Impact of Gold Nanoparticles on Amyloid Î² ... - ACS Publications

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Impact of Gold Nanoparticles on Amyloid β‑Induced Alzheimer’s Disease in a Rat Animal Model: Involvement of STIM Proteins Mehdi Sanati,† Fariba Khodagholi,‡ Samaneh Aminyavari,§ Forough Ghasemi,∥ Mahdi Gholami,† Abbas Kebriaeezadeh,† Omid Sabzevari,† Mohammad Javad Hajipour,⊥,# Mohammad Imani,∇ Morteza Mahmoudi,*,○ and Mohammad Sharifzadeh*,†

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Department of Toxicology & Pharmacology, Faculty of Pharmacy; Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences, Tehran 1416753955, Iran ‡ Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran § Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran 1416753955, Iran ∥ Department of Nanotechnology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO), Karaj 3135933151, Iran ⊥ The Persian Gulf Biomedical Sciences Research Institute, Persian Gulf Marine Biotechnology Research Center, Bushehr University of Medical Sciences, Bushehr 47263, Iran # Non-Communicable Diseases Research Center, Endocrinology and Metabolism Population Sciences Institute, Tehran University of Medical Sciences, Tehran 1416753955, Iran ∇ Department of Novel Drug Delivery Systems, Iran Polymer and Petrochemical Institute, Tehran, Iran ○ Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1416753955, Iran ABSTRACT: Alzheimer’s disease (AD) is the most common type of neurodegenerative amyloid disorder causing progressive cognitive decline and memory loss. A considerable number of therapies for AD rely on inhibition/delay/ dissociation of amyloid beta (Aβ) oligomers and fibrils. In this case, nanoparticles (NPs) demonstrated substantial effects on the Aβ fibrillation process; however, their effects on progressive cognitive decline and memory have been poorly investigated in vivo. In this study, acquisition and retention of spatial learning and memory are studied in a rat animal model of AD after intrahippocampal (IH) and intraperitoneal (IP) injections of a model NP, i.e., gold NPs (AuNPs). The outcomes revealed that the AuNPs could improve the acquisition and retention of spatial learning and memory in Aβ treated rats as indicated by decreased time (Aβ: 39.60 ± 3.23 s vs Aβ+AuNPs: 25.78 ± 2.80 s) and distance (Aβ: 917.98 ± 50.81 cm vs Aβ+AuNPs: 589.09 ± 65.96 cm) of finding the hidden platform during training days and by increased time spent in the target quadrant (Aβ: 19.40 ± 0.98 s vs Aβ+AuNPs: 29.36 ± 1.14 s) in the probe test in Morris water maze (MWM). Expression of brain-derived neurotrophic factor, BDNF, cAMP response element binding protein, CREB, and stromal interaction molecules, e.g., STIM1 and STIM2 was also increased, supporting improved neural survival. Our outcomes may pave a way for mechanistic insights toward the role of NPs on retrieval of the deteriorated behavioral functions in brain tissue after AD outbreak. KEYWORDS: Alzheimer’s disease, learning, memory, rat animal model, gold nanoparticles It is broadly accepted that the fibrillation process in AD starts with a nucleation or lag phase (rate limiting step) in which non-monomeric Aβ units interact to create initial nucleolus acting as template.9 Thereafter, in the oligomerization phase, self-assembly accelerates to form soluble oligomers. In the last step, i.e., elongation phase, insoluble fibrils are

1. INTRODUCTION Alzheimer’s disease (AD) is one of the most growing concerns in the aging population and is dramatically linked to mortality.1−4 Insoluble extracellular deposits, known as senile plaques, resulting from aggregation of amyloid beta (Aβ) and tau proteins, are attributed to AD pathophysiology.5,6 The interactions occurring between senile plaques and cellular components and mechanisms lead to dementia and progressive cognitive decline.7,8 © XXXX American Chemical Society

