Serotonin content and uptake in platelets can distinguish Parkinson's disease from control patients and essential tremor

Abstract

We have demonstrated that platelets from Parkinson's disease patients exhibit impaired serotonin uptake and release. This seems to be caused by a dysfunction in secretory vesicles (SecVs), which share the mechanisms of accumulation with dopaminergic neurons. As a working hypothesis, this dysfunction may create an excess of dopamine causing neurotoxicity a process absent in other conditions like Essential Tremor or Essential Tremor Plus. The serotonin functional analysis in platelets is a potential tool to reduce misdiagnosis.

To validate this, we performed a functional study using liquid chromatography (UHPLC) to analyze serotonin content, uptake, and secretion in platelets from 50 Parkinson's disease patients, 23 essential tremor, 17 essential tremor plus, and 50 controls.

Parkinson's disease patients show impaired platelet serotonin content and uptake vs. Controls (P < 0.001), essential tremor (P < 0.01) and essential tremor plus (P < 0.05), ANOVA. Notably, no significant differences were found between controls and essential tremor nor essential tremor plus; although, essential tremor plus manifested mild serotonin storage dysfunction.

Platelet serotonin dysfunction reflects distinct pathological origins between Parkinson's disease and other tremor syndromes, suggesting that platelet serotonin analysis is a viable and objective tool for differential diagnosis between Parkinson's disease and tremor syndromes.

Plain Language Summary

A test in blood platelets can distinguish Parkinson's from other tremors

Parkinson's disease can be difficult to distinguish from other tremor disorders, especially when symptoms first appear. In our study, we discovered that platelets (a type of blood cell) from people with Parkinson's disease show an abnormal ability to handle serotonin, a chemical messenger stored in cells using mechanisms similar to those for dopamine. This change was not found in people with essential tremor. Based on this difference, we describe a simple blood-based test that may help doctors distinguish Parkinson's disease from other tremor conditions. This serotonin-based test has potential as a biomarker to improve early and accurate diagnosis.

Introduction

Essential tremor and Parkinson's disease are the two most prevalent movement disorders worldwide. ¹ In the absence of definitive biological markers, their diagnosis remains primarily reliant on clinical history and physical examination findings.^2,3

The diagnosis of Essential tremor relies on the Consensus Statements of the International Parkinson and Movement Disorder Society.^3,4 This consensus recognized a subgroup of patients with Essential tremor with a more complex phenotype and introduced a new classification: Essential tremor plus. Those patients exhibit Essential tremor features alongside subtle neurological signs (e.g., resting tremor, dystonia, impaired tandem gait, memory impairment) that are insufficient to establish an alternative diagnosis.^5,6

Resting tremor is present in 63% to 89% of essential tremor patients^5,7 while between 39% and 89% of Essential tremor cases meet the criteria for Essential tremor plus.

Action tremor, which is characteristic of Essential tremor,^3,4 is also observed in Parkinson's disease. ⁸ Age-related slowing of motor function may be mistaken for bradykinesia, ⁹ and in patients with pre-existing Essential tremor, this may lead to a misdiagnosis of Parkinson's disease. ⁹ The co-occurrence of Parkinson's disease and Essential tremor has been reported in several clinical and epidemiological studies.^1,10,11 Due to overlapping phenotypes, clinical distinction between Essential tremor and Parkinson's disease can be challenging in some cases. ¹² While functional imaging, such as dopamine transporter scans (DaTSCAN), can aid in differential diagnosis with good accuracy, ¹³ its limited accessibility and high cost prevent its routine use in general neurological practice. ¹⁴ Consequently, there is an urgent need for accessible, objective laboratory tools to distinguish these overlapping phenotypes.

Secretory vesicles (SecVs) serve as the primary reservoirs for neurotransmitters, including the biological amines dopamine and serotonin. Dysfunctional SecVs storage could lead to excessive free cytosolic dopamine, a key factor in Parkinson's disease pathogenesis. ¹⁵ We have accumulated ample experience with SecVs from chromaffin cells^16–19 which has now been applied to other cell types. Importantly, the mechanisms for serotonin accumulation in SecVs are shared with other amine-storing cells, such as dopamine in dopaminergic neurons. Given these functional similarities, we hypothesize that similar impairments in SecVs function could occur in platelets of Parkinson's disease patients.

