and to even form DNA-protein cross-links (through various adduction mechanisms) has been reported (Nair et al., 1986; Brooks and Theruvathu, 2005). Aldehyde-protein adducts typically involve sulfhydryl groups of cysteine residues and amino groups of lysine residues but can also involve other amino acid side chains including histidine and arginine (Sayre et al., 2001). Aldehydes adduct proteins by various mechanisms including Michael addition-type reactions and Schiff base-type condensation reactions. Aldehyde-nucleic acid adducts primarily involve amino groups of both purines and pyrimidines (Brooks and Theruvathu, 2005). Aldehydes have also been shown to be involved in the adduction of coenzymes, leading to their inactivation and depletion by mechanisms such as Knoevenagel condensation (Farrant et al., 2001). Accordingly, the adduction of aldehydes with various cellular components is believed to be the primary mechanism underlying their toxicity. Subsequent biological effects of aldehyde adduction can be cytotoxic, mutagenic, and even carcinogenic (Krokan et al., 1985; Esterbauer et al., 1991), involving the rapid depletion of GSH and protein thiols and the inactivation of enzymes (Chio and Tappel, 1969) with subsequent alterations in signal transduction pathways (Leonarduzzi et al., 2004), gene expression (Kumagai et al., 2000) and DNA repair (Feng et al., 2004).
Although many enzyme systems exist to detoxify aldehydes, perturbations in aldehyde metabolism do occur and contribute to a variety of disease states. Indeed, the accumulation of aldehydes from inborn errors of aldehyde metabolism has been associated with many pathological conditions (Vasiliou and Pappa, 2000). For example, the impaired metabolism of various endogenous aldehydes is causally associated with many diseases, including Sjögren-Larsson syndrome (Rizzo and Carney, 2005), type II hyperprolinemia (Valle et al., 1976), -hydroxybutyric aciduria (Pearl et al., 2003), pyridoxine-dependent seizures (Mills et al., 2006), and hyperammonemia and hypoprolinemia (Baumgartner et al., 2000). In addition, lipid-derived aldehydes, such as 4-HNE, acrolein, and MDA, have been implicated in alcohol-related diseases, including alcoholic liver disease, fibrosis, and atherosclerosis (Poli, 2000; Sun et al., 2001), and neurological diseases, such as PD and AD (Yoritaka et al., 1996; Lovell et al., 2001). Similarly, impaired metabolism of the ethanol metabolite acetaldehyde has been implicated in many alcohol-related diseases, including cirrhosis (Enomoto et al., 1991; Chao et al., 1994) and numerous head and neck cancers (Muto et al., 2000; Yokoyama et al., 2001), and late onset AD (Kamino et al., 2000).
Oxidative deamination of various biogenic amines (including indoleamines and catecholamines) results in the formation of "biogenic aldehydes." The indoleamines serotonin and tryptamine generate the biogenic aldehydes 5-hydroxyindole-3-acetaldehyde and indole-3-acetaldehyde, respectively. The catecholamines, dopamine, norepinephrine, and epinephrine, also give rise to biogenic aldehydes upon deamination (Fig. 2). Dopamine generates DOPAL, whereas both norepinephrine and epinephrine are deaminated to form DOPEGAL. Despite Blaschko's hypothesis in the early 1950s that indoleamine- and catecholamine-derived aldehydes may be toxic (Blaschko, 1952), biogenic aldehydes were originally believed to be innocuous intermediates in biogenic amine metabolism. However, early studies investigating their possible role in the pharmacological actions of ethanol (levels of biogenic aldehydes may increase during ethanol metabolism) and their ability to form isoquinoline-derived condensation products with their parent amine (Deitrich and Erwin, 1975; Tipton et al., 1977) led to the discovery that biogenic aldehydes actually have distinct physiological properties of their own. In fact, the indoleamine-derived aldehydes 5-hydroxyindole-3-acetaldehyde and indole-3-acetaldehyde have been shown to illicit many biological effects including neurotransmitter-like actions in the CNS (Sabelli et al., 1969; Palmer et al., 1986) and the inhibition of various enzymes (Tabakoff, 1974; Erwin et al., 1975).
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). Many important functions of the brain including memory, learning, movement, and behavior are thought to be mediated by catecholamines. Accordingly, the loss of specific populations of catecholaminergic neurons and subsequent deficits in related brain function are the basis of various neurodegenerative pathological conditions, including PD and AD. The specific vulnerability and death of these neurons have been hypothesized to involve toxic catecholamine metabolites or compounds that are selectively produced by, or accumulated in, catecholamine neurons such as DOPAL and DOPEGAL (Li et al., 2001; Burke et al., 2004). Indeed, there is an increasing body of evidence demonstrating the neurotoxic properties of the catecholamine-derived aldehydes DOPAL and DOPEGAL by various cytotoxic mechanisms including the generation of free radicals and initiation of apoptosis (Burke et al., 1998; Li et al., 2001). Along those lines, it has been suggested that DOPAL and DOPEGAL represent endogenous neurotoxins that may play a significant role in cell death associated with neurodegenerative diseases (Kristal et al., 2001; Eisenhofer et al., 2004).
