(1) Decreased vesicular uptake: A common theme in catecholaminergic neurodegeneration: 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. 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 dopamine/ (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 3,4-dihydroxyphenylglycol/norepinephrine 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 is 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). (2) Decreased vesicular storage and aldehyde dehydrogenase activity in MSA: In Parkinson disease (PD) evidence has accrued for a vesicular storage defect and aldehyde dehydrogenase (ALDH) inhibition in residual dopaminergic terminals, resulting in 3,4-dihydroxyphenylacetaldehyde (DOPAL) accumulation. In this study we asked whether multiple system atrophy (MSA) entails a similar abnormal neurochemical pattern. MSA and PD groups had similarly decreased putamen DA and DOPAC levels and decreased DOPAC/DOPAL ratios, with increased DOPAL/DA ratios. MSA and PD therefore both entail a catecholamine metabolic profile indicating impaired vesicular storage, decreased ALDH activity, and DOPAL buildup, which may be part of a common pathway in catecholamine neuronal death. Goldstein DS, Sullivan P, Holmes C, Kopin IJ, Sharabi Y, Mash DC. Decreased vesicular storage and aldehyde dehydrogenase in multiple system atrophy. Park Rel Dis 2015;21:567-572. (3) Constructing a mouse model of a double hit involving decreased vesicular storage and decreased aldehyde dehydrogenase activity: We are collaborating with Huaibin Cai (NIA) to develop an animal model of PD based on a double hit of decreased type 2 vesicular monoamine transporter (VMAT2) and ALDH1A1 activity. We have generated recombinant adeno-associated viral vectors that express Cre-dependent VMAT2 and ALDH1A1 shRNAs to knock down the expression of ALDH1A1 and VMAT2 in dopaminergic neurons. We have injected the viral vectors into the midbrain of dopamine transporter (DAT)-Cre knock-in mice that selectively express Cre and turn on the expression of shRNAs in the midbrain dopaminergic neurons. We are now determining whether the animals develop catecholaminergic neurodegeneration and motor abnormalities resembling those in PD. (4) Simultaneous measurement of parent and cysteinyl catechols indicates decreased L-aromatic-amino-acid decarboxylase activity and vesicular storage in the synucleinopathies PD and MSA. The severity of putamen DA depletion in parkinsonian synucleinopathies exceeds that explained by denervation alone. Decreased activities of tyrosine hydroxylase (TH) or L-aromatic-amino-acid decarboxylase (LAAAD) or decreased vesicular storage in residual terminals could make up the difference. Cysteinyl-DOPA (Cys-DOPA) and cysteinyl-dopamine (Cys-DA) are formed from spontaneous oxidation of the parent compounds in the neuronal cytoplasm. We assayed levels of endogenous catechols including DA, its cytoplasmic metabolites (Cys-DA, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylethanol, and 3,4-dihydroxyphenylacetaldehyde), and 2 tyrosine hydroxylation products proximate to DA (DOPA and Cys-DOPA) in post-mortem putamen tissue samples from 17 PD and 25 MSA patients and 30 controls. In the synucleinopathy patients an index of vesicular storage of DA (the ratio of DA to the sum of its cytoplasmic metabolites) averaged 54% of control (p=0.001), an index of L-aromatic-amino-acid decarboxylase (LAAAD) activity (the ratio of DA and the sum of its cytoplasmic metabolites to the sum of DOPA+Cys-DOPA) averaged 21% of control (p<0.0001), and an index of innervation (the sum of DOPA+Cys-DOPA) averaged 63% of control (p=0.01). Preliminarily, putamen DA depletion in these diseases therefore seems to reflect not only nigrostriatal denervation but also 2 functional abnormalities in the residual terminals--decreased vesicular storage and decreased LAAAD activity. (5) Role of oxidation in DOPAL-induced oligomerization of alpha-synuclein: PD has long been known to involve the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies, which contain abundant deposits of alpha-synuclein. The autotoxic DA metabolite DOPAL might link these two pathological features, because DOPAL potently oligomerizes and aggregates alpha-synuclein. Alpha-synuclein contains multiple lysine residues. The laboratory of Ad Bax (NIDDK) recently determined the structure of the dominant product in reactions between DOPAL and alpha-synuclein--a dicatechol pyrrole lysine adduct (Werner-Allen JW, Dumond JF, Bax A. Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of -Synuclein. Angew Chem Int Ed Engl. 2016;55:7374-7378). This novel modification results from the addition of two DOPAL molecules to the lysine sidechain amine through their aldehyde moieties and the formation of a new carbon-carbon bond between their alkyl chains to generate a pyrrole ring. The discovery should provide a valuable chemical basis for future studies of DOPAL-induced crosslinking of alpha-synuclein. We have established a collaboration with the Bax lab, to conduct cellular studies evaluating the role of DOPAL oxidation in mediating alpha-synuclein oligomerization. (6) Immunofluorescence confocal microscopy to visualize sympathetic noradrenergic nerves and alpha-synuclein in PD and related disorders: As a member of the Autonomic Rare Diseases Clinical Research Consortium, we are providing fixed skin biopsy tissues to collaborators at Harvard, to assess alpha-synuclein deposition and autonomic nerve fiber densities in patients with autonomic synucleinopathies. Within our Section we have begun performing tyrosine hydroxylase (TH) and alpha-synuclein immunofluorescence confocal microscopy in skin biopsies, sympathetic ganglion tissue, and myocardium, to determine whether alpha-synuclein deposition occurs in sympathetic noradrenergic neurons in Lewy body disease. (7) DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells: 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 obtained evidence that DOPAL transmitted to glial cells augments intra-cellular 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. (Jinsmaa Y, Sullivan P, Sharabi Y, Goldstein DS. DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells. Auton Neurosci 2016;194:46-51).
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