Direct pharmacological Akt activation rescues Alzheimer's disease like memory impairments and aberrant synaptic plasticity

Accepted Manuscript Direct pharmacological Akt activation rescues Alzheimer's disease like memory impairments and aberrant synaptic plasticity Jee Hyun Yi, Soo Ji Baek, Sunghoo Heo, Hye Jin Park, Huiyoung Kwon, Seungheon Lee, Jiwook Jung, Se Jin Jeon, Byung C. Kim, Young Choon Lee, Jong Hoon Ryu, Dong Hyun Kim PII:

S0028-3908(17)30494-X

DOI:

10.1016/j.neuropharm.2017.10.028

Reference:

NP 6913

To appear in:

Neuropharmacology

Received Date: 22 March 2017 Revised Date:

23 September 2017

Accepted Date: 21 October 2017

Please cite this article as: Yi, J.H., Baek, S.J., Heo, S., Park, H.J., Kwon, H., Lee, S., Jung, J., Jeon, S.J., Kim, B.C., Lee, Y.C., Ryu, J.H., Kim, D.H., Direct pharmacological Akt activation rescues Alzheimer's disease like memory impairments and aberrant synaptic plasticity, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2017.10.028. 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.

ACCEPTED MANUSCRIPT Direct pharmacological Akt activation rescues Alzheimer’s disease like memory impairments and aberrant synaptic plasticity

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Jee Hyun Yi1,#, Soo Ji Baek2,#, Sunghoo Heo2, Hye Jin Park3, Huiyoung Kwon3, Seungheon Lee4, Jiwook Jung5, Se Jin Jeon6, Byung C. Kim2, Young Choon Lee3,9,

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Jong Hoon Ryu7,8,*, Dong Hyun Kim3,9,*

School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol,

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Bristol, UK.

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

Republic of Korea 3

Department of Medicinal Biotechnology, College of Health Sciences, Dong-A

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University, Busan, Republic of Korea

Department of Aquatic Biomedical Sciences, School of Marine Biomedical Science,

College of Ocean Science, Jeju National University, Jeju, Republic of Korea Department of Herbal Medicinal Pharmacology, College of Herbal Bio-industry,

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Daegu Haany University, Kyungsan, Republic of Korea School of Natural Resources and Environmental Science, Kangwon National

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University, ChoonCheon, Republic of Korea 7

Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee

University, 1 Hoeki-dong, Dongdaemoon-Gu, Seoul, Republic of Korea 8

Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 1

Hoeki-dong, Dongdaemoon-Gu, Seoul, Korea. 9

Institute of Convergence Bio-Health, Dong-A University, Busan, Republic of Korea

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ACCEPTED MANUSCRIPT #

These authors are contributed equally in this study.

*Corresponding authors: Dong Hyun Kim

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Email: [email protected] Tel: +82-51-200-7583 Fax: +82-51-200-7588

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Jong Hoon Ryu Email: [email protected]

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Tel: +82-2-961-9230

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Fax: +82-2-961-0369

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ACCEPTED MANUSCRIPT Abstract Amyloid β (Aβ) is a key mediator for synaptic dysfunction and cognitive impairment implicated in Alzheimer’s disease (AD). However, the precise mechanism of the toxic

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effect of Aβ is still not completely understood. Moreover, there is currently no treatment for AD. Protein kinase B (PKB, also termed Akt) is known to be aberrantly regulated in the AD brain. However, its potential function as a therapeutic target for

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AD-associated memory impairment has not been studied. Here, we examined the role of a direct Akt activator, SC79, in hippocampus-dependent memory impairments

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using Aβ-injected as well as 5XFAD AD model mice. Oligomeric Aβ injections into the 3rd ventricle caused concentration-dependent and time-dependent impairments in learning/memory and synaptic plasticity. Moreover, Aβ aberrantly regulated caspase-3, GSK-3β, and Akt signaling, which interact with each other in the

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hippocampus. Caspase-3 and GSK-3β inhibitor ameliorated memory impairments and synaptic deficits in Aβ-injected AD model mice. We also found that pharmacological activation of Akt rescued memory impairments and aberrant

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synaptic plasticity in both Aβ-treated and 5XFAD mice. These results suggest that

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Akt could be a therapeutic target for memory impairment observed in AD.

