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1.  Hypertension Increases Cerebral 6-18F-Fluorodopa–Derived Radioactivity 
6-18F-fluorodopa PET depicts the striatal dopaminergic lesion characterizing Parkinson disease (PD); however, striatal uptake of 6-18F-fluorodopa–derived radioactivity can be normal. Supine hypertension (SH) might increase 6-18F-fluorodopa uptake.
Methods
We measured putamen, caudate, and occipital cortex 6-18F-fluorodopa–derived radioactivity and supine blood pressure in patients with PD + SH (systolic pressure ≥ 180 mm Hg, n = 8), patients with PD without SH (PD − SH, n = 19), patients with pure autonomic failure (n = 8), and controls (n = 16).
Results
Peak putamen radioactivity correlated with supine systolic pressure across all subjects and among PD patients and was higher in PD + SH than in PD − SH (P = 0.01). Both subgroups had rapid fractional declines in radioactivity between the peak and late values (P < 0.0001, compared with controls). Arterial 6-18F-fluorodopa concentrations were similar in the compared groups.
Conclusion
In PD, SH is associated with augmented striatal 6-18F-fluorodopa–derived radioactivity. Regardless of SH, retention of 6-18F-fluorodopa–derived radioactivity is markedly reduced. A model-independent approach can identify striatal dopaminergic denervation in PD.
doi:10.2967/jnumed.109.062869
PMCID: PMC4164388  PMID: 19690020
fluorodopa; Parkinson; pure autonomic failure; orthostatic hypotension; supine hypertension
2.  Clinical laboratory evaluation of autoimmune autonomic ganglionopathy: Preliminary observations 
Several forms of chronic autonomic failure manifest as neurogenic orthostatic hypotension, including autoimmune autonomic ganglionopathy (AAG) and pure autonomic failure (PAF). AAG and PAF are thought to differ in pathogenesis, AAG reflecting decreased ganglionic neurotransmission due to circulating antibodies to the neuronal nicotinic receptor and PAF being a Lewy body disease with prominent loss of sympathetic noradrenergic nerves. AAG therefore would be expected to differ from PAF in terms of clinical laboratory findings indicating postganglionic noradrenergic denervation. Both diseases are rare. Here we report preliminary observations about clinical physiologic, neuropharmacologic, neurochemical, and neuroimaging data that seem to fit with the hypothesized pathogenetic difference between AAG and PAF. Patients with either condition have evidence of baroreflex–sympathoneural and baroreflex–cardiovagal failure. Both disorders feature low plasma levels of catecholamines during supine rest, but plasma levels of the other endogenous catechols, dihydroxyphenylalanine (DOPA), dihydroxyphenylacetic acid (DOPAC), and dihydroxyphenylglycol (DHPG), seem to be lower in PAF than in AAG, probably reflecting decreased norepinephrine synthesis and turnover in PAF, due to diffuse sympathetic noradrenergic denervation. PAF entails cardiac sympathetic denervation, whereas cardiac sympathetic neuroimaging by thoracic 6-[18F]fluorodopamine scanning indicates intact myocardial sympathetic innervation in AAG.
doi:10.1016/j.autneu.2008.12.004
PMCID: PMC4164394  PMID: 19155193
Autonomic failure; Dysautonomia; Sympathetic nervous system; Norepinephrine; Fluorodopamine
3.  Temporary elimination of orthostatic hypotension by norepinephrine infusion 
A cardinal manifestation of chronic autonomic failure is neurogenic orthostatic hypotension (OH), which often is associated with supine hypertension, posing a therapeutic dilemma. We report here success in a first step toward development of a “prosthetic baroreceptor system” to maintain blood pressure during orthostasis without worsening supine hypertension. In all of four patients with neurogenic OH, titrated i.v. NE infusion kept directly recorded intra-arterial pressure at or above baseline during progressive head-up tilt. We conclude that titrated i.v. NE infusion temporarily eliminates OH.
doi:10.1007/s10286-012-0176-4
PMCID: PMC4118053  PMID: 22983778
Norepinephrine; Orthostatic hypotension; Sympathetic nervous system; Baroreflex
4.  Determinants of buildup of the toxic dopamine metabolite DOPAL in Parkinson’s disease 
Journal of neurochemistry  2013;126(5):591-603.
Intra-neuronal metabolism of dopamine (DA) begins with production of 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is toxic. According to the ‘catecholaldehyde hypothesis,’ DOPAL destroys nigrostriatal DA terminals and contributes to the profound putamen DA deficiency that characterizes Parkinson’s disease (PD). We tested the feasibility of using post-mortem patterns of putamen tissue catechols to examine contributions of altered activities of the type 2 vesicular monoamine transporter (VMAT2) and aldehyde dehydrogenase (ALDH) to the increased DOPAL levels found in PD. Theoretically, the DA : DOPA concentration ratio indicates vesicular uptake, and the 3,4-dihydroxyphenylacetic acid : DOPAL ratio indicates ALDH activity. We validated these indices in transgenic mice with very low vesicular uptake (VMAT2-Lo) or with knockouts of the genes encoding ALDH1A1 and ALDH2 (ALDH1A1,2 KO), applied these indices in PD putamen, and estimated the percent decreases in vesicular uptake and ALDH activity in PD. VMAT2-Lo mice had markedly decreased DA:DOPA (50 vs. 1377, p < 0.0001), and ALDH1A1,2 KO mice had decreased 3,4-dihydroxyphenylacetic acid:DOPAL (1.0 vs. 11.2, p < 0.0001). In PD putamen, vesicular uptake was estimated to be decreased by 89% and ALDH activity by 70%. Elevated DOPAL levels in PD putamen reflect a combination of decreased vesicular uptake of cytosolic DA and decreased DOPAL detoxification by ALDH.
