Evaluation of ferritin and TfR level in plasma neural-derived exosomes as potential markers of Parkinson's disease

Abstract

Introduction: Early diagnosis of Parkinson's disease (PD) remains challenging. It has been suggested that abnormal brain iron metabolism leads to excessive iron accumulation in PD, although the mechanism of iron deposition is not yet fully understood. Ferritin and transferrin receptor (TfR) are involved in iron metabolism, and the exosome pathway is one mechanism by which ferritin is transported and regulated. While the blood of healthy animals contains a plentiful supply of TfR-positive exosomes, no studies have examined ferritin and TfR in plasma neural-derived exosomes.

Methods: Plasma exosomes were obtained from 43 patients with PD and 34 healthy controls. Neural-derived exosomes were isolated with anti-human L1CAM antibody immunoabsorption. Transmission electron microscopy and western blotting were used to identify the exosomes. ELISAs were used to quantify ferritin and TfR levels in plasma neural-derived exosomes of patients with PD and controls. Receivers operating characteristic (ROC) curves were applied to map the diagnostic accuracy of ferritin and TfR. Independent predictors of the disease were identified using logistic regression models.

Results: Neural-derived exosomes exhibited the typical exosomal morphology and expressed the specific exosome marker CD63. Ferritin and TfR levels in plasma neural-derived exosomes were significantly higher in patients with PD than controls (406.46 ± 241.86 vs. 245.62 ± 165.47 ng/μg, P = 0.001 and 1728.94 ± 766.71 vs. 1153.92 ± 539.30 ng/μg, P < 0.001, respectively). There were significant positive correlations between ferritin and TfR levels in plasma neural-derived exosomes in control group, PD group and all the individuals (rs = 0.744, 0.700, and 0.752, respectively). The level of TfR was independently associated with the disease (adjusted odds ratio 1.002; 95% CI 1.000-1.003). ROC performances of ferritin, TfR, and their combination were moderate (0.730, 0.812, and 0.808, respectively). However, no relationship was found between the biomarkers and disease progression.

Conclusion: It is hypothesized that ferritin and TfR in plasma neural-derived exosomes may be potential biomarkers for PD, and that they may participate in the mechanism of excessive iron deposition in PD.

Keywords: Parkinson’s disease; biomarker; exosome; ferritin; transferrin receptor.

FIGURE 1

Flowchart outlining the experimental procedures described in this figure. Ab, antibody.

FIGURE 2

Identification of plasma neural-derived exosomes. Representative transmission electron microscopy image showing the morphological characteristics of the exosomes (black arrows) (A). Western blot images (B) showing that the exosomal marker CD63 was expressed in exosomal samples.

FIGURE 3

Comparation of ferritin and TfR in PD and controls, and ROC analysis of biomarkers for PD diagnosis. The box plots showed the comparison of the levels of ferritin (A) and TfR (B) in the two groups. *, P < 0.01. In the whole cohort, ferritin in plasma neural-derived exosomes provided an AUC of 0.73 (sensitivity = 67.4%, specificity = 79.4%) for PD versus controls (C). TfR performed similarly (AUC = 0.812, sensitivity = 86.0%, specificity = 70.6%) in the whole cohort (D). And, the combination of ferritin and TfR did not obviously improve the performance with AUC of 0.808 (sensitivity = 86.0%, specificity = 73.5%) (E).

FIGURE 4

Correlation analysis between ferritin and TfR in plasma neural-derived exosomes. A significant positive correlation between ferritin and TfR was observed in controls (rs = 0.744, p < 0.001) (A), patients with PD (rs = 0.700, p < 0.001) (B), and all the individuals (rs = 0.752, p < 0.001) (C).