Platelets, irregularly shaped anucleate cell fragments circulating in the bloodstream, are involved primarily in coagulation but also in storage and release of serotonin via SecVs (dense- or δ-granules). We previously demonstrated impaired serotonin content and uptake capacity, decreased secretion, and increased spontaneous leak-out in Parkinson's disease patient platelets compared to matched controls. ²⁰ Our current functional tests in platelets can blindly identify Parkinson's disease vs. control with ≈90% of reliability, suggesting a SecVs functional failure and a performance comparable to α-synuclein seeding assay in cerebrospinal fluid. ²¹ It is therefore expected that a serotonin-based test could serve as a useful tool for the differential diagnosis of tremors.

Since the etiology of tremor syndromes differ fundamentally from the neurodegenerative pathway of Parkinson's disease, we postulate that SecVs function should remain unaffected in those syndromes, Therefore, measuring platelet serotonin turnover could provide an objective physiological signature for differential diagnosis. In this study, we demonstrate that serotonin levels are normal in Essential tremor, partially affected in Essential tremor plus, and significantly altered in Parkinson's disease. To our knowledge, this is the first laboratory tool capable of distinguishing these tremor-associated diseases.

Materials and methods

A detailed description of the Methods can be found in the Supplementary material (SM).

Study oversight and patient consents. This study was approved by the Ethical Committees at the University of La Laguna (CEIBA2017-0244) and the Canary Islands Health Services (CHUC202080) Written informed consent was obtained from all participants. The study enrolled 23 essential tremor and 17 essential tremor plus patients. To ensure balanced statistical comparisons, we reduced our growing current database of 173 Parkinson's disease patients and 288 controls to 50 each, using a pseudorandomization routine (RAND function, Microsoft Excel; see SM for details). Gender and age restriction were not considered for the final pseudo-randomization procedure, since introducing those factors did not significantly alter the results. Based on the ratio of control and ET + patients being around 3.125, a sample size study suggests a minimum number of 35 controls/10 ET + for the AUC we obtained (MedCalc Software Ltd Ostend, Belgium). Tremor-syndrome patients were recruited specifically for this study, and their data were compared against this sub-cohort of Parkinson's disease patients and controls. Demographic and clinical assignments are summarized in Table 1 of SM. DaTSCAN imaging was utilized as a complementary diagnostic tool in cases of clinical uncertainty (i.e., early-stage Parkinson's disease, differential diagnosis between Parkinson's disease and other tremor syndromes, or in patients with atypical symptoms and/or lack of response to treatment). Recent literature does not provide evidence that Parkinson's medications such as L-DOPA, rasagiline, safinamide, apomorphine, deprenyl, and pramipexole alter platelet serotonin handling, including in studies of co-administration with antidepressants.^22,23

Platelet preparation. Two blood samples of 9 mL were obtained by venipuncture (BD-Vacutainer^®, with 18 mg K₂-EDTA: Plymouth, UK). Platelets were purified at room temperature by sequential centrifugations and maintained in dark and a humidified atmosphere at room temperature by gentle rotation. All assays were conducted within 24–48 h of isolation.

Serotonin uptake and release assays. Platelets were incubated in the dark at room temperature (22°C) with increasing concentrations of serotonin (0, 3 nM, 10 nM, 33 nM, 100 nM, 333 nM, 1 µM, 3 µM, 10 µM and 33 µM) for 2 h. To estimate the non-vesicular uptake (non-specific component) platelets were pretreated with 1 µM reserpine for 30 min prior and during serotonin incubations. Specific (net) uptake was defined as the difference between total and non-specific uptake. In release experiments, platelets were incubated with thrombin (0–4 U/mL) for 90 s, collecting both the supernatant containing the released serotonin and remaining platelets pellet.

Quantification of serotonin. Serotonin was quantified using an isocratic UHPLC system (Shimadzu S40 series, Kyoto, Japan) coupled to an electrochemical detector set at +700 mV (DECADE Elite, Antec Scientific, Alphen Rijn, Netherlands). 10 µL samples containing 200 nM of isoproterenol (internal standard) were injected. Chromatograms were recorded using the LabSolutions^® software (Shimadzu).

Protein analysis and diagnostic threshold. Serotonin concentrations were normalized to total protein content (BCA method, Sigma-Aldrich), using BSA for the calibration curve. Results are expressed in nmol serotonin/mg protein. Diagnostic thresholds were established as the mean minus one standard deviation of the control group. Validated threshold values (nmol/mg) were: Basal content < 1.72; Total uptake < 7.49; and Specific uptake < 5.98 (see SM for ROC analysis).

