The Diagnostic Value of CSF α-Synuclein in the Differential Diagnosis of Dementia with Lewy Bodies vs. Normal Subjects and Patients with Alzheimer’s Disease

<div><p>The detection of α-synuclein (α-syn) in the cerebrospinal fluid (CSF) of patients with synucleinopathy has yielded promising but inconclusive results. The aim of the present study was to determine the diagnostic value of α-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimer’s disease (AD), after strict control of several recognized confounders. Sixteen patients with DLB, 18 patients with AD and 22 age- and sex-matched normal controls (CTRL) were recruited. The levels of total α-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF α-syn levels in DLB patients as compared to the CTRL and AD groups (P= 0.049 and 0.01 respectively). ROC analysis revealed that increased α-syn was 81.8% specific for the discrimination of DLB vs. CTRL and 90% vs. AD. However, sensitivity was lower (56.2 % and 50% respectively). These findings provide evidence for a possible diagnostic role of α-syn as a surrogate biomarker for DLB.</p> </div

Receiver operating characteristic (ROC) curves of CSF α-Syn for the discrimination between DLB and either CTRL or AD.

<p>Receiver operating characteristic (ROC) curves of CSF α-Syn for the discrimination between DLB and either CTRL or AD.</p

Analytical characteristics of α-Syn ELISA.

<p>(A) Generation of a standard curve of the α-Syn ELISA in the low concentration range (0.1-2.7 ng/ml) showing the assay sensitivity (5 pg/ml). (B) Assessment of assay specificity by measuring different concentrations of recombinant α-Syn and β-Syn (0.1-0.9 ng/ml). (C) ELISA specificity was tested by measuring brain homogenates from KO, WT and α-Syn Tg mice. No α-Syn was detected in the KO animals. α-Syn levels in Tg brain extracts were 3.7-fold higher than in WT (***: p<0.001, n=4, 1-way ANOVA test followed by Tukey’s test) (D) Calibration graphs for the assessment of the day-to-day reproducibility of the assay during a period of 6 months (n=7 curves). The average of the S/B ratios for each concentration were plotted vs. the input of recombinant α-Syn. Error bars correspond to the reproducibility of the assay.</p

Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

<p>The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct “autophagosome-exosome-like” profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.</p