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

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.


Introduction
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder that is characterized by progressive memory impairment and cognitive dysfunction. The hallmarks of AD are the formation of intracellular neurofibrillary tangles composed of hyper-phosphorylated tau and extracellular amyloid plaques mainly composed of amyloid b (Ab). Ab is generated through sequential cleavage of amyloid precursor protein (APP) by band c-secretases [1,2].
Ab exists as soluble monomers and oligomers, and insoluble fibrils. Which of these forms of Ab is the active species that are responsible for synaptic loss and neurodegeneration in AD is controversial [3,4]. Neither monomeric nor fibrillar forms of Ab appear to be responsible [4,5]. Rather, a number of studies indicate that oligomeric Ab is the most potent neurotoxic species in association with AD [6,7,8,9,10]. For example, oligomeric Ab reduces neuronal viability approximately 10-fold more efficiently than fibrillar Ab [11].
Ab levels in healthy brain are delicately regulated by a dynamic equilibrium between production of Ab from APP and clearance of Ab via perivascular drainage or enzymatic degradation. The cytotoxic process of AD is closely linked to an imbalance between the production and clearance of Ab [12]. Therefore, restoration of this balance by increasing the degradation of Ab might be a valid therapeutic modality for the treatment of AD [13]. Several endogenous proteases can degrade Ab. Among these, neprilysin (NEP) is considered to be the physiological regulator of the Ab levels in the brain parenchyma [14,15,16]. Intracerebral injections of a recombinant lentivirus expressing human NEP reduce Ab deposits and neurodegenerative alterations in a mouse model of amyloidosis [17]. Implantation of primary fibroblast cells that express a secreted form of human NEP also significantly reduces plaque burdens in the mouse brain [18]. Consistently, the genetic ablation of NEP in mice markedly increases Ab levels in whole brain and plasma, increases plaque burdens in the hippocampus, and leads to the development of AD-like neuropathology [19]. Lentivirus-mediated long-term expression of NEP improves behavioral performances and ameliorates neurodegenerative pathology in APP mice [20]. However, the therapeutic potential of NEP is controversial as over-expression of NEP failed to reduce the toxic oligomeric Ab levels nor improve cognitive deficits in AD mice although it did reduce plaque formation [21].
Nuclear inclusion a (NIa) of turnip mosaic virus is a cytosolic protease with a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln [22]. In an in vitro study, we demonstrated that NIa specifically cleaves Ab, which contains the Val-His-His-Gln sequence near to its putative a-secretase cleavage site [23]. We further showed that lentivirus-mediated expression of NIa in the brain of AD mice significantly reduced Ab pathology and improved behavioral deficits [23,24].
Several lines of evidence have suggested that the progression of AD may be associated with mitochondrial dysfunction [25,26]. Ab inhibits import of nuclear-encoded mitochondrial proteins, and subsequently impairs mitochondrial functions and morphology [27]. In neurons, the overexpression of Ab results in mitochondrial fragmentation and an abnormal subcellular distribution of mitochondria by evoking an imbalance between mitochondrial fusion and fission [28]. Furthermore, Ab impairs oxidative phosphorylation and ATP production in transgenic AD mice [29].
Here, we compared the functions of NIa and NEP, and found that NIa, but not NEP, cleaved oligomeric Ab and prevented Abinduced cytotoxicity and mitochondrial dysfunction in human neuroblastoma cells. By tracing exogenously added Ab, we determined that NIa prevents localization of endocytosed Ab to mitochondria. Our study suggests that disruption of Ab trafficking to mitochondria via intracellular degradation of Ab is a valuable approach for preventing Ab-induced cytotoxicity.

