highlights three recent advances from our group in the area of mechanisms of catecholaminergic neurodegeneration. First, we completed and published data from many experiments on protein modifications produced by the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL). Second, we developed and applied novel immunofluorescence confocal microscopic methods to quanitfy deposition of the protein alpha-synuclein (AS) within sympathetic noradrenergic neurons. Our results suggest that such deposition is a common pathophysiologic feature of Lewy body forms of neurogenic orthostatic hypotension (nOH). Third, we obtained neuroimaging data confirming that a vesicular storage defect in sympathetic noradrenergic nerves contributes to myocardial norepinephrine depletion. (1) DOPAL modifies multiple proteins related to catecholaminergic neurodegeneration: The catecholaldehyde hypothesis posits that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate of intraneuronal dopamine metabolism, is an autotoxin that challenges neuronal homeostasis in catecholaminergic neurons. DOPAL induces many protein modifications, such as oligomerization of alpha-synuclein (AS) We examined DOPAL-induced protein-quinone adduct formation (quinonization), ubiquitination, and aggregation in cultured cells and in test tube experiments. Using near infrared fluorescence spectroscopy we detected spontaneous oxidation of DOPAL to DOPAL-quinone and obtained the first evidence that DOPAL quinonizes proteins related to catecholaminergic neurodegeneration, including AS, the type 2 vesicular monoamine transporter, glucocerebrosidase, ubiquitin, and L-aromatic-amino-acid decarboxylase (LAAAD, required for dopamine synthesis). We noted that DOPAL oligomerizes ubiquitin and LAAAD, inactivates LAAAD, evokes substantial intracellular protein ubiquitination, and aggregates intracellular AS. N-Acetylcysteine (NAC) attenuated or prevented all these protein modifications. We are extending from wells to cells by testing whether DOPAL quinonizes intracellular proteins as visualized by near-infrared fluorescence microscopy. The results from this project have inspired a new disease modification strategy based on decreasing the formation and oxidation of DOPAL, with the goal of slowing or preventing catecholaminergic neurodegeneration (Project Z1A NS003125). (2) Alpha-synuclein deposition within sympathetic noradrenergic neurons is a common feature of Lewy body diseases: Lewy body diseases are associated with sympathetic noradrenergic deficiency and AS deposition in sympathetic ganglion neurons (Goldstein et al., Clin Auton Res 2017;27:57-62; Isonaka et al., Clin Auton Res 2017;27:97-101; Isonaka et al., Clin Auton Res 2018;28:223-230). The results are consistent with a pathogenic role of AS within sympathetic noradrenergic nerves; however, post-mortem studies in isolation cannot test a pathogenetic hypothesis rigorously. Therefore, we have validated and are now applying a novel method to quantify colocalization of AS with tyrosine hydroxylase (TH, a marker of catecholaminergic neurons) within sympathetic noradrenergically innervated constituents in skin biopsies from patients with Lewy body nOH. Data about cardiac sympathetic noradrenergic innervation are being obtained by 18F-dopamine positron emission tomography in the same patients. Preliminarily, the results show that all patients with Lewy body nOH have increased AS/TH colocalization and decreased myocardial 18F-dopamine-derived radioactivity, supporting a pathogenic role of intracellular AS in catecholaminergic neurodegeneration. (3) Sick but not dead: Further evidence for functional abnormalities in sympathetic noradrenergic neurons in Lewy body nOH: It has been almost axiomatic that in Lewy body forms of nOH noradrenergic deficiency directly and solely reflects loss of sympathetic noradrenergic nerves. In an article in the Journal of Clinical Investigation in 2011 we reported the first evidence for functional abnormalities within extant cardiac sympathetic neurons in Lewy body diseases. Additional in vivo and post-mortem data have supported the notion that a substantial proportion of catecholaminergic neurons in these diseases are sick but not dead (Goldstein & Sharabi, Brain Res 2018 (in press)). In this reporting period we obtained further data that fit with this concept. We conducted positron emission tomographic scanning after administration of 18F-dopamine and on a separate day 11C-methylreboxetine. Both are radioligands for the cell membrane norepinephrine transporter, but after neuronal uptake 18F-dopamine is stored in vesicles, whereas 11C- methylreboxetine does not enter neurons. A vesicular storage defect would produce a greater fall in 18F-dopamine-derived than in 11C- methylreboxetine-derived radioactivity. Patients with nOH from a Lewy body disease (pure autonomic failure or Parkinson disease with orthostatic hypotension) and control subjects without evidence of central neurodegeneration or nOH underwent 18F-dopamine and 11C-methylreboxetine scanning. Subjects with cardiac transplants were included, to adjust for non-specific binding of 11C-methylreboxetine in the absence of sympathetic innervation. We found preliminarily that cardiac 18F-dopamine/11C-methylreboxetine ratios are decreased in Lewy body nOH, consistent with a vesicular storage defect in the residual cardiac sympathetic nerves. (4) Collaborations: (a) Computer models of stress, allostasis, and allostatic load in catecholaminergic neurodegeneration: Evolving concepts link chronic stress with catecholamine autotoxicity and catecholaminergic neurodegeneration in diseases such as PD. We have proposed that coordination of catecholaminergic systems mediates adjustments maintaining health and that senescence-related disintegration of these systems leads to disorders of regulation and to neurodegenerative diseases. Chronically repeated episodes of stress-related catecholamine release and reuptake, with attendant increases in formation of the toxic dopamine metabolite DOPAL, might accelerate this process (Goldstein & Kopin, Cell Mol Neurobiol 2018;38:13-24). Extending on previous and recently published (Goldstein & Kopin, Auton Neurosci 2017;208:1528) conceptual models, in collaboration with Mark Pekker (Univ. of Alabama), Yehonatan Sharabi (Tel Aviv University, Israel), and Graeme Eisenhofer (Univ. of Dresden, Germany) we are constructing a detailed kinetic model incorporating negative feedback regulation, catecholamine autotoxicity via DOPAL-AS interactions, and allostatic load in sympathetic noradrenergic neurons that predicts aging-related myocardial norepinephrine deficiency, a feature of Lewy body diseases. (b) Pleiotropic neuropathological and biochemical alterations associated with Myo5a mutation in a rat model: In a collaborative study (Landrock et al., Brain Res 2018;1679:155-170) we analyzed biochemical alterations involved in the pathogenesis of a neurodegenerative/movement disorder in juvenile rats with a mutant Myosin5a (Myo5a) gene. 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. In the affected animals we found hyperphosphorylation of AS and tau and region-specific buildup of DOPAL related to decreased aldehyde dehydrogenase activity. We propose a pleiotropic pathogenesis that links the Myo5a gene mutation to deficient neuronal development.
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