Multiple pathogenic proteins implicated in neuronopathic Gaucher disease mice

Abstract

Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid β-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of β-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of β-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28-40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aβ-aggregates) in nGD.

Figure 1.

APP accumulation in the brains of 4L/ and 9H/PS-NA mice. (A) Serial brain sections from 12-week WT, 4L/PS-NA and 9H/PS-NA mice were processed for immunohistochemistry analysis using rabbit polyclonal anti-APP antibodies. Multiple APP aggregates (brown particles) were seen in cerebral cortex (Cor), caudate putamen (CPu), hippocampus (Hp) and substantia nigra (SN) regions of 4L/PS-NA and 9H/PS-NA brains. The inset in each image is the enlarged region pointed by arrows. The nuclei of neural cells were counterstained with hematoxylin (blue). The images were captured with 40× objective lens. The scale bars are 40 μm and only show on the first image (Cor, WT). (B) Immunoblots of APP in WT (lanes 1–3), 4L/PS-NA (lanes 4–6) and 9H/PS-NA (lanes 7–9) cerebral cortex (50 μg lysate protein) from 12-week mice (n = 3) were developed with rabbit polyclonal anti-APP antibody. The migrated APP size was ∼112 kDa according to the protein molecular weight markers at the left. The blot was stripped and re-probed with β-actin antibody as the loading control (bottom panel). (C) Quantification of immunoblots was conducted using ImageQuant 5.2 software. The scanned density values relative to WT (open bar, 100%), 4L/PS-NA (gray bar) and 9H/PS-NA (black bar) were plotted at Y-axis after normalization to the density of β-actin at each loading.

Figure 2.

APP accumulation in the various types of neural cells of 9H/PS-NA brain and overlapped with α-synuclein. (A) Brain sections from 12-week WT and 9H/PS-NA mice were processed for dual-antibody immunostaining using mouse monoclonal anti-APP with specific neural cell markers: anti-NeuN for neurons (20× objective lens), anti-GFAP for astrocytes (20×) and anti-CNPase for oligodendrocytes (40×) as indicated. The APP signals (red) were associated with NeuN signals (green), GFAP (green) and CNPase (green) signals indicated by arrows. (B) Brain sections from 12-week 9H/PS-NA (left panels) and WT (right panels) mice were processed for immunofluorescence analysis with anti-α-synuclein (red) and anti-APP (green) antibodies. Images showed multiple overlapped α-synuclein and APP signals (yellow) in the 9H/PS-NA brains as indicated by the arrow, but no aggregates of APP and α-synuclein were seen in WT brains. Neural cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). The pixel scatter diagrams are attached to show the analysis of co-localization of APP and α-synuclein signals. The images were captured with 40× objective lens. The scale bars are 20 μm.

Figure 3.

APP/α-synuclein cellular compartments in the brains of 9H/PS-NA mice. (A) Brain sections from 12-week 9H/PS-NA (top) and WT (bottom) mice were processed for dual-antibody immunostaining using anti-APP with anti-P62 (left), anti-LC3 (center) and anti-TOM40 (right) as indicated. APP signals are pointed by arrows. The pixel scatter diagrams for APP/TOM40 signals are shown. (B) Brain sections were from 12-week 9H/PS-NA (top) and WT (bottom) mice and processed for dual-antibody immunostaining using anti-α-synuclein with anti-P62 (left column), anti-LC3 (center column) and anti-TOM20 (right column). α-synuclein signals are pointed by arrows. The pixel scatter diagrams for α-synuclein/TOM20 signals are shown. The images were captured with 40× objective lens. The scale bars are 20 μm.

Figure 4.

