Figure 1.
γ-Secretase components and activity in a discontinuous iodixanol gradient.
Postmitochondrial supernatant from rat brain was layered on a 2.5–30% iodixanol gradient and fractions were collected from the bottom of the tube. A) Western blots showing the γ-secretase components, the substrate APP and the subcellular markers N-cadherin (plasma membrane), syntaxin 13 (early endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC53 (ER-Golgi intermediate compartment) and KDEL (endoplasmatic reticulum). Each marker was analyzed in 3–4 gradients. B) Protein concentration in the fractions. Note that the protein concentration does not peak in the same fraction as the γ-secretase components or activity. The line is added just to guide the eye. C) Aβ40 production from endogenous substrate. The fractions were incubated over night at 37°C with or without L-685,458 and the Aβ40 concentration was measured by ELISA. Production was calculated as concentration without inhibitor minus concentration with inhibitor. D) Aβ40 production with added exogenous substrate (C99-FLAG). The fractions were incubated as above but with the addition of C99-FLAG. Due to slight discrepancies in the peak fraction between experiments which resulted in large standard deviations for each fraction, we have chosen to show a representative experiment rather than the mean value. The peak fraction was, however, always fraction 5, 6 or 7 and the enrichment in the peak fraction was always at least three-fold. Each experiment was repeated 5 times. E) Quantification of the subcellular markers in the different fractions. The fractions with highest γ-secretase activity are indicated by the dotted box. Mean values (% of optical density (OD) in the fraction with the highest density for each marker) are plotted (n = 3–4). Again, the standard deviations were high due to shifts in the gradient and have been removed to avoid a too disordered picture. The shift of fractions with the highest density of different markers also results in that the mean values don't reach 100% in most cases. The lines were added just to guide the eye.
Figure 2.
Preparation of synaptic membranes and synaptic vesicles from rat brain.
A) Western blot for γ-secretase components and subcellular markers in the purification steps. synaptophysin (synaptic vesicles), PSD-95 (post-synaptic membrane), N-cadherin (plasma membrane), syntaxin13 (endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC-53 (ER-Golgi intermediate compartment), KDEL (ER), H, homogenate; P2, 17 000×g pellet; Syn, Synaptosomes; LP1, lysed synapstosomes pellet, SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet from 17 000×g supernatant. Each marker was analyzed in 3–7 preparations. B) Electron micrographs of the synaptic membrane fraction showing structures resembling emptied synaptosomes with occasional attached post-synaptic densities (arrow). C) Electron micrograph of the synaptic vesicle fraction showing small vesicles of 40–50 nm but also some larger vesicles. Scale bar = 100 nm.
Figure 3.
γ-Secretase activity in synaptic fractions.
A) Aβ40 production from endogenous substrate was assessed by incubating the fractions at 37°C for 16 h with or without the γ-secretase inhibitor L-685,458. The Aβ40 levels were analyzed by ELISA and the levels in the samples with L-685,458 were subtracted from the levels without L-685,458. B) Aβ40 production after the addition of 20 ng of an exogenous substrate, C99-FLAG, was obtained as above with the difference that 20 ng of C99-FLAG was added prior to incubation. C) AICD production was assessed as above and the samples without L-685,458 were analyzed with immunoblotting. D) Degradation of AICD was investigated by adding synthetic AICD to the sample and incubating at 37°C for 16 h in the presence of L-685,458. H, homogenate; SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet; C = control (buffer and AICD only). Data are presented as mean values +/− SD (n = 4). *, p<0.05; **, p<0.01.
Figure 4.
Presence of the γ-secretase components in a highly pure synaptic vesicle fraction.
Highly pure synaptic vesicles were prepared using a protocol including controlled pored glass chromatography (CPG-SV) and analyzed for the presence of γ-secretase components and subcellular markers (see Figure 2), using Western blot. H, homogenate; Crude SV, synaptic vesicles used in Figure 2 & 3; P3, 100 000×g pellet.
Figure 5.
Labelling of γ-secretase active sites in rat brain sections.
A) Labelling by GCB (γ-secretase inhibitor with a cleavable biotin moiety, green) is specific since it is competed out by a 100 x excess of the “cold” inhibitor L-685,458 (right panel). DAPI-staining (nucleus) in blue. B) Double labelling of the endosomal marker Rab5 (red) and GCB (green). C) Double labelling of the cis-Golgi marker GM130 (red) and GCB (green). Each experiment was performed 3 times. Scale bar = 10 µM.
Figure 6.
Labelling of γ-secretase active sites in mouse primary cortical neurons.
A) Labelling by GCB (green) is specific since it is competed out by a 100 x excess of the “cold” inhibitor L-685,458 (right panel). DAPI-staining in blue. Double labelling of GCB (green) with B) the endosomal marker Rab5, C) the cis-Golgi marker GM130, D) the pre-synaptic marker synapsin and E) the post-synaptic marker PSD-95 (red). Each experiment was performed 3 times. Scale bar = 10 µM.