Received: November 9, 2018 Accepted: March 21, 2019

A

DOI: 10.1021/acschemneuro.8b00622 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Variation of 4 day mean of escape latency (1), traveled distance (2), swimming speed (3), and time spent in target quadrant (4) and four consecutive days demonstration of escape latency (5) in MWM. Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPih (1, 10, and 100 μg·mL−1) (A). Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPip (200 μg·mL−1) or Buca+AuNPip (200 μg·mL−1) (B). Representative traces of rats in MWM during training days (C). Intrahippocampal infusion of Aβ significantly increased traveled path during training days in comparison to control. AuNPs ameliorated spatial acquisition impairment in Aβ-treated animals. **p < 0.01, ***p < 0.001, and ****p < 0.0001 significantly different compared to control group; #p < 0.05, ##p < 0.01, and ####p < 0.0001 significantly different compared to Aβih treated animals. Results expressed as mean ± SEM (N = 6 rats in each group).

formed. Both soluble oligomers and insoluble fibrils can interfere with the normal functioning of neurons. Therefore, approaches concentrating on inhibition/delay/dissociation of oligomers and fibrils as well as prevention/alleviation of neurodegeneration are widely considered to find new therapeutic solutions for AD.7,8,10 Numerous biomolecules and biological events are associated with the progress and pathogenesis of AD. Due to its complicated/different clinical, anatomic, and physiological features, multifactorial therapeutic approaches should be considered for AD.11−14 It is well-understood that multiple biochemical actors work together to survive neurons under

adverse conditions.15−17 For example, brain-derived neurotrophic factor (BDNF) stimulates and controls neural growth and plasticity, which are critical for learning and memory processes.18,19 Due to its structural homology to nerve growth factor, BDNF has high affinity to tyrosine kinase B (TrkB) receptor.20,21 Binding of BDNF to TrkB receptor has a unique capacity to trigger a signaling cascade resulting in activation of cAMP response-element binding protein (CREB) and/or CREB-binding protein (CBP) transcription factor tuning the expression of proteins engaged in the neural growth, resistance, plasticity, and survival.22 B


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Figure 2. Representation of Aβ aggregates in the CA1 area of the hippocampus. Coronal sections stained with thioflavin S to probe Aβ plaques. No obvious aggregation found in control group (A), while significant plaques (arrow) probed in AD model (B). IH injection of AuNPs (10 and 100 μg·mL−1) significantly inhibited Aβ aggregation (C and D).

adsorbing Aβ monomers and interfere with the fibrillation process.39 For example, it was shown that negatively charged gold nanoparticles (AuNPs) could inhibit the Aβ fibrillation process and dissociate the formed fibrils in vitro.40 Although inhibiting the fibrillation process or disaggregating the performed fibrils by AuNPs are successfully documented in vitro, to date, their consequent behavioral impact are poorly investigated in vivo.40−43 In this study, we assessed the effect of commercially available citrate-conjugated AuNPs (Sigma-Aldrich, St. Louis, MO) alone or in combination with bucladesine (a membranepermeable cAMP analog) on a male Wistar rat model of AD [developed by a single intrahippocampal (IH) injection of Aβ(1−42)]. A systematic experimental approach was adopted to investigate the learning/memory-related behavior and the levels of STIM1, STIM2, p-CREB, and BDNF factors in the hippocampus before and after IH and intraperitoneal (IP) injections of AuNPs.

Calcium (Ca2+) homeostasis is another important factor in regulating neuron function.23 Neurons benefit from a highly developed Ca2+ machinery that regulates the Ca2+ signaling pathway.24 Endoplasmic reticulum (ER) is known as the main source of endogenous Ca2+.23 Stromal interaction molecules 1 and 2 (STIM1 and STIM2) are dynamic Ca2+ sensors that recognize slight changes in ER Ca2+ storage and then couple with the mechanisms responsible for Ca2+ inflow in ER-plasma membrane (ER-PM) junction, which has been comprehensively reviewed by Soboloff et al. elsewhere.25 Altered expression level of STIM1 and STIM2 proteins and disrupted Ca2+ homeostasis have been previously reported in AD models, e.g., familial Alzheimer’s disease (FAD) presenilin1 (PS1) mutant mice,26 amyloid precursor protein (APP) knock-in mice,27 and hippocampal28 or brain endothelial29 culture treatment with Aβ. It is noteworthy that regulation of cytosolic Ca2+ concentration following ER Ca2+ homeostasis is critical for extracellular signal-related protein kinase (ERK)-dependent sustained CREB phosphorylation/activation and consequently regulation of important biological processes including synaptic plasticity.16,30 Recently, it was found that STIM2, in addition to working with STIM1 in the development of Ca 2+ homeostasis, cooperates with protein kinase A (PKA) in regulating AMPA glutamate receptors (AMPARs)-dependent function of neural excitatory synapses in a Ca2+-independent manner.31,32 Despite numerous efforts to find out AD therapy, fewer than expected drugs have made it to clinical trial and many of them could not reach the market.33−36 One of the current promising strategy for treatment of AD is based on development of multifunctional nanoparticles (NPs).37,38 Due to their high surface to volume ratio, NPs demonstrate a unique capacity in

