Two-month-old no carrier (control mice) and B6SJL-Tg(APPSwFlLon, PSEN1* M146L*L286V)6799Vas/Mmjax (5XFAD Tg mice) were used for producing the focal cerebral ischemia.

Experimental procedures involving the use of live animals have been carried out in accordance with the guidelines established by the European Community Directive 86/609/EEC (November 24, 1986), Italian Ministry of Health and the University of Turin institutional guidelines on animal welfare (law 116/92 on Care and Protection of living animals undergoing experimental or other scientific procedures; authorization number 17/2010-B, June 30, 2010). Moreover, the Ethical Committee of the University of Turin approved this type of studies.

The animals were maintained under 12-h light/dark cycles and were provided with water and food “ad libitum” (standard mouse chow 4RF25-GLP, Mucedola srl, Settimo Milanese, Italy). Specifically, all the procedures were carried out in order to minimize the pain and distress in the animals and we used the fewest number of animals required to obtain statistically significant data.

Focal ischemia was performed as previously described (Labat-gest and Tomasi, 2013). Briefly, 10 min before surgery, the animals were i.p injected with Rose Bengal (15 mg/ml) according to their body weight (10 μl/g). Then, we used 4% isoflurane (Isoflurane-Vet 100%, Liquid, Merial Italy, Milan, Italy) vaporized in O₂/N₂O 50:50 to anesthetize wt and tg mice. The anesthesia was maintained at 1.5%–2.5% isoflurane while mice were in the stereotaxic apparatus for small rodents (Stoelting, Wood Dale, IL, USA). The skull was exposed and an optical fiber cable (150 W) was placed in close contact with a small region (approximately 30 mm²) 2 mm lateral to the Bregma, corresponding to the sensory motor cortex (Vogt and Paxinos, 2014). After 15 min of illumination the mice were sutured and placed in a warm cage for recovery.

After 24 h the animals were killed by using an overdose of anesthetic, the brains were collected and cut with a mouse brain matrix in 1 mm coronal slices; brain extracts were removed from lesioned and controlateral areas. Then the samples were prepared for Triphenyl-Tetrazolium Chloride (TTC) staining. The TTC staining was used to stain and allow dissection of the lesioned areas and not to quantify the amount of the ischemic damage. The TTC reaction was stopped with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4). The brain slices were maintained in the fixative for 2 weeks and then processed for the isotropic fractionator.

Isotropic fractionator was executed according to Herculano-Houzel and Lent (2005) following a TTC staining as described before (Repetto et al., 2016). Briefly, after the neural tissue was properly fixed, a single brain slice per animal was collocated into a glass tissue grinder, a saline-detergent solution consisting of 40 mM sodium citrate and 1% Triton™ X-100 (Sigma-Aldrich, St. Louis, MO, USA) was added and the tissue was carefully homogenizated to obtain a nuclear suspension. The nuclear suspension obtained was stained with the fluorescent DNA dye 4′-6-diamino-2-phenylindole dihydrochloride (DAPI; DAPI, dilactate, D9564, Sigma-Aldrich, St. Louis, MO, USA). Aliquots from the isotropic suspension were loaded into a hemeocytometer (Neubauer chamber) and observed under fluorescence microscopy (Nikon Eclipse 80i). The nuclei density was evaluated by counting the number of nuclei within sectors of the hemeocytometer coverslipped (1 mm² area; 0.1 mm depth) of four aliquots for sample. To recognize the fraction of neuronal nuclei among the total number of DAPI-stained nuclei, another aliquot of the isotropic suspension was collected and stained with mouse primary antibody for the neuronal nuclear protein NeuN (MAB377, Chemicon, Single Oak Drive, Temecula, CA, USA, 1:200 in PBS, overnight incubation at room temperature (RT)). Then, the nuclei were washed in saline and incubated at RT for at least 2 h with the secondary Cy3 conjugated anti-mouse donkey antibody (Chemicon, Single Oak Drive, Temecula, CA, USA; 1:200 in PBS) and normal donkey serum (1:10; D9663, Sigma-Aldrich, St. Louis, MO, USA). The non-neuronal cells were obtained as the difference between the total number of cells and the total number of neurons.

TAT-fused Uch-L1 was provided by Dr. Ottavio Arancio (Professor at Columbia University), the construct was obtained as described by Gong et al. (2006).

Briefly, TAT vectors were transformed into E. Coli BL21(DE3) pLysS competent cells (Novagen), and the obtained colonies were grown as 1 ml overnight cultures in Luria broth (LB) medium (Sigma-Aldrich) with 100 mg ampicillin, in the presence of 100 mM IPTG. Then the cultures were transferred to 500 ml LB ampicillin plus 200 mM IPTG to obtain large-scale preparations. Fusion proteins were purified according to ProBond purification system (Invitrogen).