Results

Figure 1 illustrates the dose-dependent serotonin uptake by platelets from Parkinson's disease, essential tremor, essential tremor plus, alongside healthy controls after 2 h serotonin incubation. Net serotonin uptake was determined by subtracting the non- specific component in the presence of the VMAT blocker reserpine. While total serotonin uptake is presented in Figure S1 (Supplementary Material, SM). Total uptake is larger than net uptake as it accounts for net content plus unspecific serotonin binding.

Figure 1.

The serotonin management tests can differentiate Parkinson's disease (pd) from tremor syndromes. Platelets were isolated using our optimized protocol for measuring serotonin (5-HT). Specific (net) serotonin uptake was calculated after subtracting the non-specific component obtained by incubating platelets with the same serotonin concentrations in the presence of 1 µM reserpine (30 min prior and during serotonin incubation). Pooled data are shown as means ± 95% confidence interval from 50 Parkinson's disease patients (PD, red dots), 23 essential tremor (ET, dark blue dots), 17 essential tremor plus (ET+, light blue dots) and 50 controls (Ctrl, black dots), representing the average of duplicate measurements normalized to total protein. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA). Standard deviation was used for statistical purposes and for establish the threshold of “pathological values”. No significant differences were found neither between control and essential tremor nor essential tremor plus (p = 0.52). See data points in Figure 2 of SM.

Since our preliminary report involving 75 Parkinson's disease patients and 116 controls, ²⁰ we have expanded our cohort to 173 Parkinson's disease patients and 288 controls. This robust dataset corroborates our previous findings, confirming a significant impairment in secretory vesicle (SecVs) serotonin accumulation in the Parkinson's disease group.

Following the International Parkinson and Movement Disorder Society criteria we categorized tremor syndromes into essential tremor and essential tremor plus ³ and analyzed the serotonin management in each group. Notably, serotonin management in both essential tremor and essential tremor plus are indistinguishable from controls (p = 0.99 and 0.52 respectively) but drastically differ from Parkinson's disease (Figure 1). In the Supplementary Material, we show detailed information of all patients enrolled in the study.

Comparative analyses revealed differences in naïve (basal) serotonin content and significantly net uptake (at 10 µM serotonin concentradiatetion) between Parkinson's disease and both tremor groups (Figure. 2(A) and (B) SM). ROC/AUC analysis yielded a high discriminatory potential for net serotonin uptake between essential tremor plus and Parkinson's disease, with an Area Under the Curve (AUC) of 0.8319 (95% CI: 0.7214–0.9423), despite the relatively low number of essential tremor plus patients enrolled (See Supplementary Material). These results suggest that the serotonin test is a promising tool for differential diagnosis, with even greater statistical power expected as the tremors sample sizes increase.

Figure 2.

Secretory features of platelets. A, After centrifugation of the platelet suspension, serotonin (5-HT) was analyzed in the pellet (platelet content) and in the supernatant (Sup) both from quiescent platelets (Basal) and after triggering release with 4 U/mL of thrombin for 90 s. B, The fractional release [ = serotonin in supernatant/(serotonin released + serotonin in supernatant)] showed a significant increase in the serotonin leakage only on Parkinson's disease (PD) platelets under basal conditions. Conversely, evoked release of platelets from Parkinson's disease was significantly lower than controls and essential tremor (ET). *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Students’ t test). The other comparisons resulted in non-significant differences. n = 39 Control (Ctrl), 40 Parkinson's disease, 21 essential tremor, and 15 essential tremor plus (ET+).

To assess functional storage, we evaluated the secretory properties of platelets under resting conditions (Basal) and after stimulation with the specific secretagogue thrombin (Throm), comparing tremor syndromes platelet behaviour with those from Parkinson's disease patients and controls (Figure 2(A)).

Platelets from Parkinson's disease patients released quantitatively less serotonin than control. However, when serotonin release is normalized to the total serotonin content, a serotonin leakage is evidenced only in resting (unloaded) platelets from Parkinson's disease patients (Figure 2(B)). This aberrant leak-out of serotonin indicates a failure in the sequestering capacity of platelet δ-granules in Parkinson's disease patients leading to an inability to retain amines within the vesicle and their subsequent accumulation in the cytosol. This phenomenon was absent in essential tremor cases while, interestingly, essential tremor plus patients exhibited intermediate kinetics response to serotonin incubation, further evidencing the biological heterogeneity of this subgroup.