Key words: Amyloid β; Alzheimer’s disease; Akt; Long-term potentiation; Memory

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Introduction Alzheimer’s disease (AD) is a neurodegenerative disease, whose main symptom

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is memory impairment. Amyloid β (Aβ) and Tau pathologies are known as main

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processes in the progression of AD and blocking of these pathologies has long been

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believed to be the therapeutic goal for AD. However, there is still no medical

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approach approved to target these hallmarks of the disease.

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Aβ is aggregated and deposited in the AD brain and directly suppresses functions

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of neurons (Palop and Mucke, 2010) and causes inflammation (Tuppo and Arias,

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2005) and oxidative stress (Butterfield, 2002) as a secondary effect. Because

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previous studies revealed that Aβ induces synaptic dysfunction and memory

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impairment (Haass and Selkoe, 2007; Selkoe, 2008; Shankar et al., 2008) at an

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early stage of AD, studies investigating the precise mechanisms and ways of

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blocking the effects of Aβ have long been believed to help curing AD symptoms

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(Klein, 2002; Klein et al., 2001; Kotilinek et al., 2008; Wang et al., 2016b). Therefore,

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an excessive number of studies have been conducted to uncover the toxic

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mechanisms of Aβ.

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Akt, protein kinase B, is an enzyme that plays important roles in cellular processes,

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including glucose metabolism (Kohn et al., 1996; Sakoda et al., 2003), apoptosis

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(Song et al., 2005), and cell proliferation (Vivanco and Sawyers, 2002). Activation of

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Akt is induced via phosphorylation by mTORC2 (Sarbassov et al., 2005) and PDK1

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(Franke, 2008) and this activation has been reported to play an important role in

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synaptic plasticity and memory formation (Horwood et al., 2006). Aberrant regulation

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of Akt has reported in AD brain. Several studies revealed increased activation of Akt

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in AD (Griffin et al., 2005; Rickle et al., 2004; Talbot et al., 2012) but, opposite result

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is also exist (Lee et al., 2009). However, Aβ, itself, may suppress the activity of Akt

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ACCEPTED MANUSCRIPT (Abbott et al., 2008; Chen et al., 2009; Magrane et al., 2005) and several effective

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drugs, which are reported as candidates for the treatment of AD, activate Akt

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indirectly (Ali and Kim, 2015; Jin et al., 2005; Yi et al., 2016). Aβ induces Akt

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cleavage through caspase-3 activation, resulting in activation of GSK-3β (Jo et al.,

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2011). Moreover, because activation of Akt-related molecules with drugs ameliorates

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Aβ toxicity (Ahmad et al., 2016; Baki et al., 2004; Ma et al., 2009; Xing et al., 2011),

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the regulation of Akt might be a good target for investigation. However, the effect of

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the direct regulation of Akt on AD pathology has not been studied.

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In the present study, we examined the effect of AC79, a direct activator of Akt, on

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Aβ-induced synaptic plasticity and memory impairments using an Aβ injection model

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as well as a 5XFAD transgenic AD mouse model.

12 Methods and Materials

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1. Animals

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Male C57BL6 mice (30 - 34 g, 10 weeks) were purchased from the SAMTAKO

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biokorea (Osansi, Korea), and kept in the University Animal Care Unit for 1 week

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prior to the experiments. The animals were housed 4 per cage, allowed access to

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water and food ad libitum; the environment was maintained at a constant

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temperature (23 ± 1 °C) and humidity (60 ± 10%) under a 12-h light/dark cycle (the

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lights were on from 07:30 to 19:30). 5XFAD male mice were received from Jackson

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Laboratory (Jackson Laboratory, USA) and crossbred with hybrid B6SJLF1 (from

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Taconic) female mice. The offspring, heterozygous transgenic and littermate wild

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type (WT) male mice, were used. The mice were housed in individual ventilated

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cages (IVC) with access to water and food ad libitum, under a 12-h light/dark cycle

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(the lights were on from 07:30 to 19:30). The treatment and maintenance of the

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ACCEPTED MANUSCRIPT animals were carried out in accordance with the Animal Care and Use Guidelines

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Dong-A University, Korea. All of the experimental protocols using animals were

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approved by the Institutional Animal Care and Use Committee of Dong-A University,

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Korea.