2. RESULTS AND DISCUSSION 2.1. Memory Performance. Since memory deficit is considered as a pathognomonic symptom of AD, we employed a Morris water maze (MWM) apparatus to demonstrate spatial memory impairment during the development of AD in the animal model. We found that following IH administration Aβ protein significantly impaired spatial learning and memory formation evident in Aβih treatment group after 21 days as indicated by increased time (p < 0.001) and distance (p < 0.0001) of finding the hidden platform during training days and by decreased time spent in the target quadrant (p < 0.01) in the probe trial test compared to the control animals (Figure 1). The data obtained from the swimming speed test showed C


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Figure 3. Variation of hippocampal pyramidal cells. (A) control, (B) Aβih, and (C) Aβih+AuNPih: qualitative (A−C) and quantitative (D) assessment. Aβih (1 μg·μL−1) altered integrity and linear shape as well as cell count of pyramidal cells in CA1 subfield of the hippocampus after 21 days. AuNPih (10 μg·mL−1) remarkably prevented the destructive effect of Aβih. ****p < 0.0001 significantly different compared to control group; #### p < 0.01 significantly different compared to Aβih treated animals. Data presented as mean ± SEM (N = 4 samples in each group).

there is no significant difference among control and Aβihinjected group (Figure 1A and B) demonstrating no motor or musculoskeletal damage. To ascertain the development of in vivo model of neurodegeneration, in parallel with spatial memory impairment, we carried out thioflavin S and Nissl staining of hippocampal slices. Results showed Aβ plaques as well as significant neural loss (p < 0.0001) in the Aβih test group which supported the behavioral findings (Figures 2 and 3). In another part of the study, we examined the in vivo impact of the citrate-conjugated AuNPs on Aβ-induced learning and memory disturbances. The negative surface potential of the NPs (−47.7 ± 10.9 mV) was determined by using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) before treatment. We showed that AuNPsih, AuNPsip, and Bucla +AuNPsip had no significant effect (p > 0.05) on spatial learning and memory of normal rats. However, in the case of Aβih-treated animals, AuNPsih (10 and 100 μg·mL−1), AuNPsip, and Bucla+AuNPsip significantly reduced time and distance of finding the hidden platform and increased time spent in the target quadrant (Figure 1). Also, thioflavin S and Nissl staining of hippocampal slices confirmed the protective effect of AuNPs against Aβ fibrillation process and its neurodegenerative consequences, respectively (Figures 2 and 3).

According to a large body of experiments, hippocampus contributes to different types of learning and memory processes and hippocampal lesions disrupt memory skills expansion.44 In agreement with our findings, numerous investigations have shown spatial memory impairment after a delayed time-point following local infusion of Aβ into the CA1 subfield of the dorsal hippocampus.45,46 Also, a meaningful correlation between Aβ accumulation in the brain and spatial memory impairment has been suggested in an animal model of AD.47 Moreover, it has been previously shown that IH injection of Aβ mediates significant neural loss as well as cognitive impairment.48 It is noteworthy that the majority of in vitro studies demonstrated that NPs have the potential to reduce/dissociate Aβ oligomers and fibrils by misleading the aggregation process.49 Also, monitoring of Aβ fibrillationinduced cytotoxicity in vitro has been shown to reduce neurotoxicity in the presence of different concentrations of AuNPs.40 In keeping with the in vitro reports, our findings demonstrated that IH injection of AuNPs with negative surface potential significantly inhibits Aβ aggregation in the hippocampus tissue in vivo (Figure 2). Furthermore, we showed that the IH/IP injection of these NPs improves the acquisition and retention of spatial learning and memory in rats injected with Aβ(1−42) ( Figure 1). D


