Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain.

Tissue and immunostaining Massachusetts Alzheimer Disease Research Center (ADRC) Brain Bank provided free floating human brain sections (50 µm) from patients homozygous for APOEε3 or APOEε4 and with postmortem confirmed AD. All sections were pretreated with 10 mM citrate buffer pH6 for 10 minutes at 95°C before immunostaining. Sections were incubated with either C-terminal specific ApoE antibody, 3H1 (aa 243-272, 1∶100, Ottawa Heart Institute); or 3D6, which is directed against Aβ (1∶500, Elan Pharmaceuticals) antibodies and visualized with secondary antibody conjugated to Alexafluor 488 (A488) (Invitrogen). After sequential washing, the sections were incubated with 6C5 antibody (ApoE NT, aa 1-15, 1∶1000) (Ottawa Heart Institute) directly conjugated to Cyanine 3 (Cy3) (GE Healthcare). To probe the interaction between Aβ and ApoE CT; Aβ was labeled as above and ApoE CT was labeled using 3H1, directly conjugated to Cy3 [44].

Fluorescence Lifetime Microscopy

Sections were imaged using a Zeiss LSM-510 microscope, which has excitation and emission channels for confocal, near infrared and FLIM imaging. Cortical neuritic senile plaques were initially located using visual inspection of both fluorophores under epifluorescent wide field microscopy. Upon identification of a plaque, multi-track confocal images of A488 (green) and Cy3 (red) were obtained to confirm the co-localization of the two fluorophores, followed by FLIM imaging. Excitation of A488 for FLIM was achieved with a picosecond 2-photon laser (Tsunami, Spectra-Physics). Images were taken with the laser tuned to 760 nm, which we have found to be the most efficient two photon wavelength for selective excitation of A488 while minimizing autofluorescent contribution. Analysis was performed using a combination of SPCImage v2.9.5 (Becker and Hickl, GmbH) and previously described in house post analysis programs; χ 2 filter and MUGLE [27] , written in MATLAB (Mathworks, MA, USA). In brief, the analysis method is a multi-stage process. In the first instance, all images were fit with mono-exponential functions in SPCImage. Matrices of brightness, lifetime, and goodness of fit parameter (χ r 2 ), are imported into the program chifilt, in which χ r 2 is used as a pseudo-contrast for autofluorescence. A cutoff for χ r 2 is selected by comparing all images; binary masks are created and saved. A baseline fluorescent lifetime for the donor fluorophore is obtained by analyzing the data from sections stained only with donor and used as a fixed prior lifetime for bi-exponential fits of sections stained with both donor and acceptor. During both of these later two stages, pixels are discarded by both the use of a region of interest around the plaque, and the masks created in the first stage of analysis. The intensity weighted histogram of the lifetimes is fit to one or more Gaussians using MUGLE. Differences in interacting fraction; A F /(A F +A NF ), where A is the pre-exponential factor and the subscripts F and NF refer to the FRET quenched and unquenched components were calculated along with normalized inter-terminal distance based on FRET efficiency where R 0 is the Förster radius, at which 50% of energy is transferred from donor to acceptor [26] . Results were analyzed for significance by analysis of variance (ANOVA) and Bonferoni-Dunn post hoc tests.

Preparation of Brain extracts

Cortical gray matter from the temporal lobe of AD and non-demented control patients was homogenized in RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche, cat #11836153001), and then subjected to centrifugation at 16,000×g for 20 min at 4°C. The supernatant was used for SDS-PAGE and Western blot. For fractional brain extracts, samples were sequentially homogenized in 5 volumes of; TBSI (Tris-buffered saline containing a protease inhibitor cocktail [Roche]), 2% Triton X-100, and 2% SDS, with 25 strokes on a mechanical Dounce homogenizer, and subjected to centrifugation at 260,000×g for 20 min at 4 degrees for each fractional extract. In each case, the supernatant was drawn off and used for Western blot analysis. For the final extraction, SDS insoluble pellets were sonicated in 70% formic acid, centrifuged at 260,000×g for 30 minutes at 4 degrees. The supernatant was evaporated and resolubilized in dimethyl dulfoxide (DMSO) for Western blot analysis.

SDS-PAGE and Western Blot

Protein concentrations were determined by BCA assay. Equal amounts of total protein were loaded per well and electrophoresed through 10–20% Tricine or Tris-Glycine gradient gels (Invitrogen, catalog #E66255BOX and #EE61355BOX) and then transferred to PVDF membrane (PerkinElmer, catalog #NEF1002001). The membranes were incubated in blocking buffer (5% nonfat dried milk in TBS containing 0.01% Tween-20) for 1 h at room temperature, and then incubated with primary antibody diluted in blocking buffer for 1–2 h at room temperature. Primary antibodies used were: goat anti-ApoE (Calbiochem, catalog #179478), 3H1 mouse anti-C-terminal ApoE (Ottawa Heart Institute), and 6C5 mouse anti-N-terminal ApoE (Ottawa Heart Institute). Membranes were then incubated with HRP-conjugated horse anti-goat (Vector, catalog #PI-9500) or goat anti-mouse (Bio-Rad, catalog #170-6516) secondary antibody diluted in blocking buffer for 1 h at room temperature and protein detected by enhanced chemiluminescence (PerkinElmer, catalog #NEL102001).