Infliximab ameliorates AD-associated object recognition memory impairment

Accepted Manuscript Title: Infliximab ameliorates AD-associated object recognition memory impairment Author: Dong Hyun Kim Seong-Min Choi Jihoon Jho Man-Seok Park Jisu Kang Se Jin Park Jong Hoon Ryu Jihoon Jo Hyun Hee Kim Byeong C. Kim PII: DOI: Reference:

S0166-4328(16)30355-2 http://dx.doi.org/doi:10.1016/j.bbr.2016.06.001 BBR 10244

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

10-12-2015 30-5-2016 1-6-2016

Please cite this article as: Kim Dong Hyun, Choi Seong-Min, Jho Jihoon, Park Man-Seok, Kang Jisu, Park Se Jin, Ryu Jong Hoon, Jo Jihoon, Kim Hyun Hee, Kim Byeong C.Infliximab ameliorates AD-associated object recognition memory impairment.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2016.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Infliximab ameliorates AD-associated object recognition memory impairment

Dong Hyun Kim1*, Seong-Min Choi2,3*, Jihoon Jho2,4, Man-Seok Park2,3, Jisu Kang1, Se Jin Park5$, Jong Hoon Ryu5, Jihoon Jo2,3,4, Hyun Hee Kim6 , Byeong C. Kim2,3,4

1

Department of Medicinal Biotechnology, College of Health Sciences and Institute of

Convergence Bio-Health, Dong-A University, Busan, 604-714, Republic of Korea 2

Chonnam-Bristol Frontier Laboratory, Biomedical Research Institute, Chonnam

National University Hospital, Gwangju, 61469, Republic of Korea 3

Department of Neurology, Chonnam National University Medical School, Gwangju,

61469, Republic of Korea 4

Department of Biomedical Sciences, Chonnam National University Medical School,

Gwangju, 61469, Republic of Korea 5

Departments of Life and Nanopharmaceutical Sciences and of Oriental

Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130701, Republic of Korea 6

DNA Damage Response Center, Chosun University School of Medicine, Gwangju

501-759, Republic of Korea

* These authors contributed equally to this work. $

Current address: Life Science Research Institute, Daewoong Pharmaceutical Co.,

Ltd., Gyeonggi-Do, Republic of Korea Address for Correspondence Byeong C. Kim, MD, PhD. Department of Neurology, Chonnam National University Medical School, 42 Jebongro, Dong-gu, Gwangju, 61469, Republic of Korea Tel: +82-62-220-6123 E-mail: [email protected]

Jihoon Jo, PhD. 1

Department of Biomedical Sciences, Chonnam National University Medical School, 42 Jebongro, Dong-gu, Gwangju, 61469, Republic of Korea Tel: +82-62-220-4419 E-mail: [email protected]

Research Highlight

 The administration of A oligomers caused visual recognition memory impairment  A oligomers perturbed mAChR-LTD in mouse PRh slices  Infliximab improved visual recognition memory impaired by pre-administered A oligomers  Infliximab improved the detrimental A effect on mAChR-LTD.

Abstract Dysfunctions in the perirhinal cortex (PRh) are associated with visual recognition memory deficit, which is frequently detected in the early stage of Alzheimer’s disease. Muscarinic acetylcholine receptor–dependent long-term depression (mAChR-LTD) of synaptic transmission is known as a key pathway in eliciting this type of memory, and Tg2576 mice expressing enhanced levels of A oligomers are found to have impaired mAChR-LTD in this brain area at as early as 3 months of age. We found that the administration of A oligomers in young normal mice also induced visual recognition memory impairment and perturbed mAChR-LTD in mouse PRh slices. In addition, when mice were treated with infliximab, a monoclonal antibody against TNF-, visual recognition memory impaired by pre-administered A oligomers dramatically improved and the detrimental A effect on mAChR-LTD was annulled. Taken 2

together, these findings suggest that A-induced inflammation is mediated through TNF- signaling cascades, disturbing synaptic transmission in the PRh, and leading to visual recognition memory deficits.

