Vesicular sequestration limits the buildup of toxic products of enzymatic and spontaneous oxidation of catecholamines. A vesicular storage defect therefore could play a pathogenic role in the death of catecholaminergic neurons in a variety of neurodegenerative diseases. In putamen, deficient vesicular storage is revealed in vivo by accelerated loss of 18F-DOPA-derived radioactivity and post-mortem by decreased tissue dopamine (DA):DOPA ratios; in myocardium in vivo by accelerated loss of 18F-DA-derived radioactivity and post-mortem by increased 3,4-dihydroxyphenylglycol (DHPG):norepinephrine (NE) ratios; and in sympathetic noradrenergic nerves overall in vivo by increased plasma F-3,4-dihydroxyphenylacetic acid (F-DOPAC):DHPG ratios. We retrospectively analyzed data from 20 conditions with decreased or intact catecholaminergic innervation, involving different etiologies, pathogenetic mechanisms, and lesion locations. All conditions involving parkinsonism had accelerated loss of putamen 18F-DOPA-derived radioactivity; in those with post-mortem data there were also decreased putamen DA:DOPA ratios. All conditions involving cardiac sympathetic denervation had accelerated loss of myocardial 18F-dopamine-derived radioactivity; in those with post-mortem data there were increased myocardial DHPG:NE ratios. All conditions involving localized loss of catecholaminergic innervation had evidence of decreased vesicular storage specifically in the denervated regions. Thus, across neurodegenerative diseases, loss of catecholaminergic neurons seems to be associated with decreased vesicular storage in the residual neurons (Goldstein DS, Holmes C, Mash D, Sidransky E, Stefani A, Kopin IJ, Sharabi Y. Deficient vesicular storage: A common theme in catecholaminergic neurodegeneration. Parkinsonism Relat Disord 2015;21:1013-1022). In Parkinson disease (PD), alpha-synuclein is found characteristically in Lewy bodies, whereas in MSA the protein is found in glial cytoplasmic inclusions (GCIs). In one or our patients who clinically had the parkinsonian form of MSA, brain PET scanning showed bilaterally decreased putamen 18F-DOPA-derived radioactivity, and thoracic PET scanning showed markedly decreased 18F-DA-derived radioactivity throughout the left ventricular myocardium. At autopsy the patient had putamen atrophy and drastic myocardial NE and putamen DA depletion. Alpha-synuclein deposits were noted in glia, but there was no alpha-synuclein deposition in brain neurons or in sympathetic ganglia. Putamen DA:DOPA and DOPAC:3,4-dihydroxyphenylacetaldehyde (DOPAL) ratios were low and DOPAL:DA high. This important and so far unique case demonstrates that in MSA catecholamine depletion can occur without Lewy bodies or intra-neuronal alpha-synuclein deposition in the sympathetic nervous system or brain. Meanwhile, the pattern of catechol ratios suggests the same triad of a sequestration-to-oxidative deamination shift, DOPAL buildup, and decreased DOPAL detoxification by aldehyde dehydrogenase (ALDH) as we have found in sporadic PD. Therefore, in MSA, the putative catecholamine autotoxicity pattern can occur without intra-neuronal synucleinopathy (Cook GA, Sullivan P, Holmes C, Goldstein DS. Cardiac sympathetic denervation without Lewy bodies in a case of multiple system atrophy. Park Rel Dis 2014;20:926-928). In collaborative studies with G. Miller (Emory) on neuropathologic consequences of altered vesicular uptake mediated by the type 2 vesicular monoamine transporter (VMAT2), we obtained evidence for acceleration of aging-related loss of noradrenergic neurons in the locus ceruleus and the heart in VMAT2-Lo mice (Taylor TN, Alter SP, Wang M, Goldstein DS, Miller GW. Reduced vesicular storage of norepinephrine causes progressive degeneration in the locus ceruleus of mice. Neuropharmacology 2014;76:A97-A105). Mice with elevated VMAT2 activity, on the other hand, have enhanced DA release and decreased PD-related neurodegeneration in vivo (Lohr KM, Bernstein AI, Stout KA, Dunn AR, Lazo CR, Alter SP, Wang M, Li Y, Fan X, Hess EJ, Yi H, Vecchio LM, Goldstein DS, Guillot TS, Salahpour A, Miller GW. Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson's disease-related neurodegeneration in vivo. PNAS 2014;111:9977-9982). The findings in these mouse models fit with the catecholamine autotoxicity theory. We previously obtained evidence that a shift from vesicular sequestration to oxidative deamination of cytosolic catecholamines contributes to putamen DA depletion in PD. We hypothesized that an analogous shift takes place in sympathetic nerves in the heart. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites DHPG and DOPAC from PD patients and controls. We devised, validated, and applied 5 neurochemical indices of the sequestration-deamination shift and used a kinetic model to estimate the extent of the vesicular storage defect. The majority of PD patients (70%) had profound NE depletion (mean 2% of control), and in this subgroup all 5 indices of a sequestration-deamination shift were increased compared to controls. Vesicular storage in residual nerves was estimated to be decreased by 84-91% in this subgroup. Therefore, most PD patients have severe myocardial NE depletion, which is due to both sympathetic denervation and decreased vesicular storage in the residual nerves (Goldstein DS, Sullivan P, Holmes C, Miller GW, Sharabi Y, Kopin IJ. A vesicular sequestration to oxidative deamination shift in myocardial sympathetic nerves in Parkinson disease. J Neurochem 2014:131:219-228). A potentially important link between catecholaldehydes and synucleinopathy in the pathogenesis of PD is DOPAL-induced oligomerization of alpha-synuclein. Divalent metal cations, especially Cu+2, augment this oligomerization. We found that the aldehyde of serotonin, 5-hydroxyindolealdehyde, also potently oligomerizes alpha- synuclein (Jinsmaa Y, Cooney A, Sullivan P, Sharabi Y, Goldstein DS. The serotonin aldehyde, 5-HIAL, oligomerizes alpha-synuclein. Neurosci Lett 2015;590:134-137). This finding may help explain the occurrence of Lewy bodies in serotonergic neurons and the relatively selective losses of monoaminergic neurons in PD. Glial cells express little if any alpha-synuclein, and so bases for glial cytoplasmic inclusions in MSA have been obscure. Alpha-synuclein can spread cell to cell in a prion-like manner to glial cells. We are exploring whether DOPAL also can be transmitted and whether in glial cells DOPAL augments oligomerization of alpha-synuclein. We are carrying out co-incubation experiments involving PC12 cells treated to maximize endogenous DOPAL production and human glioblastoma cells or oligodendrocytes separated by a membrane from the PC12 cells so that the 2 cell types share the medium. Preliminarily, DOPAL can exit the cells in which it is produced and undergo uptake by adjacent glial cells. Moreover, both human glioblastoma cells and oligodendrocytes take up DOPAL and alpha-synuclein from the medium, and in both cell types intracellular DOPAL augments intracellular oligomerization of alpha-synuclein. Cell to cell spread of both DOPAL and alpha-synuclein and intracellular interactions between DOPAL and alpha-synuclein provide a novel explanation for the glial cytoplasmic inclusions found in MSA. DA oxidation products can bind covalently with proteins, altering functional characteristics and threatening neuronal integrity. We have found preliminarily that DOPAL binds to both lysine and cysteine, and we have successfully measured cysteinyl-DOPAL for the first time. In order to track interactions of DA oxidation products with cellular proteins, we are planning to conduct experiments involving incubation of PC12 cells with 14C-DA with or without blockade of DOPAL formation or of vesicular uptake.
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