Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Elevated COUP-TFII expression in dopaminergic neurons accelerates the progression of Parkinson's disease through mitochondrial dysfunction.

Parkinson's disease (PD) is a neurodegenerative disorder featuring progressive loss of midbrain dopaminergic (DA) neurons that leads to motor symptoms. The etiology and pathogenesis of PD are not clear. We found that expression of COUP-TFII, an orphan nuclear receptor, in DA neurons is upregulated in PD patients through the analysis of public datasets. We show here that through epigenetic regulation, COUP-TFII contributes to oxidative stress, suggesting that COUP-TFII may play a role in PD pathogenesis. Elevated COUP-TFII expression specifically in DA neurons evokes DA neuronal loss in mice and accelerates the progression of phenotypes in a PD mouse model, MitoPark. Compared to control mice, those with elevated COUP-TFII expression displayed reduced cristae in mitochondria and enhanced cellular electron-dense vacuoles in the substantia nigra pars compacta. Mechanistically, we found that overexpression of COUP-TFII disturbs mitochondrial pathways, resulting in mitochondrial dysfunction. In particular, there is repressed expression of genes encoding cytosolic aldehyde dehydrogenases, which could enhance oxidative stress and interfere with mitochondrial function via 3,4-dihydroxyphenylacetaldehyde (DOPAL) buildup in DA neurons. Importantly, under-expression of COUP-TFII in DA neurons slowed the deterioration in motor functions of MitoPark mice. Taken together, our results suggest that COUP-TFII may be an important contributor to PD development and a potential therapeutic target.

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

In Parkinson's disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of H2 O2 and reduced glutathione levels. In addition, TH-HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4-dihydroxyphenylacetaldehyde and 5-S-cysteinyl-dopamine and were more susceptible than wild-type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.

Characterization of Catecholaldehyde Adducts with Carnosine and l-Cysteine Reveals Their Potential as Biomarkers of Catecholaminergic Stress.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of dopamine and norepinephrine to produce 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), respectively. Both of these aldehydes are potently cytotoxic and have been implicated in pathogenesis of neurodegenerative and cardiometabolic disorders. Previous work has demonstrated that both the catechol and aldehyde moieties of DOPAL are reactive and cytotoxic via their propensity to cause macromolecular cross-linking. With certain amines, DOPAL likely reacts via a Schiff base before oxidative activation of the catechol and rearrangement to a stable indole product. Our current work expands on this reactivity and includes the less-studied DOPEGAL. Although we confirmed that antioxidants mediated DOPAL's reactivity with carnosine and N-acetyl-l-lysine, antioxidants had no effect on reactivity with l-cysteine. Therefore, we propose a non-oxidative mechanism where, following Schiff base formation, the thiol of l-cysteine reacts to form a thiazolidine. Similarly, we demonstrate that DOPEGAL forms a putative thiazolidine conjugate with l-cysteine. We identified and characterized both l-cysteine conjugates via HPLC-MS and additionally identified a DOPEGAL adduct with carnosine, which is likely an Amadori product. Furthermore, we were able to demonstrate that these conjugates are produced in biological systems via MAO after treatment of the cell lysate with norepinephrine or dopamine along with the corresponding nucleophiles (i.e., l-cysteine and carnosine). As it has been established that metabolic and oxidative stress leads to increased MAO activity and accumulation of DOPAL and DOPEGAL, it is conceivable that conjugation of these aldehydes to carnosine or l-cysteine is a newly identified detoxification pathway. Furthermore, the ability to characterize these adducts via analytical techniques reveals their potential for use as biomarkers of dopamine or norepinephrine metabolic disruption.

Oxidative Transformations of 3,4-Dihydroxyphenylacetaldehyde Generate Potential Reactive Intermediates as Causative Agents for Its Neurotoxicity.