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2. Cannulae implantation

Cannulae were implanted into the 3rd ventricle. Mice were anesthetized with Zoletil

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50 (10 mg/kg, i.m.) and placed in a stereotaxic instrument (David Kopf Instruments,

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Tujunga, CA). Guide cannulae (26 G) were aimed at the 3rd ventricle (stereotaxic

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coordinates: AP, -2.00 mm; ML, 0 mm; DV, -2.00 mm) using an atlas of the mouse

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brain (Paxinos and Franklin, 2004). The guide cannulae were fixed to the skul with

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dental cement and covered with dummy cannulae. To inject drugs, mice were

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carefully restrained by hand and infused with drugs through an injector cannula (30

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G) at 0.25 µl/min). After 2 min of infusion, the infusion needle remained in the guide

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cannula for 1 min more to ensure proper delivery of the drugs.

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Add 1.0% NH4OH directly to the Aβ1-42 (35 - 40 µl to 0.5 mg peptide or 70-80 µl to 1

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mg peptide). Then this solution was immediately diluted with 1 X PBS to a

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concentration of 1 mg/ml. The solution was gently vortexed and sonicated at room

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temperature until fully imscible. Aβ1-42 (10 µM) was incubated at 37 °C for 1 week,

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and then 3 µl of Aβ or vehicle were acutely injected into the intracerebroventricle. Z-

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DEVD-fmk, SC79, and TCS2002 were purchased from Tocris Bioscience (Ellisville,

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MO). Z-DEVD-fmk (cell-permaeble, irreversible inhibitor of caspase-3/CPP32, Tocris

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Bioscience, 30 µM/3 µl) (Fan et al., 2014), TCS2002 (Potent GSK-3β inhibitor, Tocris

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ACCEPTED MANUSCRIPT Bioscience, 10 µM/3 µl) (Saitoh et al., 2009) and SC79 (Akt activator, Tocris

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Bioscience, 100 µg/3 µl) (Wang et al., 2016a) was dissolved in 0.4%

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dimethylsulphoxide (DMSO, MC/B, Norwood, OH, U.S.A.) in 0.1 M phosphate-

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buffered saline (PBS; pH 7.4). Drugs were injected into 3rd ventricle.

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4. Inhibitory avoidance test

We assessed training and test trials for 2 days using the inhibitory avoidance task.

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Testing was performed in a box consisting of two identical chambers (20 × 20 × 20

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cm) with one chamber illuminated with a 50-W bulb and another non-illuminated; the

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two chambers were separated by a guillotine door (5 × 5 cm). The floor of the non-

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illuminated compartment was comprised of 2-mm stainless steel rods spaced 1 cm

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apart as described previously (Kim et al., 2006). In the training trial, the mice were

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initially placed in the illuminated compartment. The door between the two

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compartments was opened 10 s later and was automatically closed when the mice

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entered the non-illuminated compartment. A 3 s electrical foot shock (0.5 mA) was

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then delivered through the stainless steel rods. The test trial was conducted 24 h

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after the acquisition trial by returning the individual mice to the illuminated

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compartment. In both trials, latency to enter was defined as the time it took for the

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mouse to enter the dark compartment after the door was opened. During the

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acquisition trial, the mice that did not enter the non-illuminated compartment within

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60 s of the door opening the door were gently introduced into the dark chamber, and

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the latency to enter was recorded as 60 s. Latencies were recorded for up to 300 s

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during the retention trial.

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5. Y-maze test

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ACCEPTED MANUSCRIPT The Y-maze is a three-arm horizontal maze (40 cm long and 3 cm wide with 12 cm

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high walls) in which the arms are symmetrically disposed at 120° angles to each

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other. The maze floor and walls were constructed from dark opaque polyvinyl plastic

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as previously described (Kim et al., 2006). Mice were initially placed in one arm, and

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the sequence and numbers of arm entries were manually recorded for each mouse

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over an 8-min period. An alternation was defined as the consecutive entry into all

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three arms (i.e., ABC, CAB, or BCA, but not BAB). The maze arms were thoroughly

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cleaned with water between each test to remove residual odors and residues. The

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alternation score (%) for each mouse was defined as the ratio of the actual number

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of alternations to the total number possible (defined as the total number of arm

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entries minus two) multiplied by 100 as shown by the following equation: %

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Alternation = [(Number of alternations) / (Total arm entries – 2)] × 100. The number

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of arm entries was used as an indicator of locomotor activity.

