Pure autonomic failure (PAF) is a rare sporadic disorder characterized by autonomic failure in the absence of a movement disorder or dementia and is associated with very low plasma norepinephrine (NE) levels—suggesting widespread sympathetic denervation, however due to its rarity the pathology remains poorly elucidated. We sought to correlate clinical and neurochemical findings with sympathetic nerve protein abundances, accessed by way of a forearm vein biopsy, in patients with PAF and in healthy controls and patients with multiple systems atrophy (MSA) in whom sympathetic nerves are considered intact. The abundance of sympathetic nerve proteins, extracted from forearm vein biopsy specimens, in 11 patients with PAF, 8 patients with MSA and 9 age-matched healthy control participants was performed following a clinical evaluation and detailed evaluation of sympathetic nervous system function, which included head-up tilt (HUT) testing with measurement of plasma catecholamines and muscle sympathetic nerve activity (MSNA) in addition to haemodynamic assessment to confirm the clinical phenotype. PAF participants were found to have normal abundance of the NE transporter (NET) protein, together with very low levels of tyrosine hydroxylase (TH) (P<0.0001) and reduced vesicular monoamine transporter 2 (VMAT2) (P<0.05) protein expression compared with control and MSA participants. These findings were associated with a significantly higher ratio of plasma 3,4-dihydroxyphenylglycol (DHPG):NE in PAF participants when compared with controls (P<0.05). The finding of normal NET abundance in PAF suggests intact sympathetic nerves but with reduced NE synthesis. The finding of elevated plasma ratio of DHPG:NE and reduced VMAT2 in PAF indicates a shift towards intraneuronal NE metabolism over sequestration in sympathetic nerves and suggests that sympathetic dysfunction may occur ahead of denervation.

Introduction

Pure autonomic failure (PAF) is a rare sporadic disorder characterized by profound orthostatic hypotension in association with other features of autonomic failure, but in the absence of neurological features of the other neurodegenerative disorders associated with autonomic failure such as multiple systems atrophy (MSA), Parkinson’s disease (PD) or Lewy body dementia (LBD) [1]. Pathologically, PAF is related to PD and LBD by the finding of the abnormal accumulation of α-synuclein, in the form of Lewy bodies, and thus is now considered a Lewy body disease [2]. In PAF, Lewy bodies have been identified within both peripheral and central nervous system structures, but with preponderance for the peripheral autonomic nervous system, including the sympathetic ganglia and distal sympathetic nerve axons [3,4]. Despite the association of these conditions with the abnormal accumulation of α-synuclein, clinical presentation is varied and the underlying pathophysiology has not been fully elucidated. Adding to the complexity of diagnosis, any of these disorders may present initially with idiopathic orthostatic hypotension and other features of autonomic failure. Progression and prognosis vary considerably between these disorders with PAF being the rarest, but providing the best prognosis. Thus attempts have been made to differentiate these conditions in order to study pathology, define prognosis and guide therapy.

In order to further define the pathobiology of PAF, the study of carefully characterized clinical cohorts is required. In vivo studies to date have focused on differentiating between PAF and other neurodegenerative disorders associated with autonomic failure. PAF, in contrast with PD with orthostatic hypotension (PD + OH) and MSA, has been associated with significantly lower urinary excretion of the sympathetic nervous system neurotransmitter norepinephrine (NE) [5], together with very low circulating levels of NE [68]. These findings have been incorporated into consensus guidelines for the diagnosis of PAF [1].

Functional imaging studies of cardiac sympathetic innervation using (123) I-metaiodobenzylguanidine (MIBG) or [18F] fluorodopamine, as substrates for the NE transporter (NET), a monoamine transporter found in presynaptic sympathetic nerve terminals [9], have demonstrated impaired uptake and release of NE in the heart of patients with PAF and PD + OH, but not in MSA [10,11]. These findings are supported by catheter studies using radiolabelled NE (tritiated NE, [3H]-NE) that have demonstrated a marked reduction in the cardiac release and extraction of NE in PAF [12]. The results of these studies have been interpreted as demonstrating cardiac sympathetic denervation in PAF and PD, but not in MSA [10,11]. Data obtained using post-mortem cardiac biopsies have been consistent with this finding, demonstrating reduced staining for tyrosine hydroxylase (TH); the rate-limiting enzyme in catecholamine synthesis; in the myocardium of patients with a history of PD or PAF, but not in those with MSA. The presence of TH staining has been used as a marker for sympathetic neurons. Reduction or absence of TH staining in this context has been interpreted as sympathetic denervation [13,14]. Consistent with this construct, a post-mortem cardiac biopsy study additionally staining for neuronal elements has confirmed sympathetic denervation in a PD cohort [15].

Pathological studies in life have attempted to better define the underlying pathology in PAF. Skin autonomic innervation assessed using immunofluorescence methods in well-defined cohorts of patients with MSA and PAF demonstrated a reduction in immunoreactivity of dopamine-β hydroxylase (DβH), a membrane-bound enzyme in vesicles within sympathetic nerve terminals, and of a pan-neuronal marker protein gene product 9.5 in PAF, but not in MSA patients when compared with controls [16]. Skin biopsies using DβH as a marker of sympathetic nerves, in addition to specific neuronal markers and calculation of epidermal nerve fibre density, have shown a reduction in the density of sympathetic nerve fibres in PD with good correlation with reduced I-123 MIBG uptake on cardiac scintigraphy [17].

Overall the findings to date indicate skin and cardiac sympathetic denervation in PAF and PD, but not in MSA. As systemic catecholamine levels are significantly lower in PAF than idiopathic PD [7,8], the collective interpretation of the findings has been that there is widespread post-ganglionic autonomic denervation in PAF, compared with a restricted post-ganglionic involvement in PD, and a predominant central defect in autonomic function in MSA [18].

In contrast, an EM study of perivascular sympathetic nerve endings in PAF and MSA has shown intact nerve endings, but with a reduction in catecholamine containing vesicles, which was more marked in the PAF group [19]. A skin biopsy study in a PAF patient demonstrated morphologically normal sympathetic nerves containing α-synuclein aggregates, but absent staining for TH [20], and in a PAF patient, a cardiac biopsy study demonstrated normal nerve morphology in the absence of TH staining [14]—these studies suggested that sympathetic nerves may be present but associated with reduced or absent neurotransmitter content in PAF patients.