Neurogenerative diseases, such as Parkinson's disease, are associated, not only with the selective loss of dopamine (DA), but also with the accumulation of reactive catechol-aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is formed as the immediate oxidation product of cytoplasmic DA by monoamine oxidase. DOPAL is well known to exhibit toxic effects on neuronal cells. Both catecholic and aldehyde groups seem to be associated with the neurotoxicity of DOPAL. However, the exact cause of toxicity caused by this compound remains unknown. Since the reactivity of DOPAL could be attributed to its immediate oxidation product, DOPAL-quinone, we examined the potential reactions of this toxic metabolite. The oxidation of DOPAL by mushroom tyrosinase at pH 5.3 produced conventional DOPAL-quinone, but oxidation at pH 7.4 produced the tautomeric quinone-methide, which gave rise to 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxybenzaldehyde as products through a series of reactions. When the oxidation reaction was performed in the presence of ascorbic acid, two additional products were detected, which were tentatively identified as the cyclized products, 5,6-dihydroxybenzofuran and 3,5,6-trihydroxybenzofuran. Physiological concentrations of Cu(II) ions could also cause the oxidation of DOPAL to DOPAL-quinone. DOPAL-quinone exhibited reactivity towards the cysteine residues of serum albumin. DOPAL-oligomer, the oxidation product of DOPAL, exhibited pro-oxidant activity oxidizing GSH to GSSG and producing hydrogen peroxide. These results indicate that DOPAL-quinone generates several toxic compounds that could augment the neurotoxicity of DOPAL.

Structural Features and Toxicity of α-Synuclein Oligomers Grown in the Presence of DOPAC.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing α-Synuclein.

Lewy body diseases such as Parkinson's disease involve intraneuronal deposition of the protein α-synuclein (AS) and depletion of nigrostriatal dopamine (DA). Interactions of AS with DA oxidation products may link these neurohistopathologic and neurochemical abnormalities via two potential pathways: spontaneous oxidation of DA to dopamine-quinone and enzymatic oxidation of DA catalyzed by monoamine oxidase to form 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is then oxidized to DOPAL-Q. We compared these two pathways in terms of the ability of DA and DOPAL to modify AS. DOPAL was far more potent than DA both in oligomerizing and forming quinone-protein adducts with (quinonizing) AS. The DOPAL-induced protein modifications were enhanced similarly by pro-oxidation with Cu(II) or tyrosinase and inhibited similarly by antioxidation with N-acetylcysteine. Dopamine oxidation evoked by Cu(II) or tyrosinase did not quinonize AS. In cultured MO3.13 human oligodendrocytes DOPAL resulted in the formation of numerous intracellular quinoproteins that were visualized by near-infrared spectroscopy. We conclude that of the two routes by which oxidation of DA modifies AS and other proteins the route via DOPAL is more prominent. The results support developing experimental therapeutic strategies that might mitigate deleterious modifications of proteins such as AS in Lewy body diseases by targeting DOPAL formation and oxidation. SIGNIFICANCE STATEMENT: Interactions of the protein α-synuclein with products of dopamine oxidation in the neuronal cytoplasm may link two hallmark abnormalities of Parkinson disease: Lewy bodies (which contain abundant AS) and nigrostriatal DA depletion (which produces the characteristic movement disorder). Of the two potential routes by which DA oxidation may alter AS and other proteins, the route via the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde is more prominent; the results support experimental therapeutic strategies targeting DOPAL formation and DOPAL-induced protein modifications.

The catecholaldehyde hypothesis: where MAO fits in.

Monoamine oxidase (MAO) plays a central role in the metabolism of the neurotransmitters dopamine, norepinephrine, and serotonin. This brief review focuses on 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is the immediate product of MAO acting on cytoplasmic dopamine. DOPAL is toxic; however, normally DOPAL is converted via aldehyde dehydrogenase (ALDH) to 3,4-dihydroxyphenylacetic acid (DOPAC), which rapidly exits the neurons. In addition to vesicular uptake of dopamine via the vesicular monoamine transporter (VMAT), the two-enzyme sequence of MAO and ALDH keeps cytoplasmic dopamine levels low. Dopamine oxidizes readily to form toxic products that could threaten neuronal homeostasis. The catecholaldehyde hypothesis posits that diseases featuring catecholaminergic neurodegeneration result from harmful interactions between DOPAL and the protein alpha-synuclein, a major component of Lewy bodies in diseases such as Parkinson disease, dementia with Lewy bodies, and pure autonomic failure. DOPAL potently oligomerizes alpha-synuclein, and alpha-synuclein oligomers impede vesicular functions, shifting the fate of cytoplasmic dopamine toward MAO-catalyzed formation of DOPAL-a vicious cycle. When MAO deaminates dopamine to form DOPAL, hydrogen peroxide is generated; and DOPAL, hydrogen peroxide, and divalent metal cations react to form hydroxyl radicals, which peroxidate lipid membranes. Lipid peroxidation products in turn inhibit ALDH, causing DOPAL to accumulate-another vicious cycle. MAO inhibition decreases DOPAL formation but concurrently increases the spontaneous oxidation of dopamine, potentially trading off one form of toxicity for another. These considerations rationalize a neuroprotection strategy based on concurrent treatment with an MAO inhibitor and an anti-oxidant.