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III. 3,4-Dihydroxyphenylacetaldehyde and 3,4-Dihydroxyphenylglycolaldehyde in the Central Nervous System |
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Oxidative deamination of catecholamines to form aldehyde metabolites was first described in the mid-1930s (Richter, 1937). Dopamine is deaminated to form DOPAL, whereas norepinephrine and epinephrine are both deaminated to form DOPEGAL (Fig. 2). A common misconception is that catecholamines are metabolized at sites distant from those of their synthesis and release (for a review of common fallacies about catecholamine metabolism, see Eisenhofer et al., 2004). Rather, most catecholamine metabolism has been shown to take place in the same cells in which they are produced and without prior release (Kopin, 1964; Maas et al., 1970). Accordingly, the formation of DOPAL and DOPEGAL is believed to occur primarily in the cytoplasm of the neurons that synthesize their parent catecholamines (Eisenhofer et al., 1992). This process is thought to occur primarily after the passive leakage of catecholamines into the cytoplasm from storage vesicles or, as a minor pathway, following their reuptake into the nerve terminal (Kopin, 1964; Eisenhofer et al., 1992). Monoamine transporters are responsible for sequestering catecholamines into storage vesicles within neurons, but it is estimated that 
10% of catecholamines escape into the neuronal cytoplasm and are subsequently metabolized (Eisenhofer et al., 1992). Indeed, 70 to 75% of norepinephrine turnover seems to occur from intraneuronal metabolism of norepinephrine leaking from storage vesicles, with the remainder made up by intraneuronal metabolism after reuptake, extraneuronal uptake and metabolism or loss of norepinephrine to the circulation (Eisenhofer et al., 1996b; Eisenhofer et al., 1998). Numerous in vitro and in vivo studies in animals and humans have demonstrated that leakage of catecholamines from storage vesicles is the primary pathway leading to catecholamine catabolism (Goldstein et al., 1988; Halbrugge et al., 1989; Tyce et al., 1995). The drug reserpine, which blocks the sequestration of catecholamines into storage vesicles, has been shown to cause depletion of catecholamine stores due to the rapid metabolism of unsequestered catecholamines (Kopin and Gordon, 1962). Studies with rat brain synaptic vesicles have shown dopamine turnover to be more rapid than that of norepinephrine (Floor et al., 1995), suggesting that leakage of dopamine from storage vesicles into the neuronal cytoplasm may be even more significant than it is for norepinephrine.
Intraneuronal formation of DOPAL and DOPEGAL is catalyzed by the actions of MAO (Fig. 2). Whereas other enzymes are involved in the degradation of catecholamines, MAO metabolism seems to be the principal intraneuronal pathway (Rivett et al., 1982). MAO is a flavin-containing, particle-bound enzyme localized primarily in the outer mitochondrial membrane (Schnaitman et al., 1967). In the brain, the enzyme is almost exclusively localized in nerve terminals (Westlund et al., 1985, 1993) where it catalyzes the oxidative deamination of dopamine, norepinephrine, and epinephrine to their respective aldehydes. The MAO reaction first involves the formation of an imine, which subsequently undergoes a nonenzymatic conversion to the corresponding aldehyde. MAO exists as two distinct genetic isoforms, MAO-A and MAO-B, both of which are catalytically active with catecholamines (Youdim et al., 2006). However, studies using preferential inhibitors of MAO-A and MAO-B have indicated that the production of DOPAL and DOPEGAL from dopamine, norepinephrine, and epinephrine can be attributed primarily to MAO-A (Waldmeier et al., 1976; Fowler and Benedetti, 1983; Fornai et al., 2000). The CNS distribution of the two MAO isozymes is consistent with this contention in that catecholaminergic neurons in the LC, RVLM, and SN primarily contain MAO-A whereas MAO-B is localized within serotonergic neurons in the dorsal raphe nucleus and superior central nucleus (Westlund et al., 1985).
After intraneuronal formation, DOPAL and DOPEGAL may leave nerve terminals by simple diffusion and reenter cells through various transport processes. DOPAL is taken up by rat neostriatal synaptosomes, and studies using mazindol, a selective dopamine uptake inhibitor, have suggested that the aldehyde reenters dopaminergic nerve terminals via the dopamine transporter (DAT) (Mattammal et al., 1995). The DAT is expressed in presynaptic terminals of SN neurons where it primarily mediates the reuptake of neuronally released dopamine (Reith et al., 1997; Jones et al., 1998). The DAT is also responsible for the uptake of 1-methyl-4-phenylpyridine (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an exogenous dopaminergic neurotoxin that induces parkinsonian symptoms in affected individuals and is used as a model of PD in animal studies (Javitch et al., 1985). DOPEGAL is taken up by catecholaminergic PC-12 cells and studies using desipramine, which blocks the neuronal catecholamine uptake transporter, have suggested that DOPEGAL is actively transported by this mechanism (Burke et al., 2001).
