Patti Sullivan

Determinants of buildup of the toxic dopamine metabolite DOPAL in Parkinson's disease.

Intra‐neuronal metabolism of dopamine (DA) begins with production of 3,4‐dihydroxyphenylacetaldehyde (DOPAL), which is toxic. According to the ‘catecholaldehyde hypothesis,’ DOPAL destroys nigrostriatal DA terminals and contributes to the profound putamen DA deficiency that characterizes Parkinsons disease (PD). We tested the feasibility of using post‐mortem patterns of putamen tissue catechols to examine contributions of altered activities of the type 2 vesicular monoamine transporter (VMAT2) and aldehyde dehydrogenase (ALDH) to the increased DOPAL levels found in PD. Theoretically, the DA : DOPA concentration ratio indicates vesicular uptake, and the 3,4‐dihydroxyphenylacetic acid : DOPAL ratio indicates ALDH activity. We validated these indices in transgenic mice with very low vesicular uptake (VMAT2‐Lo) or with knockouts of the genes encoding ALDH1A1 and ALDH2 (ALDH1A1,2 KO), applied these indices in PD putamen, and estimated the percent decreases in vesicular uptake and ALDH activity in PD. VMAT2‐Lo mice had markedly decreased DA:DOPA (50 vs. 1377, p < 0.0001), and ALDH1A1,2 KO mice had decreased 3,4‐dihydroxyphenylacetic acid:DOPAL (1.0 vs. 11.2, p < 0.0001). In PD putamen, vesicular uptake was estimated to be decreased by 89% and ALDH activity by 70%. Elevated DOPAL levels in PD putamen reflect a combination of decreased vesicular uptake of cytosolic DA and decreased DOPAL detoxification by ALDH.

Vesicular uptake blockade generates the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde in PC12 cells: relevance to the pathogenesis of Parkinson's disease

Parkinsons disease entails profound loss of nigrostriatal dopaminergic terminals, decreased vesicular uptake of intraneuronal catecholamines, and relatively increased putamen tissue concentrations of the toxic dopamine metabolite, 3,4‐dihydroxyphenylacetaldehyde (DOPAL). The objective of this study was to test whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, as α‐synuclein oligomers may be pathogenetic in Parkinsons disease, oligomerizes α‐synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPAL—daidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by a method based on 6F‐ or 13C‐dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL‐induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L‐aromatic‐amino‐acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95–97% and rapidly increased cell DOPAL content (p = 0.0008). Daidzein+AL1576 augmented DOPAL responses to reserpine (p = 0.004). Intracellular DOPAL contributed to dopamine‐evoked apoptosis and DOPA‐evoked synuclein dimerization. The findings fit with the ‘catecholaldehyde hypothesis,’ according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes Parkinsons disease.

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.

Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease

Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinsons disease (PD). Mechanisms of relatively selective rotenone‐induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the ‘catecholaldehyde hypothesis,’ buildup of the autotoxic dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F‐labeled catechols were measured in PC12 cells incubated with rotenone (0–1000 nM, 180 min), without or with F‐dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F‐DOPAL, and 3,4‐dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD+ was supplied, whereas the direct‐acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone‐induced toxicity in dopaminergic neurons.

Benomyl, aldehyde dehydrogenase, DOPAL, and the catecholaldehyde hypothesis for the pathogenesis of Parkinson's disease.

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is detoxified mainly by aldehyde dehydrogenase (ALDH). We find that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resemble those noted previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related Parkinson’s disease (PD). Exposure to pesticides that inhibit ALDH may therefore increase PD risk via DOPAL buildup. This study lends support to the “catecholaldehyde hypothesis” that the autotoxic dopamine metabolite DOPAL plays a pathogenic role in PD.

Cardiac sympathetic denervation without Lewy bodies in a case of multiple system atrophy

Objective: To report a case of autopsy-proven multiple system atrophy without intra-neuronal alpha-synuclein deposits with profound cardiac sympathetic denervation. Background: Differentiating multiple system atrophy (MSA) from Parkinson disease with orthostatic hypotension (PD+OH) can be very difficult clinically. Neuroimaging studies have indicated that all patients with PD+OH have cardiac sympathetic denervation, whereas most MSA patients have intact innervation. In PD+OH immunoreactive alpha-synuclein (SYN-ir) is found in intra-neuronal cytoplasmic inclusion_Lewy bodies_whereas in MSA SYN-ir is in glial cytoplasmic inclusions (GCIs). A minority of MSA patients have both GCIs and cardiac sympathetic denervation, perhaps reflecting a “hybrid disease.” Methods: We report a case of autopsy-proven MSA and cardiac sympathetic denervation without Lewy bodies. Results: A Greek man had progressive, levodopa-unresponsive Parkinsonism and autonomic dysfunction without resting tremor or cerebellar signs. There were OH, baroreflex failure, low CSF norepinephrine and 3,4-dihydroxyphenylacetic acid (DOPAC), and decreased cardiac 18F-dopamine-derived radioactivity and putamen 18F-DOPA-derived radioactivity. At autopsy about one year later there was putamen atrophy, with SYN-ir deposits in glia but not in neurons or sympathetic ganglia. Apical myocardial norepinephrine and putamen dopamine were markedly decreased. Putamen dopamine:DOPA and DOPAC:3,4-dihydroxyphenylacetaldehyde (DOPAL) ratios were low and DOPAL:dopamine high. Conclusions: In MSA cardiac catecholamine depletion can occur even without Lewy bodies or alpha-synuclein deposition in the sympathetic nervous system. The pattern of catechol ratios fits with a pathogenetic shift from vesicular uptake to oxidative deamination of cytosolic catecholamines, coupled with decreased DOPAL detoxification by aldehyde dehydrogenase, as has been reported in sporadic PD. Parkinsonism, neurogenic OH, and profound catecholamine depletion in the putamen and heart can result from a common pathogenetic mechanism in catecholamine neurons, independently of neuronal alpha-synucleinopathy. Disclosure: Dr. Cook has nothing to disclose. Dr. Sullivan has nothing to disclose. Dr. Holmes has nothing to disclose. Dr. Goldstein has nothing to disclose.