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ACS Chemical Neuroscience Table 1. Measurement of ROS and MDA in the Hippocampus Tissuea AuNPs −1

ROS (μg·mg protein) MDA (μg·mg−1 protein)

control

1 μg·mL−1

10 μg·mL−1

100 μg·mL−1

1.17 ± 0.17 0.98 ± 0.07

1.35 ± 0.16 1.07 ± 0.05

1.49 ± 0.14 1.20 ± 0.08

1.37 ± 0.21 1.15 ± 0.06

Animals received intra-CA1 injections of AuNPs (1, 10, and 100 μg·mL−1). No significant difference observed in comparison to control group. Results expressed as mean ± SEM (N = 4 samples in each group). a

results are consistent with the recent studies showing that Aβ altered STIM1 and STIM2 proteins expression level.26,28,29 Also, it has been demonstrated that Aβ oligomers cause downregulation of CREB and BDNF mRNA in neuroblastoma cells.22,62 AuNPsih (10 and 100 μg·mL−1), AuNPsip, and Bucla +AuNPsip showed reverse effects and increased the level of STIM1, STIM2, BDNF, and p-CREB proteins in the hippocampus of Aβih-treated animals (Figures 4−7). STIMs are ER-resident Ca2+ sensors and play an essential role in the regulation of cellular Ca2+ homeostasis. Any reduction in the expression or function of these proteins may affect the neural growth and survival.63,64 It is known that STIM1 and STIM2 both play a synergistic role in activating cAMP-dependent PKA.32 In other words, the process of cAMP production by adenylyl cyclase (AC) which is essential for PKA activation and long-term potentiation (LTP) is highly Ca2+-sensitive.65 cAMP/PKA signaling pathway indirectly regulates neural synaptic plasticity and affect learning and memory-related processes through activation/inhibition of different targets.66−68 CREB is a substantial target of PKA that controls important genes transcription including those involved in neuron survival.69 On the other hand, cross-talk between ERK and PKA is critical for CREB translocation to the nucleus which is highly dependent on the cytosolic Ca2+ level.16 Thereby, it could be suggested that Aβ reduces cAMP production, inhibits PKA activity, and disrupts ERK-dependent CREB translocation indirectly through STIMs dysregulation, and in turn reduces the expression of important factors and markers including neurotrophic factors, e.g., BDNF and cholinergic markers which correlate Aβ to synaptic loss and memory deficit. BDNF is a major factor in synaptic plasticity, particularly LTP, and plays a crucial role in learning and memory formation. In this regard, Arancibia et al. have revealed the protective effect of BDNF against Aβ-induced neurotoxicity in the culture of rat hippocampal neurons.70 In addition, cholinergic system hypofunction is critically involved in AD pathophysiology. It has been also shown that bucladesine-nicotine combination could synergistically improve memory through enhancement of cAMP/PKA pathway and cholinergic system function.71 Interestingly, it was found that AuNPs have a potential acetylcholine esterase (AChE) inhibitory effect in neurons culture which in turn enhances acetylcholine (ACh) availability within synaptic cleft and thus, improves cholinergic transmission.72 Synaptic dysfunction is the primary cause of Aβ-induced cognitive impairment. The impact of Aβ on synapses in vulnerable structures including hippocampus has become one of the major concerns in AD research.73,74 It has been reported that the determinant effect of Aβ on synapse activity is partly due to a decrease in postsynaptic density protein 95 (PSD-95) which is responsible to keep and employ AMPA type glutamate receptor subunit GluA1 (GluR1) at the postsynaptic site.74 In brain tissue, AMPA receptor AMPARs trafficking is critical for fast excitatory transmission. Significant reduction in