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6. Object recognition test

Mice were habituated to the open field (25 cm x 25 cm x 25 cm) with an internal

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cue on one of the four walls for 10 min. Thirty minutes after the habituation; mice

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were re-placed in the same box with two distinct objects. The objects consisted of a

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glass box and plastic cylinder. Mice were allowed to freely explore the objects for 10

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min. After 2 h, mice were placed back in the same box for the test phase. The two

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objects were again present, but one object was now displaced to a novel spatial

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location. Mice were allowed to freely explore the environment and the objects for 5

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min. Time spent exploring the displaced and non-displaced objects were measured.

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Preference ratio was calculated by following formula: Tdisplaced or Tnon-displaced/(Tdisplaced

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+ Tnon-displaced) x 100. Tdisplaced, time spent exploring displaced object. Tnon-displaced, time

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spent exploring non-displaced object.

3 7. Electrophysiology

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Mouse hippocampal slices were prepared using micro-vibratome (Lafayette-

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campden neuroscience TM). The brain was rapidly removed and placed in ice-cold

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artificial cerebrospinal fluid (ACSF; bubbled with 95% O2/5% CO2), which comprised:

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(mM) NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; D-

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glucose, 10. Transverse hippocampal slices (400 µm thick) were prepared.

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Hippocampal slices were submerged in ACSF (20–25 °C) for 2h before transfer to

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the recording chamber (28–30 °C, flow rate ∼3 ml/min) as required. Field recordings

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were made from stratum pyramidale in area CA1. Stimulating electrode was placed

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in the Schaffer collateral-commissural pathway. Stimuli (constant voltage) were

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delivered at 30 s intervals. To induce LTP, theta burst stimulation (5 trains of 4

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pulses at 100 Hz) was delivered. The slope of the evoked field potential responses

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were averaged from four consecutive recordings (fEPSPs) evoked at 30 s intervals.

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8. Western blot analysis

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For the preparation of Western blot samples, coronal-sliced hippocampal tissues

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were made using micro-vibratome (Lafayette-campden neuroscience TM). Then the

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stratum radiatum, enriched in CA1 dendrites, were microdissected and homogenized

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in an ice-cold Tris–HCl buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 mM

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EDTA, 1 mM EGTA, 1 mM PMSF, a Complete Protease Inhibitor Cocktail (1

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tablet/50 ml) and a PhosSTOP Phosphatase Inhibitor Cocktail (1 tablet/10 ml).

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Samples of the homogenates (30 µg of protein) were then subjected to SDS-PAGE

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ACCEPTED MANUSCRIPT (8%) under reducing conditions. The proteins were transferred to PVDF membranes

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in a transfer buffer [25 mM Tris–HCl buffer (pH 7.4) containing 192 mM glycine and

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20% v/v methanol] and further separated at 400 mA for 2 h at 4 °C. The Western

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blots were then incubated for 1 h with a blocking solution (2% BSA or 5% skim milk),

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then with rabbit anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA,

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1:1000), rabbit anti-caspase-3 (Cell Signaling Technology, Beverly, MA, 1:1000),

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rabbit anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), rabbit anti-

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pAkt1 (Ser473) (Cell Signaling Technology, Beverly, MA, 1:1000), rabbit anti-Akt1

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(Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), rabbit anti-pGSK3β (Ser9)

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(Cell Signaling Technology, Beverly, MA, 1:1000), rabbit anti-GSK3β (Santa Cruz

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Biotechnology, Santa Cruz, CA, 1:1000) antibody overnight at 4 °C, washed ten

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times with Tween20/Tris-buffered saline (TTBS), incubated with a 1:2000 dilution of

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horseradish peroxidase-conjugated secondary antibodies for 2 h at room

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temperature, washed ten times with TTBS, and finally developed by enhanced

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chemiluminescence (Amersham LifeScience, Arlington Heights, IL).

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9. Statistics

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The values are expressed as the means ± S.E.M. The data were analyzed using

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one-way analysis of variance (ANOVA) followed by Student Newman–Keuls test for

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multiple comparisons. The data from drug experiments were analyzed using two-way

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ANOVA followed by Bonferroni test for multiple comparisons. The statistical

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significance was set at P < 0.05.