Key words: Alzheimer’s disease; Infliximab; Muscarinic acetylcholine receptordependent long-term depression; Visual recognition memory.

1. Introduction

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly [13]. AD is a progressive brain disorder, which initially affects the brain regions controlling memory and cognitive functions, spreading to adjacent regions involved in learning, communication, and behavior [4]. The major neuropathological hallmarks of AD are the extracellular amyloid neuritic plaques, intracellular neurofibrillary tangles, and neuronal degeneration [5]. Amyloid peptide (A) is the principal component of the extracellular amyloid neuritic plaques and interacts with the activated microglia [6] and reactive astrocytes surrounding these plaques [7-9]. A number of studies have demonstrated local increases in pro-inflammatory mediators in AD, which are generally assumed to exacerbate the progression of AD [10-12]. In multiple epidemiological studies, a significant reduction of AD risk has been observed in longterm as opposed to short-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) [13, 14]. Neuroinflammation plays a central role in the causation of AD by occurring in pathologically vulnerable regions of the brain and is more characteristic of chronic than acute inflammation, as it is not accompanied by swelling, heat, and pain [10, 15]. 3

Given that inflammation in the central nervous system precedes neuronal damage in AD, the signs of chronic inflammation may serve as more sensitive markers for prodromal disease compared to the presence of neuritic plaques and neurofibrillary tangles. A growing body of evidence suggests that the three major pro-inflammatory cytokines, interleukin (IL)-1-, IL-6, and tumor necrosis factor (TNF)- can mediate neurodegeneration and neuronal dysfunction. A general pattern of upregulation in the serum levels of these proinflammatory cytokines has been noticed in patients with AD [16, 17], especially with depression [18]. TNF-, which can lead to apoptosis, has been implicated in the neurodegenerative changes associated with AD: increased levels occurred in the brains and plasma of patients with AD (Fillit et al., 1991; Tarkowski, 2002), while the expression of TNF receptor type 1 (TNF-R1) was also increased (Li et al., 2004). The activation of microglia by A was demonstrated to result in the production of TNF- (Chao et al., 1994; Meda et al., 1995). Moreover, TNF- was identified as the principal neurotoxic agent resulting from A-induced pro-inflammatory transcriptional changes (Combs et al., 2001; Floden et al., 2005). In addition,  inhibition of long-term potentiation (LTP) of synaptic transmission in the mouse hippocampus was shown to be mediated via TNF- [19]. Amyloid neuritic plaques and neurofibrillary tangles initially develop in the anterior subhippocampal area encompassing the perirhinal cortex (PRh) and entorhinal cortex before the hippocampal pathology begins [20, 21]. Lesions in this subhippocampal area are regarded as diagnostic signs for the preclinical and prodromal stage of AD, and the earliest diagnosis can be made at this time, which is critical for maximizing the efficacy of therapeutic interventions using diseasemodifying agents, but the pathological changes are still minimal [22]. The PRh, 4

located in the medial temporal area is involved in visual recognition memory [23, 24]. Mechanisms behind long-term depression (LTD) in the PRh, play a significant role in generating visual recognition memory [25-29] by reducing responsiveness following repetitive exposure to visual stimuli. This appears to involve various receptor types, including N-methyl-D-aspartate (NMDA) receptors, metabotropic glutamate receptors, and muscarinic acetylcholine receptors [25, 30]. The endocytosis of α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors has also been reported to be a notable mediator of LTD in the PRh [28, 31]. There have not been many studies addressing whether A oligomers disrupt LTD in the PRh leading to impaired visual recognition memory, but in a recent study, perturbed synaptic transmission and LTD were observed in the PRh of Tg2576 mice at 3 months of age, suggesting that A has the potential to cause LTD impairment and related memory deficits in the PRh [32]. The present study demonstrated that infliximab, a Food and Drug Administration (FDA)-approved TNF- antibody, ameliorated A-induced muscarinic acetylcholine receptor-dependent LTD (mAChR-LTD) impairment in the PRh and related memory deficit, supporting a hypothesis that A oligomers employ TNF--mediated inflammatory responses to elicit synaptic deficits in the PRh, thereby deteriorating visual recognition memory, an early symptom of AD.