The "Sick-but-not-Dead" Phenomenon Applied to Catecholamine Deficiency in Neurodegenerative Diseases.

The catecholamines dopamine and norepinephrine are key central neurotransmitters that participate in many neurobehavioral processes and disease states. Norepinephrine is also the main neurotransmitter mediating regulation of the circulation by the sympathetic nervous system. Several neurodegenerative disorders feature catecholamine deficiency. The most common is Parkinson's disease (PD), in which putamen dopamine content is drastically reduced. PD also entails severely decreased myocardial norepinephrine content, a feature that characterizes two other Lewy body diseases-pure autonomic failure and dementia with Lewy bodies. It is widely presumed that tissue catecholamine depletion in these conditions results directly from loss of catecholaminergic neurons; however, as highlighted in this review, there are also important functional abnormalities in extant residual catecholaminergic neurons. We refer to this as the "sick-but-not-dead" phenomenon. The malfunctions include diminished dopamine biosynthesis via tyrosine hydroxylase (TH) and L-aromatic-amino-acid decarboxylase (LAAAD), inefficient vesicular sequestration of cytoplasmic catecholamines, and attenuated neuronal reuptake via cell membrane catecholamine transporters. A unifying explanation for catecholaminergic neurodegeneration is autotoxicity exerted by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in cytoplasmic dopamine metabolism. In PD, putamen DOPAL is built up with respect to dopamine, associated with a vesicular storage defect and decreased aldehyde dehydrogenase activity. Probably via spontaneous oxidation, DOPAL potently oligomerizes and forms quinone-protein adducts with ("quinonizes") α-synuclein (AS), a major constituent in Lewy bodies, and DOPAL-induced AS oligomers impede vesicular storage. DOPAL also quinonizes numerous intracellular proteins and inhibits enzymatic activities of TH and LAAAD. Treatments targeting DOPAL formation and oxidation therefore might rescue sick-but-not-dead catecholaminergic neurons in Lewy body diseases.

Formation of large oligomers of DOPAL-modified α-synuclein is modulated by the oxidation of methionine residues located at C-terminal domain.

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

Biochemical characterization of the catecholaldehyde reactivity of L-carnosine and its therapeutic potential in human myocardium.

Oxidative deamination of norepinephrine (NE) and dopamine (DA) by monoamine oxidase (MAO) generates the catecholaldehydes 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and 3,4-dihydroxyphenylacetaldehyde (DOPAL), respectively, and H2O2. Catecholaldehydes are highly reactive electrophiles that have been implicated as causal factors in the etiology of neurodegenerative diseases and cardiac injury from ischemia and diabetes. The reactivity of both catechol and aldehyde groups enables the catecholaldehdyes to cross-link proteins and other biological molecules. Carnosine is a ß-alanyl-histidine dipeptide found in millimolar concentrations in brain and myocardium. It is well known to detoxify aldehydes formed from oxidized lipids and sugars, yet the reactivity of carnosine with catecholaldehydes has never been reported. Here, we investigated the ability of carnosine to form conjugates with DOPAL and DOPEGAL. Both catecholaldehydes were highly reactive towards L-cysteine (L-Cys), as well as carnosine; however, glutathione (GSH) showed essentially no reactivity towards DOPAL. In contrast, GSH readily reacted with the lipid peroxidation product 4-hydroxy-2-nonenal (4HNE), while carnosine showed low reactivity to 4HNE by comparison. To determine whether carnosine mitigates catecholaldehyde toxicity, samples of atrial myocardium were collected from patients undergoing elective cardiac surgery. Using permeabilized myofibers prepared from this tissue, mitochondrial respiration analysis revealed a concentration-dependent decrease in ADP-stimulated respiration with DOPAL. Pre-incubation with carnosine, but not GSH or L-Cys, significantly reduced this effect (p < 0.05). Carnosine was also able to block formation of catecholaldehyde protein adducts in isolated human cardiac mitochondria treated with NE. These findings demonstrate the unique reactivity of carnosine towards catecholaldehydes and, therefore, suggest a novel and distinct biological role for histidine dipeptides in this detoxification reaction. The therapeutic potential of carnosine in diseases associated with catecholamine-related toxicity is worthy of further examination.

Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of α-Synuclein.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic and reactive product of dopamine catabolism. In the catecholaldehyde hypothesis for Parkinson's disease, it is a critical driver of the selective loss of dopaminergic neurons that characterizes the disease. DOPAL also cross-links α-synuclein, the main component of Lewy bodies, which are a pathological hallmark of the disease. We previously described the initial adduct formed in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine (DCPL). Here, we examine the chemical basis for DOPAL-based cross-linking. We find that autoxidation of DCPL's catechol rings spurs its decomposition, yielding an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings to give an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. This product is formed by an intermolecular carbon-carbon bond between the isoindole rings of the two DCILs that generates two structurally nonequivalent and separable atropisomers. Using α-synuclein, we demonstrate that the DOPAL-catalyzed formation of oligomers can be separated into two steps. The initial adduct formation occurs robustly within an hour, with DCPL as the main product, and the second step cross-links α-synuclein molecules. Exploiting this two-stage reaction, we use an isotopic labeling approach to show the predominant cross-linking mechanism is an interadduct reaction. Finally, we confirm that a mass consistent with a di-DCIL linkage can be observed in dimeric α-synuclein by mass spectrometry. Our work elucidates previously unknown pathways of catechol-based oxidative protein damage and will facilitate efforts to detect DOPAL-based cross-links in disease-state neurons.

Exploring the role of methionine residues on the oligomerization and neurotoxic properties of DOPAL-modified α-synuclein.

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.

3,4-Dihydroxyphenylacetaldehyde-Induced Protein Modifications and Their Mitigation by N-Acetylcysteine.

The catecholaldehyde hypothesis posits that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediary metabolite of dopamine, is an autotoxin that challenges neuronal homeostasis in catecholaminergic neurons. DOPAL toxicity may involve protein modifications, such as oligomerization of α-synuclein (AS). Potential interactions between DOPAL and other proteins related to catecholaminergic neurodegeneration, however, have not been systemically explored. This study examined DOPAL-induced protein-quinone adduct formation ("quinonization") and protein oligomerization, ubiquitination, and aggregation in cultured MO3.13 human oligodendrocytes and PC12 rat pheochromocytoma cells and in test tube experiments. Using near-infrared fluorescence spectroscopy, we detected spontaneous DOPAL oxidation to DOPAL-quinone, DOPAL-induced quinonization of intracellular proteins in both cell lines, and DOPAL-induced quinonization of several proteins related to catecholaminergic neurodegeneration, including AS, the type 2 vesicular monoamine transporter, glucocerebrosidase, ubiquitin, and l-aromatic-amino-acid decarboxylase (LAAAD). DOPAL also oligomerized AS, ubiquitin, and LAAAD; inactivated LAAAD (IC50 54 µM); evoked substantial intracellular protein ubiquitination; and aggregated intracellular AS. Remarkably, N-acetylcysteine, which decreases DOPAL-quinone formation, attenuated or prevented all of these protein modifications and functional changes. The results fit with the proposal that treatments based on decreasing the formation and oxidation of DOPAL may slow or prevent catecholaminergic neurodegeneration.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde.

BACKGROUND: The aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an endogenous neurotoxin implicated in Parkinson's Disease. Elucidating protein targets of DOPAL is essential in understanding it's pathology. The enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of DOPAL. METHODS: GAPDH activity was measured via reduction of NAD+ cofactor (340 nm). Protein aggregation was assessed with SDS-PAGE methods and specific modification via chemical probes. RESULTS: Low micromolar levels of DOPAL caused extensive GAPDH aggregation and irreversibly inhibited enzyme activity. The inactivation of GAPDH was dependent on both the catechol and aldehyde moieties of DOPAL. It is suggested that Cys are modified and oxidized by DOPAL. CONCLUSIONS: The mechanism by which DOPAL modifies GAPDH can serve as a mechanistic explanation to the pathological events in Parkinson's Disease.

