Neuroprotective effects of the anticancer drug NVP-BEZ235 (dactolisib) on amyloid-β 1–42 induced neurotoxicity and memory impairment

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease and the main cause of dementia. Substantial evidences indicate that there is over-activation of the PI3K/Akt/mTOR axis in AD. Therefore, the aim of the present study was to investigate the effects of NVP-BEZ235 (BEZ; dactolisib), a dual PI3K/mTOR inhibitor that is under phase I/II clinical trials for the treatment of some types of cancer, in hippocampal neuronal cultures stimulated with amyloid-β (Aβ) 1–42 and in mice injected with Aβ 1–42 in the hippocampus. In cell cultures, BEZ reduced neuronal death induced by Aβ. BEZ, but not rapamycin, a mTOR inhibitor, or LY294002, a PI3K inhibitor that also inhibits mTOR, reduced the memory impairment induced by Aβ. The effect induced by Aβ was also prevented in PI3Kγ−/− mice. Neuronal death and microgliosis induced by Aβ were reduced by BEZ. In addition, the compound increased IL-10 and TNF-α levels in the hippocampus. Finally, BEZ did not change the phosphorylation of Akt and p70s6K, suggesting that the involvement of PI3K and mTOR in the effects induced by BEZ remains controversial. Therefore, BEZ represents a potential strategy to prevent the pathological outcomes induced by Aβ and should be investigated in other models of neurodegenerative conditions.


BEZ prevents Aβ induced memory deficits in the Object Recognition Task. As we demonstrated
a reduction of neuronal cell death induced by BEZ in vitro, we further investigated whether this compound could improve the memory deficits induced by Aβ . To assess the memory impairment of mice submitted to the hippocampal Aβ injection, the new object recognition task was performed. Animals that received Aβ and were treated with vehicle presented memory deficits (51.980 ± 5.214%, n = 14), but no memory impairment was observed in groups PBS + vehicle (69.830 ± 3.616%; p < 0.001, n = 14) and PBS + BEZ 25 mg/kg (70.760 ± 4.589%; p < 0.05, n = 4). Moreover, memory deficit induced by Aβ was prevented by the higher dose of BEZ (25 mg/kg; 71.690 ± 2.365%; p < 0.001, n = 11), but not by the lower dose of the compound (5 mg/kg; 62.320 ± 5.901%, n = 8) (Fig. 2A). The total distance travelled did not differ between the groups (Fig. 2D). In order to investigate the involvement of PI3K and mTOR pathway inhibition in the effects mediated by BEZ, we also treated a group of animals, which underwent Aβ injection, with LY294002 (25 mg/kg), a well-known pan PI3K inhibitor that also inhibits mTOR 24,37 . We further used another group with rapamycin (5 mg/kg) treatment, a mTOR inhibitor, as well as PI3Kγ knockout (PI3Kγ −/− ) mice. Memory impairment was not prevented both by LY294002 (64.190 ± 8.174%, n = 7), in spite of the trend to recovery (p = 0.133), and rapamycin (46.070 ± 6.628%; p = 0.57, n = 9). However, PI3Kγ −/− mice did not reveal memory deficit (68.450 ± 4.124; p < 0.01, n = 9) after Aβ injection. The total distance travelled by the animals treated with LY294002 and rapamycin was not statistically different from the other group (Fig. 2B,E), indicating that different recognition indexes of new object over the groups were not related to motor disability. On the other hand, PI3Kγ −/− mice traveled a longer distance (11.360 ± 1.017, n = 9) in comparison with the Aβ + vehicle (4.548 ± 1.030; p < 0.001, n = 4) and PBS + vehicle (7.681 ± 0.404; p < 0.01, n = 5) groups (Fig. 2C,F).
The treatment of the wild-type animals with BEZ, rapamycin or LY294002, as well as the injection of amyloid-β in PI3Kγ −/− mice did not induce body weight (g) changes at any administered doses (data not shown).
Scientific RepoRts | 6:25226 | DOI: 10.1038/srep25226 BEZ prevents hippocampal neuronal death induced by Aβ. One mechanism by which BEZ could prevent the memory impairment induced by Aβ would be the reduction of neuronal cell death induced by the peptide. To investigate this issue, we stained hippocampal slices with Fluoro-Jade C (FJC), a gold standard marker for degenerating neurons 38 . Indeed, previously published data demonstrated that FJC staining is increased at 24 h, 8 and 15 days after the injection of the peptide 39,40 . Intrahippocampal injection of Aβ induced neuronal death (FJC positive cells; pixels/μ m 2 ) in the CA1 layer of the ipsilateral hippocampus of animals treated with vehicle (2.481 ± 0.673, n = 11), when compared with groups PBS + vehicle (0.579 ± 0.094; p < 0.05, n = 8) and PBS + BEZ 25 mg/kg (0.598 ± 0.042; p < 0.05, n = 5). Treatment with BEZ 5 mg/kg (1.101 ± 0.168; p < 0.05, Aβ + BEZ 20 nM (E,F). Bar graphs summarizing the results of the quantification of memantine and BEZ on the cell death induced by Aβ (G). Results are expressed as mean ± SEM. *p < 0.05 and **p < 0.01 as compared to cultures treated with Aβ + vehicle (one-way ANOVA followed by Newman-Keuls test).

