Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinson's disease (PD). However, previous work demonstrated 3,4-dihydroxyphenylacetaldehyde (DOPAL) to be more toxic than DA. DOPAL is generated as a part of DA catabolism via the activity of monoamine oxidase and the mechanism of DOPAL toxicity is proposed to involve protein modification. Previous studies have demonstrated protein reactivity via the aldehyde moiety; however, DOPAL contains two reactive functional groups (catechol and aldehyde) both with the potential for protein adduction. The goal of this work was to determine whether protein modification by DOPAL occurs via a thiol-reactive quinone generated from oxidation of the catechol, which is known to occur for DA, or if the aldehyde forms adducts with amine nucleophiles. To accomplish this objective, the reactivity of DOPAL towards N-acetyl-lysine (NAL), N-acetyl-cysteine (NAC) and two model proteins was determined. In addition, several DOPAL analogues were obtained and used for comparison of reactivity. Results demonstrate that at pH 7.4 and 37°C, the order of DOPAL reactivity is NAL ≫ NAC and the product of NAL and DOPAL is stable in the absence of reducing agent. Moreover, DOPAL will react with model proteins, but in the presence of amine-selective modifiers citraconic anhydride and 2-iminothiolane hydrochloride, the reactivity of DOPAL towards the proteins is diminished. In addition, DOPAL-mediated protein cross-linking is observed when a model protein or a protein mixture (i.e. mitochondria lysate) are treated with DOPAL at concentrations of 5-100 μM. Protein cross-linking was diminished in the presence of ascorbate, suggesting the involvement of a quinone in DOPAL-mediated protein modification. These data indicate DOPAL to be highly reactive towards protein nucleophiles with the potential for protein cross-linking.

Recent work indicates that oxidative stress is a factor in Parkinson's disease (PD); however, it is unknown how this condition causes selective dopaminergic cell death. The neurotransmitter dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration. DA undergoes catabolism by monoamine oxidase (MAO) to the reactive intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further oxidized to 3,4-dihydroxyphenylacetic (DOPAC) acid via mitochondrial aldehyde dehydrogenase (ALDH). Previous studies found DOPAL to be more toxic than DA, and the major lipid peroxidation products, that is, 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), potently inhibit ALDH. The hypothesis of this work is that lipid peroxidation products inhibit DOPAL oxidation, yielding aberrant levels of the reactive aldehyde intermediate. Treatment of striatal synaptosomes with 2-100 microM 4HNE or 2-50 microM MDA impaired DOPAL oxidation, resulting in elevated [DOPAL]. The aberrant concentration of DOPAL yielded an increase in protein modification by the DA-derived aldehyde, evident via staining of proteins with nitroblue tetrazolium (NBT). Pretreatment of synaptosomes with an MAO inhibitor significantly decreased NBT staining. On the basis of NBT staining, the order of protein reactivity for DA and metabolites was found to be DOPAL>>DOPAC>DA. Mass spectrometric analysis of a model peptide reacted with DOPAL revealed the adduct to be a Schiff base product. In summary, these data demonstrate the sensitivity of DA catabolism to the lipid peroxidation products 4HNE and MDA even at low, physiologic levels and suggest a mechanistic link between oxidative stress and generation of aberrant levels of an endogenous and protein reactive dopaminergic toxin relevant to PD.

Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinson’s disease (PD). In addition, oxidative stress and lipid peroxidation are hypothesized culprits in PD pathogenesis. DA undergoes catabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH). As a minor and compensatory metabolic pathway, DOPAL can be reduced to 3,4-dihydroxyphenylethanol (DOPET) via cytosolic aldehyde or aldose reductase (AR). Previous studies have found DOPAL to be significantly more toxic to DA cells than DA and that the major lipid peroxidation products, i.e. 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), potently inhibit DOPAL oxidation via ALDH. The hypothesis of this work is that lipid peroxidation products inhibit DOPAL oxidation, yielding aberrant levels of the toxic aldehyde intermediate. To test this hypothesis, nerve-growth factor differentiated PC6-3 cells were used as a model for DA neurons. Cell viability in the presence of 4HNE and MDA (2-100 μM) was measured by MTT assay and it was found that only 100 μM 4HNE exhibited significant cytotoxicity. Treatment of cells with varying concentrations of 4HNE and MDA resulted in reduced DOPAC production and significant elevation of DOPAL levels, suggesting inhibition of ALDH. In cells treated with 4HNE that exhibited elevated DOPAL, there was a significant increase in DOPET. However, elevated DOPET was not observed for the cells treated with MDA, suggesting MDA to be an inhibitor of AR. Using isolated cytosolic AR, it was found that MDA but not 4HNE inhibited reductase activity toward DOPAL, surprisingly. These data demonstrate that the oxidative stress products 4HNE and MDA inhibit the aldehyde biotransformation step of DA catabolism yielding elevated levels of the endogenous neurotoxin DOPAL, which may link oxidative stress to selective neurodegeneration as seen in PD.

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective loss of dopaminergic neurons, leading to a decrease of the neurotransmitter dopamine (DA). DA is metabolized by monoamine oxidase to 3,4-dihydroxyphenyacetaldehyde (DOPAL). While the mechanism of pathogenesis of PD is unknown, DOPAL has demonstrated the ability to covalently modify proteins and cause cell death at concentrations elevated from physiologic levels. Currently, the identities of protein targets of the aldehyde are unknown, but previous studies have demonstrated the ability of catechols and other DA-catabolism products to interact with and inhibit tyrosine hydroxylase (TH). Given that DOPAL is structurally related to DA and is a highly reactive electrophile, it was hypothesized to modify and inhibit TH. The data presented in this study positively identified TH as a protein target of DOPAL modification and inhibition. Furthermore, western blot analysis demonstrated a concentration-dependent decrease in antibody recognition of TH. DOPAL in cell lysate significantly inhibited TH activity as measured by decreased L-DOPA production. Inhibition of TH was semi-reversible, with the recovery of activity being time and concentration-dependent upon removal of DOPAL. These data indicate DOPAL to be a reactive DA-metabolite with the capability of modifying and inhibiting an enzyme important to DA synthesis.

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