C. Identification and Quantification in Biological Samples
The presence and activity of biogenic aldehydes in biological tissues was first supported by the finding that tetrahydropapaveroline (THP), the tetrahydroisoquinoline alkaloid condensation product of a Pictet-Spengler condensation reaction between dopamine and DOPAL (Fig. 3), was present in the urine of L-3,4-dihydroxyphenylalanine (L-dopa)-treated PD patients (Sandler et al., 1973). Since then, other studies have confirmed the presence of both DOPAL and DOPEGAL in various tissues, including human brain. In normal human tissues, these aldehydes have been quantitated using chemically synthesized and purified standards (Burke et al., 1999a). In this study, DOPAL and DOPEGAL were separated from 12 catecholamines (and other metabolites), and their levels were analyzed in normal plasma, urine, and postmortem human brain regions. The concentration of DOPEGAL in normal postmortem LC was estimated to be 1.4 µM, a level 50% of that of 3-methoxy-4-hydroxyphenylglycol (MHPG), a major metabolite of norepinephrine and epinephrine (Burke et al., 1999a, 2004). Normal postmortem human brain SN levels of DOPAL were estimated to be 2.3 µM, a level 25% higher than that of homovanillic acid (HVA), a major dopamine metabolite. Nominal levels were seen in urine for both DOPAL and DOPEGAL. Plasma levels of DOPAL were minor, whereas those of DOPEGAL were similar to that of epinephrine. Other studies have examined the presence of DOPAL and DOPEGAL in animal tissues. DOPAL has been identified in vivo in rat striatum using trans-striatal microdialysis in freely moving rats (Colzi et al., 1996), and DOPEGAL has been detected in pulverized rat adrenal glands (Burke et al., 1995). These studies demonstrate physiological production of DOPAL and DOPEGAL at levels approaching or exceeding those observed for more well-established catecholamine metabolites.
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IV. Toxicity of 3,4-Dihydroxyphenylacetaldehyde and 3,4-Dihydroxyphenylglycolaldehyde in the Central Nervous System |
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). Since then, these aldehydes have been shown to be active compounds with physiological properties distinct from those of their parent amines (Palmer et al., 1986). The body of research reporting on investigations of the biogenic aldehydes DOPAL and DOPEGAL that began in the 1950s is not as large as one would expect considering the many molecular and genomic advances that have occurred in the ensuing period. This is due, in part, to the unstable and reactive properties of these catecholamine-derived compounds, making them difficult to synthesize (either chemically or enzymatically) in pure form and/or in large enough quantities to be useful for experimental purposes. Recent advances, however, in the chemical synthesis of DOPAL and DOPEGAL (Narayanan et al., 2003) and the growing number of studies indicating that they may be important neurotoxins will no doubt lead to increased interest in and focus on these compounds in future investigations. Nonetheless, a substantial and convincing body of relatively recent evidence does exist, which indicates the toxicity and reactivity of these compounds in the CNS. On the basis of these studies, a role for DOPAL and DOPEGAL in neurodegenerative diseases has been proposed (Mattammal et al., 1995; Lamensdorf et al., 2000b; Burke et al., 2004).
Concentrations of DOPAL and DOPEGAL >6 µM induce dose-dependent toxicity in various cell lines, including differentiated PC-12 cells (Mattammal et al., 1995; Burke et al., 1996; Kristal et al., 2001). Levels of the aldehydes identified as being toxic in vitro have been reported to be close to physiological levels found in normal human postmortem brain (Burke et al., 1999a; Kristal et al., 2001). For example, DOPAL at 6.6 µM, a concentration close to the physiological levels reported in normal human autopsy specimens of the SN (Burke et al., 1999a), induced significant cytotoxicity in PC-12 cells and reduced viability by 30% (Kristal et al., 2001). DOPAL at 66 µM caused cell death in 67% of neuronally differentiated PC-12 cells after 72 h, whereas cultures incubated with equivalent concentrations of dopamine, HVA, or THP were indistinguishable from controls (Kristal et al., 2001). These results are consistent with previous findings demonstrating that PC-12 cells are resistant to dopamine concentrations <1 mM (Cantuti-Castelvetri and Joseph, 1999). On the basis of these findings, it has been suggested that DOPAL is 100-fold more toxic to PC-12 cells than is dopamine (Kristal et al., 2001).
DOPAL-induced PC-12 cell damage elicits concentration-dependent lactic acid dehydrogenase (LDH) release, a measure of cytotoxicity. PC-12 cells incubated with 33 µM DOPAL for 8 h produced a 6-fold increase in LDH (Mattammal et al., 1995). In the same study, incubation for 24 h with 6.5 µM DOPAL induced both significant degeneration of the neuritic processes and a decrease in the number of viable cells. Incubation for 24 h with 33 µM DOPAL resulted in almost no cell survival. Short term (5 min) incubation of PC-12 cells with DOPAL (100 µM) also results in significant LDH release (Hashimoto and Yabe-Nishimura, 2002).
DOPAL is also toxic to neuroblastoma SK-N-SH and SH-SY5Y cells, fetal rat mesencephalic cultures and rat neostriatal synaptosomal preparations. In SK-N-SH cells, exposure to DOPAL (1-500 µM) for 24 h produced a concentration-dependent increase in LDH leakage into the cell medium with toxicity being pronounced at 100 µM (Lamensdorf et al., 2000a). In catecholaminergic SH-SY5Y cells, early increased levels of DOPAL induced by dopamine treatment and ALDH inhibition produced delayed cell toxicity and cell losses that increased with time (Legros et al., 2004a). Dopaminergic cultures, prepared from the ventral mesencephalon of rat embryos, exposed to 1 to 5 µM DOPAL for 24 h showed no toxicity as measured by the disappearance of tyrosine hydroxylase (TH) immunoreactivity (Mattammal et al., 1995). Between 7.5 and 20 µM DOPAL, a gradual reduction in dopamine uptake was seen without a reduction in the number of TH-immunoreactive cells. However, treatment of cultures with 33 µM DOPAL resulted in the disappearance of TH immunoreactivity, with the surviving TH-immunoreactive cells showing rounded cell bodies and highly fragmented fiber networks. These morphological changes were specific to dopaminergic neurons and were not evident in other CNS cells. In rat neostriatal synaptosomes, treatment with DOPAL for 30 min (10-100 µM), produced a concentration-dependent decrease in the number of living cells and a concomitant increase in the release of LDH (Mattammal et al., 1995).