Discussion
In the present study, we demonstrated that Aβ caused neuronal death both in vitro and in vivo, as well as memory impairment and increased microgliosis in hippocampus of mice. We showed that the treatment with BEZ, an anticancer drug, prevented all these pathological alterations. In addition, BEZ increased the levels of IL-10 and TNF-α and there was a trend toward an increase in IL-6 levels in the hippocampus following injection with Aβ .
After the diagnosis of AD, the disease generally leads patients to cognitive impairments and death into about 3 to 9 years 13 . Memory impairment and other cognitive deficits are associated with increased dependence and incapacity in AD 42 . These symptoms are related to the functions of cerebral structures affected, specially hippocampus and neocortical areas 15 . Once there is no effective therapy against AD progression, there is a great need of pharmacological development in this area 43 .
The PI3K/Akt/mTOR pathway plays an important role in the integration of synaptic signaling 20 . A series of evidences suggest that the increase in cell cycle events, loss of neuronal processes and neurotoxicity after exposition to Aβ depends on the activation of PI3K pathway. A previous study showed that the blockade of PI3K with wortmannin in mixed neuron-glia cultures treated with Aβ reduces microglia activation 44 . Therefore, the inhibition of this pathway might represent a potential therapeutic strategy for the treatment of AD 45 . The inhibition with a drug targeting both PI3K and mTOR would be useful, once it would avoid retrograde activation of Akt usually observed after treatment with mTOR inhibitors 26 .
According to previous reports, Aβ injection led to memory impairment 10,39,40,46 , which was prevented by the BEZ treatment. However, whether PI3K and mTOR inhibition mediates the effects on memory induced BEZ is still controversial. First, we demonstrated that LY294002, a compound known to inhibit PI3K and mTOR, as well as rapamycin, which inhibits mTOR, did not avoid memory impairment in this context. Second, BEZ also Quantification of IL-2, IL-4, IFN-γ and IL17A in ipsilateral and contralateral hippocampi (K). Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to Aβ + vehicle; # p < 0.05 as compared to PBS + vehicle (one-way ANOVA followed by Newman-Keuls test). did not alter the phosphorylation of Akt and p70s6K in the hippocampus 4 h after the treatment. On the other hand, the PI3Kγ −/− mice were resistant to the memory impairment induced by Aβ , a result that corroborates a previous study which showed that a sole PI3Kγ inhibition (with AS605240) prevented learning deficits induced by Aβ 1-40 in the Morris Water Maze 10 . Nevertheless, further studies would be necessary to investigate whether the inhibition of PI3Kγ is responsible for the beneficial effects of BEZ in this model. Besides, other currently unknown mechanisms may also be involved.
We also demonstrated that BEZ reduced in vitro and in vivo neuronal cell death induced by Aβ . Interestingly, LY294002 also reduced FJC staining induced by Aβ , albeit it did not prevent memory impairment. Indeed, different studies demonstrated that LY294002 reduced memory improvement induced by other drugs, albeit it has no effect on memory per se 47,48 . Thus, even though a reduction in neuronal cell death induced by LY294002 would improve memory deficits, this effect would be counteracted by its direct effect on cognition. On the other hand, PI3Kγ −/− mice were resistant to the memory impairment, but not neuronal death, induced by Aβ . Importantly, the neuronal cell death observed in the present model is not as intense as observed in other models, such as in neurodegeneration induced by excitotoxic stimuli. Thus, considering that there is a dichotomy between neuronal death induced by Aβ injection and memory, it is possible that different mechanisms underlie the beneficial effects of BEZ.
Since BDNF and NGF promote proliferation, differentiation and survival of neurons and glial cells, as well as mediate cognitive and behavioral responses, we measured their levels in the hippocampus. Other studies have shown a decrease in neurotrophin receptors in cholinergic neurons of patients with AD 13 . It has also been shown that BDNF is reduced both in brains of patients with AD and in cell cultures treated with Aβ , what appears to be dependent on the age and pathology progression 49 . Moreover, treatment with either BDNF or NGF in animal models of AD is able to improve some features associated with the disease, like the memory impairment 13 . Notwithstanding, in our model no difference in the levels of these neurotrophins were detected 7 days after the peptide infusion or the drug treatment. This discrepancy might be related to the disease models used in different studies and to the different periods between the peptide infusion and the determination of the neurotrophins levels. In addition to this, we observed that cleaved and total caspase-3 levels were not changed by BEZ, suggesting that the neuronal cell death induced by Aβ and the effect of BEZ might not be related to reduction of this molecule.