APP distribution in cellular compartments of cortical neural cells from 9H/PS-NA mouse brains. (A) Co-localization of APP with cellular organelles. Cortical neural cells isolated from 12-week of 9H/PS-NA (top panels) and WT (bottom panels) mice were applied to dual-antibody immunostaining using rhodamine-conjugated anti-APP with anti-cellular organelle markers: FITC (green)-conjugated mitochondrial markers TOM40 and Cox IV, autophagosome marker p62 and lysosome marker LAMP1 as indicated. The merged images show various degree of co-localization of APP with each marker (yellow, arrows). Neural cell nuclei were stained by DAPI (blue). The images were captured with 63× objective lens. The scale bars are 20 μm. (B) Co-localization analyses. Pearson correlation coefficient was conducted for co-localized Rhodamine (APP) and FITC (each cellular organelle marker) signals in (A). The bar graphs show mean Pearson correlation coefficient (PCC, r) at Y-axis plotted to each dual-antibody set (X-axis). The PCC for APP-TOM40 was 0.76 (SEM ± 0.045, n = 14); APP-Cox IV, 0.76 (SEM ± 0.029, n = 25); APP-P62, 0.51 (SEM ± 0.033, n = 33); APP-LAMP1, 0.23 (SEM ± 0.029, n = 26). Pixel scatter diagrams for each image are shown in Supplementary Material, Figure S4. The results of Pearson analysis demonstrate mitochondrial preference of APP distribution in 9H/PS-NA cortical neural cells.

Figure 5.

α-Synuclein distribution in cellular compartments of cortical neural cells from 9H/PS-NA mouse brains. (A) Co-localization of α-synuclein with cellular organelles. Cortical neural cells isolated from 12-week of 9H/PS-NA (top panels) and WT (bottom panels) mice were applied to dual-antibody immunostaining using rhodamine-conjugated anti-α-synuclein with organelle markers: FITC (green)-conjugated TOM20, TOM40, Cox IV, P62 and LAMP1 as in Figure 4. The merged images show various degree of co-localization of α-synuclein with each marker (yellow). Neural cell nuclei were stained by DAPI (blue). The images were captured with 63× objective lens. The scale bars are 20 μm. (B) PCC analysis was conducted from co-localized Rhodamine (α-synuclein) and FITC (each organelle marker) signals in (A). The bar graphs show mean PCC (r) at Y-axis plotted to each dual-antibody set (X-axis). The PCC for αSyn-TOM20 was 0.76 (SEM ± 0.040, n = 21); αSyn-TOM40 was 0.35 (SEM ± 0.056, n = 30); αSyn-Cox IV, 0.28 (SEM ± 0.061, n = 13); αSyn-P62, 0.33 (SEM ± 0.046, n = 40); αSyn-LAMP1, 0.14 (SEM ± 0.028, n = 41). Pixel scatter diagrams for each image are shown in Supplementary Material, Figure S5. The results of Pearson analysis demonstrate preferential mitochondrial association of α-synuclein in 9H/PS-NA cortical neural cells.

Figure 6.

CBE-induced GC/GS increase in newborn cerebral cortical neural cells. The neural cells isolated from mouse newborn cerebral cortices were cultured in the medium with 2 mm GCase-irreversible inhibitor CBE (+) or without CBE (−) for 7 days. Neural cells were harvest for GC/GS (A–B) and immunofluorescence (A–B, C) analyses. (A) Electrospray ionization-liquid chromatography-tandem mass spectrometry analysis shows significantly increased total cellular GC (>15-fold, P = 0.0397) and GS (>28-fold, P = 0.0085) levels in CBE-treated (+) compared with un-treated CBE (−) cells. (B) The proportion of GC species. GC18-0 was dominant species indicating majority of neurons in the neural cells. (C) Population of neural cells. Bar graphs present percentage of each neural cell types in CBE (+) and CBE (−) cultures. Cell type specific marker for neurons, astrocytes and oligodendrocytes were Map2, GFAP and CNPase, respectively. CBE treatment had equal effect on each cell type survival.

Figure 7.

APP accumulation in CBE-induced newborn cerebral cortical neural cells. (A) Cell type with accumulation of APP. Dual-antibody immunostaining using anti-APP (Rhodamine-red) with FITC (green)-conjugated neural cell markers Map2, GFAP and CNPase, respectively. The merged images show co-localized signals of APP with cell markers in CBE (+) cells (yellow, bottom), but not in CBE (−) cells (top). (B) Quantitation of percentage cells with APP accumulation showing in (A). Bar graphs are plotted from (A) and show percentage of APP aggregates in each type of neural cells (neurons, astrocytes and oligodendrocytes) with (CBE+) or without CBE-treatment (CBE−) (n = 8–16 slides). (C) Co-localization of APP with cellular organelles in mouse newborn cerebral cortical neural cells with CBE (+) (bottom) or without CBE (−) (top). Panels are merged images of APP (Rhodamine-red) with organelle markers TOM40, Cox IV, P62 and LAMP1 (FITC-green), respectively. Cell nuclei were stained by DAPI (blue). The images were captured with 63× objective lens. The scale bars are 20 μm. (D) PCC analysis of APP co-localization with cellular organelle markers in the neural cells post CBE treatment (images in C). The bar graph shows mean PCC(r) at Y-axis plotted to each dual-antibody set (X-axis) (n = 5–8 slides). The PCC for APP-TOM40 was 0.64 (SEM ± 0.048, n = 5); APP-Cox IV, 0.30 (SEM ± 0.035, n = 8); APP-P62, 0.45 (SEM ± 0.077, n = 8); APP-LAMP1, 0.27 (SEM ± 0.046, n = 7). Pixel scatter diagrams for each image are shown in Supplementary Material, Figure S6.

