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Results: 8

1.
Figure 1

Figure 1. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Quantification of LC3 and p62 Levels in the presence or absence of bafilomycin A1 in cultured WT-ES, PS1ko-ES and PSdko-ES Cells. A, LC3 levels detected by Western blotting. B, Quantification of LC3 II levels normalized to actin (*p<0.06, **p<0.01, ***p<0.05, n=3). C, Autophagic flux of LC3II in WT-ES, PS1ko-ES and PSdko-ES cells. D, p62 levels detected by Western blotting. E, Quantification of p62 levels normalized to actin (*p<0.01, **p<0.01, ***p<0.05, n=5). F, Autophagic flux of p62 in WT-ES, PS1ko-ES and PSdko-ES cells. G, Detection of PS1 expression in WT-ES, PS1ko-ES and PSdko-ES cells. H, Detection of PS2 expression by RT-PCR in WT-ES, PS1ko-ES and PSdko-ES cells.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
2.
Figure 4

Figure 4. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

V0a1 is glycosylated in the absence of presenilin(s). A, Stably expressed human V0a1-6His is glycosylated in cultured PSdko MEF. The treatment of the lysates with PNGase F or Endo H is indicated above lanes 2, 3, 5 and 6. The respective presence and absence of PS1 expression in cultured WT and PSdko MEF is shown in the lower panel. B, Transiently expressed human V0a1-6His is glycosylated in the absence of presenilin(s) in ES cells. PNGase F treatment is indicated above lanes 2, 4 and 6. C, Endogenous V0a1 is glycosylated in the hippocampi of PScdko mice. The treatment with PNGase F or EndoH is indicated above lanes 2, 3, 5 and 6 respectively.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
3.
Figure 3

Figure 3. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Processing of cathepsin D in the absence of presenilin(s). A, Processing of cathepsin D in cultured WT-ES, PS1ko-ES and PSdko-ES cells. Intermediate and heavy chain of CatD are shown in the upper two panels respectively; actin is used as an internal loading control. B, Quantification of cathepsin D expression by quantitative PCR analysis in cultured WT-ES, PS1ko-ES and PSdko-ES cells. The results represent the mean± SEM of four sets of reactions for each ES cell type (p>0.07 and p>0.3 respectively, n=4). NS: non-significant. C, Cathepsin D processing in PScdko mice. Intermediate and heavy chain of CatD are shown in the top two panels and PS1 NTF and APP CTFs levels are shown in the lower panels.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
4.
Figure 7

Figure 7. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

V0a1 is glycosylated when either STT3A or STT3B is downregulated in HEK 293 Cells. A, HEK 293 cells were transfected with control siRNA (lanes 1 to 3), STT3A siRNA (lanes 4 and 5) or STT3B siRNA (lanes 6 and 7) followed by a transient transfection with V0a1-6His cDNA. V0a1 panel shows the expression and the glycosylation state of V0a1 in these transfected cells. STT3A panel confirms the depletion of STT3A following the STT3A siRNA transfection (lanes 4 and 5). PSAP panel shows the selective reduction of the glycosylation of a STT3A substrate PSAP (lanes 4 and 5), which further confirms the loss of STT3A activity following STT3A siRNA transfection. STT3B panel confirms the depletion of STT3B following the STT3B siRNA transfection (lanes 6 and 7). The proCatC panel shows the selective reduction of the glycosylation of a STT3B substrate procathepsin C (lanes 6 and 7), which further confirms the loss of STT3B activity following STT3B siRNA treatment. B, Pulse-labeling of HEK293 cells cotransfected with V0a1-6His and either control siRNA, STT3A or STT3B siRNA, and cells transfected with control siRNA alone was used as a control for V0a1-6His immunoprecipitation. Labeled V0a1 panel shows the level and glycosylation state of labeled V0a1 after immunoprecipitation with anti-6XHis antibody under each condition. STT3B and proCatC panels show the Western blot results of the labeled samples to confirm the depletion of STT3B following the STT3B siRNA transfection (STT3B panel, lane 4) and the reduction of proCatC glycosylation after STT3B siRNA treatment (proCatC panel lane 4). proCatC band is indicated with an arrow.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
5.
Figure 5

