Intracellular Cleavage of Amyloid β by a Viral Protease NIa Prevents Amyloid β-Mediated Cytotoxicity

<div><p>Nuclear inclusion a (NIa) of turnip mosaic virus is a cytosolic protease that cleaves amyloid β (Aβ) when heterologously overexpressed. Lentivirus-mediated expression of NIa in the brains of APP(sw)/PS1 mice significantly reduces cerebral Aβ levels and plaque depositions, and improves behavioral deficits. Here, the effects of NIa and neprilysin (NEP), a well-known Aβ-cleaving protease, on oligomeric Aβ-induced cell death were evaluated. NIa cleaved monomeric and oligomeric Aβ at a similar rate, whereas NEP only cleaved monomeric Aβ. Oligomeric Aβ-induced cytotoxicity and mitochondrial dysfunction were significantly ameliorated by NIa, but not by NEP. Endocytosed fluorescently-labeled Aβ localized to mitochondria, and this was significantly reduced by NIa, but not by NEP. These data suggest that NIa may exerts its protective roles by degrading Aβ and thus preventing mitochondrial deposition of Aβ.</p></div

In vitro cleavage of Aβ by NIa and NEP.

<p>For the cleavage assay, 2.5 µM of monomeric (<b>A</b>) and oligomeric Aβ (<b>B</b>) were incubated with 0.5 µM of purified NIa or NEP for 1, 2, and 3 h. The reaction mixture was separated on a PeptiGel (Elpis Biotech), blotted, and probed with the anti-Aβ 6E10 antibody. The densities of the intact Aβ bands were quantified using NIH ImageJ software and plotted. Each data point and error bar represents the mean ± SD (n = 3).</p

NIa, but not NEP, restores Aβ-mediated mitochondrial dysfunction.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 10 µM of oligomeric Aβ for an additional 48 h. (<b>A</b>) Cells were treated with 2.5 µM of JC-1, an indicator of Ψm, for 15 min at 37°C and visualized by confocal microscopy. The intensities of red and green JC-1 fluorescence were quantitated using the MetaMorph imaging software and their ratios were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 7); *<i>p</i><0.05, **<i>p</i><0.01. (<b>B</b>) Cells were incubated with 30 µM of DHE, an indicator of ROS for 30 min. Low (panels a–d) and high (panels a’–d’) magnification images of cells were obtained using a confocal microscope. Data represent the number of fluorescent cells as a percentage of the total number of cells in the observed field. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 5); *<i>p</i><0.05, **<i>p</i><0.01.</p

Endocytosed oligomeric Aβ accumulates in lysosomes and mitochondria.

<p>Human neuroblastoma SH-SY5Y cells were treated with 2.5 µM of Alexa Fluor-labeled Aβ oligomers for 90 min (pulse) and were further incubated in fresh media for 90, 270, or 630 min (chase). Cells were co-stained with LysoTracker and MitoTracker, and observed under a confocal microscope. The images indicated by the open boxes are shown in a higher magnification in the adjacent columns. The yellow color in the merged image indicates co-localization of green (Alexa Fluor 488-labeled Aβ) and red (LysoTracker, Red/MitoTracker, Deep Red) fluorescence. The percentages of lysosomes (white bars) or mitochondria (gray bars) that co-localized with Aβ were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 10). DIC, differential interference contrast.</p

NIa, but not NEP, prevents accumulation of Aβ in mitochondria.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 2.5 µM of Alexa Fluor 488-labeled Aβ oligomers for an additional 18 h. Cells were stained with LysoTracker (<b>A</b>) or MitoTracker (<b>B</b>), and observed by confocal microscopy. White arrowheads in panel A indicate Aβ that co-localized with lysosomes and open arrowheads in panel B indicate Aβ that co-localized with mitochondria. The percentages of lysosomes or mitochondria that co-localized with Aβ were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 10); **<i>p</i><0.01.</p