DOPEGAL is also toxic in vitro. DOPEGAL concentrations of 5.9 and 59.5 µM decreased PC-12 cell viability (by 23 and 61%, respectively), with most of the cell loss occurring after 2 days of exposure (Burke et al., 1996). Epinephrine itself was also slightly toxic to these cells, reducing viability by 17%, but no other oxidative or methylated metabolite of epinephrine (aside from DOPEGAL) was toxic to PC-12 cells. The results of this study also underscored the importance of the terminal carbonyl group in the toxicity of DOPEGAL. Converting the terminal carbonyl moiety of DOPEGAL to a hydroxyl group to form its tautomer, 2',3,4-trihydroxyacetophenone, diminished toxicity significantly.
In vivo cytotoxicity of DOPAL has been reported in neurons and glia in the SN and VTA (Burke et al., 2003). DOPAL, dopamine, and oxidative, reduced, and methylated metabolites of dopamine were injected into rat SN and VTA. Five days after treatment, these regions were evaluated by Nissl preparation and cell-specific immunoreactivities. At doses of 100 ng, DOPAL was most toxic to SN neurons, followed by VTA neurons and, finally, glial cells. Neurons of the SN were consistently more affected, indicating the selective toxicity of DOPAL toward dopaminergic nerves. Neither dopamine nor its other metabolites elicited evidence of neurotoxicity, even when injected at 5-fold higher doses than that shown for DOPAL to cause toxicity. In contrast, rats treated systemically for 30 days with L-dopa, which is enzymatically converted to dopamine (by L-dopa decarboxylase), exhibited a 3-fold increase in the levels of DOPAL in the brain but showed no evidence of nigrostriatal dopaminergic cytotoxicity as measured by striatal dopamine content (Legros et al., 2004b).
In vivo toxicity of DOPEGAL has also been demonstrated—in rat RVLM (Burke et al., 2001). DOPEGAL was injected into adrenergic neurons in the RVLM and apoptosis of these adrenergic neurons (identified immunohistochemically by their content of the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase) was evaluated. Fifty nanograms of DOPEGAL caused apoptotic loss of epinephrine neurons after 18 h, as evaluated by in situ terminal deoxynucleotidyl-transferase mediated dUTP nick-end label staining. The degree of neurotoxicity was both dose- and time-dependent. Ten-fold higher doses of DOPEGAL produced necrosis of these neurons. Neither epinephrine nor MHPG was shown to be cytotoxic in this study.
Various mechanisms have been suggested to explain the observed cytotoxicity of DOPAL and DOPEGAL. These include protein adduction, isoquinoline formation, and free radical generation.
It is well known that aldehydes react with proteins to form various adducts that can disrupt protein function and cause cellular damage. It has been suggested that biogenic aldehydes react with proteins to form both unstable and stable adducts (Esterbauer et al., 1991). Evidence of this formation has been illustrated in reactions between DOPAL and the protein hemoglobin (Helander and Tottmar, 1989). DOPAL initially reacts with hemoglobin to form Schiff bases involving its aldehyde group and the free amino groups of lysine, tyrosine, or valine residues (Fig. 4). Schiff bases are inherently unstable and the binding that involves them can be reversed or further converted into more stable products by physiological reducing agents, such as glutathione or ascorbate (Tuma et al., 1984). Stable adducts formed between DOPAL and hemoglobin rendered the aldehyde unable to serve as a substrate for ALDH, whereas the DOPAL that was more loosely bound (i.e., Schiff base) could. Interestingly, at concentrations exceeding 5 µM, DOPAL actually inactivated ALDH by interacting with the surface of the protein through irreversible covalent modification in areas deemed important for enzyme activity (MacKerell and Pietruszko, 1987). Exhaustive dialysis and reducing agents did not reverse the inactivation. The substrate analog, chloral, protected ALDH against DOPAL-mediated inactivation. The authors suggested that DOPAL inactivates ALDH by formation of a covalent bond involving its catechol ring rather than its aldehyde group. A recent study supports this observation of substrate-mediated inhibition of ALDH by DOPAL, although the mechanism was not investigated (Florang et al., 2006).
