GCase promotes Aβ_1–42 oligomer degradation.

<p>(A) Primary cultured mouse cortical neurons were infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i>, and 5 μM oligomeric Aβ_1–42 or PBS was added for 48 h at 10 days <i>in vitro</i>. 10 μg/ml of E-64d and 10 μg/ml of PepA were treated 4 h before the treatment of Aβ_1–42 oligomers. The monomeric and oligomeric Aβ expressions were measured with anti-Aβ antibody (4G8) 24 h after the treatment of oligomeric Aβ_1–42. (B) The quantification of Aβ levels is shown (Student’s t test, <i>P</i>* < 0.05, <i>P</i>*** < 0.001).</p

GCase protein levels and activity are reduced in sporadic AD brain tissues and Aβ_1–42 oligomer-treated primary neurons.

<p>(A and B) GCase expression was decreased in the hippocampal region of brains affected by Alzheimer’s disease (n = 6), as opposed to controls (n = 6). GCase expression levels were normalized to β-actin and quantified. (C) The GCase enzymatic activities were measured using lysosome-enriched fractions for the brains affected by Alzheimer’s disease and the controls (n = 6 per group). (D) Expression of <i>GBA1</i> gene was determined in AD hippocampal regions compared to the controls using real-time PCR. The β-actin mRNA was used as an internal reference control to normalize relative mRNA levels (n = 6 per group). (E) Oligomeric and monomeric Aβ_1–42 were identified by SDS-PAGE using anti-Aβ antibody (4G8). (F) Primary cortical neurons were incubated with 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. The cell lysates were subjected to immunoblot using GCase and β-actin antibodies. (G) The expression level of GCase was quantified. Data are expressed as the mean ± SEM (Student’s t test, *<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Overexpression of GCase increases intracellular pH levels reduced by Aβ_1–42 oligomers in primary neurons.

<p>(A) Primary mouse cortical neurons were incubated with pHrodo™ Red or pHrodo™ Green at 37°C for 30 minutes. The fluorescence intensities in the intracellular area (dotted outline) were measured using various pH ranges (4.5, 5.5, 6.5, and 7.5) of intracellular pH calibration buffers. The fluorescence units of pHrodo™ Red and pHrodo™ Green signals are represented as a standard curve. (B) Primary mouse cortical neurons infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i> were incubated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. The neurons were loaded with pHrodo™ Red and pHrodo™ Green and live cell imaging solution, and further incubated at 37°C for 30 minutes. (C and D) The fluorescence units were quantified and represented as a graph. (E) The fluorescence units of pHrodo™ Green were converted to pH and represented as a graph (two-way ANOVA, Bonferroni posttest, **<i>P</i> < 0.01, ***<i>P</i> < 0.001).</p

Lysosomal Enzyme Glucocerebrosidase Protects against Aβ_1-42 Oligomer-Induced Neurotoxicity

<div><p>Glucocerebrosidase (GCase) functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher’s disease (GD), Parkinson’s disease (PD), and Dementia with Lewy Bodies (DLB). However, there is little information about the role of GCase in the pathogenesis of Alzheimer’s disease (AD). Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ_1–42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ_1–42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ_1–42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD.</p></div

Overexpression of GCase restores impairment of lysosomal biogenesis induced by Aβ_1–42 oligomers in neurons.

<p>(A) Primary mouse cortical neurons were infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i>. 72 h after the infection, the cells were further incubated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h. The plasma membranes were labeled with CellLight plasma membrane-CFP (PM, blue) and the lysosomes were labeled with LysoTracker Red DND-99 (Lysosome, red). (B) The relative area of lysosomes in plasma membrane (dotted outline) was measured (two-way ANOVA, Bonferroni posttest, <i>P</i>* < 0.05, <i>P</i>*** < 0.001). (C) The diameters of LysoTracker positive-puncta were quantified and represented as a scatter plot (two-way ANOVA, Bonferroni posttest, *<i>P</i> < 0.05, **<i>P</i> < 0.01) versus control (LV-Cont, PBS).</p

Ectopic expression of GCase restores lysosomal cathepsin D activity reduced by Aβ_1–42 oligomer treatment in primary neurons.

<p>(A) The localization of cathepsin D is represented using lysosomal marker (LAMP1) and DAPI. (B) The cathepsin D activity levels were measured in lysosome-enriched and cytoplasmic fractions of lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus infected primary mouse cortical neurons treated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. (C) Immunoblot analysis for heavy chain subunit of mature cathepsin D (CatD heavy chain) in cytoplasmic and lysosome-enriched fractions was performed using anti-cathepsin D antibody. The quality of cytosolic and lysosome-enriched fractions was confirmed with immunoblotting using anti-MEK1/2 and LAMP1 antibodies, respectively. The values are the mean ± SEM of three independent experiments (two-way ANOVA, Bonferroni posttest, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001).</p