Blood extracellular vesicles carrying synaptic function- and brain-related proteins as potential biomarkers for Alzheimer's disease.

INTRODUCTION: Objective and accessible markers for Alzheimer's disease (AD) and other dementias are critically needed. METHODS: We identified NMDAR2A, a protein related to synaptic function, as a novel marker of central nervous system (CNS)-derived plasma extracellular vesicles (EVs) and developed a flow cytometry-based technology for detecting such plasma EVs readily. The assay was initially tested in our local cross-sectional study to distinguish AD patients from healthy controls (HCs) or from Parkinson's disease (PD) patients, followed by a validation study using an independent cohort collected from multiple medical centers (the Alzheimer's Disease Neuroimaging Initiative). Cerebrospinal fluid AD molecular signature was used to confirm diagnoses of all AD participants. RESULTS: Likely CNS-derived EVs in plasma were significantly reduced in AD compared to HCs in both cohorts. Integrative models including CNS-derived EV markers and AD markers present on EVs reached area under the curve of 0.915 in discovery cohort and 0.810 in validation cohort. DISCUSSION: This study demonstrated that robust and rapid analysis of individual neuron-derived synaptic function-related EVs in peripheral blood may serve as a helpful marker of synaptic dysfunction in AD and dementia.

Development of a Sensitive Diagnostic Assay for Parkinson Disease Quantifying α-Synuclein-Containing Extracellular Vesicles.

OBJECTIVE: To develop a reliable and fast assay to quantify the α-synuclein (α-syn)-containing extracellular vesicles (EVs) in CSF and to assess their diagnostic potential for Parkinson disease (PD). METHODS: A cross-sectional, multicenter study was designed, including 170 patients with PD and 131 healthy controls (HCs) with a similar distribution of age and sex recruited from existing center studies at the University of Washington and Oregon Health and Science University. CSF EVs carrying α-syn or aggregated α-syn were quantified using antibodies against total or aggregated α-syn, respectively, and highly specific, sensitive, and rapid assays based on the novel Apogee nanoscale flow cytometry technology. RESULTS: No significant differences in the number and size distribution of total EVs between patients with PD and HCs in CSF were observed. When examining the total α-syn-positive and aggregated α-syn-positive EV subpopulations, the proportions of both among all detected CSF EVs were significantly lower in patients with PD compared to HCs (p < 0.0001). While each EV subpopulation showed better diagnostic sensitivity and specificity than total CSF α-syn measured directly with an immunoassay, a combination of the 2 EV subpopulations demonstrated a diagnostic accuracy that attained clinical relevance (area under curve 0.819, sensitivity 80%, specificity 71%). CONCLUSION: Using newly established, sensitive nanoscale flow cytometry assays, we have demonstrated that total α-syn-positive and aggregated α-syn-positive EVs in CSF may serve as a helpful tool in PD diagnosis. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that total and aggregated α-syn-positive EVs in CSF identify patients with PD.

Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson's disease.

Peripheral biomarkers indicative of brain pathology are critically needed for early detection of Parkinson's disease (PD). In this study, using NanoString and digital PCR technologies, we began by screening for alterations in genes associated with PD or atypical Parkinsonism in erythrocytes of PD patients, in which PD-related changes have been reported, and which contain ~ 99% of blood α-synuclein. Erythrocytic CHCHD2 mRNA was significantly reduced even at the early stages of the disease. A significant reduction in protein and/or mRNA expression of CHCHD2 was confirmed in PD brains collected at autopsy as well as in the brains of a PD animal model overexpressing α-synuclein, in addition to seeing a reduction of CHCHD2 in erythrocytes of the same animals. Overexpression of α-synuclein in cellular models of PD also resulted in reduced CHCHD2, via mechanisms likely involving altered subcellular localization of p300 histone acetyltransferase. Finally, the utility of reduced CHCHD2 mRNA as a biomarker for detecting PD, including early-stage PD, was validated in a larger cohort of 205 PD patients and 135 normal controls, with a receiver operating characteristic analysis demonstrating > 80% sensitivity and specificity.

Erythrocytic α-synuclein contained in microvesicles regulates astrocytic glutamate homeostasis: a new perspective on Parkinson's disease pathogenesis.