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Carbonyl compounds can react with -arylethylamines, such as dopamine, in a rapid, nonenzymatic Pictet-Spengler condensation reaction to form isoquinoline alkaloid derivatives (Yamanaka et al., 1970; Sandler et al., 1973). For example, the reaction between acetaldehyde and dopamine results in the formation of salsolinol (Fig. 5) (Sandler et al., 1973), whereas that of DOPAL and dopamine generates THP (Davis and Walsh, 1970; Cohen, 1976) (Fig. 3). THP, salsolinol, and other isoquinoline derivatives are structurally related to the selective dopaminergic neurotoxin and PD-inducing agent MPTP/MPP+ and have been suggested to play a role in dopaminergic cell death characterized by PD (McNaught et al., 1998; Storch et al., 2002). Indeed, THP and salsolinol, have been demonstrated to be selective dopaminergic neurotoxins (Goto et al., 1997; Storch et al., 2002). Salsolinol and THP are found in high concentrations in the urine of PD patients on L-dopa treatment (Sandler et al., 1973) and salsolinol is found in the cerebrospinal fluid of untreated PD patients (Maruyama et al., 1996). The primary mechanism underlying MPTP/MPP+ dopaminergic toxicity (after MPP+ uptake by the DAT into dopamine neurons) is MPP+-induced inhibition of complex I of the mitochondrial respiratory chain, leading to depleted ATP, enhanced ROS production and ultimately dopaminergic cell death (Brooks et al., 1989; Cleeter et al., 1992). Similarly, THP is also a complex I inhibitor (Suzuki et al., 1990; McNaught et al., 1995). Inhibition of complex I also leads to the decreased availability of NAD+, the required cofactor in ALDH-mediated DOPAL metabolism. This could result in the accumulation of DOPAL and increased generation of THP. As mentioned, the dopaminergic specificity of MPTP/MPP+ relies on the uptake of MPP+ by the DAT, which transports this toxin selectively into dopaminergic cells. In vitro, THP has been shown to inhibit dopamine uptake through the DAT (Okada et al., 1998), indicating that it may also be a DAT substrate. THP also inhibits dopamine biosynthesis through mechanisms involving increased oxidative stress and the inhibition of TH activity (Kim et al., 2005). Indeed, many isoquinolines exhibit pharmacological actions and can act as false neurotransmitters or inhibitors of physiological mechanisms that regulate the actions of catecholamines (Cohen, 1976). For example, THP has been suggested to be responsible for some of the hypotensive and -adrenergic actions of L-dopa (Sandler et al., 1973). Accordingly, it is possible that some of the neurotoxic effects reported for DOPAL may be due, in part, to the generation of THP.
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Like aldehydes, free radicals and reactive oxygen species (ROS) are known to damage cellular components and interrupt physiological functions, which can lead to various disease states, including cell death and cancer. The reactions catalyzed by MAO to form DOPAL and DOPEGAL produce hydrogen peroxide (H2O2), which can generate other ROS and free radicals. Recently, DOPAL itself has been reported to generate free radicals, specifically the hydroxyl radical, in the presence of H2O2 (Li et al., 2001). This free hydroxyl radical production was not observed with DOPEGAL, dopamine, or other dopamine metabolites. The authors speculated that DOPAL may act as a cofactor in the generation of hydroxyl radicals. In support of this hypothesis, DOPAL did not produce the hydroxyl radical in the absence of H2O2 or in the presence of Fe2+ alone. However, H2O2 in the presence of either Fe2+ or DOPAL did result in hydroxyl radical formation. Thus, DOPAL may have a role similar to that of Fe2+ in the formation of hydroxyl radicals from H2O2 wherein DOPAL functions as a reducing agent and is oxidized in the process. In addition to its electrophilic carbonyl carbon, DOPAL contains two easily oxidizable phenolic groups. Auto-oxidation of DOPAL to the DOPAL-o-quinone, a process reported for dopamine (Hasegawa et al., 2006), may also produce free hydroxyl radicals in the presence of H2O2, similar to that reported for the dopamine derivative, 6-hydroxydopamine (Cohen and Heikkila, 1974). Furthermore, it was suggested that the ability of DOPAL to produce the hydroxyl radical may be due to its lower redox potential in relation to DOPEGAL or other dopamine metabolites (Cohen and Heikkila, 1974; Liu and Mori, 1993).
DOPEGAL has also been reported to generate a free radical in the presence of H2O2. In this case, the free radical seems to be a DOPEGAL radical that may involve the side-chain -hydroxyl group (Burke et al., 1998). According to electron paramagnetic resonance analyses, this radical elicited a signal different from that for the hydroxyl radical, leading the authors to suggest that DOPEGAL itself has a free radical form (Li et al., 2001). This observation was not seen with norepinephrine. It is intriguing that DOPAL and DOPEGAL have the capacity to generate free radicals, and they seem to do so by different pathways. Generation of ROS by DOPAL and DOPEGAL could have serious functional significance arising from GSH depletion and increased cellular oxidative stress. Further study of these processes is necessary to elucidate the exact mechanisms involved.