doi:10.1111/jnc.12345
PMCID: PMC4096629  PMID: 23786406
DOPAC; DOPAL; dopamine; DOPET; monoamine oxidase; Parkinson’s disease
5.  Vesicular Uptake Blockade Generates the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells: Relevance to the Pathogenesis of Parkinson Disease 
Journal of neurochemistry  2012;123(6):932-943.
Parkinson disease entails profound loss of nigrostriatal dopaminergic terminals, decreased vesicular uptake of intra-neuronal catecholamines, and relatively increased putamen tissue concentrations of the toxic dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL). The objective of this study was to test whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, since alpha-synuclein oligomers may be pathogenetic in Parkinson disease, oligomerizes alpha-synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPAL—daidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by a method based on 6F- or 13C-dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL-induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L-aromatic-amino-acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95–97% and rapidly increased cell DOPAL content (p=0.0008). Daidzein+AL1576 augmented DOPAL responses to reserpine (p=0.004). Intracellular DOPAL contributed to dopamine-evoked apoptosis and DOPA-evoked synuclein dimerization. The findings fit with the “catecholaldehyde hypothesis,” according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes Parkinson disease.
doi:10.1111/j.1471-4159.2012.07924.x
PMCID: PMC3514596  PMID: 22906103
Dihydroxyphenylacetaldehyde; DOPAL; aldehyde dehydrogenase; reserpine; Parkinson disease; monoamine oxidase
6.  In utero copper treatment for Menkes disease associated with a severe ATP7A mutation 
Molecular genetics and metabolism  2012;107(1-2):222-228.
Menkes disease is a lethal X-linked recessive neurodegenerative disorder of copper transport caused by mutations in ATP7A, which encodes a copper-transporting ATPase. Early postnatal treatment with copper injections often improves clinical outcomes in affected infants. While Menkes disease newborns appear normal neurologically, analyses of fetal tissues including placenta indicate abnormal copper distribution and suggest a prenatal onset of the metal transport defect. In an affected fetus whose parents found termination unacceptable and who understood the associated risks, we began in utero copper histidine treatment at 31.5 weeks gestational age. Copper histidine (900 μg per dose) was administered directly to the fetus by intramuscular injection (fetal quadriceps or gluteus) under ultrasound guidance. Percutaneous umbilical blood sampling enabled serial measurement of fetal copper and ceruloplasmin levels that were used to guide therapy over a four-week period. Fetal copper levels rose from 17 μg/dL prior to treatment to 45 μg/dL, and ceruloplasmin levels from 39 mg/L to 122 mg/L. After pulmonary maturity was confirmed biochemically, the baby was delivered at 35.5 weeks and daily copper histidine therapy (250 μg sc b.i.d.) was begun. Despite this very early intervention with copper, the infant showed hypotonia, developmental delay, and electroencephalographic abnormalities and died of respiratory failure at 5.5 months of age. The patient’s ATP7A mutation, which severely disrupted mRNA splicing, resulted in complete absence of ATP7A protein on Western blots. These investigations suggest that prenatally initiated copper replacement is inadequate to correct Menkes disease caused by severe loss-of-function mutations, and that postnatal ATP7A gene addition represents a rational approach in such circumstances.
doi:10.1016/j.ymgme.2012.05.008
PMCID: PMC3444639  PMID: 22695177
7.  Adrenomedullary Response to Glucagon in Patients with Primary Sjögren’s Syndrome 
Several studies showed signs of autonomic dysfunction in patients with primary Sjögren's syndrome (pSS). Adrenomedullary function might be of importance for pSS pathogenesis by affecting salivary gland functions and modulating immune responses. The aim of the study was to evaluate the adrenomedullary hormonal system in patients with pSS.
The glucagon test (1 mg i.v.) was performed in 18 pSS patients and 13 control subjects. During the testing each patient had electrocardiographic and impedance cardiographic monitoring. Plasma epinephrine and norepinephrine were assayed by liquid chromatography with electrochemical detection after batch alumina extraction.