2. Materials and Methods 2.1. Animals Male ICR mice (25–30 g, 6 weeks old) were purchased from the Orient Co. Ltd., a branch of the Charles River Laboratories (Seoul, Korea). Mice were fed with food and water ad libitum and housed under a 12 h light/dark cycle (light on between 07:30– 5

19:30 h) at 23 ± 1 °C with 60 ± 10% humidity. Animal treatment and maintenance were performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85–23, revised 1985) and the Animal Care and Use Guidelines issued by Chonnam National University, Korea.

2.2. Cannula implantation Mice were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) under Zoletil 50 anesthesia (10 mg/kg, i.m.), and guide cannulae (26 G) were aimed at the 3rd ventricle region (AP, 2.10mm from bregma; ML, 0.00 mm from midline; DV, 1.50mm from the dura) using an atlas of the mouse brain [33]. The guide cannulae were fixed to the skull with dental cement and covered with dummy cannulae. Following surgery, mice were allowed to recover for 7 days. For the behavioral experiment, infliximab alone (2 μg/6μl) or infliximab (2 μg/3μl) + Aβ1–42 (54 ng/3μl) were administered 24 h before the behavioral experiments. Mice were carefully restrained by hand and infused with drugs or vehicle through injector cannulae (30 G) extended 1.0 mm beyond the tips of guide cannulae. The infusion volume was 6 μl, and the infusion rate was 1 μl/min. After the infusion, the infusion needle was left in the guide cannula for 1 min to ensure proper delivery of the reagents

2.3. Aβ1–42 injection and drug administration Aβ oligomers were prepared as follow the method in previous report [34]. The amyloid-β 1-42 peptide (Aβ1-42, Abcam, Bristol, UK) was initially dissolved at a concentration of 1 mg/ml in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) (SigmaAldrich). This solution was then incubated at room temperature for 1 h with

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occasional vortexing at a moderate speed. Next, the solution was sonicated for 10 min in a water bath sonicator. The HFIP/peptide solution was then dried under a gentle stream of nitrogen gas. 100% DMSO was then used to re-suspend the peptide, which was then incubated at room temperature for 12 min with occasional vortexing. The final solution was aliquoted into smaller volumes and stored at –80°C. For a working solution, 500 – 1000 µl (depending on the final concentration to be used) D-PBS (Invitrogen, UK) was added to the peptide stock solution and incubated for 2 h at room temperature to allow for peptide aggregation. Aβ1–42 oligomer (54 ng/6 μl) or DPBS (6 μl) injected into the third ventricle using Hamilton's microsyringe at stereotactic coordinates (anterior-posterior, − 2.00 mm from bregma; dorso-ventral, − 1.50 mm from the dura mater; medial-lateral, 0 mm from the midline) according to a mouse brain atlas [33] under anesthesia using Zoletil 50® (10 mg/kg, i.m.). The needle was removed after 5 min using three intermediate steps with a 1-min inter-step delay to minimize backflow. Mice were placed on a thermal pad (32 – 33 °C) until they awakened. Control animals were injected with the same amount of sterile saline (6 μl) in an identical manner. Infliximab (Remicade®) was dissolved in 0.9% saline. Infliximab alone (2 μg/6μl) or infliximab (2 μg/3μl) + Aβ1–42 (54 ng/3μl) were administered 24 h before the experiments.

2.4. Novel object recognition test Twenty-four hours after i.c.v. injection, behavioral test was conducted. The experimental apparatus consisted of a black rectangular open field (25 cm x 25 cm x 25 cm). The novel object recognition task was carried out as described elsewhere [35, 36] with slight modifications. Habituation training took place by exposing the animal to the experimental apparatus for 5 min in the absence of objects for four days.