Apoptosis is a form of programmed cell death associated with a disruption in Ca2+ homeostasis (Orrenius and Nicotera, 1994) and the activation of caspase protease proteins (Scarlett and Murphy, 1997). Mitochondria are known to play a role in apoptotic neuron death (Wallace, 1999), and recent evidence has associated apoptosis with the induction of the mitochondrial membrane permeability transition (PT). Indeed, mitochondrial PT activation is believed to be a critical factor in the development of neurotoxicity and neurodegeneration (Bachurin et al., 2003), and it has been linked to the apoptotic type of catecholaminergic neuron death seen in PD and other neurodegenerative diseases (Lassmann et al., 1995; Anglade et al., 1997). Induction of the PT is characterized by matrix swelling, outer membrane rupture, release of apoptotic signaling molecules from the intermembrane space, and a collapse of the mitochondrial membrane potential due to increased permeability of the inner membrane (Bachurin et al., 2003; Fiskum et al., 2003). PT activation is thought to be caused by the opening of PT pores on the inner mitochondrial membrane. These pores control the transport of Ca2+ ions and small compounds (up to 1.5 kDa) in and out of the mitochondria and thereby function to maintain Ca2+ homeostasis in the cell. PT induction also seems to be involved in releasing the apoptotic initiation factors, cytochrome c and Apaf-1, which activate downstream caspases, leading to apoptosis (Kluck et al., 1997; Scarlett and Murphy, 1997). Various reactive species, such as free radicals, ROS, and aldehydes, are known to activate the mitochondrial PT (Kristal et al., 1996; Packer et al., 1997). DOPAL and DOPEGAL, both of which are generated by MAO on the outer mitochondrial membrane in close proximity to mitochondrial PT pores, have been reported to induce the Ca2+-mediated activation of the mitochondrial PT (Burke et al., 1998; Kristal et al., 2001). In isolated, energetically compromised liver mitochondria, very low DOPAL concentrations (
0.125 µM) increased the rate of PT induction at physiological Ca2+ concentrations, as measured by mitochondrial swelling (Kristal et al., 2001). In contrast, dopamine at high concentrations (up to 500 µM) had no effect on PT induction. DOPAL activation of mitochondrial PT was blocked by specific PT inhibitors, including cyclosporine A and trifluoperazine. These inhibitors also protected differentiated PC-12 cells from DOPAL-induced cytotoxicity, indicating that mitochondrial PT activation is one mechanism involved in DOPAL-induced cell death (Kristal et al., 2001). In contrast, actively respiring mitochondria were shown to be highly resistant to PT induction by DOPAL, suggesting that mitochondrial dysfunction may need to precede DOPAL-mediated toxicity.
DOPEGAL at low concentrations (6 µM) has also been reported to induce the mitochondrial PT in isolated liver mitochondria, whereas norepinephrine had no such effect (Burke et al., 1998). DOPEGAL may also disrupt Ca2+ homeostasis. Exposure of PC-12 cells to 0.5 µM DOPEGAL produced a 12-fold increase in cytosolic Ca2+ shortly after exposure (Burke et al., 2000). PC-12 cells cultured in the presence of very high concentrations of DOPEGAL (30 mM) showed increased activity of caspase 3, a key protease in the apoptotic process. In the same study, boc-aspartate-fluor-O-methyl-ketone, a caspase inhibitor, blocked DOPEGAL-induced (3 µM) cytotoxicity in cultured sympathetic neurons. DOPEGAL (20 µM) was also shown to trigger the release of Ca2+ from isolated mitochondria. Based on these findings, it was suggested that DOPEGAL may induce apoptotic cell death in neurons through the disruption of calcium homeostasis (Burke et al., 1997). Indeed, DOPAL and DOPEGAL may induce apoptotic cell death by activating the mitochondrial PT, resulting in the release of Ca2+ and the activation of caspases. Activation of the mitochondrial PT by DOPAL and DOPEGAL could be a result of the free radical species generated by these compounds, as has been reported for other aldehyde species (Ka et al., 2003) or, alternatively, by mechanisms involving their inherent reactivity as aldehydes, as was reported for 4-HNE (Kristal et al., 1996). Another mechanism of apoptosis that may be pertinent is complex I inhibition. THP, like the PD-inducing agent MPTP/MPP+(Cleeter et al., 1992), is a complex I inhibitor and has been shown to induce apoptosis in vitro in dopaminergic cell lines (Seaton et al., 1997). Further investigation of cell death mechanisms initiated by DOPAL and DOPEGAL is warranted.
F. Potential Role in Neurodegeneration
Given the increasing evidence demonstrating the neurotoxic properties of DOPAL and DOPEGAL, it is not surprising that their role as endogenous neurotoxins and involvement in selective neuron death associated with neurodegenerative diseases, such as PD and AD, have been proposed (Mattammal et al., 1995; Eisenhofer et al., 2000; Burke et al., 2004). PD is a progressive neurodegenerative disease characterized by bradykinesia, rigidity, resting tremor, and ataxia. Although all catecholaminergic neuron subtypes are lost in PD (Gai et al., 1993; Lang and Lozano, 1998), the major symptoms are associated primarily with the selective loss of dopaminergic neurons in the SN pars compacta (SNpc) region and norepinephrine neurons in the LC region. PD is also characterized by the formation of Lewy bodies in the SN, which are cytoplasmic inclusions made up of ubiquitin and -synuclein proteins (Piao et al., 2000). The mechanisms involved in these processes are not well established. Many hypotheses on the etiology of PD exist, including alterations in -synuclein (Zarranz et al., 2004), increased free radicals and oxidative stress (Przedborski and Ischiropoulos, 2005), DAT dysfunction (Storch and Schwarz, 2000), mitochondrial dysfunction (Cassarino and Bennett, 1999), and environmental toxins (Betarbet et al., 2000). More recently, it has been suggested that endogenous neurotoxins produced specifically by catecholaminergic neurons, such as DOPAL and DOPEGAL, may contribute to the selective vulnerability of these cells to degeneration in the development of PD (Kristal et al., 2001; Burke et al., 2004).