Pure autonomic failure without synucleinopathy

Pure autonomic failure is a rare form of chronic autonomic failure manifesting with neurogenic orthostatic hypotension and evidence of sympathetic noradrenergic denervation unaccompanied by signs of central neurodegeneration. It has been proposed that pure autonomic failure is a Lewy body disease characterized by intra-neuronal deposition of the protein alpha-synuclein in Lewy bodies and neurites. A middle-aged man with previously diagnosed pure autonomic failure experienced a sudden, fatal cardiac arrest. He was autopsied, and tissues were harvested for neurochemical and immunofluorescence studies. Post-mortem microscopic neuropathology showed no Lewy bodies, Lewy neurites, or alpha-synuclein deposition by immunohistochemistry anywhere in the brain. The patient had markedly decreased immunofluorescent tyrosine hydroxylase in sympathetic ganglion tissue without detectable alpha-synuclein even in rare residual nests of tyrosine hydroxylase-containing ganglionic fibers. In pure autonomic failure, sympathetic noradrenergic denervation can occur without concurrent Lewy bodies or alpha-synuclein deposition in the brain or sympathetic ganglion tissue.

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.

Pleiotropic neuropathological and biochemical alterations associated with Myo5a mutation in a rat Model

In this study, we analyze the neuropathological and biochemical alterations involved in the pathogenesis of a neurodegenerative/movement disorder during different developmental stages in juvenile rats with a mutant Myosin5a (Myo5a). In mutant rats, a spontaneous autosomal recessive mutation characterized by the absence of Myo5a protein expression in the brain is associated with a syndrome of locomotor dysfunction, altered coat color, and neuroendocrine abnormalities. Myo5a encodes a myosin motor protein required for transport and proper distribution of subcellular organelles in somatodendritic processes in neurons. Here we report marked hyperphosphorylation of alpha-synuclein and tau, as well as region-specific buildup of the autotoxic dopamine metabolite, 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), related to decreased aldehyde dehydrogenases activity and neurodegeneration in mutant rats. Alpha-synuclein accumulation in mitochondria of dopaminergic neurons is associated with impaired enzymatic respiratory complex I and IV activity. The behavioral and biochemical lesions progress after 15 days postnatal, and by 30-40 days the animals must be euthanized because of neurological impairment. Based on the obtained results, we propose a pleiotropic pathogenesis that links the Myo5a gene mutation to deficient neuronal development and progressive neurodegeneration. This potential model of a neurodevelopmental disorder with neurodegeneration and motor deficits may provide further insight into molecular motors and their associated proteins responsible for altered neurogenesis and neuronal disease pathogenesis.

Is pure autonomic failure a distinct nosologic entity

To the Editor: We are responding to the editorial by Drs. Coon and Low about our case report, ‘‘Pure autonomic failure without synucleinopathy’’ [1]. Herbert Spencer’s concept of progress from an indefinite, incoherent homogeneity toward a definite, coherent heterogeneity indeed applies well to the issue of whether pure autonomic failure (PAF) is a single nosologic entity. Here we provide historical perspective and hopefully open up a conversation about the clinical and scientific meaning of PAF. Before the consensus definition published in 1996 [3], a major focus of research in autonomic medicine was on distinguishing PAF (previously called Bradbury-Eggleston syndrome, idiopathic orthostatic hypotension, or primary orthostatic hypotension) from multiple system atrophy (MSA, previously called the Shy-Drager syndrome, striatonigral degeneration, or olivopontocerebellar atrophy). The main question at the time was this: How does the pathophysiology of chronic autonomic failure without central neurodegeneration differ from that with central neurodegeneration? A report by Ziegler et al. in the New England Journal of Medicine in 1977 [6] showed that primary orthostatic hypotension involves low plasma levels of norepinephrine (NE), whereas the Shy-Drager syndrome does not. Our subsequent work expanded on this by noting that plasma levels of both NE and its main neuronal metabolite 3,4dihydroxyphenylglycol (DHPG) are low in PAF and normal in MSA. These findings indicated that peripheral sympathetic noradrenergic deficiency differentiates PAF from MSA. At the time, chronic autonomic failure from Parkinson’s disease (PD) was not part of the picture. The 1996 consensus definition mentions PD with autonomic failure, but without a discussion of clinical laboratory means to distinguish among PAF, PD with autonomic failure, and MSA. The next year saw the publication of three relevant articles. First was the report that a rare form of familial PD in which the disease is transmitted as an autosomal dominant trait results from mutation of the gene encoding the protein, alpha-synuclein [4]. The second described the discovery that Lewy bodies, a pathologic feature of sporadic PD, contain abundant deposits of alpha-synuclein [5]. Subsequently, PAF, PD with autonomic failure, and MSA all were reported to involve synucleinopathy (Lewy bodies in PAF and PD, glial cytoplasmic inclusions in MSA). A new scheme was advanced classifying the three conditions as autonomic synucleinopathies. Also in 1997, we reported evidence that PD involves sympathetic noradrenergic denervation resembling that in PAF [2]. The findings in our patient pose a question arising from this brief chronology: How does one classify a patient who according to the 1996 consensus criteria had PAF but who had no evidence of synucleinopathy? & David S. Goldstein goldsteind@ninds.nih.gov