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During the training phase, mice were placed in the experimental apparatus in the presence of two identical objects and allowed to explore for 5 min. After a retention interval of 24 h, mice were placed again in the apparatus, where a novel one replaced one of the objects. Mice were allowed to explore for 5 min. Preference for the novel and familiar object were expressed as the percent time spent exploring the novel object relative to the total time spent exploring both objects. The objects were a metal cylinder and a metal triangular pyramid with approximately the same height. Exploration was recorded in a 10-min trial by an investigator blinded to the treatment. Sniffing, touching, and stretching the head toward the object at a distance not more than 2 cm were scored as object investigation. Each group’s ability to recognize the novel object was determined by dividing the mean time exploring the novel object by the mean of the total time exploring both objects during the test session. This value was multiplied by 100 to obtain a percentage preference for the novel object (Tnovel/Ttotal x 100, Tfamiliar/Ttotal x 100). Discrimination ratio was calculated by following formula. [(Tnovel - Tfamiliar)/Ttotal ].

2.5. Western blot analysis To investigate the expression levels of OX-42, COX-2 and iNOS in PRh after Aβ1– 42

injection, mice were sacrificed at 24 h after Aβ1–42 (54 ng /6 μl) or LPS (5 μg/6 μl)

injection. Sham group was treated D-PBS (6 μl). Isolated PRh tissues were homogenized in ice-chilled Tris-HCl buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 μg/ml aprotinin, 15 μg/ml leupeptin, 10 μg/ml bacitracin, 10 μg/ml pepstatin, 15 μg/ml trypsin inhibitor, 1 mM NaF, and 1 mM Na3VO4 and incubated with 30 min at 4 °C. Debris was removed by microcentrifugation (4200 x g, 20 min), followed by quick freezing of the

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supernatants. The protein concentration was determined using the Bio-Rad protein assay reagent according to the manufacture's instruction. Samples (15 μg of protein) were then subjected to SDS-PAGE (8% gel) under reducing conditions. Proteins were transferred to PDVF membranes using transfer buffer [25 mM Tris-HCl (pH 7.4) containing 192 mM glycine and 20% v/v methanol] at 400 mA for 2 h (4 °C). Next, blots were incubated for 2 h with blocking solution (5% skimmed milk) and then placed at 4 °C overnight with rat anti-OX-42 (1:2000, Serotex), goat anti-COX-2 (1:1000, Santa Cruz Biotechnology Inc.) or rabbit anti-iNOS (1:1000, Santa Cruz Biotechnology Inc.) antibody. Blots were washed six times with Tween 20/Trisbuffered saline (TTBS), incubated with a 1:5000 dilution of horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology Inc.) for 1 h at room temperature, washed six times with TTBS, and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). Blots were then stripped and incubated with anti-tubulin or anti-ERK antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA). Film densitometry was performed using Quantity One Image Analysis System version 4.6.3 (Bio-Rad Laboratories, CA). Expression levels were normalized to GAPDH levels in the same membrane.

2.6. Electrophysiology Acute PRh slices were prepared from 6-week-old male mice. Animals were anesthetized with halothane, decapitated, and the brain rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF; bubbled with 95% O2 /5% CO2) which comprised: (mM) NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; D-glucose, 10. A mid-sagital section was made, with the rostral and caudal parts of the brain being removed by single scalpel cuts made at approximately 45° to