Another mechanism related to AD is neuroinflammation. Immune cells, such as microglia, migrate and accumulate in the vicinity of Aβ plaques, leading to plaque phagocytosis and Aβ degradation 16,17,50 . However, due to a sustained activation, microglia also releases pro-inflammatory cytokines, neurotoxins and other substances that can lead to neuronal death 9,51 . The chronic inflammatory process, associated with the production of inflammatory mediators and cellular stress, increases APP amyloidogenic processing, causing a vicious cycle 52 . As in a previous study 50 , we demonstrated here that Aβ increased microgliosis, which was prevented by BEZ treatment. This effect could contribute to the reduced neurotoxicity, and could be associated with the cognitive improvement after the drug treatment.
Different studies have shown that cytokines may be involved in the pathogenesis of AD and cognitive dysfunction. IL-10 is a neuroprotective cytokine that interacts with cell surface receptors, especially in glia 52 . IL-10 has some behavioral effects arising from pro-inflammatory cytokines inhibition, showing its potential to ameliorate neuroinflammation, cognitive deficits and neurodegeneration. Previous data showed that this cytokine is able to reduce microgliosis, improve spatial learning in the radial arm water maze and enhance neurogenesis in a transgenic model of AD 53 . Therefore, the improvement in Object Recognition Task could be related to IL-10 increased levels after treatment with BEZ. TNF-α plays a central role in cytokine production cascade during the inflammatory response, being predominantly produced by microglia in AD 52 . Several authors have demonstrated that increased expression of TNF-α participate in the neuroinflammation associated with AD [54][55][56] . On the other hand, it has been demonstrated a neuroprotective role of this cytokine against glutamate, free radicals and Aβ induced toxicity in cultured neurons 52,57 . In AD, IL-6 levels are also altered, with increased expression in the vicinity of Aβ plaques and in the cerebrospinal fluid of patients. In spite of stimulating the synthesis of APP in glial cell cultures, increasing the damage in cortical neurons cultures stimulated with Aβ 58 , IL-6 may also have beneficial roles. Studies with transgenic models for AD showed that IL-6 was important to promote gliosis, leading to clearance of amyloid plaques [58][59][60] . Herein, BEZ increased TNF-α and showed a trend to increase in IL-6 levels in the ipsilateral hippocampus. Is has been previously shown that BEZ enhances the levels of other inflammatory mediators, such as COX-2, and prostaglandins, such as PGE 2 and PGD 2 in LPS-stimulated microglia 35,36 . Nevertheless, no difference in the levels of these cytokines was detected 7 days after the peptide infusion. This could be related to variations between the model and the protocol adopted by our study in comparison with the others.
In this study, we demonstrated that the treatment with BEZ, a PI3K and mTOR inhibitor with anticancer properties, improves mice performance in the object recognition task after intrahippocampal Aβ administration. In parallel with this cognitive effect, treatment induces neuroprotective effects, preventing cell death and reducing microgliosis. These effects might be related to the change in the production of different cytokines, albeit the mechanism remains unclear. Thus, BEZ might represent a potential drug to prevent the pathological outcomes induced by Aβ . However, more studies are necessary in order to investigate the protective mechanism promoted by BEZ in transgenic AD models.

Methods
Culture of primary hippocampal neurons. Neuronal cultures were prepared from the hippocampus of C57Bl/6 mice neonates up to 1 day of age. After dissection, hippocampal tissue was subjected to digestion with trypsin, followed by cell dissociation. Cells were added to the Neurobasal medium supplemented with N2 and B27, GlutaMAX (2.0 mM), penicillin (50.0 μ g/ml) and streptomycin (50.0 mg/ml), and then plated on previously prepared poly-L-ornithine four well plates. The cells were incubated at 37 °C and 5% CO 2 in a humidified incubator and cultured for 8 days, with medium change every 4 days. Importantly, we have previously established that 95% of the cells in these cultures are neurons 61  Photographs were taken with the microscope EVOS ® FLoid ® Cell Imaging Station, using 488 nm filter for green images (Calcein-AM) and 633 nm for red images (ethidium homodimer-1). Images were analyzed with ImageJ software. The number of dead cells was expressed as a percentage of total cell number.

Animals.