Some properties of NPs are important considering their effect on Aβ fibrillation process, e.g., concentration and surface potential.49 In the present study, we found that coadministration of citrate-conjugated AuNPs with Aβ(1−42) monomers rescued the spatial memory impairment and neural loss induced by Aβ(1−42) alone in a dose-dependent manner (Figures 1−3). Co-incubation of carboxyl-conjugated AuNPs with Aβ monomers has been shown to produce smaller fibrils and spherical oligomers with remarkably reduced in vitro neurotoxicity in a concentration-dependent manner. It has been proposed that negative surface of particles can interfere with the fibrillation process of Aβ through electrostatic interactions and hydrogen bonding.40 Therefore, we hypothesized that the use of safely (in terms of toxicity) higher concentrations of AuNPs may provide better capacity to redirect Aβ fibrillation.40,42 Since induction of oxidative stress is one of the main mechanisms of metallic NPs neurotoxicity,50 we assessed the level of ROS and MDA, as important oxidative stress indices, in the hippocampus of animals that received IH injection of AuNPs alone. Our findings support the safety of applied AuNPs concentrations (Table 1). There are important restrictions for drug delivery to the brain including the blood-brain barrier (BBB) as an impassable obstacle for many active agents.51,52 Accordingly, engineered NP-based drug delivery platforms are promising candidates to overcome this limitation.53 Modified 5 nm AuNPs, for example, have been successfully incorporated for doxorubicin delivery to a brain tumor.51 Physicochemical characteristics of NPs influence their application in drug delivery to the brain.52 As the positive surface charge of NPs causes toxicity to the BBB, most NPs that are used in brain drug delivery platforms have a negative surface potential.54−56 The size of NPs is negatively correlated with their permeability to the BBB.57,58 It has been revealed that 5 nm AuNPs provide greater brain concentration in comparison to larger AuNPs.59 Thereby, development of NPs with particular characteristics proportionate to this application is of importance. Hepato-biliary clearance is another important restriction for NPs delivery to the brain.59 The behavior of NPs in the biological environments is affected by their unique specifications.59,60 Due to the dynamic protein bindings, the organ distribution of negatively charged AuNPs alters in a size/ surface area dependent manner.59,60 The amount of AuNPs accumulated in the liver is higher than that in other organs, which significantly increases with the size of AuNPs (from 50% 1.4 nm AuNPs to >99% 200 nm AuNPs).59 Hence, most of the larger AuNPs are trapped in the liver and eliminated. Considering the NPs pharmacokinetics is critical to design specified NPs with improved delivery to the target site.61 2.2. Western Blot Analysis. The data obtained from Western blot analysis showed that Aβih causes a significant reduction in the expression of STIM1, STIM2, and BDNF proteins along with lower p-CREB/CREB (p < 0.0001) ratio in the hippocampus of rats in comparison to control animals. Our E


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Figure 4. Variation of STIM1/β-actin (A) and STIM2/β-actin (B) ratio in the hippocampus. Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPih (10 and 100 μg· mL−1). **p < 0.01 and ****p < 0.0001 significantly different compared to control group; #p < 0.5 and ###p < 0.001 significantly different compared to Aβih treated animals. Data expressed as mean ± SEM (N = 4 samples in each group).

Figure 5. Variation of STIM1/β-actin (A) and STIM2/β-actin (B) ratio in the hippocampus. Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPip (200 μg·mL−1) or Buca+AuNPip (200 μg·mL−1). **p < 0.01 and ****p < 0.0001 significantly different compared to control group; ##p < 0.01 and ####p < 0.0001 significantly different compared to Aβih treated animals. Data expressed as mean ± SEM (N = 4 samples in each group).

the number and/or activity of hippocampal AMPARs is related to synaptic dysfunction and cognitive decline.75 Interestingly, recent findings indicate the important role of STIM2 in the regulation of synaptic plasticity as well as GluR1 receptor function.31,76 For example, it has been shown that silencing of the STIM2 gene in extracted hippocampal neurons leads to the reduction of dendritic spines density.28 Also, Several studies have shown the link between Aβ-induced downregulation of STIM2 and synaptic loss in AD pathophysiology.18,19 In 2015, Garcia-Alvarez et al. showed that cAMP triggers STIM2 migration to ER−PM junctions which are connected to excitatory synapses.31 These investigators showed that this function is carried out in a Ca2+-independent manner. They suggested that the role of STIM2, as a downstream target of