In the present study, we postulated that perivascular sympathetic nerves may be present but dysfunctional in PAF. We extensively evaluated the sympathetic nervous system profile of participants presenting with presumed PAF, probable MSA and healthy controls participants, prior to comparing the relative quantities of key sympathetic nerve proteins in forearm vein specimens via Western blot analysis. The evaluation of participants before final group assignation and subsequent Western blot analysis of sympathetic nerve proteins included a clinical screening assessment and subsequent comprehensive assessment of sympathetic nervous system function. Final allocation to the PAF group was made if the detailed study protocol below confirmed autonomic failure in association with very low plasma NE concentrations (supine plasma NE concentration <120 pg/ml), consistent with the 1996 consensus statement definition of PAF [1].

The study protocol was approved by the Alfred Health Human Ethics Committee and conformed to the appropriate National Health and Medical Research Council guidelines on ethical conduct of human research. Written informed consent was obtained from all participants prior to entry into the study.

Materials and methods

The study protocol has been summarized in a flow diagram (Figure 1).

Study flow diagram

Figure 1
Study flow diagram

Abbreviations: BP, blood pressure; HR, heart rate.

Figure 1
Study flow diagram

Abbreviations: BP, blood pressure; HR, heart rate.

Clinical screening

Participants with PAF and MSA were recruited by S.J.C. and M.D.E. from patients referred to their clinical practices. All MSA and PAF participants had undergone assessment by a specialist movement disorders neurologist prior to enrolment in the study. Baseline clinical screening involved a comprehensive systems review questionnaire covering past medical history, medications and questions specifically screening for autonomic failure symptoms and secondary causes of autonomic failure. Results of prior neurological assessment and investigations relevant to the diagnosis of autonomic failure were reviewed. Clinic records and primary care physician records were obtained and reviewed to corroborate the historical duration of symptoms. A clinical examination including assessment of clinic supine and standing blood pressure (BP) and heart rates (HR) was performed. Patients were suspected to have PAF if they had longstanding, persistent, orthostatic hypotension without compensatory HR response, in the absence of confounding medication, in addition to features consistent with generalized autonomic failure, including gastrointestinal, genitourinary and sudomotor dysfunction. Suspected PAF participants had no clinical features of a movement disorder or cognitive impairment following neurological assessment and secondary causes of autonomic failure had been excluded. MSA participants had autonomic failure in association with either Parkinsonism and/or cerebellar dysfunction, with a clinical picture conforming to the 2008 consensus statement for diagnosis of probable MSA [21]. Healthy control participants were recruited using poster advertising at local chapters of the University of the Third Age (U3A). Screening assessments were negative for symptoms of autonomic failure and participants had normal postural HR and BP responses to standing. Control participants were not taking any medications known to effect the cardiovascular or autonomic nervous system and had no history of diabetes, hypertension, cardiovascular diseases or any disorder affecting the autonomic nervous system. Participants in all three groups were in sinus rhythm at the time of testing.

Sympathetic nervous system evaluation

Haemodynamic and sympathetic neurochemical and electrophysiological measurements were made in all three groups. In the presumed PAF and probable MSA group, these findings were used to confirm autonomic failure, and specifically, in the case of PAF participants, to confirm low plasma NE levels, prior to final group assignation. In two patients, who were involved in a pilot study, and who underwent repeat vein biopsy for the present study, total body NE spillover and regional sympathetic activity in the heart had previously been measured. In the current study, haemodynamic, arterial plasma NE, total body NE spillover and muscle sympathetic nerve activity (MSNA) measurements were made during supine rest and head-up tilting (HUT) in order to quantify the sympathetic response to upright posture. In preparation for the study, PAF and MSA participants had medications affecting the cardiovascular or autonomic nervous system withheld prior to the study days whenever possible. All short-acting pressor and antihypertensive agents were withheld for 24–48 h prior depending on their half-life. Fludrocortisone was withheld for 5 days when possible. No MSA participants were taking medications for management of Parkinsonism at the time of the study. For all participants, over the counter preparations including analgesics and vitamins were excluded for 48 h and caffeine, alcohol and heavy exertion were excluded for 24 h prior to the study days. Participants were fasted from 6 am on the study day. All studies were commenced at 9.30 am and performed in a temperature controlled (21°C), quiet, dimly lit room.

Supine rest and HUT table testing

HUT testing was performed to study the effects of gravity on BP, HR, NE release and MSNA. In autonomic failure syndromes, cardiovascular symptoms are most prominent during standing. Incremental HUT was performed after a 30-min post-instrumentation period of supine rest using a motorized tilt bed with a footboard (ABCO Healthcare Dual Action tilt table). A single lead III electrocardiogram (EKG) was recorded continuously throughout the study. A 3 French arterial catheter was placed percutaneously in the non-dominant brachial artery for continuous monitoring of arterial BP and collection of blood samples during the study. A 21-gauge venous cannula was placed in an antecubital vein for a continuous fixed rate infusion of [3H]-NE following a priming bolus. MSNA in post-ganglionic fibres distributed to the skeletal muscle vasculature were recorded by clinical microneurography as described.

HR, BP and MSNA recordings collected during the last 5 min of supine rest were used for analysis. Blood samples were collected at the conclusion of the 30-min rest period, prior to tilting. Participants then underwent progressive HUT at 20, 30, 40 and 60 angles, for 10 min at each angle. Blood samples were collected at the end of each 10-min period. The duration of the supine rest and tilt periods is determined by the time necessary for the [3H]-NE to reach steady state at each time point.

Participants were returned to the supine position at the conclusion of the 60° tilt period or earlier if severe presyncope occurred in association with a systolic BP (SBP) <90 mmHg. All participants were returned to the supine position if SBP fell to <70 mmHg, irrespective of symptoms. As participants in the autonomic failure groups were often unable to continue tilt testing to higher angles, BP, HR and MSNA values reported, and used for comparison, are an average of the values over the last 90–120 s of stable recording taken at the highest tilt angle, prior to returning the participant to supine. Plasma catecholamine levels from blood taken at the highest tilt angle at which steady state was achieved were used as end-tilt values.