In PD, SNpc neurons are more vulnerable to neurodegeneration than VTA and hypothalamic arcuate dopaminergic neurons (Storch and Schwarz, 2000). Likewise, DOPAL has been shown to be significantly more toxic to dopaminergic SN neurons than dopaminergic VTA neurons and glia in vivo (Burke et al., 2003; Burke et al., 2006). Furthermore, these studies demonstrate that DOPAL is significantly more toxic to these neurons than dopamine and other metabolites. Stereotactic injections of DOPAL, dopamine, and other metabolites into the SN of Sprague-Dawley rats demonstrated that only DOPAL, at concentrations within the physiological range of 2 to 3 µM, causes neurodegeneration. Physiological concentrations of dopamine and other metabolites did not cause cytotoxicity. However, dopamine at 200-fold greater doses than DOPAL did produce lesions in the SN and VTA.
As mentioned, the selective dopaminergic toxicity of MPTP is due to DAT-mediated uptake of MPP+ and, similarly, dopaminergic neurodegeneration in PD has been hypothesized to involve the cellular accumulation of dopamine-like molecules into dopamine neurons by the presynaptic DAT (Lee et al., 2001). DOPAL has been implicated as a substrate for the DAT and could accumulate in dopamine neurons by this mechanism (Mattammal et al., 1995). Moreover, the DAT is expressed in higher concentrations in SNpc neurons than in other dopaminergic neurons, and this mechanism may explain the increased sensitivity of SNpc neurons to DOPAL (Storch and Schwarz, 2000). As mentioned, THP, may also serve as a DAT substrate. The preferential uptake of DOPAL and/or THP into dopaminergic neurons by the DAT may enhance dopaminergic neurotoxicity.
Compared with other neuronal subpopulations, SNpc neurons may have increased cytosolic Ca2+ concentrations and be more susceptible to free radical damage (Hirsch et al., 1997). As mentioned, both DOPAL and DOPEGAL have been suggested to initiate apoptosis through Ca2+-mediated processes (Burke et al., 1998; Kristal et al., 2001) and to generated free radical species (Burke et al., 1998; Li et al., 2001). The specific vulnerability of SNpc neurons coupled with these mechanisms could also lead to enhanced cytotoxicity.
Excess dopamine release from dopaminergic neurons is another mechanism thought to be involved in PD neurodegeneration. Indeed, the PD-inducing agent MPTP/MPP+ is a potent dopamine-releasing agent (Obata, 2002). DOPAL is also reported to increase dopamine release in PC-12 cells and rat striatal synaptosomes. In PC-12 cells, DOPAL (10 µM) caused a >2-fold increase in the release of dopamine (Hashimoto and Yabe-Nishimura, 2002). This effect was shown to be Ca2+-independent. Other reactive aldehydes tested (i.e., 4-HNE) did not initiate dopamine release, suggesting that the effect was specific to DOPAL. In rat striatal synaptosomes, 33 µM DOPAL caused a significant increase in dopamine release (>55%) (Mattammal et al., 1995). This effect was specific to dopamine-containing terminals as opposed to those containing GABA. Excess DOPAL-induced dopamine release may be one mechanism underlying the neurotoxicity of DOPAL and may be a factor in dopaminergic neurodegeneration characterized by PD.
Central to the hypothesis that endogenous neurotoxins, such as DOPAL and DOPEGAL, are involved in neurodegenerative diseases is the requirement that they accumulate to levels that become toxic to catecholamine neurons. Many mechanisms could lead to increased intraneuronal concentrations of these aldehydes including, as mentioned, the preferential uptake of DOPAL by the DAT. In addition, the increased synthesis of DOPAL and DOPEGAL could influence their levels. Accordingly, age-related increases in MAO have been reported (Oreland and Gottfries, 1986), which could lead to increased formation of these aldehydes. Furthermore, pharmacological treatment with the dopamine precursor, L-dopa, used to treat PD, has been shown to elevate rat brain DOPAL levels by as much as 18-fold (Fornai et al., 2000; Legros et al., 2004b).
Impaired metabolism of DOPAL may also affect its accumulation. Indeed, in catecholaminergic neuroblastoma SH-SY5Y cells, significant accumulation of DOPAL was achieved by treatment with dopamine (1 mM) along with the inhibition of DOPAL metabolism by the ALDH inhibitor disulfiram (10 µM) (Bonnet et al., 2004; Legros et al., 2004a). Under these conditions, increased cytotoxicity was also demonstrated. Similarly, mitochondrial dysfunction of complex I and the resulting decreased availability of NAD+, the required cofactor for ALDH-mediated oxidation of aldehydes, may lead to increased levels of both DOPAL and DOPEGAL. Indeed, complex I inhibition by rotenone leads to significant DOPAL accumulation in PC-12 cells (Lamensdorf et al., 2000a). Under these conditions, the further inhibition of DOPAL metabolism by specific inhibitors of ALDH and aldose reductase (AR) increased DOPAL levels even more (12-fold that of control) (Lamensdorf et al., 2000b). DOPAL-mediated substrate inhibition of ALDH (MacKerell and Pietruszko, 1987; Florang et al., 2006) and age-related ALDH deficiencies (Chen and Yu, 1996) may also play a role in DOPAL accumulation in the CNS.