cAMP, is necessary for PKA-dependent phosphorylation of GluR1 as well as its recruitment in postsynaptic membrane. More specifically, STIM2-dependent PKA/GluR1 complex formation at the ER−PM junction has an important impact on synaptic plasticity and function. Moreover, it has been demonstrated that cAMP/PKA pathway improves GluR1 trafficking by affecting PSD-95 synaptic levels.77 On the other hand, this signaling pathway enhances CREB activity and in turn, employs BDNF to maintain synaptic AMPARs function during learning and memory formation.78 Taken together, it is supposed that AuNPs, in addition to enhancing the level of STIM2, p-CERB and BDNF expression, may play a F


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to smaller and less toxic species; (ii) AuNPs increase STIM expression levels and subsequently potentiate the cAMP/PKA signaling cascade in a Ca2+-dependent manner; (iii) AuNPs can directly enhance the cAMP levels and thus recruit STIM2 and PKA to potentiate GluR1-dependent synaptic plasticity; (iv) AuNPs have the capacity to inhibit cholinesterase enzyme and enhance cholinergic transmission in nerve terminals.

3. CONCLUSION In this paper we uncovered the potential capacity of AuNPs on relieving memory impairment and neural damage in an animal model of AD. The observed positive behavioral role of AuNPs may be due to the redirection of Aβ peptide fibrillation kinetic which minimizes the destructive effects of fibrils on p-CREB, BDNF, STIM1, and STIM2. Many other behavioral and molecular approaches need to be performed in future studies to mechanistically understand the in vivo roles of AuNPs in the progression of AD. 4. METHODS 4.1. Materials. AuNPs suspension stabilized in citrate buffer (5 nm diameter, 6.02 mg·mL−1, zeta (ζ) potential = −47.7 ± 10.9 mv), Aβ(1−42), bucladesine, ketamine, and xylazine were all purchased from Sigma-Aldrich (St. Louis, MO). Aβ(1−42) was dissolved in isotonic sterile saline (0.9%). AuNPs and bucladesine stocks diluted by isotonic citrate buffer (10 mmol·L−1) and saline, respectively, and sterilized using a 0.22 μm filter. Anti-STIM1, anti-STIM2, anti-CREB, anti-p-CREB, and anti β-actin antibodies and horseradish peroxidaseconjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA). Anti-BDNF antibody was bought from Abcam (Cambridge, UK). Other chemicals were provided from Sigma-Aldrich or Merck and used as received. 4.2. Animals. Adult male Wistar rats (180−220 g) granted from the Faculty of Pharmacy, Tehran University of Medical Sciences were used in this study. Animals were housed in groups of five in stainless steel cages under standard conditions including controlled-temperature and given food and water ad libitum. A 12 h light/12 h dark cycle (lights on 07:00) was maintained, and the training and testing of animals were performed during the light cycle. All procedures were in accordance with the guidelines of the ethical committee for use and care of laboratory animals of Tehran University of Medical Sciences (No. 8069, July 2008). 4.3. Surgical and Treatment Procedures. Rats were anesthetized using an IP injection of ketamine (100 mg·kg−1) and xylazine (10 mg·kg−1). Guide cannula (22G) was implanted into the CA1 region of the dorsal hippocampus by stereotaxic surgery instrument (Stoelting, Wood Dale, IL) according to the Paxinos and Watson atlas of the stereotaxic coordinates of the rat brain (anterior−posterior, 3.8 mm; mediolateral, ±2.2 mm; dorsoventral, 2.7 mm from bregma).79 Treatments and behavioral assessments were performed after a 1 week recovery period. For bilateral IH injections, an injection needle (30G) was linked to a Hamilton syringe using a custom-made polyethylene tube. Injections (1 μL at each side) were performed during 1 min, and the injection needle was retained for a further 60 s in the CA1 site for complete discharge. IP injections were done at a volume of 1 mL.kg−1. Rats were divided into the different groups explained in Table 2. 4.4. Behavioral Test. Morris water maze (MWM) test was performed to assess spatial learning and memory of the rats. Briefly, rats were trained to find the hidden platform placed 1 cm below the water surface for 4 consecutive days. Each training session included one block of four trials. Each trial was started from a predefined point (north, east, west, and south) randomly. Probe test was performed on the fifth day when the platform was removed, and rats were allowed to swim in the pool for 90 s. The behavior of rats was recorded using a video camera placed above the water maze. Escape latency (time spent to find the platform), traveled distance (distance to find the

Figure 6. Variation of p-CREB/CREB ratio in the hippocampus. Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPih (10 μg·mL−1). ****p < 0.0001 significantly different compared to control group; ###p < 0.001 significantly different compared to Aβih treated animals. Data expressed as mean ± SEM (N = 4 samples in each group).