Microneurography: MSNA recording

In order to measure peripheral sympathetic nerve activity, recording of MSNA to the leg was undertaken. Multiunit post-ganglionic sympathetic nerve activity was recorded using a tungsten microelectrode (FHC, Bowdoinham, ME, U.S.A.) inserted percutaneously into a muscle fascicle of the peroneal nerve near the head of the fibula, and adjusted until a satisfactory spontaneous muscle sympathetic nerve signal was observed. In the absence of recordable MSNA, sampling from multiple nerve sites was attempted. Confirmation of the position of the electrode within the nerve bundle was confirmed by the presence of afferent muscle and/or skin signals in the absence of any recordable efferent signals. Attempts at obtaining a nerve signal were abandoned after 30 min if no signal was recorded following attempts made at multiple (>five) sites. MSNA bursts included for analysis were pulse synchronous, with a clear rise from the baseline to a peak and subsequent fall back to baseline. Bursts were at least twice the amplitude of the baseline fluctuations [22,23]. MSNA signals were digitized with a sampling frequency of 1000 Hz (Power Labs recording system, model ML785/8SP, AD Instruments). MSNA was analysed over a 90–120 s stable section of recording taken during the last 5 min of supine rest. If MSNA activity was obtained during supine rest recording was continued during HUT. MSNA is expressed as multiunit nerve burst firing frequency (bursts.min−1) and normalized for HR, burst incidence (bursts.100 heart beats−1).

Measurement of plasma catecholamines and whole body plasma NE clearance and spillover

Arterial blood for measurement of plasma catecholamines was collected into chilled tubes containing GSH and ethyleneglycoltetraacetic acid (EGTA). Plasma was separated by refrigerated centrifugation (4°C at 1800 rpm) and stored at −80°C for subsequent assay. Samples were assayed using HPLC as previously described [24]. A fixed rate continuous infusion of a tracer dose of [3H]-NE was used to determine the rate of clearance of NE (l/min) from the circulation. This allows adjustment of raw plasma NE concentration values for NE plasma clearance, to report the rate of spillover of NE from sympathetic nerves to plasma [25]. Adjustment of raw values is performed to account for the variation in the rate of fall in clearance of NE from the circulation with assumption of upright posture. Calculations were performed using the following equations:

 
formula
 
formula

The ratio of plasma 3,4-dihydroxyphenylglycol (DHPG), the primary intraneuronal metabolite of NE, to plasma NE was calculated from the sample taken at the conclusion of supine rest. This ratio was used to enable comparison, during basal conditions, of the relative proportion of NE undergoing intraneuronal deamination to DHPG compared with that being recycled following sequestration into sympathetic nerve vesicles [26,27].

Regional assessment of sympathetic activity

Two PAF participants who formed the basis of the pilot study, and who underwent repeat vein biopsies for the present study, underwent assessment of total body NE spillover to plasma and [3H]-NE extraction across the heart as a measure of cardiac sympathetic innervation [12]. The fractional extraction of [3H]-NE across the heart (NEEX) was calculated during a constant-rate infusion of [3H]-NE, with blood sampling from the brachial artery (A) and coronary sinus (CS) [28]. Thus, at steady state:

 
formula

Vein biopsies

To access sympathetic nerves for protein analysis, a forearm vein biopsy was performed under local anaesthesia. In order to access sympathetic nerve proteins, a 1.0–1.5 cm segment of subcutaneous forearm vein was harvested. Veins have been demonstrated to be a rich source of sympathetic nerves [29]. A small subcutaneous vein was identified and marked. Following injection of local anaesthetic, a 1.5-cm skin incision was made. The vein was identified and the segment removed following ligation at each end with absorbable sutures. The skin was closed with interrupted sutures. After removal, the vein was frozen in liquid nitrogen and stored at −80°C until analysis.

Western blot analysis of sympathetic proteins

Tissue was prepared using lysates from vein biopsy specimens. Sympathetic nerve proteins chosen for analysis in the study included NET, TH, vesicular monoamine transporter type 2 (VMAT2) and Dynamin 1. NET is a monoamine transporter found in the central nervous system, presynaptic sympathetic nerve terminals, the adrenal medulla, the lung and in the placenta. Thus in the context of a peripheral vein biopsy specimen it is a specific marker for perivascular sympathetic nerves terminals. TH is the rate-limiting enzyme in NE synthesis and is found within sympathetic nerve terminals. VMAT2 acts to sequester NE within vesicles in the sympathetic nerve terminal. Dynamin 1 is involved in vesicular endocytosis and trafficking in nerve terminals; it is not specific to sympathetic nerves. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Western blot analysis was performed on the extracted proteins. The tissue samples were homogenized in PRO-Prep protein extraction solution (17081, INtRON Biotechnology, Korea). Proteins were separated by electrophoresis on a 10% acrylamide minigel and transferred on to PVDF membranes (NEF1002, PerkinElmer Life Sciences Inc, Waltham, MA, U.S.A.), and detected by incubation with specific primary antibodies followed by further incubation with a peroxidase conjugated anti-IgG and then with ECL reagents (NEL104, PerkinElmer LAS Inc, Buckinghamshire, U.K.). Antibodies used included anti-hNET (NET17-1, MAb Technologies Inc, Neenah, WI), anti-TH (AB152, Millipore, CA, U.S.A.), anti-Dynamin I (3G4B6) (sc-53877, Santa Cruz Biotechnology Inc, Texas, U.S.A.), anti-VMAT2 (V9014, Sigma–Aldrich Inc, MO, U.S.A.) and anti-GAPDH (sc-32233, Santa Cruz Biotechnology Inc, Texas, U.S.A.). Scanned signals were analysed with Quantity One software, version 4.5.2 (Bio–Rad Laboratories Inc., Hercules, CA, U.S.A.).

Statistical analysis

Statistical analysis was performed using SigmaStat version 3.5 (Systat Software Inc, Point Richmond, CA, U.S.A.). Demographic and clinical data between groups were compared using one-way ANOVA for baseline comparisons and two-way ANOVA for repeated measures. Non-parametric data were log transformed as appropriate or analysed using one-way ANOVA on ranks (Kruskal–Wallis test). When a significant difference was found on one-way ANOVA, pairwise comparisons were performed to determine where the difference lay. Adjustments for multiple comparisons were made using the Holm–Sidak method for parametric data and Dunn’s method for non-parametric data. P-values reported for pairwise comparisons are those obtained following adjustment for multiple comparisons using the above methods. Within group comparisons of supine compared with end-tilt values were made using paired ttests. All results are reported as mean ± S.E.M. Values of P<0.05 were considered statistically significant.

Results

Clinical assessment of sympathetic nervous system function

The clinical assessment was undertaken to confirm intact sympathetic nervous system function in the healthy control population and autonomic failure in the PAF and MSA populations prior to inclusion of vein biopsy specimens for analysis. In addition, PAF participants were only included for vein biopsy analysis if supine plasma NE concentration was <120 pg/ml, consistent with the 1996 consensus statement definition of PAF [1]. Key results from the clinical assessments are summarized below and in Table 1.