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Results

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1. Intracerebroventricular (i.c.v.) injection of Aβ causes hippocampal LTP and

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memory impairment Since synthetic Aβ impair synaptic plasticity in the hippocampus and long-term

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memory (Balducci et al., 2010), we examined Aβ under different experimental

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conditions. To evaluate the precise concentration of Aβ that leads to memory

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impairment, we first conducted inhibitory avoidance and object location recognition

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tests after i.c.v. injection of various concentrations of Aβ. We found that 10 µM of Aβ

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significantly impaired inhibitory avoidance memory (F3,36 = 3.020, P < 0.05, n =

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10/group, supplementary Fig. S1A) and object location recognition memory (F3,36 =

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2.961, P < 0.05, n = 10/group, supplementary Fig. S1B). Moreover, hippocampal

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slices from Aβ (1 or 10 µM)-injected groups showed significantly lower LTP levels

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compared to those of a vehicle-injected control group [Aβ (0 µM), 193 ± 8; Aβ (0.5

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µM), 177 ± 12; Aβ (1 µM), 147 ± 10; Aβ (10 µM), 115 ± 12; F3,24 = 10.52, P < 0.05, n

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= 7/group, supplementary Fig. S1C].

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Synthetic Aβ transiently impaired hippocampus-dependent memory (Balducci et

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al., 2010). Therefore, we next examined the time course of the effect of Aβ on

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memory and synaptic plasticity. Behavioral experiments were started at 1, 3, or 7

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days after Aβ injection. The memory-impairing effect of Aβ persisted until 3 days

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after injection in the inhibitory avoidance test (day 1, t18 = 2.922, P < 0.05, n =

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10/group; day 3, t18 = 1.829, P < 0.05; day 7, t18 = 0.5026, P > 0.05, n = 10/group,

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supplementary Fig. S1D) and in the object location recognition test (day 1, t18 =

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2.100, P < 0.05; day 3, t18 = 2.618, P < 0.05; day 7, t18 = 0.6681, P > 0.05, n =

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10/group, supplementary Fig. S1E). Moreover, hippocampal LTP levels were also

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found to be impaired in the slices taken at 1, 3, or 7 days after Aβ injection (Day 1:

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control = 166 ± 12, Aβ = 114 ± 10, t12 = 3.159, P < 0.05, n = 7/group, supplementary

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ACCEPTED MANUSCRIPT Fig. S1F; Day 3: control = 187 ± 11, Aβ = 135 ± 16, t12 = 2.557, P < 0.05, n = 7/group,

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supplementary Fig. S1G; Day 7: control = 180 ± 17, Aβ = 146 ± 5, t12 = 1.889, P <

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0.05, n = 7/group, supplementary Fig. S1H). Based on these results, we conducted

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all other experiments at 3 days after Aβ (10 µM) injection.

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2. Aβ activates the CAG pathway

A previous report suggested that the caspase-3/Akt/GSK-3β cascade is important

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in Aβ-impairing synaptic plasticity (Jo et al., 2011). However, these authors

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conducted experiments with perfusion of Aβ into the recording chamber using

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hippocampal slices. Here, we examined whether this system is active in vivo as well

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as in vitro. To test this, we injected Aβ into the 3rd ventricle and first measured the

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changes in the caspase-3/Akt/GSK-3β cascade in the radiatum of the hippocampal

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CA1 region, which is mainly consists of dendrites of pyramidal neurons. Aβ injection

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lead to significantly higher cleaved caspase-3 levels (t6 = 2.143, P < 0.05, n =

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5/group, Fig. 1B and 1C). However, levels of the phosphorylated form of Akt1 and

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GSK-3β were significantly lower in Aβ-injected groups (pAkt1: t6 = 1.999, P < 0.05, n

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= 5/group; pGSK-3β: t6 = 1.994, P < 0.05, n = 5/group, Fig. 1B and 1C), while levels

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of the Akt1 and GSK-3β did not change significantly. These results suggest that Aβ

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inhibits Akt1 and activates caspase-3 and GSK-3β.