All procedures used in this study were approved and strictly followed the ethical principles of animal experimentation adopted by the Ethic Committee on Animal Use of Federal University of Minas Gerais, and institutionally approved under protocol number 336/2012. Experiments were conducted using male C57Bl/6 mice (25-30 g, 10-12 weeks of age) obtained from Animal Care Facilities of the Institute of Biological Sciences (ICB), and PI3Kγ −/− , which were a kind gift from Prof. Mauro M. Teixeira, from ICB -UFMG, Belo Horizonte, Brazil. Animals were kept under controlled room temperature (24 °C) under 12 h:12 h light-dark cycle, with free access to food and water. In total, 86 animals were used in this study.
Drug treatment protocol. Human Aβ 1-42 (Invitrogen) was prepared according to manufacturer instructions. The aggregated peptide (400 pmol/ 0.5 μ L/mice) or PBS (vehicle) was administered via intra-hippocampal route. Briefly, the animals were anesthetized with an intraperitoneal (i.p.) injection of ketamine (80 mg/kg) and xylazine (8 mg/kg) and then submitted to stereotactic surgery and intrahippocampal injection. The needle was inserted unilaterally and Aβ 1-42 or PBS solution was injected into the right hippocampus at the following coordinates from bregma 62 : anteroposterior = −1.9 mm, mediolateral = − 1.5 mm, and dorsoventral = − 2.3 mm. The confirmation of the correct placement of the needle was made using Cresyl Violet staining (data not shown).
Scientific RepoRts | 6:25226 | DOI: 10.1038/srep25226 Object Recognition Task. On the 4 th day after surgery, the Object Recognition Task was started.
The animals were habituated during 5 minutes in an acrylic square box, dimensions 380 × 380 × 15 mm (length × width × height), covered with shavings. On the 5 th and 6 th days the animals were re-exposed to the box, in which 2 equal objects were introduced diagonally. Animals were let 10 and 5 minutes inside de box, in the 2 subsequently days, respectively. On the 7 th day, one of the old objects (OO) was replaced by a new object (NO), and the animals were exposed for 5 minutes 72,73 . The records were analyzed through ANY-maze software version 4.99, and the recognition index was obtained by the formulae: time NO × 100/ (time NO + time OO). The total travelled distance was also measured as a control of the test. Animals that did not investigated the objects were not included in the analysis.
Intracardiac perfusion and brain slice preparation. In the last day of treatment, 4 hours after the drug administration a subgroup of animals were anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg) via i.p. route and then were subjected to thoracotomy to expose the heart. A hypodermic needle was inserted into the left ventricle, through which PBS and buffered paraformaldehyde (PFA) 4% were administered with the assistance of a peristaltic pump (4 mL/min). Meanwhile, an incision was made in the aortic arch to allow blood output. After completing the perfusion, the animals were decapitated, their brains were removed and stored in buffered PFA 4% overnight. Subsequently, the brains were moved to a 30% sucrose solution, until complete saturation, then were frozen in isopentane 99% and dry ice for 20 seconds and stored at − 80 °C 74 . Brains were sliced into 30-μ m-thick sections at − 20 °C with the aid of a cryostat.
Fluoro-Jade C staining. Fluoro-Jade C (FJC) is an anionic fluorescein derivate used to label degenerating neurons 38 . Although the exact mechanism is not known, different studies have demonstrated that FJC is a reliable dye used to stain dying neurons [75][76][77][78][79] . The hippocampal slices were washed 3 times in PBS for 30 minutes and mounted on gelatinized slides. After drying, slides were immersed in a basic solution of sodium hydroxide (1%) in ethanol (80%) for 5 min, EtOH (70%) for 2 minutes and rinsed with distilled water for 2 minutes. Protected from light, the slides were incubated in a solution of potassium permanganate (0.06%) for 20 minutes, washed with distilled water for 2 minutes and incubated in FJC (Millipore, Billerica, MA, USA) solution (0.0001%) in acetic acid (0.1%) for 20 minutes. Subsequently, they were washed again twice with distilled water for 1 min. After complete drying, slides were dipped in xylene for 1 minute and coverslipped with DPX (Sigma-Aldrich, St. Louis, MO, USA) 78 .
The slides were observed under fluorescence microscope Zeiss in 10× magnification lens and pictures of the CA1 layer of both hippocampi were taken for quantification of labeled cells.
The slides were observed under a Zeiss fluorescence microscope in 10× magnification lens. Pictures of the CA1 layer of both hippocampi were taken for quantification of labeled cells.
Body weight data was analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni's test for variables with parametric distribution. Behavioral, biochemical, histological and in vitro data was analyzed by one-way ANOVA followed by Newman-Keuls test for variables with parametric distribution. The data were presented as mean ± standard error of the mean (SEM). The level of significance was set at p < 0.05.