highlights recent advances from the Autonomic Medicine Section (formerly Clinical Neurocardiology Section) in the area of mechanisms of catecholaminergic neurodegeneration. We published that the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL) induces numerous protein modifications including oligomerization and quinonization of alpha-synuclein (AS) and that antioxidation with N-acetylcysteine mitigates these modifications (Jinsmaa et al., JPET 2018;366:113-124). We developed and applied novel immunofluorescence confocal microscopic methods to quanitfy AS deposition within sympathetic noradrenergic neurons (Isonaka et al., Clin Auton Res 2018;28:223-230) and showed that in Lewy body forms of neurogenic orthostatic hypotension (nOH) AS is co-localized with tyrosine hydroxylase, a marker of sympathetic noradrenergic neurons, in skin biopsies (Isonaka et al., Hypertension 2019;73:910-918). Intra-neuronal AS deposition therefore may be a common pathophysiologic feature of Lewy body nOH. Third, we developed and applied a computational model to assess comprehensively all the known pathways determining cardiac norepinephrine stores and obtained evidence for three types of functional abnormalities in residual sympathetic noradrenergic neurons. We successfully tested predictions from the model in a new cohort of patients with autopsy-proved PD (Goldstein et al., JCI Insight 2019;doi: 10.1172/jci.insight.130441) We also have collaborated with several research groups in studies related to mechanisms of catecholaminergic neurodegeneration. (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 oligomerization, protein-quinone adduct formation (quinonization), and aggregation in cultured cells and in test tube experiments. Using near infrared fluorescence (nIRF) 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 VMAT, glucocerebrosidase (decreased activity of which causes Gaucher disease), and L-aromatic-amino-acid decarboxylase (LAAAD), which is required for dopamine synthesis (Jinsmaa et al., JPET 2018;366:113-124). We also noted that DOPAL inactivates LAAAD and aggregates AS in cells. Preliminarily, we have extended from wells to cells by demonstrating that DOPAL quinonizes intracellular proteins as visualized by nIRF microscopy (Jinsmaa et al., unpublished observations). The results from this project have inspired a new disease modification strategy based on decreasing the formation and oxidation of DOPAL. (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 (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 validated and applied a novel method to quantify colocalization of AS with tyrosine hydroxylase (TH, in this situation a marker of catecholaminergic neurons) within sympathetic noradrenergically innervated constituents in skin biopsies from living patients with Lewy body and non-Lewy body forms of neurogenic orthostatic hypotension (nOH). Data about cardiac sympathetic noradrenergic innervation were obtained by 18F-dopamine positron emission tomographic (PET) scanning in the same patients. We found that all patients with Lewy body nOH have increased AS-TH colocalization and decreased myocardial 18F-dopamine-derived radioactivity, consistent with a pathogenic role of intracellular AS in catecholaminergic neurodegeneration (Isonaka et al., Hypertension 2019;73:910-918). (3) Functional abnormalities in residual sympathetic noradrenergic neurons (the sick-but-not-dead phenomenon) in Lewy body diseases: It has been almost axiomatic that in Lewy body diseases catecholamine deficiency directly and solely reflects loss of catecholaminergic neurons. Our 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 2019;1702:74-84). Most recently we applied a unique and novel computational model to assess comprehensively for the first time all the known pathways determining myocardial norepinephrine stores in Lewy body diseases. After taking denervation into account, we obtained evidence for three types functional abnormalities in residual cardiac sympathetic neuronsdecreased catecholamine biosynthesis via TH and LAAAD, decreased vesicular storage of dopamine and norepinephrine, and decreased norepinephrine recycling via neuronal reuptake (Goldstein et al., JCI Insight 2019;doi: 10.1172/jci.insight.130441). (4) Collaborations: (a) The mitochondrial complex 1 blocker rotenone produces an adult rat model of PD. In a collaborative study with Dr. Yehonatan Sharabi (Tel Aviv University) we have found preliminarily that systemic administration of the metabolic stressor rotenone, which produces behavioral abnormalities mimicking those in PD, evokes a central neurochemical pattern consistent with the sick-but-not-dead phenomenondecreased vesicular uptake of cytoplasmic catecholamines, decreased dopamine biosynthesis via LAAAD, decreased ALDH activity, and DOPAL buildup. (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 region-specific buildup of DOPAL related to decreased ALDH activity. We propose a pleiotropic pathogenesis that links the Myo5a gene mutation to deficient neuronal development. (c) Elevated COUP-TFII expression in dopaminergic neurons may accelerate the progression of PD: In a collaborative study with researchers at Baylor we have found that the expression of COUP-TFII, an orphan nuclear receptor, is upregulated in PD patients. Elevated COUP-TFII expression in dopaminergic neurons, in turn, evoked neurodegeneration in mice and accelerated phenotypic progression in a PD mouse model, MitoPark. Mechanistically, we found that COUP-TFII evokes mitochondrial dysfunctions, decreases ALDH activity, and tends to build up endogenous DOPAL. These preliminary results support the view that COUP-TFII is a contributor to PD. (d) Effects of TH over-expression on striatal dopaminergic functions: In collaboration with researchers at the Univ. of Toronto we assayed striatal and cardiac tissue samples from transgenic TH animals. Preliminarily, TH over-expression increases striatal and cardiac tissue contents of dopamine and DOPAC, confirming that transgenic TH animals have increased dopamine synthesis.
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