Baseline concentrations of epinephrine and norepinephrine were comparable between pSS and controls. Glucagon administration induced a significant increase in systolic blood pressure, diastolic blood pressure, heart rate, cardiac output (p < 0.01), stroke volume; however the changes were comparable between pSS and controls. Epinephrine levels increased (p < 0.01) in response to glucagon administration while norepinephrine concentration did not change. There was no significant difference in neurochemical responses to glucagon between pSS and controls. In conclusion, the present results suggest normal adrenomedullary function in pSS.
doi:10.1007/s10571-011-9787-x
PMCID: PMC3378756  PMID: 22350211
Primary Sjögren’s syndrome; epinephrine; adrenal medulla; norepinephrine
8.  Cerebrospinal fluid biomarkers of central catecholamine deficiency in Parkinson’s disease and other synucleinopathies 
Brain  2012;135(6):1900-1913.
Central catecholamine deficiency characterizes α-synucleinopathies such as Parkinson’s disease. We hypothesized that cerebrospinal fluid levels of neuronal metabolites of catecholamines provide neurochemical biomarkers of these disorders. To test this hypothesis we measured cerebrospinal fluid levels of catechols including dopamine, norepinephrine and their main respective neuronal metabolites dihydroxyphenylacetic acid and dihydroxyphenylglycol in Parkinson’s disease and two other synucleinopathies, multiple system atrophy and pure autonomic failure. Cerebrospinal fluid catechols were assayed in 146 subjects—108 synucleinopathy patients (34 Parkinson’s disease, 54 multiple system atrophy, 20 pure autonomic failure) and 38 controls. In 14 patients cerebrospinal fluid was obtained before or within 2 years after the onset of parkinsonism. The Parkinson’s disease, multiple system atrophy and pure autonomic failure groups all had lower cerebrospinal fluid dihydroxyphenylacetic acid [0.86 ± 0.09 (SEM), 1.00 ± 0.09, 1.32 ± 0.12 nmol/l] than controls (2.15 ± 0.18 nmol/l; P < 0.0001; P < 0.0001; P = 0.0002). Dihydroxyphenylglycol was also lower in the three synucleinopathies (8.82 ± 0.44, 7.75 ± 0.42, 5.82 ± 0.65 nmol/l) than controls (11.0 ± 0.62 nmol/l; P = 0.009, P < 0.0001, P < 0.0001). Dihydroxyphenylacetic acid was lower and dihydroxyphenylglycol higher in Parkinson’s disease than in pure autonomic failure. Dihydroxyphenylacetic acid was 100% sensitive at 89% specificity in separating patients with recent onset of parkinsonism from controls but was of no value in differentiating Parkinson’s disease from multiple system atrophy. Synucleinopathies feature cerebrospinal fluid neurochemical evidence for central dopamine and norepinephrine deficiency. Parkinson’s disease and pure autonomic failure involve differential dopaminergic versus noradrenergic lesions. Cerebrospinal fluid dihydroxyphenylacetic acid seems to provide a sensitive means to identify even early Parkinson’s disease.
doi:10.1093/brain/aws055
PMCID: PMC3359749  PMID: 22451506
Parkinson's; dopamine; norepinephrine; DHPG; DOPAC; biomarker
9.  Neonatal Diagnosis and Treatment of Menkes Disease 
The New England journal of medicine  2008;358(6):605-614.
BACKGROUND
Menkes disease is a fatal neurodegenerative disorder of infancy caused by diverse mutations in a copper-transport gene, ATP7A. Early treatment with copper injections may prevent death and illness, but presymptomatic detection is hindered by the inadequate sensitivity and specificity of diagnostic tests. Exploiting the deficiency of a copper enzyme, dopamine-β-hydroxylase, we prospectively evaluated the diagnostic usefulness of plasma neurochemical levels, assessed the clinical effect of early detection, and investigated the molecular bases for treatment outcomes.
METHODS
Between May 1997 and July 2005, we measured plasma dopamine, norepinephrine, dihydroxyphenylacetic acid, and dihydroxyphenylglycol in 81 infants at risk. In 12 newborns who met the eligibility criteria and began copper-replacement therapy within 22 days after birth, we tracked survival and neurodevelopment longitudinally for 1.5 to 8 years. We characterized ATP7A mutations using yeast complementation, reverse-transcriptase–polymerase-chain-reaction analysis, and immunohistochemical analysis.
RESULTS
Of 81 infants at risk, 46 had abnormal neurochemical findings indicating low dopamine-β-hydroxylase activity. On the basis of longitudinal follow-up, patients were classified as affected or unaffected by Menkes disease, and the neurochemical profiles were shown to have high sensitivity and specificity for detecting disease. Among 12 newborns with positive screening tests who were treated early with copper, survival at a median follow-up of 4.6 years was 92%, as compared with 13% at a median follow-up of 1.8 years for a historical control group of 15 late-diagnosis and late-treatment patients. Two of the 12 patients had normal neurodevelopment and brain myelination; 1 of these patients had a mutation that complemented a Saccharomyces cerevisiae copper-transport mutation, indicating partial ATPase activity, and the other had a mutation that allowed some correct ATP7A splicing.