Parkinson's disease is a neurodegenerative disorder characterized by the transmission and accumulation of toxic species of α-synuclein (α-syn). Extracellular vesicles (EVs) are believed to play a vital role in the spread of toxic α-syn species. Recently, peripheral α-syn pathology has been investigated, but little attention has been devoted to erythrocytes, which contain abundant α-syn. In this study, we first demonstrated that erythrocyte-derived EVs isolated from Parkinson's disease patients carried elevated levels of oligomeric α-syn, compared to those from healthy controls. Moreover, human erythrocyte-derived EVs, when injected into peripheral blood in a mouse model of Parkinson's disease, were found to readily cross the blood-brain barrier (BBB). These EVs accumulated in astrocyte endfeet, a component of the BBB, where they impaired glutamate uptake, likely via interaction between excitatory amino acid transporter 2 (EAAT2) and oligomeric α-syn. These data suggest that erythrocyte-derived EVs and the oligomeric α-syn carried in them may play critical roles in the progression or even initiation of Parkinson's disease. Additionally, the mechanisms involved are attributable at least in part to dysfunction of astrocytes induced by these EVs. These observations provide new insight into the understanding of the mechanisms involved in Parkinson's disease.

Extracellular microvesicles-derived from microglia treated with unaggregated α-synuclein attenuate mitochondrial fission and toxicity-induced by Parkinsonian toxin MPP.

Biological functions of extracellular vesicles (EVs) are being discovered to be critical in neurodegenerative disorders, including Parkinson's disease (PD). A previous study using cellular models of PD has suggested that EVs derived from microglia exposed to aggregated α-synuclein (α-Syn) leads to enhanced neurotoxicity. However, the function of EVs derived from microglia not treated with aggregated a-Syn or treated with monomeric α-Syn are unclear. Here, employing a widely used cellular model of PD, i.e. SH-SY5Y cells treated with MPP+, a well-established parkinsonian toxicant, we revealed that microglial EVs, when not stimulated by aggregated α-Syn, appeared to be protective, and the mechanisms, though remain to be defined further, appeared to involve mitochondrial dynamics, especially mitochondrial fission.

Erythrocytic α-Synuclein as a potential biomarker for Parkinson's disease.

BACKGROUND: Erythrocytes are a major source of peripheral α-synuclein (α-Syn). The goal of the current investigation is to evaluate erythrocytic total, oligomeric/aggregated, and phosphorylated α-Syn species as biomarkers of Parkinson's disease (PD). PD and healthy control blood samples were collected along with extensive clinical history to determine whether total, phosphorylated, or aggregated α-Syn derived from erythrocytes (the major source of blood α-Syn) are more promising and consistent biomarkers for PD than are free α-Syn species in serum or plasma. METHODS: Using newly developed electrochemiluminescence assays, concentrations of erythrocytic total, aggregated and phosphorylated at Ser129 (pS129) α-Syn, separated into membrane and cytosolic components, were measured in 225 PD patients and 133 healthy controls and analyzed with extensive clinical measures. RESULTS: The total and aggregated α-Syn levels were significantly higher in the membrane fraction of PD patients compared to healthy controls, but without alterations in the cytosolic component. The pS129 level was remarkably higher in PD subjects than in controls in the cytosolic fraction, and to a lesser extent, higher in the membrane fraction. Combining age, erythrocytic membrane aggregated α-Syn, and cytosolic pS129 levels, a model generated by using logistic regression analysis was able to discriminate patients with PD from neurologically normal controls, with a sensitivity and a specificity of 72 and 68%, respectively. CONCLUSIONS: These results suggest that total, aggregated and phosphorylated α-Syn levels are altered in PD erythrocytes and peripheral erythrocytic α-Syn is a potential PD biomarker that needs further validation.

Impact of Pre-Analytical Differences on Biomarkers in the ADNI and PPMI Studies: Implications in the Era of Classifying Disease Based on Biomarkers.

BACKGROUND: Neurodegenerative diseases require characterization based on underlying biology using biochemical biomarkers. Mixed pathology complicates discovery of biomarkers and characterization of cohorts, but inclusion of greater numbers of patients with different, related diseases with frequently co-occurring pathology could allow better accuracy. Combining cohorts collected from different studies would be a more efficient use of resources than recruiting subjects from each population of interest for each study. OBJECTIVE: To explore the possibility of combining existing datasets by controlling pre-analytic variables in the Alzheimer's Disease Neuroimaging Initiative (ADNI) and Parkinson's Progression Markers Initiative (PPMI) studies. METHODS: Cerebrospinal fluid (CSF) was collected and processed from 30 subjects according to both the ADNI and PPMI protocols. Relationships between reported levels of Alzheimer's disease (AD) and Parkinson's disease (PD) biomarkers in the same subject under each protocol were examined. RESULTS: Protocol-related differences were observed for Aß, but not t-tau or α-syn, and trended different for p-tau and pS129. Values of α-syn differed by platform. Conversion of α-syn values between ADNI and PPMI platforms did not completely eliminate differences in distribution. DISCUSSION: Factors not captured in the pre-analytical sample handling influence reported biomarker values. Assay standardization and better harmonized characterization of cohorts should be included in future studies of CSF biomarkers.