Mitochondrial dysfunctions, including complex I inhibition, are associated with neurodegenerative diseases (Robinson, 1998; Olanow and Tatton, 1999). Complex I inhibitors, including rotenone, MPP+, isoquinoline, and THP, induce apoptosis in dopaminergic cell lines (Seaton et al., 1997). A mitochondrial complex I deficit has been identified in the SN of PD patients (Schapira et al., 1990) and hypothesized to result from genetic mutations and/or environmental toxins (Bachurin et al., 2003; Fiskum et al., 2003). Chronic complex I inhibition by rotenone is used to create a model of PD in rats that involves a selective loss of SN and LC neurons, as well as Lewy bodies in the SN (Betarbet et al., 2000). Accumulation of DOPAL along with preexisting mitochondrial dysfunctions may act synergistically, leading to enhanced neurotoxicity. Indeed, at minimally toxic concentrations, rotenone significantly increased DOPAL-induced cytotoxicity and death in nerve growth factor-differentiated PC-12 cells (Kristal et al., 2001). Likewise, accumulation of DOPAL by both complex I inhibition (rotenone) and ALDH/AR inhibition potentiated rotenone-induced toxicity in PC-12 cells (Lamensdorf et al., 2000b). Both the accumulation of DOPAL and the enhancement of rotenone-induced toxicity were abrogated by inhibiting the formation of DOPAL with the MAO inhibitor, clorgyline. These observations suggest that the MAO-catalyzed formation of DOPAL and its accumulation by various mechanisms may be important processes that aggravate the neurotoxicity associated with mitochondrial dysfunction. Accordingly, there is substantial documentation of a neuroprotective effect of irreversible MAO inhibitors in vitro and in animal models of PD (Tabakman et al., 2004). Furthermore, the MAO inhibitors selegiline and the newly available, rasagiline, are both currently approved by the U.S Food and Drug Administration to treat PD and, used alone or as an adjunct to L-dopa therapy, seem to be promising treatments for patients with both early and advanced PD (Henchcliffe et al., 2005). Central to the activity of MAO inhibitors is their capacity to boost dopamine levels by blocking dopamine metabolism. It is, therefore, conceivable that the concomitant blockade of the production of DOPAL and DOPEGAL may also contribute to their therapeutic effects. In support of this contention, it has been postulated that increased dopamine metabolism, occurring after an initial loss of dopamine neurons, plays a role in the progression of nigrostriatal degeneration in PD (Graham, 1978; Cohen et al., 1997).
Oxidative stress is believed to be a critical factor in neurodegenerative diseases (Sayre et al., 2001). SNpc neurons have been reported to be particularly sensitive to oxidative stress and may have increased levels of H2O2 (Hirsch et al., 1997). H2O2 is generated during the formation of DOPAL and DOPEGAL and has been shown to enhance formation of DOPAL- and DOPEGAL-mediated free radicals (Burke et al., 1998; Li et al., 2001). These processes could exacerbate oxidative stress in the SNpc and be a factor in neurodegeneration of these neurons in disease states. Similarly, the formation of Lewy bodies in PD has been suggested to involve increased oxidative stress. Aggregation of -synuclein is enhanced in the presence of free hydroxyl radicals (Hashimoto et al., 1999). Free hydroxyl radicals generated by DOPAL have also been hypothesized to be involved in the oxidative modification of -synuclein and the formation of Lewy bodies in dopaminergic SN neurons (Li et al., 2001). Physiological concentrations of DOPAL (1.5-3 µM) cause aggregation of -synuclein in catecholaminergic SH-SY5Y and dopaminergic MN9D cells (Burke et al., 2006). Moreover, it has been postulated that -synuclein may actually contribute to the neurotoxicity of DOPAL (Burke et al., 2004) in a mechanism similarly described for dopamine-induced neurotoxicity. Accordingly, -synuclein has been shown to bind to the DAT, enhancing both dopamine uptake and dopamine-induced apoptosis (Lee et al., 2001) and these processes may affect DOPAL neurotoxicity. -Synuclein also catalyzes the formation of H2O2 (Turnbull et al., 2001), which could contribute to the production of DOPAL-generated free hydroxyl radicals and subsequent aggregation of -synuclein into Lewy bodies (Li et al., 2001). Recently, it has been demonstrated that -synuclein and oxidized catechol metabolites may work synergistically to potentiate each other's toxic effects (Hasegawa et al., 2006). -Synuclein was shown to exacerbate apoptotic cell death induced by o-quinone metabolites of dopamine and L-dopa, and it was suggested that oxidized catechol metabolites may form adducts with -synuclein, leading to enhanced aggregation. Furthermore, it was suggested that the mitochondrial membrane may represent an initial target for these compounds in dopaminergic neurodegeneration. As mentioned, DOPAL may also be oxidized to its o-quinone form, and this process may facilitate the formation of free radicals and the activation of the mitochondrial PT. Therefore, it is possible that DOPAL-o-quinone may participate in reactions with -synuclein similar to those described for oxidized catechol metabolites.
AD is primarily a late-onset, progressive, age-dependent neurodegenerative disorder characterized clinically by the impairment of cognitive functions and changes in behavior and personality (Robert et al., 2005). The disease is associated with the presence of intracellular neurofibrillary tangles and extracellular amyloid plaques and the apoptotic degeneration of neuronal subpopulations, specifically norepinephrine neurons of the LC and epinephrine neurons of
