The recently reported Piedmont L34V A mutant, located outside the hot spot 21C23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.Fossati, S., Cam, J., Meyerson, J., Mezhericher, E., Romero, I. A., Miltefosine Couraud, P. O., Weksler, B. B., Ghiso, J., Rostagno, A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid- variants in cells composing the cerebral vessel walls. for CD140a 1 h with 1% nonfat milk in Tris-buffered saline, pH 7.4, containing Miltefosine 0.1% Tween 20 (TBST), followed by vacuum application and 2 subsequent washes with TBST. After removal from the dot-blot apparatus and further blocking with 5% milk in TBST (1 h, room heat), the membrane was incubated overnight with A11 antibody (1:1000) followed by HRP-conjugated anti-rabbit secondary antibody. Immunoreactivity was assessed by ECL as above. As a positive control for oligomer formation and immunoreactivity with A11, samples for each of the peptides at 1- and 3-d aggregation time points were subjected to size-exclusion chromatography (SEC) on Sephadex G-75 (10/300 GL, GE Biosciences) under isocratic conditions (PBS, pH 7.4; flow rate 0.5 ml/min), as described previously (17). Equal protein load (500 ng) of each of the SEC oligomer peaks (retention time 20 min) was subjected to dot-blot analysis and probed with A11 antibody as above. CD spectroscopy Changes in the secondary structure of the different A peptides were estimated by CD spectroscopy, as described previously (10). Spectra in the far-ultraviolet light (190C260 nm; bandwidth 1 nm; intervals 1 nm; scan rate 60 nm/min) yielded by the different peptides at each time point of aggregation were recorded at 24C with a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD, USA), using a 0.2-mm-path quartz cell and a peptide concentration of 1 1 mg/ml. For each sample, 15 consecutive spectra were obtained and averaged, and baseline was subtracted. Results are expressed in terms of mean residue ellipticity (degcm2dmol?1; ref. 18). Thioflavin T binding assay Thioflavin T binding was monitored as described previously (19). Briefly, 6-l aliquots of each of the peptide aggregation time-point samples were added to 10 l of 0.1 mM Thioflavin T (Sigma) and 50 mM Tris-HCl buffer, pH 8.5, to a final volume of 200 l. Fluorescence was recorded after 300 s in a LS-50B luminescence spectrometer (Perkin Elmer, Waltham, MA, USA) with excitation and emission wavelengths of 435 and 490 nm (slit width 10 nm), respectively, as described previously (18, 20). Each sample was analyzed in duplicate. Electron microscopy Aliquots (3 l)of each of the peptide aggregation time-point samples were placed onto carbon-coated 400-mesh Cu/Rh grids (Ted Pella, Inc., Redding, CA, USA) and stained with 1% uranyl acetate in distilled water (Polysciences, Inc., Warrington, PA, USA). Stained grids were examined in a Philips CM-12 transmission electron microscope and photographed with a Gatan (4k4k) digital camera at the Image Core Facility of the Skirball Institute Miltefosine of Biomedical Medicine (New York University School of Medicine, New York, NY, USA), as described previously (18). Cell cultures Immortalized human brain microvascular ECs hCMEC/D3 (D3; ref. 21) were cultured in complete EBM-2 medium (Lonza, Allendale, NJ, USA) containing growth supplements and 2.5% FBS. This cell line retains the morphological characteristics of primary brain ECs and expresses specific brain endothelial markers and cell surface adhesion molecules (21). Human brain vascular SMCs were purchased from ScienCell (San Diego, CA, USA) and produced in SMC medium with 10% FBS in accordance with the manufacturers specifications. Evaluation of apoptosis induction by A-variant peptides Miltefosine Cell-death ELISA The extent of apoptosis caused by the different A peptides was assessed by quantitation of nucleosome formation with the Cell Death ELISAplus kit (Roche Applied Science, Indianapolis, IN, USA). Briefly, 2 104cells/well were seeded on 24-well plates and allowed to rest for.