CONCLUSIONS
Neonatal diagnosis of Menkes disease by plasma neurochemical measurements and early treatment with copper may improve clinical outcomes. Affected newborns who have mutations that do not completely abrogate ATP7A function may be especially responsive to early copper treatment.
doi:10.1056/NEJMoa070613
PMCID: PMC3477514  PMID: 18256395
10.  Relative Efficiencies of Plasma Catechol Levels and Ratios for Neonatal Diagnosis of Menkes Disease 
Neurochemical research  2009;34(8):1464-1468.
Background
Menkes disease is an X-linked recessive neurodevelopmental disorder resulting from mutation in a copper-transporting ATPase gene. Menkes disease can be detected by relatively high concentrations of dopamine (DA) and its metabolites compared to norepinephrine (NE) and its metabolites, presumably because dopamine-beta-hydroxylase (DBH) requires copper as a co-factor. The relative diagnostic efficiencies of levels of catechol analytes, alone or in combination, in neonates at genetic risk of Menkes disease have been unknown.
Methods
Plasma from 44 at-risk neonates less than 30 days old were assayed for DA, NE, and other catechols. Of the 44, 19 were diagnosed subsequently with Menkes disease, and 25 were unaffected.
Results
Compared to unaffected at-risk infants, those with Menkes disease had high plasma DA (P < 10−6) and low NE (P < 10−6) levels. Considered alone, neither DA nor NE levels had perfect sensitivity, whereas the ratio of DA:NE was higher in all affected than in all unaffected subjects (P = 2 × 10−8). Analogously, levels of the DA metabolite, dihydroxyphenylacetic acid (DOPAC), and the NE metabolite, dihydroxyphenylglycol (DHPG), were imperfectly sensitive, whereas the DOPAC:DHPG ratio was higher in all affected than in all unaffected subjects (P = 2 × 10−4). Plasma dihydroxyphenylalanine (DOPA) and the ratio of epinephrine (EPI):NE levels were higher in affected than in unaffected neonates (P = 0.0015; P = 0.013).
Conclusions
Plasma DA:NE and DOPAC:DHPG ratios are remarkably sensitive and specific for diagnosing Menkes disease in at-risk newborns. Affected newborns also have elevated DOPA and EPI:NE ratios, which decreased DBH activity alone cannot explain.
doi:10.1007/s11064-009-9933-8
PMCID: PMC3477515  PMID: 19234788
Menkes; Dopamine; Norepinephrine; Dopamine-β-hydroxylase; DHPG; DOPAC; Diagnosis
11.  Diagnosis of Copper Transport Disorders 
Techniques for the diagnosis of copper transport disorders are increasingly important due to recent recognition of previously unappreciated clinical phenotypes and emerging advances in the treatment of these conditions. Here, we collate the diagnostic approaches and techniques currently employed for biochemical and molecular assessment of at-risk individuals in whom abnormal copper metabolism is suspected.
doi:10.1002/0471142905.hg1709s70
PMCID: PMC3156244  PMID: 21735378
12.  Intra-neuronal vesicular uptake of catecholamines is decreased in patients with Lewy body diseases 
The Journal of Clinical Investigation  2011;121(8):3320-3330.
Several neurodegenerative disorders, including Parkinson disease (PD), are characterized by the presence of Lewy bodies — cytoplasmic inclusions containing α-synuclein protein aggregates — in the affected neurons. A poorly understood feature of Lewy body diseases is loss of sympathetic nerves in the heart and other organs, manifesting as orthostatic hypotension (OH; also known as postural hypotension). We asked whether sympathetic denervation is associated with decreased uptake of catecholamines, such as dopamine and norepinephrine, into storage vesicles within sympathetic neurons. We used 6-[18F]-dopamine (18F-DA) to track myocardial uptake and retention of catecholamines. Concurrently, the fate of intra-neuronal 18F-DA was followed by assessment of arterial plasma levels of the 18F-DA metabolite 18F-dihydroxyphenylacetic acid (18F-DOPAC). The ratio of myocardial 18F-DA to arterial 18F-DOPAC provided an index of vesicular uptake. Tracer concentrations were measured in patients with PD with or without orthostatic hypotension (PD+OH, PD-No-OH); in patients with pure autonomic failure (PAF, a Lewy body disease without parkinsonism); in patients with multiple system atrophy (MSA, a non–Lewy body synucleinopathy); and in normal controls. Patients with PD+OH or PAF had decreased vesicular 18F-DA uptake and accelerated 18F-DA loss, compared with MSA and control subjects. PD-No-OH patients could be subtyped into one of these categories based on their initial 18F-DA uptake. We conclude that sympathetic denervation in Lewy body diseases is associated with decreased vesicular uptake of neuronal catecholamines, suggesting that vesicular monoamine transport is impaired. Vesicular uptake may constitute a novel target for diagnosis, treatment, and prevention.
doi:10.1172/JCI45803
PMCID: PMC3148734  PMID: 21785221
13.  Association of anosmia with autonomic failure in Parkinson disease 
Neurology  2010;74(3):245-251.
