3,4-Dihydroxyphenylacetaldehyde is the toxic dopamine metabolite in vivo: implications for Parkinson’s disease pathogenesis

Abstract

In Parkinson’s disease (PD), there is a highly selective loss of dopamine (DA) neurons in the substantia nigra (SN) greater than in the ventral tegmental area (VTA). The simplest explanation for selective DA neuron loss in PD is that DA is toxic and, because only DA neurons contain significant amounts of DA, this highly localized synthesis of DOPAL accounts for selective vulnerability of DA neurons. However, the large concentrations of DA required to produce in vivo toxicity cast doubt on its role in PD pathogenesis. Alpha-synuclein (α-syn) is the major component of the Lewy body, the pathological marker of PD, and is genetically linked to the disease. Recent studies indicate that α-syn neurotoxicity is mediated by a free radical generating metabolite of DA. Here we test the hypothesis that 3,4-dihydroxyphenylacetaldehyde (DOPAL), the monamine oxidase metabolite of DA, mediates DA toxicity in vivo. We injected DOPAL, DA and its oxidative, reduced and methylated metabolites into rat SN and VTA. Five days post-surgery, the injection sites were evaluated in Nissl preparations and with tyrosine hydroxylase (for DA neurons), neuronal nuclear antigen (for neurons) and glial fibrillary acidic protein (for astrocytes) immunoreactivities. Lesion size in SN vs. VTA was compared using morphometry. DOPAL at concentrations as low as 100 ng was toxic to DA SN neurons>DA VTA neurons>glia. Neither DA nor its other metabolites showed evidence of neurotoxicity at fivefold higher doses. However, 20 μg of DA produced lesions in the SN and VTA. We conclude that DOPAL is the toxic DA metabolite in vivo. Implications for a unified hypothesis for PD pathogenesis are discussed.

Introduction

Parkinson’s disease (PD), the second most common neurodegenerative disease [1], affects 1 million Americans with an incidence of 50,000/year [20]. PD is characterized by resting tremor, rigidity and bradykinesia associated with losses of more than 80% of dopamine (DA) neurons in the pars compacta of the substantia nigra (SN) [15], [16], [17]. Substantially fewer DA neurons are lost in the ventral tegmental area (VTA) in PD, and most other neuronal subtypes are unaffected [14], [31].

Because only DA neurons contain significant amounts of DA, the simplest explanation for selective DA neuron loss in PD is that DA itself is toxic. In fact, over 50 publications report that DA is neurotoxic in vitro and in vivo [8]. However, the very high concentrations required for DA toxicity in vitro (300 μM) [8] and in vivo (77 μg) [10] have led to skepticism about its toxicity in PD. Contrarily, a series of recent investigations by several laboratories [3], [19], [25], [40] now implicates DOPAL (3,4-dihydroxyphenylacetaldehyde), a DA metabolite as the critical endogenous toxin that triggers DA neuron loss in PD. Other major metabolites of DA include 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET). DOPAL, the product of monamine oxidase (MAO) action on DA, is oxidized by aldehyde dehydrogenase (ALDH) to DOPAC or reduced by aldehyde reductase (ALDR) to DOPET. DOPAC is methylated by catechol-O-methyltransferase (COMT) to homovanillic acid (HVA), the major brain metabolite of DA.

Mutations in the gene for α-synuclein (α-syn) are found in autosomal dominant early onset PD [29] providing strong genetic evidence for α-syn involvement in nigral DA neuron loss in PD. Contrariwise, α-syn is widely distributed in brain neurons and glia [28] and therefore by itself cannot explain the highly selective loss of DA neurons in PD [14], [31]. However, recent studies show that aggregation of α-syn is necessary for its toxicity [40]. Furthermore, Xu et al. [40] showed that aggregation of α-syn to a 53–83 kDa complex is toxic to cultured α-syn transfected DA neurons, but not non-DA neurons, which suggests that toxicity of α-syn aggregates depends on synthesis of DA. Inhibiting DA synthesis in these cultured DA neurons completely prevented α-syn induced neuron death. The α-syn transfected neurons also exhibited a marked increase in free radical generation, which, together with the accompanying cell death, was attenuated by antioxidants, leading to the conclusion that accumulation of α-syn aggregates renders endogenous levels of DA toxic by facilitating the production of a free radical generating DA metabolite [40].

In 1952 Blashko predicted that the aldehyde metabolites of amines would be toxic to cells in which they are formed [3]. Almost 50 years passed before it was shown that synthetic DOPAL [24], the monoamine oxidase-A (MAO-A) metabolite of DA, but not DA itself or its oxidative or methylated metabolites, is neurotoxic in an in vitro model of DA neurons [19] and generates a free [–OH] radical under physiological conditions [25]. The experiments described here were done to determine if DOPAL is toxic in vivo.