Superoxide is the critical driver of DOPAL autoxidation, lysyl adduct formation, and crosslinking of α-synuclein.

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomerize α-synuclein, the main component of protein aggregates in Lewy bodies. In this work we examine the connection between reactive oxygen species and DOPAL autoxidation. We show that superoxide propagates a chain reaction oxidation, and that this reaction is dramatically inhibited by superoxide dismutase. Moreover, superoxide dismutase prevents DOPAL from forming dicatechol pyrrole adducts with lysine and from covalently crosslinking α-synuclein. Given that superoxide is a major radical byproduct of impaired cellular respiration, our results provide a possible mechanistic link between mitochondrial dysfunction and synuclein aggregation in dopaminergic neurons.

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL).

Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.

Comparison of Monoamine Oxidase Inhibitors in Decreasing Production of the Autotoxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells.

According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson's disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The "cheese effect"-paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson's disease.

Antioxidant-Mediated Modulation of Protein Reactivity for 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is an endogenously produced toxic aldehyde. It is a bifunctional electrophile implicated in the loss of dopaminergic cells concomitant with Parkinson's disease and neurodegeneration. DOPAL is known to react with proteins and amino acids such as N-acetyl lysine (NAL); oxidation of the catechol moiety to the quinone of DOPAL increases this reactivity. Here, we demonstrate the ability of the antioxidants N-acetylcysteine, glutathione, and ascorbic acid to mitigate the reactivity of DOPAL with proteins and amino acids in a dose-dependent fashion. Conversely, Trolox did not lessen the observed reactivity with proteins. Interestingly, use of tricine, a buffer and reducing agent, in these systems also decreased the reactivity of DOPAL with amines, yielding tricine-derived free radical species. Modification of amines with aldehydes typically involves Schiff base chemistry; however, the observance of free radicals suggests that an oxidative step is involved in the reaction of DOPAL with lysine. Furthermore, while Schiff base formation is usually optimal at pH 5, the reaction rate of DOPAL with NAL is negligible at pH 5 and is enhanced under basic conditions (e.g., pH 9). Conditions of high pH are also favorable for catechol auto-oxidation, known to occur for DOPAL. The antioxidant-mediated protection demonstrated here suggests that oxidative stress may impart cellular vulnerability to protein modification by DOPAL. Therefore, depleted antioxidants and increased levels of lipid peroxidation products, known to prevent the detoxifying metabolism of DOPAL, may present a survival challenge to dopaminergic cells targeted in Parkinson's disease.

3,4-Dihydroxyphenylethanol (Hydroxytyrosol) Mitigates the Increase in Spontaneous Oxidation of Dopamine During Monoamine Oxidase Inhibition in PC12 Cells.

The catecholaldehyde hypothesis predicts that monoamine oxidase (MAO) inhibition should slow the progression of Parkinson's disease, by decreasing production of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). Inhibiting MAO, however, diverts the fate of cytoplasmic dopamine toward potentially harmful spontaneous oxidation products, indicated by increased 5-S-cysteinyl-dopamine (Cys-DA) levels. 3,4-Dihydroxyphenylethanol (hydroxytyrosol) is an abundant anti-oxidant phenol in constituents of the Mediterranean diet. Whether hydroxytyrosol alters enzymatic or spontaneous oxidation of dopamine has been unknown. Rat pheochromocytoma PC12 cells were incubated with hydroxytyrosol (10 µM, 180 min) alone or with the MAO-A inhibitor clorgyline (1 nM) or the MAO-B inhibitors rasagiline or selegiline (0.5 µM). Hydroxytyrosol decreased levels of DOPAL by 30 % and Cys-DA by 49 % (p < 0.0001 each). Co-incubation with hydroxytyrosol prevented the increases in Cys-DA seen with all 3 MAO inhibitors. Hydroxytyrosol therefore inhibits both enzymatic and spontaneous oxidation of endogenous dopamine and mitigates the increase in spontaneous oxidation during MAO inhibition.