Table 1
Demographic and HUT data. Mean values for HR, BP and plasma catecholamines during supine rest and at end tilt
 Controls PAF MSA Pbetween groups 
Sex, female/male 5/6 2/11 5/3 0.135 
Age, years (range (years)) 66 ± 1.7 (57–76) 64 ± 2.6 (42–79) 64 ± 3.9 (46–74) 0.730 
OH symptom duration, years 5.8 ± 1.1§ 2.1 ± 0.6 0.016 
HUT testing Supine End tilt Supine End tilt Supine End tilt Supine Δ 
SBP, mmHg 125 ± 5 114 ± 4*** 170 ± 9††† 96 ± 6*** 156 ± 11 96 ± 7*** 0.001  
ΔSBP, mmHg  -11 ± 3  -75 ± 9  -60 ± 14  <0.001 
DBP, mmHg 75 ± 3 71 ± 3 91 ± 3††† 58 ± 3*** 83 ± 4 59 ± 6*** 0.004  
ΔDBP, mmHg  -4 ± 2  - 33 ± 4  -24 ± 7  <0.001 
HR, bpm 61 ± 3 72 ± 3*** 61 ± 4§§ 69 ± 3 77 ± 4†† 84 ±3 0.012  
ΔHR, bpm  11 ± 1  8 ± 3  8 ± 3  0.376 
Plasma NE conc., pg/ml 338 ± 30 487 ± 33** 56 ± 8†,§ 89 ± 17 196 ± 38 328 ± 87** <0.001  
ΔPlasma NE conc., pg/ml  148 ± 37  33 ± 11  132 ± 67  0.082 
Total NE spillover, ng/min 636 ± 75 654 ± 60 118 ± 20†,§ 124 ± 23 445 ± 83 431 ± 94 <0.001  
ΔNE spillover, ng/min  18 ± 47  6 ±12  -14 ± 52  0.755 
Total NE clearance,l/min 1.86 ± 0.13 1.34 ± 0.11*** 2.06 ± 0.14 1.45 ± 0.1*** 2.31 ± 0.23 1.41 ± 0.17*** 0.179  
ΔTotal NE clearance, l/min  -0.52 ± 0.11  -0. 61 ±0.09  -0.89 ± 0.18  0.113 
Plasma DHPG, pg/ml 1349 ± 89 1680 ± 74*** 702 ± 74†††,§§ 756 ± 78 1138 ± 76 1356 ± 98* <0.001  
Δ Plasma DHPG, pg/ml  331 ± 65  55 ± 28  218 ± 70  0.002 
Plasma DHPG/NE ratio 4.3 ± 0.5  14.2 ± 1.7  8.4 ± 2.2  <0.001  
 Controls PAF MSA Pbetween groups 
Sex, female/male 5/6 2/11 5/3 0.135 
Age, years (range (years)) 66 ± 1.7 (57–76) 64 ± 2.6 (42–79) 64 ± 3.9 (46–74) 0.730 
OH symptom duration, years 5.8 ± 1.1§ 2.1 ± 0.6 0.016 
HUT testing Supine End tilt Supine End tilt Supine End tilt Supine Δ 
SBP, mmHg 125 ± 5 114 ± 4*** 170 ± 9††† 96 ± 6*** 156 ± 11 96 ± 7*** 0.001  
ΔSBP, mmHg  -11 ± 3  -75 ± 9  -60 ± 14  <0.001 
DBP, mmHg 75 ± 3 71 ± 3 91 ± 3††† 58 ± 3*** 83 ± 4 59 ± 6*** 0.004  
ΔDBP, mmHg  -4 ± 2  - 33 ± 4  -24 ± 7  <0.001 
HR, bpm 61 ± 3 72 ± 3*** 61 ± 4§§ 69 ± 3 77 ± 4†† 84 ±3 0.012  
ΔHR, bpm  11 ± 1  8 ± 3  8 ± 3  0.376 
Plasma NE conc., pg/ml 338 ± 30 487 ± 33** 56 ± 8†,§ 89 ± 17 196 ± 38 328 ± 87** <0.001  
ΔPlasma NE conc., pg/ml  148 ± 37  33 ± 11  132 ± 67  0.082 
Total NE spillover, ng/min 636 ± 75 654 ± 60 118 ± 20†,§ 124 ± 23 445 ± 83 431 ± 94 <0.001  
ΔNE spillover, ng/min  18 ± 47  6 ±12  -14 ± 52  0.755 
Total NE clearance,l/min 1.86 ± 0.13 1.34 ± 0.11*** 2.06 ± 0.14 1.45 ± 0.1*** 2.31 ± 0.23 1.41 ± 0.17*** 0.179  
ΔTotal NE clearance, l/min  -0.52 ± 0.11  -0. 61 ±0.09  -0.89 ± 0.18  0.113 
Plasma DHPG, pg/ml 1349 ± 89 1680 ± 74*** 702 ± 74†††,§§ 756 ± 78 1138 ± 76 1356 ± 98* <0.001  
Δ Plasma DHPG, pg/ml  331 ± 65  55 ± 28  218 ± 70  0.002 
Plasma DHPG/NE ratio 4.3 ± 0.5  14.2 ± 1.7  8.4 ± 2.2  <0.001  

*P<0.05 compared with supine, **P<0.01 compared with supine, ***P<0.001 compared with supine. Adjusted P-values for multiple comparisons: P<0.05 compared with control, ††P<0.01 compared with control, †††P<0.001 compared with control, §P<0.05 compared with MSA, §§P<0.01 compared with MSA, §§§P<0.001 compared with MSA.

Supine BP and HR measurements are the average of 90–120 s of stable recording taken during the final 5 min of a 30-min supine rest period. End-tilt BP and HR measurements are an average of the final 90–120 s of recording taken at the highest tilt angle immediately prior to returning the participant to supine. All data are expressed as mean ± S.E.M. Abbreviation: DBP, diastolic BP.

Baseline parameters

Demographic data are summarized in Table 1. Thirteen participants with PAF, two from a previous pilot study (unpublished) and eleven further participants, eight participants with MSA and eleven healthy control subjects were included. A further five participants were screened and underwent tilt testing but were not included in the study groups. These five participants had autonomic failure, but with normal supine NE. As they did not fulfill the diagnostic criteria for PAF or MSA, they were excluded from analysis in the present study. PAF patients had a longer duration of symptoms than MSA patients, consistent with the reported progression of these diseases [21,30]. There was no significant age difference between the groups.

Haemodynamic parameters and symptoms

Supine resting and end HUT haemodynamic values are summarized in Table 1.

