Several findings in this reporting period induced us to begin composing a novel, innovative disease modification trial based on combining a monoamine oxidase inhibitor (MAOI) with N-acetylcysteine (NAC), to test the catecholaldehyde hypothesis and potentially slow or prevent catecholaminergic neurodegeneration convincingly for the first time. (1) N-Acetylcysteine (NAC) prevents the increase in spontaneous oxidation of dopamine during monoamine oxidase inhibition: Dopamine in the neuronal cytoplasm can oxidize spontaneously to 5-S-cysteinyldopamine (Cys-DA). Exposure of dopaminergic cells to a MAOI decreases formation of the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL) but increases Cys-DA formation. We call this the MAOI tradeoff, because treatment with an MAOI alone may trade off one form of toxicity for another. We found that NAC prevents the MAOI tradeoff (Goldstein et al., Neurochem Res 2017;42:3289-3295). (2) NAC mitigates DOPAL-induced protein modifications: DOPAL exposure modifies many proteins in catecholaminergic cells, including alpha-synuclein (AS). NAC mitigates or prevents these alterations (Jinsmaa et al., J Pharmacol Exp Ther 2018;366:113-124). These data help rationalize MAOI+NAC treatment, because an MAOI would inhibit DOPAL formation by decreasing enzymatic oxidation of dopamine, and NAC would inhibit formation of harmful quinones by decreasing spontaneous oxidation of DOPAL and dopamine. (3) Poor plasma bioavailability of DOPET after olives ingestion: Olives contain abundant 3,4-dihydroxyphenylethanol (hydroxytyrosol, DOPET), a potential nutraceutical. After healthy volunteers ate 10 olives, plasma DOPET increased; however, there were much larger increases in plasma 3,4-dihydroxyphenylacetic acid (DOPAC), a liver metabolite of DOPET. The plasma bioavailability of ingested DOPET seems to be poor due to a hepatic first pass effect (Goldstein et al., Clin Transl Sci 2018;11:32-37). (4) Testing the catecholaldehyde hypothesis by MAOI+NAC in pure autonomic failure: Positive findings from our intramural PDRisk study (Goldstein et al., Park Rel Dis 2018; 50:108-112; Goldstein et al., Park Rel Dis 2018;52:90-93) enable us to move forward to proof of concept trials testing the catecholaldehyde hypothesis. We are constructing a new clinical protocol to test whether MAOI+NAC treatment prevents the progression of the rare Lewy body disease pure autonomic failure to PD or dementia with Lewy bodies (Kaufmann et al., Ann Neurol 2017;81:287-297). (5) Substantial renal conversion of L-threo-3,4-dihydroxyphenylserine (L-DOPS) to norepinephrine in patients with neurogenic orthostatic hypotension: The norepinephrine precursor L-DOPS is approved for treatment of symptomatic orthostatic hypotension. L-DOPS-induced increases in plasma norepinephrine levels are too small to explain the pressor effect of the drug. The kidneys contain abundant L-aromatic-amino-acid decarboxylase, which catalyzes the conversion of L-DOPS to norepinephrine. We assessed renal norepinephrine production from L-DOPS by comparing norepinephrine/L-DOPS ratios in urine to those in plasma in patients on L-DOPS. The ratio of norepinephrine/L-DOPS in urine averaged 63 times that in plasma. Thus, there is extensive renal production of norepinephrine from L-DOPS (Lamotte et al., Clin Auton Res (in press)). (6) Collaborations: (a) MAOI+NAC treatment in an animal model of the double hit: We discovered and reported previously that in synucleinopathies DOPAL buildup in the putamen reflects a double hit of decreased vesicular uptake of cytoplasmic dopamine and decreased enzymatic detoxification of DOPAL by aldehyde dehydrogenase (ALDH) (Goldstein et al., Park Rel Dis 2017;35:88-91). In collaboration with Yehonatan Sharabi (Tel Aviv University) we are testing the catecholaldehyde hypothesis in animals by assessing whether MAOI+NAC treatment prevents catecholaminergic neurodegeneration in the rotenone PD model. (b) Cerebrospinal fluid-directed rAAV9-rsATP7A plus subcutaneous copper histidinate advance survival and outcomes in a Menkes disease mouse model: Menkes disease is a rare, X-linked disorder of copper metabolism due to mutation of the gene encoding the copper ATPase, ATP7A. Because dopamine-beta-hydroxylase, which catalyzes the conversion of dopamine to norepinephrine, is a copper enzyme, Menkes disease is associated with increased levels of dopamine and its metabolites compared to norepinephrine and its metabolites. In a collaborative study of CSF-directed rAAV9-rsATP7A plus subcutaneous copper histidinate treatment in a Menkes disease mouse model, the treatment normalized brain neurochemical levels and normalized growth and neurobehavioral outcomes (Haddad et al., Mol Ther Methods Clin Dev 2018;10:165-178). (c) Potential glucocerebrosidase chaperone enhancer to treat Gaucher/PD: Among the known genetic risk factors for PD, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, human pluripotent stem cells were generated from patients with Gaucher disease with or without parkinsonism and differentiated into dopaminergic neurons. Dopaminergic neurons from patients with Gaucher disease and parkinsonism had reduced dopamine storage and dopamine reuptake via the cell membrane dopamine transporter. When the cells were treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase, the compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity, and reduced AS levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for PD treatment (Aflaki et al., J Neurosci 2016;36:7441-7452). (d) Mesenchymal stem cells to treat multiple system atrophy: Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by chronic autonomic failure and AS deposition in glial cytoplasmic inclusions in the brain. Intrathecal injection of mesenchymal stem cells might exert beneficial neurotrophic effects in this otherwise invariably lethal disease. In a collaborative study with investigators at the Mayo Clinic we are tracking cerebrospinal fluid catechols as a neurochemical biomarker of the treatment. (e) Renal sympathetic ablation to treat refractory hypertension: Renal sympathetic ablation is a possible non-pharmacologic treatment for refractory hypertension. In a collaborative study with Benjamin Levine (Univ. of Texas) under NIH Clinical Protocol 03-N-0004 we are using 18F-dopamine and 11C-methylreboxetine scanning to assess the extent of local denervation in a clinical trial of renal sympathetic ablation to treat refractory essential hypertension. (f) Atomoxetine to treat mild cognitive impairment: Mild cognitive impairment might improve by augmenting norepinephrine delivery to its receptors in the brain via treatment with atomoxetine, an inhibitor of the cell membrane norepinephrine transporter. In a collaborative study with Allan I. Levey (Emory) we are performing assays of plasma and cerebrospinal fluid levels of catechols in a clinical trial of atomoxetine in subjects with mild cognitive impairment. (g) Pyridostigmine to treat heart failure: Heart failure is associated with decreased parasympathetic cardiovagal outflow, which is associated with worse outcome. In a collaborative study with Stuart Katz (NYU) we assayed plasma catechols for a project on augmentation of parasympathetic signaling with pyridostigmine.