Cathepsin D genetic depletion in MEF cells does not cause cholesterol nor lysosomal proteins accumulation.

<p>(A) Confocal microscopy of CtsD KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsD KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01).</p

Cathepsin B/L genetic depletion in MEF cells causes accumulation of cholesterol and lysosomal proteins.

<p>(A) Confocal microscopy of CtsB KO, CtsL KO and CtsB/L double KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsB KO, CtsL KO and CtsB/L double KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in SH-SY5Y cells.

<p>(A) Confocal microscopy of SH-SY5Y control and PADK treated cells. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (*** p < 0.001). (D) RT-PCR Expression of the cholesterol egress genes in the control, PADK, U18666A- and Leu/NH₄Cl-treated SH-SY5Y cells, normalized to β-actin and quantified by 2−ΔΔCt method using control sample as calibrator. ATP-Binding Cassette sub-family A member 1 (ABCA1) and Niemann-Pick C1 protein (NPC1) mRNA levels presented as a fold change. The error bars present the mean ± SEM (* p < 0.05).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in CHO cells.

<p>(A) Confocal microscopy of CHOwt cells treated with different inhibitors. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. β-Actin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction.

<p>(A) EGFR degradation assay. Western blot analysis of EGFR in SH-SY5Y cells treated with different inhibitors at different time points. α-Tubulin was used as a loading control. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01). (C) Confocal microscopy of CHOwt cells treated with different inhibitors and CHO <i>NPC1</i>-null cells. LysoTracker (red) and Hoechst (blue). (D) Western blot of CHOwt cells treated with different inhibitors and CHO <i>NPC1</i>-null cells using LC3 antibody. β-Actin was used as a loading control. (E) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01).</p

Sez6L co-staining with BACE1 reveals their increased immunoreactivity in the cell body of NPC1 vs. wt mouse primary cortical neurons.

<p>Representative images of primary mouse cortical neurons stained with Sez6L antibody co-stained with BACE1. In NPC1^-/- (NPC1) cortical neurons, in contrast to NPC1^+/+ (wt) neurons, we observed an increased punctate staining of Sez6L in the cell body and in the neuronal processes. The cell body co-staining of Sez6L and BACE1 was enhanced in NPC1 vs. wt neurons (indicated by arrows).</p

The levels of BACE1-generated soluble APPβ fragments are increased in the cortex of 4-weeks old NPC1 vs. wt mice.

<p>(A) Western blot analysis of soluble APPβ (sAPPβ) and actin (Actin-DEA) protein levels in DEA fractions of the cortex collected from 4-weeks old wt (NPC1^+/+; N = 6) and NPC1 (NPC1^-/-; N = 6) mice. A standard protein lysate (S) was included twice on each gel. (B) A graph representing quantified protein signals for soluble APPβ (sAPPβ) in the cortex of 4-weeks old animals which were normalized against actin protein levels (Actin-DEA).</p

The increased levels of soluble Sez6, Sez6L and APP ectodomains reveal enhanced proteolysis by BACE1 in 4-weeks old NPC1 vs. wt mouse brains.

<p>(A-C) Western blot analyses of soluble Sez6 (sSez6), soluble Sez6L (sSez6L), total soluble APP (sAPPt) and actin (Actin-DEA) in DEA fractions of the cortex (A), hippocampus (B) and cerebellum (C) collected from 4-weeks old wt (NPC1^+/+; N = 6) and NPC1 (NPC1^-/-; N = 6) mice. A standard protein lysate (S) was included twice on each gel. (D-F) Graphs representing quantified protein signals of sSez6 (D), sSez6L (E) and sAPPt (F) which were normalized against actin (Actin-DEA) in the cortex (CX), hippocampus (HP) and cerebellum (CB) of 4-weeks old wt- and NPC1-mice.</p

BACE1 distribution is similar between 10-weeks old NPC1 vs. wt mouse brains.

<p>BACE1 (green) is intensely staining mossy fibers in the hippocampus of both wt and NPC1 mice. DAPI (blue) was used to counterstain all nuclei. There may be a slight decrease of BACE1 immunoreactivity in NPC1 mouse brains most likely due to profound neuropathological processes at this stage.</p

Sez6L immunoreactivity is lost in Purkinje neurons upon their neurodegeneration in 10-weeks old NPC1 vs. wt mouse brains.

<p>Sez6L (green) is specifically expressed in neurons of the cornu ammonis (CA) and granular layer of dentate gyrus (DG) of the hippocampus, as well as in deep layer cortical neurons and Purkinje cells in the cerebellum in 10-weeks old wt mice. Due to profound neurodegeneration, 10-weeks old NPC1 mice show a substantial loss of Sez6L intensity in Purkinje neurons. DAPI (blue) was used to counterstain all nuclei. ca1, cornu ammonis 1; mo, molecular layer; gr, granular layer.</p