Figure 7. Variation of BDNF/β-actin ratio in the hippocampus. Animals received an intra-CA1 injection of Aβ (1 μg·μL−1) alone or in combination with AuNPih (10 μg·mL−1). ***p < 0.001 significantly different compared to control group; ##p < 0.01 significantly different compared to Aβih treated animals. Data expressed as mean ± SEM (N = 4 samples in each group).

role in improving synaptic function in AD model by direct potentiating cAMP-dependent signaling pathways. Finally, according to the existing evidence31,32,40,65,72,76 and our findings, we can bring forward several hypotheses regarding the ameliorating effect of AuNPs on Aβ-induced spatial memory impairment: (i) AuNPs inhibit/delay the elongation phase of Aβ fibrillation or dissociate existing fibrils G


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ACS Chemical Neuroscience Table 2. Experimental Design (n = 7 Rats in Each Group) group code

injection route

day(s)

MWM training

treatment(s)

Aβih AuNPsih

IH IH

1 1, 2, 3, and 4

AuNPsip Bucla+AuNPsip

IP IP

1, 2, 3, and 4 1, 2, 3, and 4

AuNPsih + Aβih

IH

1, 4, 8, 12, and 16

AuNPsip + Aβih

IP

1, 4, 8, 12, and 16

Bucla+AuNPsip+Aβih

IP

1, 4, 8, 12, and 16

control

IH and/ or IP

1, 2, 3, and 4

Aβ in saline (1 μg·μL−1) AuNPs in citrate buffer (1, 10, and 100 μg·mL−1) each day 30 min before training in MWMa AuNPs in citrate buffer (200 μg·mL−1) each day 30 min before training in MWM co-injection of AuNPs in citrate buffer (200 μg·mL−1) + bucladesine in saline (1 μmol· L−1) each day 30 min before training in MWM AuNPs in citrate buffer (1, 10, and 100 μg·mL−1) every 4 days after IH injection of Aβ in saline (1 μg·μL−1) AuNPs in citrate buffer (200 μg·mL−1) every 4 days after IH injection of Aβ in saline (1 μg·μL−1) Co-injection of AuNPs (200 μg·mL−1) in citrate buffer + bucladesine in saline (1 μmol·L−1) every 4 days after IH injection of Aβ in saline (1 μg·μL−1) citrate buffer (10 mmol·L−1) and/or saline each day 30 min before training in MWM

1, 4, 8, 12, and 16

citrate buffer (10 mmol·L−1) and/or saline every 4 days

days 21−24 days 1−4 days 1−4 days 1−4 days 21−24 days 21−24 days 21−24 days 1−4 days 21−24

a

Morris water maze. esterase enzymes and then reacts with ROS to produce highly fluorescent DFC. The level of ROS was measured by quantification of DFC fluorescence intensity using an ELISA F-2000 fluorescence spectrometer at 485 nm (excitation) and 525 nm (emission) wavelengths. 4.7.2. Lipid Peroxidation (LPO) Measurement. Thiobarbituric acid assay carried out to determine the level malondialdehyde (MDA) as an important marker for lipid peroxidation in the hippocampus tissue.85,86 In brief, hippocampus homogenate was mixed with an equal volume of trichloroacetic acid (10%) and was cooled in ice. The mixture was then centrifuged at 276g (T = 0 °C) for 25 min and the supernatant was collected. The supernatant was mixed with an equal volume of thiobarbituric acid (0.67%) and was heated in boiling water for 10 min. Finally, the mixture was cooled in ice and the developed color was measured spectrophotometrically at 532 nm wavelength in comparison to reagent blank. 4.8. Statistical Analysis. Average of each memory performance related parameter in MWM test including escape latency, traveled distance, swimming speed, and time spent in target quadrant and protein expression was calculated and analyzed using two-way analysis of variance for measurement of NP effects. One-way analysis of variance was used for comparing quantitative histological and oxidative stress changes. The Tukey’s post hoc test was used for multiple comparisons. A p value of 0.05 or less was considered statistically significant.