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the dorso-ventral axis and each half glued by its caudal end to a vibroslice stage (Leica, Nussloch, Germany). Slices (400 μm) that included perirhinal, entorhinal and temporal cortices were stored submerged in aCSF (20 - 25°C) for 1 - 2 h before transferring to the recording chamber. A single slice was placed in a submerged recording chamber (28-30°C, flow rate ~ 2 ml/min) when required. Standard field recordings were used during this study. Stimulating electrodes were placed either side of the rhinal sulcus. One stimulating electrode was placed dorsorostrally on the temporal cortex side (area 35/36) and one ventro-caudally on the entorhinal cortex side (area 35/entorhinal cortex) of the rhinal sulcus. Stimuli (constant voltage) were delivered alternately to the two electrodes (each electrode 0.033Hz) (Fig. 2). Recordings electrodes were filled with 3M NaCl and placed within layer II/III. Experiments in which there was greater than 10% drift in baseline were excluded. The amplitude of the excitatory postsynaptic currents or evoked field potential responses was measured, each of 4 consecutive responses were averaged and expressed relative to the normalized pre-conditioning baseline, an average of 15 points before 5 Hz stimulation. To induce LTD, 5Hz stimulation was delivered for 10 min to the entorhinal side alone. Effects of 5Hz stimulation were measured at 55 min (averaged over a 5 min period) after delivering 5 Hz stimulation.

2.7. Statistical analysis Western blot The values are expressed as the means ± S.E.M. The results of electrophysiology (control input vs. LTD input) and object preference ratio of object recognition test (novel object vs. familiar object) were analyzed using Student’s t-test. Total exploration time and discrimination ratio of object recognition test were

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analyzed using one-way analysis of variance (ANOVA) followed by Turkey’s posthoc test for multiple comparison. The statistical significance was set at P < 0.05.

Results

1. Effect of infliximab on Aβ1-42-induced object recognition memory impairment. To test the effect of infliximab on AD-like memory impairment, we first examined the effect of infliximab on Aβ1-42–induced novel object recognition memory impairment. Mice treated with Aβ1-42 (54 ng/6 μl, i.c.v.) [37] were subjected to behavioral test 24 h after. Infliximab (2 μg/6 μl, i.c.v.) [38] was injected with or without Aβ1-42. Control mice showed significantly more exploration time to novel object compared to familiar object (t14 = 4.876, P = 0.0002, n = 8/group, Fig. 1A). In contrast, Aβ1-42-treated mice failed to distinguish the objects (t14 = 1.171, P = 0.2613, n = 8/group, Fig. 1A). Infliximab treatment blocked Aβ1-42-induced impairment of recognition memory without affecting normal recognition memory (Aβ1-42 + infliximab, t14 = 2.916, P = 0.0113, n = 8/group; infliximab, t14 = 4.042, P = 0.0012, n = 8/group, Fig. 1A). Discrimination ratio also significantly decreased in Aβ1-42-treated mice and this was restored by infliximab treatment (F3,28 = 6.792, P = 0.0014, n = 8/group, Fig. 1B). These results were not due to the changes of exploration behavior and interest on objects during training sessions (F3,28 = 0.6595, P = 0.5839, n = 8/group, Fig. 1C) and test (F3,28 = 0.2452, P = 0.8640, n = 8/group, Fig. 1D). These results suggest that infliximab improves AD-associated recognition memory impairment.

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2. Muscarinic receptor dependent LTD in the perirhinal cortex. Previous report indicated that LTD in the rat PRh induced by 5 Hz is mAChRdependent [39]. To confirm this in mice, we conducted the same experiment in the mice PRh (Fig. 2). Five Hz stimulation induced LTD in the control mice PRh (t12 = 3.219, P = 0.0037, Fig. 3A). Scopolamine, a mAChR antagonist, blocked LTD (t12 = 1.364, P = 0.0989, Fig. 3B). However, AP5, an NMDAR antagonist, failed to block LTD (t12 = 2.759, P = 0.0025, Fig. 3C). These results suggest that 5 Hz-induced LTD is mAChR-dependent.