NIa, but not NEP, Cleaves Oligomeric Ab In vitro
We first performed an in vitro cleavage assay to compare the proteolytic activities of NIa and NEP for Ab. Monomeric and oligomeric Ab were incubated with the same amounts of purified NIa and NEP, and were then analyzed by Western blotting. Cleavage of Ab was discerned by the disappearance of protein bands corresponding to intact monomeric and oligomeric Ab. As expected, monomeric Ab was efficiently cleaved by both NIa and NEP ( Figure 1). However, oligomeric Ab was only cleaved by NIa, not by NEP ( Figure 1B). Notably, NIa cleaved both monomeric and oligomeric Ab indistinguishably with a similar catalytic activity. To the best of our knowledge, NIa is the only cytosolic protease that can cleave both monomeric and oligomeric Ab with a strict substrate specificity.

NIa, but not NEP, Prevents Oligomeric Ab-mediated Cytotoxicity
We next examined whether NIa or NEP can inhibit oligomeric Ab-mediated cytotoxicity in human neuroblastoma SH-SY5Y cells. The cells were transformed with plasmids expressing HAtagged NIa or NEP. Expression of NIa and NEP was assessed by Western blotting with an anti-HA antibody (Figure 2A). The amounts of plasmids used for cell transformations were adjusted so that the expression levels of NIa and NEP were almost equal in all the subsequent experiments.
Treatment of SH-SY5Y cells with oligomeric Ab for 48 h reduced cell viability in a dose-dependent manner ( Figure S1). The most prominent effects were seen with 10-20 mM Ab. Thus, 10 mM oligomeric Ab was used to observe the cytotoxic effects of Ab in all the subsequent experiments. Under these conditions, Ab reduced cell viability by ,35% as assessed by 3-[4,5-dimethylthizaol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays ( Figure 2B). This effect was significantly inhibited by NIa (,16% reduction vs. control) but not by NEP ( Figure 2B). Abmediated cytotoxicity can also be monitored by nuclear fragmentation and condensation, a phenomenon known as pyknosis. Under control conditions, only 1-2% of cells underwent pyknosis as observed using a fluorescence microscope. In line with previous reports [30,31], Ab increased the percentage of pyknotic cells to ,17%. This Ab-mediated pyknosis was significantly reduced by NIa (,7%), but not by NEP ( Figure 2C). Collectively, these results indicate that NIa, but not NEP, prevents oligomeric Ab-mediated cytotoxicity.

NIa, but not NEP, Ameliorates Oligomeric Ab-mediated Mitochondrial Dysfunction
Ab reportedly is associated with mitochondrial dysfunction [27]. Thus, we examined whether NIa or NEP affect Ab-induced mitochondrial dysfunction in SH-SY5Y cells. To monitor mitochondrial membrane potential (Ym) using a confocal microscope, we utilized JC-1, which exists as a green-fluorescent J-monomer at depolarized membrane potentials and as a red-fluorescent Jaggregate at hyperpolarized membrane potentials. A decrease in the ratio of red fluorescence to green fluorescence indicates a decrease in Ym. As expected, Ab significantly reduced Ym, which was reversed by NIa, but not by NEP ( Figure 3A).
Next, we utilized the cell-permeable fluorescent dye dihydroethidium (DHE) to monitor the production of reactive oxygen species (ROS). When DHE is oxidized by superoxide anions to oxoethidium, it intercalates into DNA and generates red fluorescence [32]. In line with previous reports [33], Ab increased the percentage of cells with red fluorescence (,33% vs. ,12% in control), which was significantly attenuated by NIa (,21%), but not by NEP ( Figure 3B). Taken together, these data indicate that NIa, but not NEP, ameliorates Ab-mediated mitochondrial dysfunction.