Figure 5. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

V0a1 is glycosylated and cathepsin D is processed into mature forms in cells and brains of mice expressing independent FAD-linked mutant PS1. A, Human V0a1-6His is glycosylated in N2a cells stably expressing FAD-linked PS1 mutants E280A and ΔE9. V0a1-6His was transiently transfected into N2a cells harboring human PS1 or FAD-linked mutant E280A or ΔE9. Untransfected or empty vector-transfected cells were used as controls (lanes 1 to 4). PNGase F treatment is indicated above the lanes. B, Cathepsin D is processed into mature forms in N2a cells stably expressing FAD-linked PS1 mutants E280A and ΔE9. Levels of both intermediate form and heavy chain of CatD are shown in the upper panel. The expression of human PS1 and FAD-linked E280A and ΔE9 are shown in the lower panel. Full length human PS1 and unproteolysed ΔE9 are indicated by gray arrows; endogenous mouse PS1NTF is indicated by a black arrow labeled with m, and the human PS1 NTF is indicated by a black arrow labeled with h. C, Endogenous V0a1 is glycosylated in the brains of transgenic mice overexpressing either wild type human PS1, or FAD-linked PS1 ΔE9 or PS1 M146L, non-transgenic (NTG) littermate is also used as a control. Treatment with PNGase F is indicated above the corresponding lanes. D, Cathepsin D is processed into mature forms in an indistinguishable pattern in non-transgenic brain, or in transgenic brains overexpressing WT human PS1, or FAD-linked PS1ΔE9 or PS1M146L. The expression of human PS1, or FAD-linked PS1ΔE9 or M146L is shown in the lower panel, full length human PS1 and unproteolysed PS1ΔE9 are indicated by gray arrows; human PS1 NTF is indicated by a black arrow.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
6.
Figure 2

Figure 2. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Mean vesicle pH measurement in cultured WT-ES, PS1ko-ES and PSdko-ES Cells. A, Representative images of cultured WT-ES cells used for ratiometric analysis using the pH sensitive probe Lysosensor-DND-160. Images were collected while ES cells were incubated in buffer clamped at various pH values (pH7.5, 6.5, 5.5, 4.5 and 3.5). ImageJ was used to generate a ratiometric image where pH is represented by a color following the scale in B. B, Graph showing the pH-sensitive nature of the dual emission probe, Lysosensor-DND-160, at acidic pH for blue (W1), but not yellow (W2), emission spectra (N=10), significant effect of wavelength; Two-way ANOVA: P<0.0001 (F=74.64, DFn=4, d=58), post-tests: W1 vs. W2; pH7.5 (P>0.05), pH6.0 (P<0.01), pH5.0, 4.5 and 4.0 (P<0.001), values are mean+SEM). C, Graph showing the standard curve (ratio=W1/W2, N=20 vesicles/pH, values are mean+SEM). D, Pseudo colored images of cultured WT-ES, PS1ko-ES and PSdko-ES cells incubated with the Lysosensor-DND-160 dye. Fifteen 60X images were collected per group and the experiment was repeated twice. The number of cells in each encircled “field” varied both within and between ES cell types because of differences in cell size and morphology. E, W1/W2 ratio distribution of all vesicles in each ES cell type. F, Vesicle pH distribution within the acidic range in WT-ES, PS1ko-ES and PSdko-ES cells. G, Mean vesicle pH in WT-ES, PS1ko-ES and PSdko-ES cells within the acidic range.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
7.
Figure 6