3. Inflammation state in the perirhinal cortex after Aβ1-42 injection. Before testing infliximab, we examined whether inflammation occurred in the PRh after Aβ1-42 injection. Lipopolysaccharide (LPS), a positive control, or Aβ1-42, was injected 24 h before sacrifice. LPS significantly increased OX-42 (t6 = 5.076, P = 0.0012, n = 4/group), iNOS (t6 = 6.893, P = 0.0002, n = 4/group), and COX-2 (t6 = 3.194, P = 0.0094, n = 4/group) immunoreactivities (Fig. 4). Aβ1-42 also significantly increase OX-42 (t6 = 5.064, P = 0.0011, n = 4/group), iNOS (t6 = 1.995, P = 0.0465, n = 4/group), and COX-2 (t6 = 3.375, P = 0.0075, n = 4/group) (Fig. 4). These results suggest that Aβ1-42 injection cause inflammation in the PRh in mice.

4. Effect of infliximab on Aβ1-42-induced muscarinic receptor dependent LTD impairment in the perirhinal cortex, ex vivo. To test the plausibility of the effect of infliximab on Aβ1-42-induced object recognition memory impairment at the cellular level, we made extracellular field recordings in the PRh slices from 6-week-old mice. Because mAChR-LTD in the PRh mediates object recognition memory [25, 28], we examined 5 Hz stimulation-induced

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LTD in the PRh, which is known to mediate mAChR at this age in mice [39]. Five Hz stimulation induced LTD in the control PRh (t12 = 7.629, P < 0.0001, Fig. 5A) but not in the Aβ1-42-treated PRh (t12 = 0.930, P = 0.3704, Fig. 5B). However, infliximab treatment blocked Aβ1-42-induced LTD impairment (t12 = 6.338, P < 0.0001, Fig. 5C) without affecting control LTD (t12 = 4.862, P = 0.0004, Fig. 5D). These results suggest that infliximab counteracts Aβ1-42 toxicity through rescuing cholinergic transmission.

5. Effect of infliximab on Aβ1-42-induced muscarinic receptor dependent LTD impairment in the perirhinal cortex, in vitro. To rule out the possibility that the injection-induced neuronal damage affects synaptic plasticity, we conducted an in vitro experiment using the PRh slices of 6week-old naïve mice. Slices were incubated with infliximab 30 min, and then subjected to Aβ1-42 (500 nM) + infliximab (25 μg/mL) [19] for 2 h. Five Hz stimulation induced LTD in the control slice (t12 = 7.240, P < 0.0001, Fig. 6A) but not in the Aβ1-42-treated slice (t12 = 0.470, P = 0.6468, Fig. 6B). However, infliximab treatment blocked Aβ1-42-induced LTD impairment (t12 = 3.920, P = 0.0020, Fig. 6C) without affecting control LTD (t12 = 3.100, P = 0.0092, Fig. 6D). These results confirm that infliximab blocks Aβ1-42 toxicity.

Discussion Present data demonstrate that treatment of mice with infliximab, a monoclonal antibody against TNF-, results in dramatic improvement in visual recognition memory which had been compromised by pre-administration of pathogenic A oligomers. Impairment in mAChR-LTD by A treatment in the mouse PRh slices, 13

involved in visual recognition memory, was also rescued by infliximab treatment, indicating that cholinergic transmission underlying visual recognition memory is one of the target functions of inflammation triggered by Aoligomers in this brain area. To our knowledge, this is the first report showing that inflammation in the PRh could lead to AD-related prodromal memory impairment since the effect of A-induced inflammation on the hippocampus was first demonstrated [19]. When it comes to inflammation in the brain, glial cells, such as microglia and astrocytes, are the main producers of inflammatory factors [40] and indeed, there are hundreds of studies demonstrating that both cell types are found surrounding the amyloid plaques [6-9, 40] and are activated by Aoligomers [40, 41]. The interaction of A oligomers with both cell types is mediated through cell surface receptors, including the receptor for advanced glycation end products (RAGE), CD14, toll-like receptor 2 (TLR2), and TLR4 [11, 42-44]. The binding of A oligomers to these receptors not only induces phagocytosis of bound A oligomers but also initiates intracellular signaling cascades, activating NF-B or p38 MAPK, leading to the enhanced expression of pro-inflammatory cytokines such as IL1-, IL-6, and TNF- [45]. Interestingly microglia and astrocytes play a pivotal role in A clearance in the brain, actively involved in removing soluble A peptides from the extracellular space of the brain via apolipoprotein E–aided proteolytic mechanisms employing two independent proteases, insulin-degrading enzyme (IDE) and neprilysin [46, 47]. Accordingly, glial cells can be thought of as the main scavengers of A that maintain the physiological levels of A in the brain by degrading de novo-generated A via clearance mechanisms and by removing pathogenic A oligomers or fibrillar A through inflammatory responses. Under normal conditions, inflammation does not