Supine rest

Supine hypertension is a common finding in autonomic failure and thus, as expected, there was a significant difference in supine SBPs, taken at the conclusion of the supine rest period pretilting, between the autonomic failure groups and control participants.

HUT

The haemodynamic findings and symptoms during the tilt studies in the autonomic failure groups (PAF and MSA) were as expected [16,30,31]. Symptoms prompting return to supine in these groups included fatigue, tiredness, sleepiness, leg weakness, chest pain, pain over the neck and upper back (coat-hanger distribution), mild restlessness, light-headedness or a feeling of heaviness in the head. Only one autonomic failure participant developed any visual disturbance prior to return to supine. No participant with MSA or PAF developed nausea or sweating during the tilt study. All MSA and PAF participants were returned to supine prior to the occurrence of syncope.

HUT testing was undertaken in 11 control participants. Seven participants completed the tilt protocol. Three participants developed typical vasovagal presyncope, with light-headedness, nausea and sweating, and were returned to supine prior to the conclusion of the protocol. One participant had tilt testing terminated early in the 60° tilt angle due to an occluded arterial line. Two control participants fulfilled the diagnostic criteria for orthostatic hypotension with a fall in SBP ≥20 mmHg or fall of diastolic BP (DBP) ≥10 mmHg at the highest tilt angle performed.

MSNA

MSNA recording was performed during supine rest pre-HUT. Recording was attempted in 10 of the 11 PAF participants who underwent HUT, all MSA and 10 of 11 control participants. Recording in one PAF and one control participant was not attempted due to equipment difficulties. In all participants recording attempts were made at no less than five sites over at least a 30-min period. If no afferent of efferent activity was detected after these attempts, further recording attempts were not undertaken. Representative traces are shown in Figure 2. None of the PAF participants had clearly identifiable efferent MSNA activity [16]. MSNA findings were heterogeneous in the MSA group. Efferent MSNA was recorded in two MSA participants with a mean burst frequency of 49 ± 2 bursts.min−1 and a burst incidence of 79 ± 0 bursts.100 heart beats−1. A further MSA participant had a mixed muscle and skin signal recorded throughout that could not be analysed. No efferent activity could be recorded in five MSA participants. MSNA was recorded in all ten control participants in whom it was attempted with a mean burst frequency of 41± 3 bursts.min−1 and a burst incidence of 67 ± 6 bursts.100 heart beats−1.

Representative tracings from control, MSA and PAF participants taken during supine rest

Figure 2
Representative tracings from control, MSA and PAF participants taken during supine rest

The control participant tracings demonstrate normal MSNA with pulse synchronous bursts coupled to oscillations in HR and intra-arterial BP (I.A. BP). Pulse-synchronous MSNA activity was present in this MSA participant, but without coupling to HR and BP oscillations as seen in control participants. Although, on occasion, rare MSNA bursts could be heard in PAF participants, they were unable to be unequivocally identified on recorded tracings. Abbreviation: bpm, beats per min.

Figure 2
Representative tracings from control, MSA and PAF participants taken during supine rest

The control participant tracings demonstrate normal MSNA with pulse synchronous bursts coupled to oscillations in HR and intra-arterial BP (I.A. BP). Pulse-synchronous MSNA activity was present in this MSA participant, but without coupling to HR and BP oscillations as seen in control participants. Although, on occasion, rare MSNA bursts could be heard in PAF participants, they were unable to be unequivocally identified on recorded tracings. Abbreviation: bpm, beats per min.

Plasma NE concentration, whole body plasma NE clearance and spillover, DHPG:NE ratio

Supine and end-tilt values for plasma NE and DHPG concentrations are displayed in Table 1.

Supine rest

Mean supine plasma NE concentration and total body NE spillover were significantly lower in PAF participants compared with control and MSA participants. The plasma concentration of DHPG, was significantly lower in PAF participants compared with MSA and healthy control participants, however the ratio of plasma DHPG:NE was significantly higher in PAF, but not MSA participants, compared with controls (Table 1.)

HUT

There was a significant increase in plasma NE from supine to end tilt in MSA and control participants but not in the PAF group. As expected, NE clearance fell significantly in all three groups between supine and end tilt [31,32]. However, following adjustment for the fall in NE clearance, mean whole body NE spillover values did not significantly increase from supine to end tilt in any of the three groups.

Regional sympathetic activity

Two participants with PAF, who formed the basis of a previous pilot study (unpublished), underwent a comprehensive catheter-based investigation of regional sympathetic activity, including assessment of total body NE spillover and cardiac extraction of NE. Both participants had very low total body NE spillover rates of 58 and 45 ng/min respectively and markedly reduced fractional extraction of [3H]-NE across the heart of 21.5 and 13.3%. Mean cardiac extraction of [3H]-NE has been demonstrated to be 74% in controls [12].

Western blot analysis

Western blot analysis was performed using forearm vein biopsy specimens obtained from 11 participants with PAF (with gels for TH in 11 and gels for the other proteins in 9), from 8 patients with MSA, and in 9 healthy controls. One PAF participant refused vein biopsy and the veins from two healthy and one PAF participants could not be used due to low protein yield and technical constraints.

Gels are displayed in Figures 3 and 4, and derived means in Figure 5. One outlier VMAT2 value, that from the PAF patient in the first gel lane in Figure 4, was excluded from analysis. This sample had low protein loading and the value was more than four S.D. greater than any other value for the PAF group. Other protein values from this patient were included in all analyses. Figure 3 shows the gels in PAF, MSA and control subjects for NET and TH and Figure 4 for VMAT2 and Dynamin 1, with GAPDH used as the reference protein.

Western blot gels for NET and TH in PAF, MSA and control participants

Figure 3
Western blot gels for NET and TH in PAF, MSA and control participants

Western blot analysis was performed using vein biopsy specimens obtained from 11 participants with PAF, 8 with MSA and in 9 healthy controls. TH abundance was tested in 11 PAF patients, with testing for the other proteins in 9 PAF patients. Two separate TH gels from the PAF patients are shown in juxtaposition in the figure. GAPDH was used as the reference protein.

Figure 3
Western blot gels for NET and TH in PAF, MSA and control participants

Western blot analysis was performed using vein biopsy specimens obtained from 11 participants with PAF, 8 with MSA and in 9 healthy controls. TH abundance was tested in 11 PAF patients, with testing for the other proteins in 9 PAF patients. Two separate TH gels from the PAF patients are shown in juxtaposition in the figure. GAPDH was used as the reference protein.