Figure 6. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Full-length PS1 does not coimmunoprecipitate with V0a1 or STT3B. A, A series of diluted lysates of cultured WT MEF and WT-ES cells or mouse hippocampi are used to assess the levels of full length PS1 and PS1 NTF. Very low level of full length PS1 is detected in MEF and ES cell lysates, while no full length PS1 is detected in hippocampus lysates under these conditions. Full length PS1 is indicated by an arrow. B, PEN-2 knockdown cell line (PEN2KD) reveals the accumulation full-length PS1 compared to naïve HEK 293 cells. The level of the remaining PEN-2 in this cell line is shown in the lower panel when naïve HEK293 cell lysate is used as a control. C, No co-immunoprecipitation of full length PS1 or PS1NTF with V0a1-6His is detected in PEN-2 knockdown cells (left panel with anti-6XHis antibody, right upper panel with anti-PS1NT antibody), while under the same conditions the co-immunoprecipitation of PS1 and NCT was confirmed (right lower panel). m: indicates mature nicastrin band, i: indicates immature nicastrin band. D, No co-immunoprecipitation of full length PS1 and STT3B is detected (left upper panel with anti-STT3B antibody, right upper panel with anti-human PS1 antibody), while under the same conditions the co-immunoprecipitation of STT3B and OST48 is confirmed (left lower panel), and the immunoprecipitated full length PS1 and PS1 NTF are shown in the right lower panel. E, With lysates from mouse N2a cells stably expressing human WT PS1, coimmunoprecipitation was performed with control IgG, anti-STT3B and anti-V0a1 antibodies. Anti-PS1 was used as a control for the immunoprecipitation. The immunoprecipitated samples were probed with anti-human PS1 antibody. No specific interaction between full length PS1 with either STT3B or V0a1 was detected.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.
8.
Figure 8

Figure 8. From: A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2.

Expression of genes associated with autophagy and lysosomal biogenesis in cells and mouse brain. A, Quantitative PCR analysis of TFEB expression in WT and PSdko MEF and ES cells. The results represent the mean± SEM of four sets of reactions for each cell type (p>0.2, n=4). B, Strategy to assess α-CamKII promoter driven cre recombinase activity in vivo. Expression of α-CamKII-cre leads to excision of loxP sites flanking mTomato red coding region and thereby activating mEGFP reporter expression within the ROSA26 locus [R26S]. Ci: Photograph of montage created by confocal z-stack tiled image of coronal brain section (×40 obj) from bigenic mice carrying CamKII-cre and Gt[ROSA]26Sortm4[ACTB-tdTomato,-EGFP]Luo transgenes; overlay of mEGFP (green), mTomato red (red), DAPI (blue) is shown, the scale bar is 500 µm. Cii and Ciii: Representative photographs of coronal brain section from single transgenic Gt[ROSA]26Sortm4[ACTB-tdTomato,-EGFP]Luo mice show expression of mTomato red (Cii), without detectable mEGFP signal (Ciii). CivCxix, Confocal z-stack projection images (×40 obj) of cells expressing mEGFP or mTomato red in specific hippocampal subfields and cortex of bigenic mice are shown in columns Civ to Cxiv and Cv to Cxvii, respectively. DAPI stained nuclei (column Cvi to Cxviii) and the overlay (column Cvii to Cxix) of mEGFP, mTomato red and DAPI signal are shown. The scale bar is 100 µm. D, Neuronal Morphology in WT and CamKII promoter-driven cre recombinase mice. Cortical or hippocampal sections from brains from WT and CamKII-cre mice were stained with antibodies to the nuclear antigen, NeuN or the cytoskeletal antigen, βIII tubulin. The scale bar is 100 µm. E, F: Transcriptome analysis of mouse Frontal Cortex (FC) and Hippocampus (HC), respectively. In both figures, autophagy genes are in blue diamonds and CLEAR network genes in red circles. X axis denotes average log2 ratio (ALR) for each gene (1 ALR = 2 fold change), Y axis corresponds to normalized log2 expression value observed on the DNA microarray (denoting the magnitude of the expression level of a gene). Differential expression values of the TFEB-dependent autophagy genes and CLEAR network genes in the HC and FC can be found in Table 3. Note that in both the frontal cortex and hippocampus the CLEAR network genes showed a highly significant groupwise overexpression (FC: p=2.0*10−19; HC: p=6.6*10−9), while the TFEB-dependent autophagy-related genes showed no populational expression shift (FC: p=0.96; HC: p=0.46). The most overexpressed genes of the CLEAR network in both the FC and HC included: Ctsd, Naglu, Gusb, Ctsz, Ctss, Hexb and Cd68.

Xulun Zhang, et al. J Neurosci. 2012 June 20;32(25):8633-8648.

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