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always result in detrimental events to neurons like neuronal apoptosis or neurodegeneration. Glial cells, especially microglia, are reported to also secrete antiinflammatory mediators and growth factors such as IL-4, IL-10, and IL-13, and transforming growth factor- as restorative mechanism after initial pro-inflammatory responses [48], which are believed to be neuroprotective by helping injured neurons recover their normal functions before neuronal damage becomes irreversible. Failure to coordinate these two apparently opposing maneuvers would cause permanent neuronal damage, leading to neuronal death. In the context of AD development, further studies are desired to assess if there are any phenotypic and functional changes in glial cells that interfere with A clearance and/or cause neurodegeneration, serving as potential risk factors for AD development. During the pro-inflammatory responses, TNF- secreted by A-activated glial cells initiates its effect on neighboring neurons which have been attacked by A oligomers, disturbing the synaptic transmission in the hippocampus and associated areas [49-51]. However, how inflammation via TNF- signaling results in impairment of synaptic transmission between PRh neurons is not well elucidated. The present study demonstrated that A oligomers caused LTD impairment of synaptic transmission in the PRh, which was reversed by treating with TNF- antibodies, suggesting that LTD deficits in the PRh can occur by way of TNF- signaling. As mentioned earlier, similar inflammatory responses triggered by A oligomers was shown to perturb synaptic functions in the hippocampus [19]. It is, therefore, not surprising that inflammation is one of the strategies that A employs to affect synaptic transmission in the hippocampus and PRh leading to related memory deficits. Another way by which A oligomers disturb synaptic functions is likely by directly attacking nearby neurons through binding to putative A receptors in the neuronal cell membrane. 15

Several A receptors in neuronal cells have been identified, including α7 nicotinic acetylcholine receptors, NMDA and AMPA receptors, insulin receptors, RAGE, cellular prion protein, and the ephrin-type B2 receptor [52-57]. Unfortunately, none of these has been successfully proved as genuine A receptors residing on neurons. Experiments that are more rigorous should be designed to assess how or whether A oligomers are internalized into neurons by using neuronal cell culture models, apart from tissue slice cultures. Meanwhile, it will be worthwhile to examine whether apparently A-induced synaptic deficits actually result from TNF- signaling cascades inside the neurons biochemically, e.g., by measuring the expression of receptors related to synaptic transmission, including AMPA receptors and/or NMDA receptors in both the hippocampus and PRh. A recent report observed that the impairment of PRh LTD underlying visual recognition memory deficit was already in progress in Tg2576 mice as early as 3 months of age [32]. Tg2576 mice are one of the most widely used transgenic mouse lines in AD research, carrying the human allele for  amyloid precursor protein with the Swedish mutation (Lys-670-Asn; Met-671-Leu) [58]. Tg2576 mice show significantly elevated levels of soluble A oligomers in the hippocampus and associated brain areas at 3 months [59]. Not surprisingly, hippocampal LTP impairment were detected at 5 months, in conjunction with compromised contextual fear conditioning [60] and visual recognition memory [61]. However, PRh LTD underlying visual recognition memory is already impaired at 3 months when the synaptic transmission in the hippocampus functions normally, strongly suggesting that the PRh is more vulnerable than the hippocampus to the early events of AD pathology. This interpretation is consistent with the observations of the impairment of visual recognition memory at initial stages of AD development as shown in AD 16

animal models [61], and patients with AD [62]. Moreover, we found that inflammation triggered by A oligomers resulted in perturbation of LTD in the PRh of even young healthy mice. It will be worthwhile to examine whether the present study can be recapitulated using Tg2576 mice, where the administration of A oligomers is not required. If so, Tg2576 could serve as a more relevant animal model for dissecting the mechanisms behind inflammation occurring in the PRh in the context of AD pathogenesis.