Background:
Olfactory dysfunction and autonomic failure are gaining recognition as nonmotor manifestations of Parkinson disease (PD). This observational study assessed whether in PD anosmia and autonomic failure are related to each other or to neuroimaging evidence of striatal dopamine deficiency.
Methods:
Olfactory function was assessed by the University of Pennsylvania Smell Identification Test (UPSIT) in 23 patients with sporadic PD. Baroreflex-cardiovagal gain was quantified from the relationship between cardiac interbeat interval and systolic pressure during the Valsalva maneuver and baroreflex-sympathoneural function by responses of systolic pressure to the Valsalva maneuver and of hemodynamics and plasma norepinephrine (NE) and dihydroxyphenylglycol (DHPG) levels to orthostasis. 6-[18F]Fluorodopamine PET and plasma and skeletal muscle microdialysate NE and DHPG were used to indicate cardiac and extracardiac noradrenergic innervation and brain 6-[18F]fluorodopa PET to indicate striatal dopaminergic innervation. Parkinsonism was assessed by UPDRS scores.
Results:
Compared to patients with PD and normal to moderately decreased sense of smell, patients with anosmic PD had lower mean baroreflex-cardiovagal gain (p = 0.04), larger falls in systolic pressure during the Valsalva maneuver and orthostasis (p = 0.04, p = 0.02), smaller orthostatic increments in plasma NE and DHPG (p = 0.003, p = 0.03), lower cardiac septal:hepatic and renal cortical:hepatic ratios of 6-[18F]fluorodopamine-derived radioactivity (p = 0.01, p = 0.06), and lower microdialysate NE and DHPG (p = 0.01; p = 0.006). Neither clinical severity of parkinsonism nor the putamen:occipital cortex ratio of 6-[18F]fluorodopa-derived radioactivity was related to the UPSIT category.
Conclusions:
In Parkinson disease, anosmia is associated with baroreflex failure and cardiac and organ-selective extracardiac noradrenergic denervation, independently of parkinsonism or striatal dopaminergic denervation.
GLOSSARY
= dihydroxyphenylglycol;
= least significant difference;
= norepinephrine;
= orthostatic hypotension;
= Parkinson disease;
= quantitative sudomotor axon reflex test;
= Unified Parkinson's Disease Rating Scale;
= University of Pennsylvania Smell Identification Test.
doi:10.1212/WNL.0b013e3181ca014c
PMCID: PMC2809034  PMID: 20083801
14.  Autoimmune autonomic ganglionopathy: treatment by plasma exchanges and rituximab 
This case report describes successful treatment of autoimmune autonomic ganglionopathy (AAG) in a 74-year-old woman by total plasma exchanges (PLEX) and rituximab. Two series of PLEX temporarily, but dramatically improved orthostatic intolerance and hypotension and baroreflex function, in a manner inversely related to ganglionic neuronal nicotinic receptor antibody titer. After rituximab treatment, the antibody titer was decreased modestly but persistently, and the patient had symptomatic and clinical laboratory evidence of continued benefit for at least 10 months, supporting the autoimmune pathogenesis of AAG.
doi:10.1007/s10286-009-0012-7
PMCID: PMC2963144  PMID: 19399547
Autoimmune autonomic ganglionopathy; Pure autonomic failure; Rituximab; Plasma exchange
15.  Dopamine Contamination of Infused Tyramine 
Clinical chemistry  2005;51(9):1733-1735.
doi:10.1373/clinchem.2005.054361
PMCID: PMC2963153  PMID: 16037411
16.  Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome 
Journal of Medical Genetics  2007;44(8):492-497.
Background
Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the underlying molecular mechanisms.
Methods
We measured biochemical markers of impaired copper transport in five patients from two unrelated families and used RNase protection, quantitative reverse transcription (RT)‐PCR, Western blot analysis and yeast complementation studies to characterise two ATP7A missense mutations, A1362D and S637L.
Results
In two brothers (family A) with A1362D, RNase protection and Western blot analyses revealed higher amounts of ATP7A transcript and protein in the older, mildly affected patient, who also had a higher plasma copper level and lower cerebrospinal fluid dihydroxyphenylalanine : dihydroxyphenylglycol ratio. These findings indicate greater gastrointestinal absorption of copper and higher activity of dopamine‐β‐hydroxylase, a copper‐dependent enzyme, respectively. In family B, three males with a missense mutation (S637L) in an exon 8 splicing enhancer showed equally reduced amounts of ATP7A transcript and protein by quantitative RT‐PCR and western blot analysis, respectively, despite a more severe phenotype in the youngest. This patient's medical history was notable for cardiac arrest as a neonate, to which we attribute his more severe neurodevelopmental outcome.
Conclusions
These families illustrate that genetic and non‐genetic mechanisms may underlie intrafamilial variability in Menkes disease and its variants.
doi:10.1136/jmg.2007.050013
PMCID: PMC2597922  PMID: 17496194
Menkes disease; intrafamilial variation; gene expression, ATP7A; residual copper transport
17.  FUNCTIONAL EFFECTS OF CARDIAC SYMPATHETIC DENERVATION IN NEUROGENIC ORTHOSTATIC HYPOTENSION 
Parkinsonism & related disorders  2008;15(2):122-127.