VUch-L1 fusion proteins were i.p. injected into mice at 0.03 g/kg, 20 min before the Rose Bengal injection and surgery procedure. After 6 or 12 h, mice were sacrificed and protein extracts were prepared and examined as described below.

The following antibodies were used for immunoblotting analyses: BACE1 (Millipore, AB5940, 1:500), pJNK1/2 (Cell Signaling Technology, #9251, 1:500); JNK1/2 (Cell Signaling Technology, #9252, 1:500); BAX (Santa Cruz Biotechnology, Sc-493, 1:100); Bcl-2 (Santa Cruz Biotechnology, Sc-509, 1:200); β actin (Sigma-Aldrich, A5441, 1:5000); Uch-L1 (Santa Cruz Biotechnology Sc-1183, 1:200).

Fresh frozen brains were homogenized in ice-cold buffer consisting of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EGTA, 1 mM EDTA, 1% Triton™-X-100, 1 mM PMSF, phosphatase and protease inhibitors and then centrifuged at 12,000 rpm for 20 min at 4°C in order to obtain soluble proteins. Lysates (20 μg) were run on 4%–12% Tris-HCl gradient PAGE gel (Invitrogen) and then transferred to nitrocellulose blotting membrane (GE Healthcare 10600008). Peroxidase-conjugated secondary antibodies were incubated 1 h at RT and revealed with Luminata Forte Western substrate (WBLUF0100, Millipore). The correct protein loading was controlled normalizing with β actin antibody.

Whole cell extracts were made in ice-cold lysis buffer (PBS, TritonX-100, SDS 10%, DTT 1 M, PMSF 0.1% and aprotinin) for 30 min and sonicated for 1 min. The lysates were centrifugated at 17,860 g for 15 min to clarify the suspensions. The protein concentration was quantified following Bradford’s method (1976). The amount of Aβ 42 was evaluated using the Human/Rat βAmyloid ELISA Kit (Wako Chemicals GmbH, Neuss, Germany) according to the manufacturer’s instructions.

BACE-1 activity was measured using a commercially available secretase kit from Calbiochem (Merck, Darmstadt, Germay), according to the manufacturer’s protocol. Briefly, samples were lysed in cold 1× Extraction Buffer (provided by the kit) to obtain a final protein concentration of 1 mg/mL.

The method is based on the ability of the enzyme to cleave a peptide conjugated with a reporter molecules (EDANS and DABCYL). The cleavage induces the release of a fluorescent signal that was detected using a fluorescence microplate reader (excitation wavelength of 355 nm and emission 510 nm) and the signal is proportional to the enzymatic activity. BACE1 activity was expressed as percentage change over activity level of control samples (Guglielmotto et al., 2012).

The hydrolase activity assay was performed using the fluorogenic ubiquitin-7-amino-4-methylcoumarin (ubiquitin-AMC; Boston Biochem, Cambridge, MA, USA) substrate diluted in an assay buffer (50 mM Tris–HCl pH 7.6, 0.5 mM EDTA, 5 mM DTT and 0.1 mg mL ovalbumin). The reaction mixture containing 400 nM substrate and 100 μg protein samples was incubated for 5 min at RT and the enzymatic activity was measured using a fluorescence spectrometer (LS55; Perkin Elmer Instruments, Waltham, MA, USA) at 25°C (EX 380 nm and EM460 nm; Guglielmotto et al., 2012).

The activity of NF-κB was measured using a commercially available kit (Active Motif, Rixensart, Belgium). The NF-κB contained in the nuclear extracts specifically binds to an oligonucleotide containing an NF-κB consensus binding site. The primary antibodies recognize epitopes on p65, p50, p52, RelB and RelC proteins upon DNA binding (Guglielmotto et al., 2012).

Statistical analyses were performed using GraphPad Prism version 4.0 (GraphPad software, San Diego). All values were presented as mean ± standard error (SEM). Means were compared by one or two-way analysis of variance (ANOVA) with Bonferroni as a post hoc test (Manassero et al., 2016).

We performed all experiments in control or 5xFAD Tg mice subjected or not to thrombotic focal cerebral ischemia mediated by photo-activation with Rose Bengal and then sacrificed up to 12 h later. Figure 1A reports the hydrolase activity; as shown, the thrombotic ischemia in control mice was followed by approximately 40% decrease of activity, whereas the ischemic injury induced a drastic decrease (−70%) of hydrolase levels in Tg mice that presented significant lower basal levels of the enzyme (−30%).