NIa, but not NEP, Prevents Accumulation of Ab in Mitochondria
We next sought to elucidate how NIa prevents Ab-mediated mitochondrial dysfunction. To this end, we traced the intracellular trafficking of exogenously added Ab in SH-SY5Y cells. Oligomeric Ab was labeled with Alexa Fluor 488 and was then added to the culture media. After 90 min of incubation (pulse), the culture media was replaced with fresh media not containing Ab and was then further incubated for 90, 270, and 630 min (chase). The cells were then fixed and co-stained with LysoTracker and Mito-Tracker. After 90 min of chase, ,10% of LysoTracker, but none of the MitoTracker, co-localized with the Alexa Flour. This suggested that the majority of the exogenously added Ab was present in endosomes, some was present in lysosomes, and none was present in mitochondria at this stage. However, the percentage of LysoTracker or MitoTracker that co-localized with Alexa fluorescence gradually increased as the duration of the chase increased. After 630 min of chase, ,35% of LysoTracker and ,5% of MitoTracker co-localized with the Alexa Flour ( Figure 4). These results are consistent with earlier observations that exogenously added Ab reaches lysosomes via endocytosis, where a portion of the peptide enters mitochondria; however, the mechanism is unknown.
We next examined whether NIa or NEP affect trafficking of oligomeric Ab to mitochondria. SH-SY5Y cells transformed with plasmids expressing NIa or NEP were treated with Alexa Flour 488-labeled oligomeric Ab for 18 h. At this time point, most of the Alexa Flour co-localized with either LysoTracker or MitoTracker. Under normal conditions, ,25% and ,5% of LysoTracker and MitoTracker, respectively, co-localized with the Alexa Flour. Neither NIa nor NEP significantly affected the co-localization of LysoTracker with the Alexa Flour ( Figure 5A). However, NIa, but not NEP, significantly reduced the percentage of MitoTracker that co-localized with the Alexa Flour (,1%) ( Figure 5B). Colocalization of MitoTracker and the Alexa Flour was further confirmed by 3-dimensional reconstruction of the confocal images ( Figure S2, Video S1-4). The Alexa Flour represents intact Ab after incubation for 18 h, supporting the validity of our experimental approach ( Figure S3). Collectively, these data suggest that NIa prevents the accumulation of oligomeric Ab in mitochondria by proteolytically degrading the peptide in the cytosol.

Discussion
The NIa protease of turnip mosaic virus has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln [22]. This protease is involved in the cleavage of viral polyproteins to generate mature viral proteins. We noticed the same Val-His-His-Gln consensus sequence in Ab near to its putative a-secretase cleavage site and surmised that this sequence could be cleaved by NIa. Indeed, NIa specifically cleaves Ab in vitro and significantly reduces Ab-induced cell death in rat neuroblastoma cells [23]. Furthermore, lentivirus-mediated expression of NIa in the brain of AD mice significantly reduces cerebral Ab levels and plaque depositions, and recovers behavioral deficits [24]. These results raised the possibility that NIa can be used as a therapeutic modality for the treatment of AD.
Currently, more than 20 endogenous Ab-cleaving enzymes have been identified [34]. Among them, NEP is considered to have a major role in the metabolism of Ab in the brain. The possible therapeutic use of NEP for AD was proposed because NEP ameliorates neurodegenerative pathology and also improves behavioral performances in APP mice [20]. However, this earlier enthusiasm has been challenged. For examples, over-expression of NEP does not improve cognitive deficits in AD mice [21]. This might be explained, at least partially, because NEP cannot cleave the more toxic oligomeric Ab, as shown here. In addition, it should be noted that NEP has diverse physiological roles in the brain. For example, overexpression of NEP causes a reduction in cAMPresponsive element-binding protein-mediated transcription, agedependent axon degeneration, and premature death in flies [35]. Sustained NEP activation may also be detrimental in mammals because NEP can degrade a wide range of circulating peptides, including enkephalin, atrial natriuretic peptide, endothelin, and substance P [36]. Therefore, NIa has certain advantages over NEP as a therapeutic modality for AD with its unique capability of cleaving the more toxic oligomeric Ab and its relatively high substrate specificity. The controversy surrounding the molecular mechanism underlying the cytotoxicity of Ab in brains has yet to be settled. Among the several hypotheses, one suggests that Ab exerts its detrimental effects partly by interfering with mitochondrial functions. Ab is internalized via raft-mediated endocytosis [37]. The internalized Ab reaches the mitochondria, where it binds to a mitochondrial enzyme called Ab-binding alcohol dehydrogenase (ABAD). It remains to be seen how the endocytosed, thus intraluminal, Ab reaches the mitochondria. The interaction between Ab and ABAD promotes leakage of ROS, mitochondrial dysfunction, and cell death [38]. Furthermore, inhibition of the Ab-ABAD interaction using a decoy peptide improves mitochondrial function in AD mice [39]. NIa did not interfere with the internalization of Ab or with the transport of Ab to lysosomes, but reduced the amounts of Ab localized in mitochondria ( Figure 5). Therefore, it appears that NIa cleaves Ab that was in transit from lysosomes to mitochondria. Our chase experiments suggest that Ab travels through endosomes and lysosomes and that a portion of Ab further travels to mitochondria ( Figure 4). Considering that NIa functions primarily in the cytosol, it is possible that Ab transiently passes the cytosol during transit from the lysosomes to mitochondria. However, we could not definitively test this hypothesis due to limitations in current imaging techniques. Interpretation of our data is also partially hampered by the fact that the traffic routes allowing the localization of Ab in mitochondria are largely unknown.
Collectively, we demonstrated that NIa prevents Ab-mediated cytotoxicity and associated mitochondrial dysfunction by reducing the amounts of Ab localized in the mitochondria. During the pathogenesis of AD, the route linking lysosomes to mitochondria can be viewed as a ''Thermopylae pass''. Annihilation of the invading Ab at this ''pass'' can be a winning strategy in the battle against AD.

