α‐Synuclein (α‐syn) has been known to be a key player of the pathogenesis of Parkinson’s disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of α‐syn into cells has also been observed. In this study, we observed that dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric α‐syn into BV‐2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of α‐syn internalization in dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol‐treated BV‐2 cells. Furthermore, inhibition of clathrin‐, caveolae‐, and dynamin‐dependent endocytosis did not prevent the internalization of α‐syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of α‐syn. In addition, we further confirmed these observations by co‐culture system of BV‐2 cells and α‐syn‐over‐expressing SH‐SY5Y cells. These findings suggest that extracellular α‐syn is internalized into microglia via GM1 as well as hitherto‐unknown protein receptors in clathrin‐, caveolae‐, and dynamin‐independent, but lipid raft‐dependent manner. Elucidation of the mechanism involved in internalization of α‐syn should be greatly helpful in the development of new treatments of α‐syn‐related neurodegenerative diseases.

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BackgroundNatural cytotoxicity, mediated by natural killer (NK) cells plays an important role in the inhibition and elimination of malignant tumor cells. To investigate the immunoregulatory role of NK cells and their potential as diagnostic markers, NK cell activity (NKA) was analyzed in prostate cancer (PCa) patients with particular focus on NK cell subset distribution.MethodsProspective data of NKA and NK cell subset distribution patterns were measured from 51 patients initially diagnosed with PCa and 54 healthy controls. NKA was represented by IFN-γ levels after stimulation of the peripheral blood with Promoca®. To determine the distribution of NK cell subsets, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies. Then, CD16+CD56dim and CD16−CD56bright cells gated on CD56+CD3− cells were analyzed using a flow-cytometer.ResultsNKA and the proportion of CD56bright cells were significantly lower in PCa patients compared to controls (430.9 pg/ml vs. 975.2 pg/ml and 2.3% vs. 3.8%, respectively; p<0.001). Both tended to gradually decrease according to cancer stage progression (p for trend = 0.001). A significantly higher CD56dim-to-CD56bright cell ratio was observed in PCa patients (41.8 vs. 30.3; p<0.001) along with a gradual increase according to cancer stage progression (p for trend = 0.001), implying a significant reduction of CD56bright cells in relation to the alteration of CD56dim cells. The sensitivity and the specificity of NKA regarding PCa detection were 72% and 74%, respectively (best cut-off value at 530.9 pg/ml, AUC = 0.786).ConclusionsReduction of CD56bright cells may precede NK cell dysfunction, leading to impaired cytotoxicity against PCa cells. These observations may explain one of the mechanisms behind NK cell dysfunction observed in PCa microenvironment and lend support to the development of future cancer immunotherapeutic strategies.

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