Background
Diseases characterized by neurogenic orthostatic hypotension (NOH), such as Parkinson disease (PD) and pure autonomic failure (PAF), are associated with cardiac sympathetic denervation, as reflected by low myocardial concentrations of 6-[18F]fluorodopamine-derived radioactivity. We studied the impact of such denervation on cardiac chronotropic and inotropic function.
Methods
Cardiac inotropic function was assessed by the preejection period index and the systolic time ratio index in response to the directly acting beta-adrenoceptor agonist, isoproterenol, and to the indirectly acting sympathomimetic amine, tyramine, in patients with PD+NOH or PAF (PD+NOH/PAF group, N=13). We compared the results to those in patients with multiple system atrophy, which usually entails NOH with normal cardiac sympathetic innervation (MSA, N=15), and in normal control subjects (N=5).
Results
The innervated and denervated groups did not differ in baseline mean preejection period index or systolic time ratio index. Tyramine increased cardiac contractility in the MSA patients and controls but not in the PD+NOH/PAF group. For similar heart rate responses, the PD+NOH/PAF group required less isoproterenol (p<0.01) and had lower plasma isoproterenol levels (p<0.01) than did the MSA group.
Conclusions
Among patients with NOH those with cardiac sympathetic denervation have an impaired inotropic response to tyramine and exaggerated responses to isoproterenol. This pattern suggests that cardiac denervation is associated with decreased ability to release endogenous norepinephrine from sympathetic nerves and with supersensitivity of cardiac beta-adrenoreceptors.
doi:10.1016/j.parkreldis.2008.04.002
PMCID: PMC2696021  PMID: 18514012
Parkinson disease; pure autonomic failure; sympathetic denervation; pre-ejection period
18.  BIOMARKERS TO DETECT CENTRAL DOPAMINE DEFICIENCY AND DISTINGUISH PARKINSON DISEASE FROM MULTIPLE SYSTEM ATROPHY 
Parkinsonism & related disorders  2008;14(8):600-607.
Objective
Biomarkers are increasingly important to diagnose and test treatments of neurodegenerative diseases such as Parkinson disease (PD). This study compared neuroimaging, neurochemical, and olfactory potential biomarkers to detect central dopamine (DA) deficiency and distinguish PD from multiple system atrophy (MSA).
Methods
In 77 PD, 57 MSA, and 87 control subjects, radioactivity concentrations in the putamen (PUT), caudate (CAU), occipital cortex (OCC), and substantia nigra (SN) were measured 2 hours after 6-[18F]fluorodopa injection, septal myocardial radioactivity measured 8 minutes after 6-[18F]fluorodopamine injection, CSF and plasma catechols assayed, or olfaction tested (University of Pennsylvania Smell Identification Test (UPSIT)). Receiver operating characteristic curves were constructed, showing test sensitivities at given specificities.
Results
PUT:OCC, CAU:OCC, and SN:OCC ratios of 6-[18F]fluorodopa-derived radioactivity were similarly low in PD and MSA (p<0.0001, p<0.0001, p=0.003 compared to controls), as were CSF dihydroxyphenylacetic acid (DOPAC) and DOPA concentrations (p<0.0001 each). PUT:SN and PUT:CAU ratios were lower in PD than in MSA (p=0.004; p=0.005). CSF DOPAC correlated positively with PUT:OCC ratios (r=0.61, p<0.0001). Myocardial 6-[18F]fluorodopamine-derived radioactivity distinguished PD from MSA (83% sensitivity at 80% specificity, 100% sensitivity among patients with neurogenic orthostatic hypotension (NOH)). Only PD patients were anosmic; only MSA patients had normal olfaction (61% sensitivity at 80% specificity).
Conclusions
PD and MSA feature low PUT:OCC ratios of 6-[18F]fluorodopa-derived radioactivity and low CSF DOPAC and DOPA concentrations, cross-validating the neuroimaging and neurochemical approaches but not distinguishing the diseases. PUT:SN and PUT:OCC ratios of 6-[18F]fluorodopa-derived radioactivity, cardiac 6-[18F]fluorodopamine-derived radioactivity, and olfactory testing separate PD from MSA.
doi:10.1016/j.parkreldis.2008.01.010
PMCID: PMC2650101  PMID: 18325818
Parkinson; multiple system atrophy; fluorodopa; fluorodopamine; DOPAC; PET; biomarker
19.  Generalized and Neurotransmitter-Selective Noradrenergic Denervation in Parkinson Disease with Orthostatic Hypotension 
Background
Patients with Parkinson disease (PD) often have manifestations of autonomic failure. About 40% have neurogenic orthostatic hypotension (NOH), and among PD+NOH patients virtually all have evidence of cardiac sympathetic denervation; however, whether PD+NOH entails extra-cardiac noradrenergic denervation has been less clear.