Preparation of Ab 42 Oligomer
Ab 42 oligomers were prepared according to the method described by Stine et al. [40]. Synthetic Ab 42 peptides were initially solubilized in 1,1,1,3,3,3-hexafluoroisopropanol (Fluka) to a concentration of 1 mM, to monomerize pre-existing aggregates. Following evaporation of the 1,1,1,3,3,3-hexafluoroisopropanol in a fume hood overnight, the resulting peptide film was stored desiccated at 220uC. Subsequently, the peptide was resuspended in anhydrous dimethyl sulfoxide to a concentration of 2.5 mM and bath sonicated for 10 min. To enrich oligomers, phenol-red free Dulbecco's modified Eagle's medium (DMEM; Gibco) was added under continuous vortexing to bring the peptide to a final concentration of 100 mM and incubated at 4uC for 24 h.

Purification of the NIa Protease and In vitro Cleavage Assay
Purification of the NIa protease was performed as described by Han et al. [23]. For the in vitro cleavage assay, 0.5 mM of purified NIa or recombinant NEP was incubated with 2.5 mM of monomeric or oligomeric Ab in a time-dependent manner. The buffers used in this reaction were as follows: NIa (20 mM HEPES [pH 7.4], 10 mM KCl, 10 mM MgCl 2 ) and NEP (50 mM Tris-HCl [pH 9.0], 0.05% Brij35). After incubation, the reaction mixture was separated on a PeptiGel (Elpis Biotech), blotted, and probed with the anti-Ab 6E10 antibody [41].

Cell Culture and DNA Transfection
Human neuroblastoma SH-SY5Y cells were grown in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Cells were transiently transfected with plasmid DNA using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions. To express the turnip mosaic virus NIa and the NEP protease in mammalian cells, codon-optimized NIa and NEP genes were subcloned into the pcDNA3 vector (Invitrogen) containing an N-terminal HA tag. A matching vector without an insert was used as a control.