Acknowledgments

This work was supported by a grant from the Brain Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning NRF-2014M3C7A1046041 (to BC Kim).

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Figure legends

Figure 1. Effect of infliximab on oligomeric Aβ1-42-induced object recognition memory impairment. Drug administration was conducted 7 days after cannulae implantation. Aβ1-42 (54 ng/6 μl), infliximab alone (2 μg/6 μl) or infliximab (2 μg/3 μl) + Aβ1–42 (54 ng/3 μl) were administered 24 h before the experiments. A. Object preference ration to both of novel and familiar objects in test session. B. Discrimination ratio in test session. C. Total exploration time in training session. D. Total exploration time in test session. Data were represented as mean ± SEM. n = 10/group. *P < 0.05.

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Figure 2. The placements of stimulating and recording electrodes.

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Figure 3. mAChR dependency of PRh-LTD in mice. Scopolamine (20 μM) or AP5 (50 μM) was perfused through baseline. LTD was evoked by low frequency stimulation (5 Hz, 900 pulses, black rectangle). A. LTD was readily induced in control slices (n = 7). B. Scopolamine (n = 7) blocked LTD. C. AP5 failed to blocked LTD (n = 7). Data were represented as mean ± SEM. White and Black circles represent control and LTD inputs, respectively. Above the graph, representative traces of fEPSPs obtained from LTD input data before (1) and 55 min after (2) low frequency stimulation.

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Figure 4. Inflammation in PRh after Aβ1–42 injection. Aβ1-42 (54 ng/6 μl) or LPS (5 μg/6 μl) or infliximab (2 μg/3 μl) + Aβ1–42 (54 ng/3 μl) were administered 24 h before sacrifice. OX-42, iNOS and COX-2 immunoreactivities were examined. Expression levels were normalized to GAPDH levels in the same membrane. Data were represented as mean ± SEM. *P < 0.05 vs sham group.

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Figure 5. Effect of infliximab on mAChR-LTD in PRh of Aβ1-42-treated mice. Aβ1-42 (54 ng/6 μl), infliximab alone (2 μg/6 μl) or infliximab (2 μg/3 μl) + Aβ1–42 (54 ng/3 μl) were administered 24 h before the experiments. mAChR-LTD was evoked by low frequency stimulation (5 Hz, 900 pulses, black rectangle). A. mAChR-LTD was readily induced in control slices (n = 7). B. Pre-injection of Aβ (n = 7) blocked mAChR-LTD. C. Infliximab blocked Aβ-induced mAChR-LTD impairment (n = 7). D. Infliximab alone does not affect mAChR-LTD. Data were represented as mean ± SEM. White and Black circles represent control and LTD inputs, respectively. Above the graph, representative traces of fEPSPs obtained from LTD input data before (1) and 55 min after (2) low frequency stimulation.

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Figure 6. Effect of infliximab on Aβ1-42-induced mAChR-LTD impairment. Acute PRh slices were incubated with infliximab (25 μg/ml) for 30 min and then more incubated with Aβ (500 nM) and infliximab for 2 h. mAChR-LTD was evoked by low frequency stimulation (5 Hz, 900 pulses, black rectangle). A. mAChR-LTD was readily induced in control slices (n = 7). B. Pre-injection of Aβ (n = 7) blocked mAChR-LTD. C. Infliximab blocked Aβ-induced mAChR-LTD impairment (n = 7). D. Infliximab alone does not affect mAChR-LTD. Data were represented as mean ± SEM. White and Black circles represent control and LTD inputs, respectively. Above the graph, representative traces of fEPSPs obtained from LTD input data before (1) and 55 min after (2) low frequency stimulation.

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