NIa, but not NEP, prevents Aβ-mediated cytotoxicity.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 10 µM of oligomeric Aβ for an additional 48 h. (<b>A</b>) Western blotting with an anti-HA antibody showed that the expression levels of NIa and NEP were similar. GAPDH (detected with an anti-GAPDH antibody) was used as the loading control. (<b>B</b>) Cell viability was determined by using the MTT assay. Each bar and error bar represents the mean ± SD (n = 3); *<i>p</i><0.05. (<b>C</b>) Cells were stained with Hoechst 33342 and viewed under a fluorescence microscope. Arrows indicate cells with pyknotic nuclei. The number of pyknotic nuclei was counted and plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 8); **<i>p</i><0.01.</p

Cytotoxic effects of cucurbitacin I in cultured neonatal rat cardiomyocytes.

<p>Cell viability was measured in cardiomyocytes treated with the DMSO (control) or cucurbitacin I at 0.1, 0.5, 1, 5, and 10 μM for 24 h (A), 48 h (B), and 72 h (C). (D) Cell viability at each concentration on a time-dependent manner. Data are expressed as the means ± S.D. from three independent experiments. Significance was determined via a two-way ANOVA. *<i>P</i> < 0.05 vs. control group. Cont, control; Cu I, cucurbitacin I.</p

Cucurbitacin I inhibits CTGF expression and MAPK signaling in hypertrophic cardiomyocytes.

<p>(A) CTGF mRNA expression levels were measured by quantitative RT-PCR in hypertrophic cardiomyocytes stimulated with PE (100 μM) for 1, 6, 12, and 24 h. (B) Cardiomyocytes were pretreated with cucurbitacin I (1 μM), and then stimulated with PE (100 μM) for 6 h. Next, CTGF mRNA expression levels were measured by quantitative RT-PCR. (C) Cardiomyocyte extracts (50 μg) were subjected to Western blot analysis of CTGF and MAPK, (ERK1/2, JNK, and p38) protein expression levels. The expression levels of CTGF, the MAPKs, and the phosphorylated forms of the MAPKs (p-ERK1/2, p-JNK, and p-p38 kinase) were estimated by measuring band densities with NIH Image J software. GAPDH was used as a loading control, and Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. Cont, control; Cu I, Cucurbitacin I.</p

Cucurbitacin I blocks the TGF-β/Smad signaling pathway in hypertrophic cardiomyocytes.

<p>Cell extracts (50 μg) were used for Western blot analysis of TGF-β and Smad (Smad2, 3, and 7) protein expression levels. The expression levels of TGF-β, Smad2, 3, and 7, and the phosphorylated forms of Smad2 and 3 (p-Smad2 and p-Smad3) were estimated by measuring band densities with NIH Image J software. GAPDH was used as a loading control. And Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. Cont, control; Cu I, Cucurbitacin I.</p

The anti-hypertrophic impact of cucurbitacin I is attenuated in the CTGF-silenced, hypertrophic cardiomyocytes.

<p>(A) Expression of CTGF was measured by western blotting in cardiomyocytes transfected with control or CTGF siRNA. (B) Representative photograph of cardiomyocytes after transfection with a scrambled siRNA or CTGF siRNA and pretreatment with cucurbitacin I (1 μM), followed by exposure to PE (100 μM). Sarcomeric organization of the cardiomyocytes was visualized by staining with an anti-α-actinin antibody. (C) Cell surface areas were measured by using NIH Image J software (n = 100 cells). Scale bars, 50 μm. (D) Quantitative RT-PCR analysis of ANF and β-MHC mRNA expression levels in scrambled siRNA- or CTGF siRNA-transfected cardiomyocytes treated with cucurbitacin I and/or PE. All experiments were performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. scr, scrambled siRNA; si-CTGF, si-RNA against CTGF; Cont, control; Cu I, Cucurbitacin I; NS, not significant.</p