Fig 1.
The increased levels of soluble Sez6, Sez6L and APP ectodomains reveal enhanced proteolysis by BACE1 in 4-weeks old NPC1 vs. wt mouse brains.
(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.
Fig 2.
The levels of BACE1-generated soluble APPβ fragments are increased in the cortex of 4-weeks old NPC1 vs. wt mice.
(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).
Fig 3.
At 10-weeks of age an enhanced BACE1-mediated proteolysis of Sez6L is present in all three brain regions of NPC1 vs. wt mice, while cleaved Sez6 is increased only in the cerebellum.
(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 10-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 10-weeks old wt (NPC1+/+; N = 6) and NPC1 (NPC1-/-; N = 6) mice.
Fig 4.
APP immunoreactivity is lost in Purkinje neurons upon their neurodegeneration in 10-weeks old NPC1 vs. wt mouse brains.
APP (green) is expressed in neurons of the cornu ammonis (CA) regions and dentate gyrus (DG) of the hippocampus, as well as in 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 APP intensity in Purkinje neurons. DAPI (blue) was used to counterstain all nuclei.
Fig 5.
BACE1 distribution is similar between 10-weeks old NPC1 vs. wt mouse brains.
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.
Fig 6.
Sez6 distribution is similar in the brains of 10-weeks old NPC1 and wt mouse.
Sez6 (green) is specifically expressed in neurons of the region cornu ammonis 1 (CA1) and hilus of dentate gyrus (DG) of the hippocampus, as well as in deep layer cortical neurons and neurons in the molecular layer of the cerebellum in both 10-weeks old wt and NPC1 mouse brains. DAPI (blue) was used to counterstain all nuclei. ca1, cornu ammonis 1; dg, dentate gyrus; mo, molecular layer; gr, granular layer.
Fig 7.
Sez6L immunoreactivity is lost in Purkinje neurons upon their neurodegeneration in 10-weeks old NPC1 vs. wt mouse brains.
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.
Fig 8.
NPC1 mouse primary cortical neurons show increased punctuate staining of Sez6 within endosomal compartments.
Representative images of primary mouse cortical neurons stained with Sez6 antibody and endolysosomal markers: transferrin receptor (TfR) and lysosomal-associated membrane protein 1 (LAMP1). Note that Sez6 in NPC1+/+ (wt) cortical neurons was localized in the soma and in neuronal processes. Upon NPC1-loss and cholesterol accumulation in NPC1-/- (NPC1) cortical neurons, its immunoreactivity showed more punctate staining in the soma (in vesicles that do not accumulate cholesterol) and in the processes (indicated by arrows).
Fig 9.
NPC1 mouse primary cortical neurons show profound Sez6L punctuate staining in neuronal processes.
Representative images of primary mouse cortical neurons stained with Sez6L antibody and endocytic markers: early endosome antigen (EEA1) and transferrin receptor (TfR). Immunoreactivity of Sez6L in NPC1+/+ (wt) cortical neurons was detected in both soma and neuronal processes. Upon NPC1-loss and cholesterol accumulation in NPC1-/- (NPC1) cortical neurons Sez6L showed more enlarged punctate staining mainly in TfR-positive vesicles in the soma and in neuronal processes (indicated by arrows).
Fig 10.
Sez6L co-staining with BACE1 reveals their increased immunoreactivity in the cell body of NPC1 vs. wt mouse primary cortical neurons.
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).