Western blot gels for VMAT2 and Dynamin 1 in PAF, MSA and control participants

Figure 4
Western blot gels for VMAT2 and Dynamin 1 in PAF, MSA and control participants

Western blot analysis was performed using vein biopsy specimens obtained from 9 participants with PAF, 8 with MSA and 9 healthy controls. GAPDH was used as the reference protein. One outlier VMAT2 value, that from the PAF patient in the first gel lane, was excluded from analysis. The value was more than four S.D. greater than any other value for the PAF group.

Figure 4
Western blot gels for VMAT2 and Dynamin 1 in PAF, MSA and control participants

Western blot analysis was performed using vein biopsy specimens obtained from 9 participants with PAF, 8 with MSA and 9 healthy controls. GAPDH was used as the reference protein. One outlier VMAT2 value, that from the PAF patient in the first gel lane, was excluded from analysis. The value was more than four S.D. greater than any other value for the PAF group.

Western blot protein abundances referenced against GAPDH, in arbitrary units

Figure 5
Western blot protein abundances referenced against GAPDH, in arbitrary units

The abundance of NET and Dynamin 1 for PAF and MSA groups were not different from control. TH and VMAT2 abundance were significantly reduced in PAF participants compared with control and MSA participants. Means ± S.E.M. are shown; *P<0.05, ****P<0.0001.

Figure 5
Western blot protein abundances referenced against GAPDH, in arbitrary units

The abundance of NET and Dynamin 1 for PAF and MSA groups were not different from control. TH and VMAT2 abundance were significantly reduced in PAF participants compared with control and MSA participants. Means ± S.E.M. are shown; *P<0.05, ****P<0.0001.

The abundance of NET and Dynamin 1 did not differ between PAF, MSA and control participants. TH and VMAT2 abundance were however significantly reduced in PAF participants compared with control and MSA participants (TH, P<0.0001; VMAT2, P<0.05). In MSA participants, the four proteins did not differ significantly from control protein abundance (Figure 5).

Discussion

Advances in elucidating the neural biology of PAF have been limited by the rarity of the disease and the difficulty in assessing neural biology in living patients who have undergone detailed autonomic assessment to confirm the clinical diagnosis. In the present study, we undertook a comprehensive assessment of participants presenting with likely PAF, probable MSA and healthy control participants to confirm the clinical phenotype, prior to assessing the relative abundance of key sympathetic proteins. Our novel findings are in contrast with much of the published literature and suggest that perivascular sympathetic nerves are present in PAF but with a functional defect.

PAF patients within our cohort had longstanding well-documented autonomic failure in the absence of a movement disorder or dementia in association with very low plasma NE concentrations together with the absence of post-ganglionic sympathetic nerve firing. The clinical findings are consistent with the consensus definition of PAF [1] and with the belief, to date, that this disorder is associated with sympathetic denervation [33]. In this PAF, cohort however we have demonstrated the presence of normal levels of NET protein in a peripheral vein specimen. NET is a key sympathetic nerve protein found in sympathetic nerves. If sympathetic denervation had occurred, low or absent NET protein levels would be expected. The normal NET protein levels were observed in association with reduced levels of VMAT2 protein, and very low levels of TH protein. Together these findings indicate that sympathetic nerve degeneration is not present in venous tissue in patients with PAF, but rather suggest there is dysfunction of sympathetic nerves.

We could not detect MSNA efferent activity in our PAF cohort, but also in five of the eight MSA participants. The absence of recordable efferent MSNA in PAF is an expected finding in this condition and to date has been considered indicative of post-ganglionic sympathetic denervation. There are few reports of MSNA findings in MSA. A recent report however demonstrated absent MSNA in 75% of a well-defined cohort of MSA patients and was considered to be the result of functional inactivity of post-ganglionic sympathetic fibres secondary to a preganglionic lesion [16]. The mechanism of absence of efferent muscle sympathetic activity in PAF and MSA thus requires further investigation, as taken together absent MSNA in these disorders cannot be equated with post-ganglionic sympathetic denervation and may alternatively be the result of nerve dysfunction.

MSNA recordings were consistently obtained in the control group. The mean supine MSNA values are consistent with those previously documented in aging populations [3436]. Three control participants developed vasovagal presyncope. False positive tilt test results are well documented in control populations [37]. The supine rest plasma NE values documented in our control cohort are consistent with previous findings in older control populations [31,38]. Total body NE spillover to plasma has previously been documented to increase significantly in response to HUT in control populations [31,39]. Although we documented an increase in plasma NE concentration, we did not demonstrate an increase in total body NE spillover in response to HUT in our control cohort. Sampling of total body NE spillover may not, however, reflect a regional increase in sympathetic outflow to the venous capacitance vessels evoked by HUT testing. Only one prior study has included plasma NE concentration and spillover responses to tilt in an older cohort [31]. Our results differ from this report and add to the body of literature in a healthy older population.

As expected in PAF, we have shown very low levels of TH protein in our PAF group compared with MSA and control participants, consistent with studies of skin and cardiac sympathetic innervation in PAF using immunolabelling techniques [14,20]. We did not measure DβH, another enzyme commonly used as a marker for sympathetic nerves [16,17]. A reduction in the expression of TH, as the rate-limiting enzyme in catecholamine synthesis, will result in a reduction in synthesis of NE. TH regulation is complex and there are multiple mechanisms by which a reduction in TH expression could occur in intact nerves, including a reduction in activation by phosphorylation or an increase in feedback inhibition, which maintains low cytosolic levels of catecholamines and thus reduces the production of reactive neurotoxic catecholamine metabolites [40]. PAF is considered a Lewy body disorder, and thus associated with accumulation of aggregated α-synuclein. α-synuclein has a role in the regulation of TH activity. Increased expression of α-synuclein has been demonstrated to result in a reduction in TH activity due to a reduction in phosphorylation at the key Ser40 residue [41]. Aggregated α-synuclein has however been associated with an increase in TH phosphorylation, suggesting that once aggregated it is no longer able to inhibit TH phosphorylation and thus TH activity increases [42]. Thus in PAF, an increase in α-synuclein expression, prior to aggregation, could result in a reduction in TH activity. Feedback inhibition in response to increased cytosolic levels of NE with subsequent down-regulation of TH, rather than sympathetic denervation, is another possible explanation for a reduction in TH protein abundance. Both dopamine and NE bind to inhibitory regulatory domains on TH. This mechanism is sensitive to increase in the cytosolic concentration of the catecholamines and is one mechanism by which the cytosolic levels of catecholamines are kept low to minimize the metabolism to toxic metabolites [43].

