A Secretory Vesicle Failure in Parkinson's Disease Occurs in Human Platelets

Abstract

Objective: The presence of elevated dopamine (DA) and its major metabolites in the cytosol of neurons has been associated with their vulnerability in Parkinson's disease (PD). Over 99% of the cell's amines are confined to secretory vesicles (SVs), making these structures fundamental in the regulation of cytosolic DA levels. SVs of platelets use similar, if not the same mechanisms to accumulate serotonin in SVs as dopaminergic neurons do to store DA. Hence, any functional defects in platelets probably mirrors events in DA neurons.

Methods: We have isolated fresh platelets from the blood of 75 PD patients, 116 matched controls and 24 patients with Parkinsonism, assaying serotonin handling (basal content, accumulation, secretion and spontaneous leakage).

Results: We found a dramatic decrease in the serotonin content and uptake by SVs, as well as decreased thrombin-induced release by platelets from PD patients but not in those from most Parkinsonism cases. Platelets from PD patients also failed to retain serotonin in SVs.

Interpretation: These findings indicate a functional impairment in the handling of amines by SVs in PD patients. This defect may serve as a biomarker of PD, and the approach described here may be potentially used for the subclinical detection of PD and to establish a platform to assay disease modifying drugs. ANN NEUROL 2022.

FIGURE 1

Secretory granules from the platelets of PD patients have less naïve serotonin and severely reduced serotonin uptake. Platelets were isolated using our optimized protocol to measure serotonin. A. The pooled data from 75 PD patients (red dots) and 116 controls (CTR, black dots), representing the average of duplicate measurements normalized to the total protein. The panel shows the basal serotonin content and its uptake (5‐HT) after 2h incubation with serotonin (10μM). The horizontal green dashed lines indicate the mean and the solid horizontal lines the standard errors: ***p < 0.001 (Mann–Whitney's test). Inset. typical chromatograms from controls and PD patients to quantify serotonin by HPLC with electrochemical detection using isoproterenol (200nM) as an internal standard. B. The serotonin uptake is reduced in PD patients in the range of 3nM to 30μM (n = 59 PD and n = 61 Controls). C. Serotonin uptake after the subtraction of the non‐specific component (1μM reserpine, n = 39 PD and n = 35 controls; Fig 1b SM): means ±95% confidence interval; ***p < 0.001 (ANOVA). D. Pooled data (means ± SEM) from 10 untreated PD patients (red) and 25 random controls (CTR, black), representing the average of the duplicates normalized to the total protein. The horizontal green dashed lines indicate the mean and the solid horizontal lines the standard errors. The panel shows the basal serotonin content and its uptake (5‐HT) after 2h incubation with serotonin (10μM): ***p < 0.001 (Mann–Whitney's test).

FIGURE 2

Secretory mechanisms are impaired in platelets from PD patients. A. Serotonin was quantified in the supernatant (Sup) and the platelet pellet was recovered by centrifugation (Pellet = serotonin not secreted). Platelets from healthy individuals (CTR) and from PD patients (PD) were stimulated for 90s with thrombin (4U/mL): ns, not significant; ***p < 0.001 (ANOVA). B. The fractional release (=serotonin release/(serotonin released + serotonin remaining)) indicated there was more serotonin leakage from PD platelets but less thrombin‐evoked release (n = 43 Control, n = 46 PD): ns, not significant; **p < 0.01; ***p < 0.001 (unpaired Student's t test).