Methods
Microdialysate concentrations of the main neuronal metabolite of norepinephrine (NE), dihydroxyphenylglycol (DHPG), were measured in skeletal muscle, and plasma concentrations of norepinephrine and DHPG were measured in response to i.v. tyramine, yohimbine, and isoproterenol, in patients with PD+NOH, patients with pure autonomic failure (PAF), which is characterized by generalized catecholaminergic denervation, and control subjects.
Results
Microdialysate DHPG concentrations were similarly low in PD+NOH and PAF, compared to control subjects (163±25, 153±27, and 304±27 pg/ml, p<0.01 each vs. control). The two groups also had similarly small plasma DHPG responses to tyramine (71±58 and 82±105 vs. 313±94 pg/ml; p<0.01 each vs. control) and NE responses to yohimbine (223±37 and 61±15 vs. 672±130 pg/ml, p<0.01 each vs. control) and virtually absent NE responses to isoproterenol (20±34 and 14±15 vs. 336±78 pg/ml, p<0.01 each vs. control). PD+NOH patients had normal bradycardia responses to edrophonium and normal epinephrine responses to glucagon
Conclusions
The results support the concept of generalized noradrenergic denervation in PD+NOH, with similar severity to that seen in PAF. In contrast, the parasympathetic cholinergic and adrenomedullary hormonal components of the autonomic nervous system seem intact in PD+NOH.
doi:10.1002/mds.22226
PMCID: PMC2667619  PMID: 18661549
Dysautonomia; Parkinson's disease; Multiple System Atrophy; Noradrenergic
20.  Neuronal Source of Plasma Dopamine 
Clinical chemistry  2008;54(11):1864-1871.
BACKGROUND
Determinants of plasma norepinephrine (NE) and epinephrine concentrations are well known; those of the third endogenous catecholamine, dopamine (DA), remain poorly understood. We tested in humans whether DA enters the plasma after corelease with NE during exocytosis from sympathetic noradrenergic nerves.
METHODS
We reviewed plasma catecholamine data from patients referred for autonomic testing and control subjects under the following experimental conditions: during supine rest and in response to orthostasis; intravenous yohimbine (YOH), isoproterenol (ISO), or glucagon (GLU), which augment exocytotic release of NE from sympathetic nerves; intravenous tri-methaphan (TRI) or pentolinium (PEN), which decrease exocytotic NE release; or intravenous tyramine (TYR), which releases NE by nonexocytotic means. We included groups of patients with pure autonomic failure (PAF), bilateral thoracic sympathectomies (SNS-x), or multiple system atrophy (MSA), since PAF and SNS-x are associated with noradrenergic denervation and MSA is not.
RESULTS
Orthostasis, YOH, ISO, and TYR increased and TRI/PEN decreased plasma DA concentrations. Individual values for changes in plasma DA concentrations correlated positively with changes in NE in response to orthostasis (r =0.72, P <0.0001), YOH (r = 0.75, P < 0.0001), ISO (r = 0.71, P < 0.0001), GLU (r = 0.47, P = 0.01), and TYR (r = 0.67, P < 0.0001). PAF and SNS-x patients had low plasma DA concentrations. We estimated that DA constitutes 2%– 4% of the catecholamine released by exocytosis from sympathetic nerves and that 50%–90% of plasma DA has a sympathoneural source.
CONCLUSIONS
Plasma DA is derived substantially from sympathetic noradrenergic nerves.
doi:10.1373/clinchem.2008.107193
PMCID: PMC2577013  PMID: 18801936
21.  Coexpression of Tyrosine Hydroxylase, GTP Cyclohydrolase I, Aromatic Amino Acid Decarboxylase, and Vesicular Monoamine Transporter 2 from a Helper Virus-Free Herpes Simplex Virus Type 1 Vector Supports High-Level, Long-Term Biochemical and Behavioral Correction of a Rat Model of Parkinson’s Disease 
Human gene therapy  2004;15(12):1177-1196.
Parkinson’s disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to l-3,4-dihydroxyphenylalanine (l -DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts l -DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper virus-free herpes simplex virus type 1 vectors that coexpress the three dopamine biosynthetic enzymes (TH, GTP CH I, and AADC; 3-gene-vector) or these three dopamine biosynthetic enzymes and the vesicular monoamine transporter (TH, GTP CH I, AADC, and VMAT-2; 4-gene-vector) were compared. Both vectors supported production of dopamine in cultured fibroblasts. These vectors were microinjected into the striatum of 6-hydroxydopamine-lesioned rats. These vectors carry a modified neurofilament gene promoter, and γ-aminobutyric acid (GABA)-ergic neuron-specific gene expression was maintained for 14 months after gene transfer. The 4-gene-vector supported higher levels of correction of apomorphine-induced rotational behavior than did the 3-gene-vector, and this correction was maintained for 6 months. Proximal to the injection sites, the 4-gene-vector, but not the 3-gene-vector, supported extracellular levels of dopamine and dihydroxyphenylacetic acid (DOPAC) that were similar to those observed in normal rats, and only the 4-gene-vector supported significant K+-dependent release of dopamine.