Discussion

The clinical differentiation of tremor syndromes remains a formidable challenge due to significant symptomatic overlap, particularly during the prodromal and early stages of Parkinson's Disease and Essential Tremor. Tremor is a key symptom in both Parkinson's disease and essential tremor, even though these disorders have different origin and affect different CNS regions. ²⁴

While laboratory tests detecting abnormal α-Syn in CSF and blood are under development, ²¹ they face limitations, such as the need for invasive lumbar punctures or interference from α-Syn content in red blood cell. ²⁵ A major challenge is the lack of reliable laboratory tools to differentiate tremor syndromes, as treatment also significantly varies. Our study addresses this diagnostic gap by validating a novel physiological approach based on the “aminergic hypothesis” of Parkinson's disease. This theory posits that neurotoxicity arises from persistently elevated levels of cytosolic dopamine, ²⁶ considering this a consequence of dysfunctional sequestration within secretory vesicles (SecVs). ¹⁵

Data from chromaffin cells show that SecVs can maintain a concentration gradient of amines towards cytosol of ≈100,000 times, as vesicular concentration of epinephrine is around 1 M^27,28 whereas free cytosolic concentrations are ≈ 10 µM. ²⁹ Although the so-called “aminergic hypothesis” as the origin of Parkinson's disease is not widely accepted, our data give support to the neurotoxicity caused by dopamine or norepinephrine when they occur freely in the cytosol. The functional tests presented here demonstrate a clear divergence in platelet serotonin management: essential tremor and essential tremor plus profiles were almost statistically indistinguishable from controls, while Parkinson's disease platelets exhibited a profound deficit (Figure 1). The intermediate kinetic behavior observed in essential tremor plus likely reflects the clinical and biological heterogeneity inherent to this classification, which often encompasses features that subtly mirror Parkinson's disease phenotypes.

Furthermore, our analysis of secretory dynamics revealed that Parkinson's disease-derived SecVs not only release significantly less serotonin upon stimulation but also exhibit an enhanced leak-out or spontaneous efflux (Figure 2(B)). This failure to retain amines within the vesicular lumen suggests a fundamental defect in the electrochemical gradient or the structural integrity of the δ-granules. Crucially, this homeostatic failure was absent in tremor syndromes cases, reinforcing the potential of SecVs competence as a differential biomarker.

From a mechanistic perspective, the sequestering capacity of SecVs relies on a complex interplay between the V-ATPase H⁺ pump, and several exchangers like H⁺/K⁺, VNUT and VMAT.^30,31 Components such as chromogranins and ATP further enhance amine storage.^18,32 A defect in SecVs would likely affect all amine-storing cells, ¹⁵ including platelets, ³³ chromaffin cells, and dopaminergic neurons, leading to dopamine neurotoxicity. ³⁴ Other cytotoxic amines like norepinephrine may also contribute to peripheral neurodegeneration in Parkinson's disease.

Platelets provide a practical model for studying amine storage, as their serotonin accumulation mechanisms mirror those for dopamine in neurons. ³⁵ Our data suggest that the systemic nature of SecVs dysfunction in Parkinson's disease leads to broad aminergic failure, explaining both motor and non-motor symptoms—a pathophysiological signature that is notably absent in essential tremor. Serotonin test shows that essential tremor plus has intermediate results between essential tremor and Parkinson's disease probably reflecting their heterogeneous basis. However, the test can distinguish essential tremor plus from Parkinson's disease. Regarding clinical translation, while certain medications—specifically selective serotonin reuptake inhibitors (SSRIs)—may modulate systemic serotonin levels, our results indicate that the core parameters of the serotonin test remain robust enough to maintain diagnostic accuracy.

In conclusion, the serotonin-based platelet test offers a cost-effective, rapid, and highly accessible diagnostic tool. By utilizing standard HPLC-ED technology available in most clinical laboratories, this method can distinguish Parkinson's disease from other tremor syndromes within hours. Beyond its diagnostic utility, this approach provides a mechanistically grounded tool to further elucidate the heterogeneous nature of tremor-related pathologies.