In our study, we found low levels of VMAT2 protein in our PAF cohort compared with control and MSA participants and a significantly elevated ratio of plasma DHPG:NE in the PAF cohort compared with control participants. The implications of these findings must be considered in the context of NE metabolic pathways. Analysis of the pattern of break down of sympathetic amines can be useful in assessing metabolic pathways and may reflect protein function. During normal sympathetic nerve function, approximately 90% of NE released from sympathetic terminals during sympathetic nerve firing is taken back up into sympathetic nerves via the action of NET, with only a very small percentage of released NE spilling over into the circulation [9]. The small percentage of NE that spills over into the circulation is metabolized in extraneuronal locations by catechol-O-methyltransferase (COMT) to normetanephrine and by the subsequent action of monoamine oxidase (MAO) and aldehyde dehydrogenase (AD) to vanillylmandelic acid (VMA). This pathway however represents only a minor pathway for NE metabolism. Following reuptake via NET, NE within sympathetic terminals is returned to sympathetic vesicles via the action of VMAT2. Passive leakage of NE from vesicles also occurs, however the cytoplasmic concentration of NE remains low due to the reuptake of 90% of NE from the cytosol into the vesicles, again by the action of VMAT2 [44]. This process limits the intraneuronal metabolism of NE, by MAO to the toxic metabolite 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and then by the action of aldehyde reductase (AR) to DHPG [44]. As most NE is taken back up into sympathetic nerve terminals after release, the majority of NE metabolism thus occurs within the cytoplasm of sympathetic nerves rather than in extraneuronal locations [44]. DHPG therefore constitutes a major circulating intermediary metabolite of NE and at rest is predominantly derived from the intraneuronal metabolism of cytosolic NE that has leaked from storage vesicles [44]. We have demonstrated low levels of VMAT2 protein, the protein responsible for vesicular sequestration of NE. A reduction in VMAT2 if it occurred as a result of denervation and thus was proportionate to a reduction in sympathetic nerves, would result in a reduction in NE and its metabolite DHPG, but with maintenance of the DHPG:NE ratio. However, a reduction in VMAT2 expression, and thus activity, in intact nerves will result in a reduction in the synthesis of NE secondary to reduced uptake of cytosolic dopamine, but also a reduction in the recycling of NE into vesicles after reuptake by NET and following the passive leakage from vesicles, the outcome of which will be an increase in the DHPG:NE ratio despite overall lower levels of NE and DHPG [45]. Consistent with this finding, a reduction in the vesicular uptake of NE in an experimental VMAT2-deficient mouse model has shown a shift from vesicular sequestration towards the intraneuronal oxidative deamination of NE via MAO and AR to DHPG, with an increased ratio of DHPG:NE in myocardial tissue of VMAT2-deficient mice [27].

Taken together the finding of normal levels of NET protein together with low levels of VMAT2 protein in association with low TH protein abundance and low plasma NE concentrations, but an increase in the ratio of plasma DHPG:NE, suggest that the defect resulting in the PAF phenotype may be a reduction in NE vesicular storage with subsequent down-regulation of TH via feedback inhibition, and thus of catecholamine synthesis within sympathetic nerves, rather than primary denervation. Our findings are consistent with the recent report by Goldstein in a Parkinson’s cohort suggesting a defect in vesicular storage of dopamine with a shift from vesicular sequestration towards intraneuronal oxidative deamination [27,45]. As PAF and PD are both characterized by the accumulation of α-synuclein in the form of Lewy bodies, this finding in the two disorders is further support of a pathophysiological link between the disorders.

The findings in the present study are, however, in contrast with the majority of the literature regarding PAF [33], and thus consideration of our findings within this context, with discussion of the discrepancies, must be made. In our study we assessed MSNA in the peroneal nerve, whole body NE spillover to plasma, whole body plasma DHPG concentration and assessed sympathetic proteins in forearm vein specimens. Differential sympathetic dysfunction in various organs and restricted denervation has been considered to occur in patients with PD, a related Lewy body disorder [18]. Previous studies in PAF have assessed whole body or cardiac-specific catecholamine kinetics, leg MSNA and/or sympathetic neuropathology of the heart, brain or skin [12,14,16,20,31]. Functional studies and tissue biopsies have not always been correlated in individual cohorts, and heart and brain specimens are, in general, obtained post mortem. Histological studies of cardiac tissue from patients with PD and PAF have demonstrated a marked reduction in TH positive nerves in post-mortem cardiac biopsies but with normal nerve fibre morphology on H&E staining [14]. Other studies have additionally demonstrated a reduction in staining for neurofilament, together with reduced TH immunoreactivity in post-mortem cardiac biopsy specimens in PD, consistent with the denervation hypothesis [15]. Biopsy specimens from post-mortem studies, by virtue of their indication, occur late in disease. As few patients assessed histologically at autopsy had undergone functional cardiac imaging in life [13] and as it is not possible to routinely obtain cardiac tissue at the time of functional studies to allow direct corelation beween imaging studies and histological findings, post-mortem findings of cardiac sympathetic denervation do not exclude dysfunction of sympathetic nerves prior to denervation at an earlier stage in the disease process.Thus the discrepancy between our findings and those in other studies may relate to methods used or a differential time course from dysfunction to denervation in different tissues. Cardiac sympathetic dysfunction is known to occur early in the neurodegenerative diseases associated with autonomic failure. Cardiac imaging studies using catecholamine analogues (123I-MIBG or 6-[18F]fluorodopamine PET) to measure cardiac uptake and subsequent washout of the analogues have been employed to assess cardiac sympathetic innervation in neurodegenerative disorders [6,10,46]. Reduced or absent uptake and/or increased washout has until recently been interpreted as indicative of cardiac sympathetic denervation. Goldstein et al. [45] have, however, recently proposed that reduced 6-[18F] fluorodopamine uptake on PET scanning in patients with PAF or PD is due to sympathetic denervation, with increased washout being secondary to impaired vesicular storage. NET dysfunction rather than sympathetic denervation has also been proposed as an explanation for the finding of reduced cardiac uptake of 123I-MIBG or reduced cardiac extraction of [3H]-NE in heart failure, stress cardiomyopathy and panic disorder—conditions associated with an increase in cardiac sympathetic drive [47].