Quantification of Cell Death/Survival
MTT (Sigma) was dissolved in phosphate buffered saline at a concentration of 2.5 mg/ml. A volume of MTT solution equivalent to 20% of the culture media volume was added to the cell culture at 37uC for 2 h. A volume of dimethyl sulfoxide (solubilization solution) equivalent to the culture media volume was added, and cells were placed on a shaker until the resulting formazan crystals were completely dissolved. The absorbance of the samples was measured at 570 nm, and the background absorbance of each well was measured at 690 nm. SH-SY5Y cells were examined for pyknotic nuclei by Hoechst 33342 staining following the methods described by Wyttenbach et al. [42] and Sellamuthu et al. [43].

Measurement of Ym
Ym was determined by staining SH-SY5Y cells with JC-1 and was measured by confocal microscopy. SH-SY5Y cells were cultured on poly-L-lysine-coated coverslips. After exposure to Ab, cells were incubated in DMEM containing 2.5 mM JC-1 for 15 min at 37uC. The cells were washed and fluorescent images were then obtained immediately using a Fluoview FV 1000 confocal laser scanning microscope. Data were analyzed with MetaMorph imaging software to quantify the intensities of red and green fluorescence. The results were expressed as the ratio of red fluorescence to green fluorescence.

Measurement of ROS Production
ROS production in SH-SY5Y cells was assayed using the oxidative fluorescent dye DHE. SH-SY5Y cells were cultured on poly-L-lysine-coated coverslips. After exposure to Ab, cells were loaded with 30 mM of DHE for 30 min at 37uC. The cells were washed to remove excess DHE and fluorescent images were captured immediately using a Fluoview FV 1000 confocal laser scanning microscope. The excitation and emission wavelengths were 510 nm and 590 nm, respectively. Images were analyzed using MetaMorph imaging software. The number of fluorescent cells were counted and represented as a percentage of the total number of cells in each image field.

Labeling of Ab 42 Oligomers
The labeling reaction was performed using the Alexa Fluor 488 Microscale Protein Labeling Kit (Invitrogen). The procedure was described by Jungbauer et al. [44] in detail.

Statistical Analysis
Results are expressed as the means 6 standard deviation (SD). Comparisons between two groups were performed using the Student's t-test. Comparisons between multiple groups were performed by one-way ANOVA with the Bonferroni correction. Statistical analyses were conducted with StatView software version 5.0 (SAS Institute Inc.). A p-value of less than 0.05 was considered statistically significant. Figure S1 Dose-dependent effects of oligomeric Ab on cell viability. SH-SY5Y cells were incubated with various concentrations of oligomeric Ab for 48 h. Cell viability was determined by using the MTT assay. Each bar and error bar represents the mean 6 SD (n = 4); **p,0.01. (EPS) Figure S2 Assessment of mitochondrial accumulation of Ab by confocal microscopy. SH-SY5Y cells were treated with 2.5 mM of Alexa Fluor-labeled Ab oligomers for 90 min and were further incubated in fresh media for 630 min. Cells were stained with MitoTracker and observed under a laser scanning confocal microscope. (A) Reconstruction of 3-D images was performed with 50-60 Z-directional slices (0.1 mm thick) of the confocal images. The 3-D images were then virtually re-sliced in YZ axis (marked by white broken lines) to obtain transversal images (a 1 , a 2 ). Open arrowheads indicate Ab that co-localized with mitochondria. Note that all the 2 yellow dots seen in XY planes (a) are also yellow when observed in YZ planes (a 1 , a 2 ). Scale bar, 20 mm. Video S1 360-degree view of the reconstituted 3-D confocal images. Video was created for the positive dot that was shown in Figure S2 (panel a 1 ).

(AVI)
Video S2 360-degree view of the reconstituted 3-D confocal images. Video was created for the positive dot that was shown in Figure S2 (panel a 2 ).

(AVI)
Video S3 360-degree view of the reconstituted 3-D confocal images. Video was created for one of the negative dots that were shown in Figure S2  Video S4 360-degree view of the reconstituted 3-D confocal images. Video was created for one of the negative dots that was shown in Figure S2