To confirm the role of Uch-L1 in the decreased hydrolase levels we performed western blot analysis using monoclonal Uch-L1 antibody and we found a significant decrease in the enzyme protein levels in both controls and Tg mice after ischemic injury (Figure 1B). We previously reported that the decrease of Uch-L1 activity corresponded to an increase in BACE1 protein levels (Guglielmotto et al., 2012). Here we confirmed that the two events are related since the vascular injury was able to significantly (approximately 2.5-fold) increase BACE1 protein levels. Tg mice have higher basal protein levels respect to controls (2-fold) but after thrombotic ischemia the BACE1 levels are increased, resulting significant not only vs. Tg controls but also respect to ischemic injured control mice (2-fold increase vs. Tg controls and 3-fold increase vs. controls ischemic injured; Figure 1C). To confirm that BACE1 was active, we measured the enzymatic activity and we confirmed that vascular injury increased BACE1 activity of 20% in control mice whereas in Tg mice produced a 50% increase; the basal activity of BACE1 in Tg mice was increased respect to control mice of approximately 25% (Figure 1D). We also measured levels of Aβ 42 in our experimental models (Figure 1E). The vascular injury in wild type mice was followed by a significant increase in Aβ 42 levels respect to control animals in which the levels were almost undetectable. As expected, the basal levels of Aβ 42 in Tg mice were significantly higher than those of control mice subjected or not to photothrombosis. The ischemic injury in Tg mice was followed by a further significant production of Aβ 42 (Figure 1D).

Thus, we observed that the decrease in Uch-L1 determined an increase of BACE1 protein levels and activity and an increased production of Aβ 42. Moreover, we can affirm that the decrease of Uch-L1 is a common event in AD and VD and that when both pathologies are present there is a cumulative effect.

We studied this pathway because it had been previously reported that Aβ 42 regulates BACE1 promoter transactivation and activity through NF-κB pathway (Buggia-Prevot et al., 2008) and that the activation of this pathway abolishes Uch-L1 gene transcription (Wang et al., 2011). We also previously found that the pharmacological inhibition of NF-κB by blocking the nuclear translocation of p50 or p65 in an in vitro model was followed by a completely protection of the Uch-L1 decrease as well as of the BACE1 increase (Guglielmotto et al., 2012). Thus, we investigated whether the focal ischemia could activate NF-κB in our experimental model. We demonstrated that the ischemic injury was able to induce nuclear activation of total NF-κB obtained by screening all NF-κB family members (Figure 2) in control mice and Tg mice exposed to Rose Bengal photo-activation. The results suggest that this pathway could be responsible for the decrease of Uch-L1.

We evaluated the neuronal density after lesion and we found that there was a significant decrease in control and Tg mice exposed to Rose Bengal photo-activation respect to not lesioned mice (Figure 3A). To determine the amount of apoptotic cell death we measured the protein levels of the proapoptotic effector BAX as well as the antiapoptotic protein Bcl-2. As reported in Figures 3B,C, the focal ischemia caused a significant increase of BAX protein (2-fold) and a parallel significant decrease in Bcl-2 (40%) in control mice. The ischemic injury induced in Tg mice was followed by a further increase in Bax (3.5-fold) and decrease in Bcl-2 (−60%) levels, that resulted significant respect to lesioned control mice (Figures 3B,C).

To foster the levels of Uch-L1 we injected control and Tg mice with a fusion protein between transduction domain of the HIV-transactivator protein (TAT), fused with an HA tag, and Uch-L1 (TAT-HA-Uch-L1; Gong et al., 2006) 30 min before ischemic injury.

To evaluate if the peptide was able to cross the blood brain barrier we performed a western blot using HA antibody, thus HA is attached to the peptide. A band is visible in cerebral homogenates after treatment with Uch-L1 peptide (data not shown). As reported in Figure 4A, the injection of TAT-HA-Uch-L1 restored normal Uch-L1 activity both in control and in Tg mice exposed to photoischemic injury (Figure 4A). The restoration of Uch-L1 was able to completely prevent the increase of BACE1 protein levels both in control and Tg mice at 6 h post injury, as reported by the representative blot and by the densitometric analysis (Figure 4B). Finally, we also found that pre-treatment with TAT-HA-Uch-L1 was able to protect against the neuronal cell death, indeed we found that the restoration of Uch-L1 significantly increases the percentage of neuronal density in pre-lesioned Tg treated animals respect to untreated lesioned ones (Figure 5A). Finally, we found that the restoration of Uch-L1 rescues Bax control levels (Figure 5B) and protects the decrease Bcl-2, both in controls and Tg mice.