The use of surrogate markers, rather than direct visualization of sympathetic nerve terminals, and the difficulty of accessing nerve tissue during life have limited the elucidation of the progression of sympathetic nerve pathology in patients with PAF. Thus due to ease of access, skin biopsy has become the predominant tissue in which sympathetic denervation has been demonstrated in the Lewy body diseases. Some but not all studies have used specific neuronal markers to correlate nerve loss with the findings of reduced staining for TH and DβH [14]. Hypohidrosis, indicative of sudomotor dysfunction, is an early clinical feature of PAF [48]. Thus, the finding of denervation in skin biopsies cannot necessarily be extrapolated to other tissues, as a differential time course from dysfunction to denervation in different tissues cannot be excluded [14].

The present study is, to our knowledge, the first to assess perivascular sympathetic nerve biology during life in patients who have undergone comprehensive clinical assessment at the same time point. The key findings from our study, in a group of well-characterized PAF patients, are the demonstration of normal levels of NET protein, in association with low levels of TH and VMAT2 proteins, low plasma NE concentrations and an increase in the plasma ratio of DHPG:NE; observations that indicate the presence of sympathetic nerves, but with a reduction in NE synthesis. The reduced VMAT2 protein together with an increased plasma ratio of DHPG:NE suggests that this may occur as a result of a reduction in NE vesicular storage, with subsequent down-regulation of TH and thus NE synthesis. These findings are in contrast with the MSA cohort, where there was no difference in the abundance of any of the key proteins when compared with controls, but a significant difference between VMAT2 and TH abundance when compared with PAF participants—consistent with the consensus that autonomic failure in MSA is predominantly secondary to a central defect with intact peripheral sympathetic nerves compared with a defect of peripheral sympathetic nerve function in PAF. The MSNA findings, with inability to record MSNA in any PAF patient, but also in five of eight MSA patients, suggests that denervation cannot be evoked as the only mechanism resulting in absence of MSNA recording in these neurodegenerative disorders.

Although our study is in contrast with many prior studies of PAF, our findings are consistent with recent work [27,45] in sporadic PD, another Lewy body disease, and with the single EM study of sympathetic nerve endings [19], thus providing further support for a construct of sympathetic neuronal dysfunction as the primary defect prior to denervation in these disorders. Accumulating data regarding the significance of VMAT2 dysfunction [45] as the early pathology resulting in sympathetic nerve dysfunction opens new avenues for research into the pathophysiology of the Lewy body disorders. There are a number of limitations to our study. The sample sizes were small. This reflects both the rarity of PAF and the difficulty of performing invasive studies off medications in both the PAF and MSA. As with most of the prior studies that have attempted to define nerve biology in autonomic failure, we have used immunolabelling techniques rather than direct visualization of nerve terminals. Future study would be strengthened by the inclusion of larger cohorts and additionally performing electron microscopic imaging of sympathetic nerve varicosities in biopsy specimens from which NET, TH and VMAT2 content has been determined, thus enabling correlation between nerve varicosity morphology, including the number and size of catecholamine containing vesicles, and relative protein abundances.

Clinical perspectives

  • The present study was undertaken to better define the neural biology of PAF, a rare degenerative neurological disorder resulting in autonomic failure in the absence of a movement disorder or dementia, in a well-defined cohort of patients.

  • They key findings of the present study are, that in the presence of very low levels of circulating NE, there was a normal abundance of the NET protein, a specific sympathetic nerve protein, together with very low TH abundance and a reduction in abundance of the vesicular monoamine transported protein abundance (VMAT2) in human forearm veins. Together with an increase in the ratio of plasma DHPG:NE in PAF participants, these findings point to a defect in the synthesis and intraneuronal recycling of NE, without denervation, in forearm veins—suggesting there may be abnormal function, ahead of denervation, in perivascular sympathetic nerves in PAF.

  • PAF has been shown to be associated with the accumulation of Lewy bodies and thus related to other Lewy body disorders associated with autonomic failure—in particular PD and LBD. The present study is therefore important as it advances our understanding of the underlying mechanisms resulting in sympathetic nerve dysfunction in a Lewy body disorder.

We thank the study participants for their time, co-operation and effort, and research nurses Donna Vizi and Jenny Starr (Alfred Baker Medical Unit) for their assistance.

Funding

This work was supported by the National Health and Medical Research Council (NHMRC) Project [grant number 1012574]; the NHMRC Fellowships [1081143 (to M.D.E.) 1042492 (to G.W.L.)].

Competing interests

Research Grants and Teaching Honoraria from Medtronic (to M.D.E.); Consultancy for Medtronic and Honoraria from Medtronic (to G.W.L.), Pfizer (to G.W.L.)]and Wyeth Pharmaceuticals for presentations (to G.W.L.); Teaching Honoraria from Medtronic Pty Ltd ( S.J.C.); and Servier Pharmaceuticals (to S.J.C.). These organizations played no role in the design, analysis or interpretation of data described here, nor in the preparation, review or approval of the manuscript.

Author contribution

L.G. conceived the study, conducted the pilot study, performed the Western Blot analyses and critically revised the manuscript, M.D.E designed the study, recruited participants, supervised the project, interpreted the results and critically revised the manuscript. C.S and E.A.L. assisted with the HUT studies, and collection and analysis of sympathetic nervous system data and critically revised the manuscript. S.P. performed the HPLC catecholamine measurements. N.E.S. critically revised the manuscript and assisted with the statistical interpretation of the data. G.W.L. interpreted the results and critically revised the manuscript. S.J.C. designed the study, recruited and performed clinical screening of participants, performed the HUT studies and vein biopsies, collected sympathetic nervous system data, analysed and interpreted the results and wrote the manuscript.

Abbreviations

     
  • AR

    aldehyde reductase

  •  
  • BP

    blood pressure

  •  
  • DHPG

    3,4-dihydroxyphenylglycol

  •  
  • DβH

    dopamine-β hydroxylase

  •  
  • GAPDH

    glyceraldehyde 3-phosphate dehydrogenase

  •  
  • HR

    heart rate

  •  
  • HUT

    head-up tilt

  •  
  • LBD

    Lewy body dementia

  •  
  • MAO

    monoamine oxidase

  •  
  • MIBG

    (123) I-metaiodobenzylguanidine

  •  
  • MSA

    multiple systems atrophy

  •  
  • MSNA

    muscle sympathetic nerve activity

  •  
  • NE

    norepinephrine

  •  
  • NET

    NE transporter

  •  
  • PAF

    pure autonomic failure

  •  
  • PD

    Parkinson’s disease

  •  
  • PD + OH

    PD with orthostatic hypotension

  •  
  • SBP

    systolic BP

  •  
  • TH

    tyrosine hydroxylase

  •  
  • VMAT2

    vesicular monoamine transporter type 2

  •  
  • [3H]-NE

    tritiated NE

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