Figure 8.

CBE-induced α-synuclein accumulation in newborn cerebral cortical neural cells. (A) Cell type accumulation of α-synuclein. Dual-antibody immunostaining using anti-α-synuclein (Rhodamine-red) with FITC (green)-conjugated neural cell markers Map2, GFAP and CNPase, respectively. The panels are merged images showing co-localized α-synuclein signals in CBE-treated (CBE+) cells (yellow, bottom), but not in untreated (CBE−) cells (top). (B) Quantitation of percentage cells with α-synuclein accumulation showing in (A). Bar graphs are plotted from (A) and showing percentage of cells having α-synuclein accumulation in each cell type with (CBE+) without (CBE−) treatment. (C) Co-localization of α-synuclein with cellular organelles in mouse newborn cerebral cortical neural cells treated with CBE (+) or no CBE (−). Panels are merged images of α-synuclein (Rhodamine-red) with cellular organelle markers TOM20, TOM40, Cox IV, P62 and LAMP1 (FITC-green), respectively. Cell nuclei were stained by DAPI (blue). The images were captured with 63× objective lens. The scale bars are 20 μm. (D) PCC analysis of α-synuclein co-localization with cellular organelle markers in the neural cells post CBE treatment (images in C). A bar graph is plotted at Y-axis of PCC (r) calculated from the fluorescence signal of captured image (C, n = 7–11 slides) and showing the degree of α-synuclein co-localization with cellular organelle markers in the neural cells post CBE treatment (panel C, CBE+). The PCC for αSyn-TOM20 was 0.42 (SEM ± 0.027, n = 11); αSyn-TOM40, 0.35 (SEM ± 0.034, n = 10); αSyn-Cox IV, 0.35 (SEM ± 0.095, n = 7); αSyn-P62, 0.15 (SEM ± 0.042, n = 7); αSyn-LAMP1, 0.12 (SEM ± 0.035, n = 9). Pixel scatter diagrams for each image are shown in Supplementary Material, Figure S7.

Figure 9.

The abnormalities of mitochondrial morphology and functions in the affected cerebral cortex neural cells. (A) Electronic micrographs of cortical neural cells in 9H/PS-NA and WT brains. Degenerating cortical neural cells from 9H/PS-NA mice (12 weeks) showed irregular cytoplasmic and nuclear membranes with condensed chromatin (n), multiple large size (up to 1.2 µm) and electronic dense mitochondria (m) in the cytoplasm. Mitochondria lost their normal fine ridge structure as compared with the 12-week WT controls. The scale bars are shown in each image. (B and C) ATP production (B) and O₂ consumption (C) in CBE-treated neural cells and Gaucher disease mouse brain cortices. Neural cells was isolated from postnatal 1–3 days WT mouse cerebral cortices and subjected to 2 mm CBE for 7 days. The brain cortices were from 12-week WT, PS-NA, 9H/9H, 9H/PS-NA, 4L/4L and 4L/PS-NA mice. ATP production (B) was reduced to 76% in CBE-treated neural cells and 77–79% in the parental mice (PS-NA, 9H9H and 4L/4L), but 62–68% in 9H/ or 4L/PS-NA nGD mouse brains compared with WT (n = 2). O₂ consumption rate (C) was decreased to 63% in CBE-treated neural cells and 66% in PS-NA, 75% in 9H/9H, 58% in 9H/PS-NA, 81% in 4L/4L and 72% in 4L/PS-NA nGD mouse brains (n = 2). Student's t-test, *P < 0.05; **P < 0.01; and ***P < 0.001.