OVERVIEW SUMMARY
Gene therapy treatments may benefit the management of Parkinson’s disease (PD). In the present study, we used a helper virus-free herpes simplex virus type 1 (HSV-1) vector system and a modified neurofilament heavy gene promoter that supports long-term expression in forebrain neurons. We coexpressed tyrosine hydroxylase, GTP cyclohydrolase I, aromatic amino acid decarboxylase, and vesicular monoamine transporter 2 in striatal cells in the 6-hydroxydopamine rat model of PD. Recombinant gene expression was maintained for 14 months in γ-aminobutyric acid (GABA)-ergic striatal neurons. Long-term behavioral (6 months) and biochemical (3 months) correction was observed with high K+-dependent release of significant levels of dopamine. These results suggest that HSV-1 vectors that coexpress multiple dopamine biosynthetic and transporter genes have promise for developing gene therapy treatments for PD.
doi:10.1089/hum.2004.15.1177
PMCID: PMC2581868  PMID: 15684695
22.  Correction of a Rat Model of Parkinson’s Disease by Coexpression of Tyrosine Hydroxylase and Aromatic Amino Acid Decarboxylase from a Helper Virus-Free Herpes Simplex Virus Type-1 Vctor 
Human gene therapy  2003;14(5):415-424.
We previously reported long-term biochemical and behavioral correction of the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease (PD) by expression of tyrosine hydroxylase (TH) in the partially denervated striatum, using a herpes simplex virus type 1 (HSV-1) vector. This study had a number of limitations, including the use of a helper virus packaging system, limited long-term expression, and expression of only TH. To address these issues, we developed a helper virus-free packaging system, a modified neurofilament gene promoter that supports long-term expression in forebrain neurons, and a vector that coexpresses TH and aromatic amino acid decarboxylase (AADC). Coexpression of TH and AADC supported high-level (80%), behavioral correction of the 6-OHDA rat model of PD for 5 weeks. Biochemical correction included increases in extracellular dopamine and DOPAC concentrations between 2 to 4 months after gene transfer. Histologic analyses demonstrated neuronal-specific coexpression of TH and AADC at 4 days to 7 months after gene transfer, and cell counts revealed 1000 to 10,000 TH positive cells per rat at 2 months after gene transfer. This improved system efficiently corrects the rat model of PD.
OVERVIEW SUMMARY
Gene therapy has potential for treating Parkinson’s disease (PD). In this study, we used a helper virus-free herpes simplex virus type 1 (HSV-1) vector system and a modified neurofilament gene promoter that supports long-term expression in forebrain neurons. We coexpressed tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC) in striatal cells in the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease (PD). Biochemical (2–4 months) and behavioral (5 weeks) correction was observed. TH and AADC were expressed for at least 7 months. These results indicate the promise of helper virus-free HSV-1 vectors for developing gene therapy of PD.
doi:10.1089/104303403321467180
PMCID: PMC2581862  PMID: 12691607
23.  Supine Low Frequency Power of Heart Rate Variability Reflects Baroreflex Function, Not Cardiac Sympathetic Innervation 
Background
Power spectral analysis of heart rate variability (HRV) has been used to indicate cardiac autonomic function. High-frequency power relates to respiratory sinus arrhythmia and therefore to parasympathetic cardiovagal tone; however, the relationship of low-frequency (LF) power to cardiac sympathetic innervation and function has been controversial. Alternatively, LF power might reflect baroreflexive modulation of autonomic outflows.
Objective
We studied normal volunteers and chronic autonomic failure syndrome patients with and without loss of cardiac noradrenergic nerves in order to examine the relationships of LF power with cardiac sympathetic innervation and baroreflex function.
Methods
We compared LF power of HRV in patients with cardiac sympathetic denervation, as indicated by low myocardial concentrations of 6-[18F] fluorodopamine-derived radioactivity or low rates of norepinephrine entry into coronary sinus plasma (cardiac norepinephrine spillover) to values in patients with intact innervation, at baseline, during infusion of yohimbine, which increases exocytotic norepinephrine release from sympathetic nerves, or during infusion of tyramine, which increases non-exocytotic release. Baroreflex-cardiovagal slope (BRS) was calculated from the cardiac interbeat interval and systolic pressure during the Valsalva maneuver.
Results
LF power was unrelated to myocardial 6-[18F] fluorodopamine-derived radioactivity or cardiac norepinephrine spillover. In contrast, the log of LF power correlated positively with the log of BRS (r=0.72, p<0.0001). Patients with low BRS (≤ 3 msec/mm Hg) had low LF power, regardless of cardiac innervation. Tyramine and yohimbine increased LF power in subjects with normal BRS but not in those with low BRS. BRS at baseline predicted LF responses to tyramine and yohimbine.
Conclusions
LF power reflects baroreflex function, not cardiac sympathetic innervation.
doi:10.1016/j.hrthm.2007.07.019
PMCID: PMC2204059  PMID: 17997358
heart rate variability; power spectral analysis; nervous system; sympathetic; fluorodopamine; baroreceptors

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