platform), swimming speed, and the time spent in the target quadrant (in probe test) were calculated via the EthoVision tracking system (Noldus Information Technology, Wageningen, The Netherlands).80 4.5. Western Blot Analysis. After completing the behavioral assessments, treated rats were sacrificed using CO2 gas and the hippocampus tissue harvested for Western blot analysis. The tissue was homogenized with a homogenizer (Micro Smash MS-100) using lysis buffer then centrifuged at 13000g (T = 4 °C) for 20 min. The supernatant was collected, and the concentration of proteins was determined by Bradford assay.81 Proteins were loaded on a 12.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, electrophoresed, and then transferred to a polyvinylidene difluoride (PVDF) membrane. In the next step, nonspecific sites were blocked with fatfree dry milk and blots were incubated with the specific primary antibody (16 h, overnight, 4 °C) and horseradish peroxidaseconjugated secondary antibody (1.5 h, room temperature), respectively. Finally, ECL kit (Amersham, Bioscience, Piscataway, NJ) was used for recording the amount of p-CREB, BDNF, STIM1, and STIM2 proteins on Biomax radiography films. ImageJ software (National Institutes of Health, Bethesda, MD) was used to calculate the protein band optical density. Finally, blots striped and incubated with anti β-actin monoclonal antibody to demonstrate the equal loading of the samples. 4.6. Histological Assessments. 4.6.1. Nissl Staining. Brain samples were fixed in paraformaldehyde and mold within paraffin after tissue processing. Sections of paraffin-embedded tissue (thickness = 7 μm) were prepared from CA1 area of the hippocampus. In the next step, sections were stained with 0.1% cresyl violet acetate. Five slices from the CA1 region were provided for histomorphometric analysis. The number of viable neurons was calculated and analyzed, using computer software Image-Pro Plus V.6 (Media Cybernetics, Inc., Silver Spring, MD). Magnification ×200 was employed for counting the neurons, and the calculation was repeated for four fields. Finally, the average number of viable neurons for these fields was recorded. 4.6.2. Thioflavin S staining. We performed thioflavin S staining to probe Aβ aggregates in the hippocampus tissue.82,83 In brief, deparaffinized sections were placed onto polylysine-coated slides and were incubated with 0.05% thioflavin S in 50% ethanol for 30 min in dark. Thereafter, sections were washed twice with 50% ethanol and once with tap water before mounting. Aβ aggregates were probed in stained sections using a fluorescence microscope (Olympus, IX71, Japan). 4.7. Oxidative Stress Assessments. 4.7.1. Reactive Oxygen Species (ROS) Measurement. To measure the level of ROS in the hippocampus tissue, we performed the dichlorofluorescein (DFC) assay as described previously.84 In brief, the hippocampus homogenate was incubated with assay buffer containing 5 μmol·L−1 dichlorodihydrofluorescein diacetate (DFCH-DA) at 37 °C for 30 min in the dark. DFCH-DA converts to DFCH by synaptosomal

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.M.). *E-mail: [email protected] (M.S.) ORCID

Morteza Mahmoudi: 0000-0002-2575-9684 Author Contributions

M.M. and M.S. designed the study and were responsible for the experimental plan. M.Sa. performed the behavioral experiments, stained the hippocampus tissues, analyzed all data presented, and wrote the manuscript. S.A. carried out Western blot experiments. F.G. helped to determine the NPs properties. M.G. assisted in behavioral experiments. F.K., A.K., and O.S. advised on the overall experimental design. M.J.H. and M.I. made a significant intellectual contribution to the manuscript. Funding

This work was supported by funding (No. 94-04-33-31230) from the Tehran University of Medical Sciences. H


Research Article

ACS Chemical Neuroscience Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Mr. Ali Kazemi for his technical assistance.

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