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3. Caspase-3 inhibition rescues Aβ-induced memory impairment

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A previous report indicated that caspase-3 knockout mice showed normal LTP in

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the presence of Aβ in an in vitro system (Jo et al., 2011). Moreover, pharmacological

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inhibition of caspase-3 rescued contextual fear memory deficits and synaptic

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dysfunctions in the Tg2576-APPswe mouse model of Alzheimer's disease (D'Amelio

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ACCEPTED MANUSCRIPT et al., 2011). We, therefore, tested the effect of inhibition of caspase-3 in avoidance

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memory, working memory, recognition memory, and long-term synaptic plasticity in

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the hippocampus. To inhibit caspase-3, we used z-DEVD-fmk, a cell-permeable and

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irreversible caspase-3/CPP32 inhibitor (IC50 = 18 µM). All experiments were

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conducted 3 days after Aβ and/or z-DEVD-fmk (30 µM/3 µl) injection into the 3rd

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ventricle. In the inhibitory avoidance test, all groups showed similar latency to enter

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in the training session (two way ANOVA, z-DEVD-fmk, F1, 35 = 0.638, P = 0.430; Aβ,

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F1, 35 = 0.035, P = 0.853; interaction, F1, 35 = 0.299, P = 0.587, n = 9 – 10/group, Fig.

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2B). In the test session, Aβ-treated mice showed significantly shorter latency to enter

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than vehicle-treated control mice. However, Aβ- and z-DEVD-fmk-co-treated mice

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showed latency to enter similar to those found for z-DEVD-fmk-treated mice (two

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way ANOVA, z-DEVD-fmk, F1, 35 = 6.209, P = 0.018; Aβ, F1, 35 = 4.778, P = 0.036;

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interaction, F1, 35 = 4.808, P = 0.035, n = 9 – 10/group, Fig. 2A). In the object location

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recognition test, total exploration time was not significantly different between groups

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(two way ANOVA, z-DEVD-fmk, F1, 35 = 0.012, P = 0.913; Aβ, F1, 35 = 0.072, P =

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0.789; interaction, F1, 35 = 0.412, P = 0.525, n = 9 – 10/group, Fig. 2D). However, Aβ-

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treated mice could not distinguish moved object and fixed object (t18 = 1.167, P >

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0.05, n = 10, Fig. 2C), while Aβ and z-DEVD-fmk-cotreated mice could distinguish

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the objects (t18 = 1.846, P < 0.05, n = 10, Fig. 2C). Moreover, Aβ-treated mice

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showed a significantly lower number of spontaneous alternations, while Aβ and z-

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DEVD-fmk-cotreated mice showed a significantly higher spontaneous alternations

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(two way ANOVA, z-DEVD-fmk, F1, 35 = 5.499, P = 0.025; Aβ, F1, 35 = 4.409, P =

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0.043; interaction, F1, 35 = 4.589, P = 0.039, n = 9-10, Fig. 2E) in the Y-maze test

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without total arm entries being affected (two way ANOVA, z-DEVD-fmk, F1, 35 = 0.118,

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P = 0.734; Aβ, F1, 35 = 0.498, P = 0.485; interaction, F1, 35 = 0.003, P = 0.959, n = 9 –

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10/group, Fig. 2F). These results suggest that caspase-3 inhibition rescued spatial

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fear memory and object location recognition memory deficits induced by Aβ. As reported above Aβ impaired LTP and facilitated LTD in the hippocampal

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Schaffer collateral pathway. However, treatment of z-DEVD-fmk with Aβ attenuated

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this effect of Aβ on LTP and LTD (LTP: two way ANOVA, z-DEVD-fmk, F1, 24 = 3.520,

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P = 0.073; Aβ, F1, 24 = 4.856, P = 0.037; interaction, F1, 24 = 4.500, P = 0.044, n =

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7/group, Fig. 2G; LTD: two way ANOVA, z-DEVD-fmk, F1, 24 = 2.529, P = 0.125; Aβ,

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F1, 24 = 21.58, P = 0.001; interaction, F1, 24 = 15.68, P = 0.006, n = 7/group, Fig. 2H).

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4. GSK-3β inhibition rescues Aβ-induced synaptic and memory dysfunction A previous report indicated that GSK-3β inhibitor rescues Aβ-induced LTP in an in

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vitro system (Jo et al., 2011). Therefore, we tested the effect of pharmacological

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inhibition of GSK-3β on avoidance memory, working memory, recognition memory

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and long-term synaptic plasticity in the hippocampus. To inhibit GSK-3β activity, we

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used a potent inhibitor of GSK-3β (IC50 = 35 nM). In the inhibitory avoidance test, all

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groups showed similar latency to enter in the training session (two way ANOVA,

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TSC2002, F1, 36 = 1.087, P = 0.3.4; Aβ, F1, 36 = 0.716, P = 0.403; interaction, F1, 36 =

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0.422, P = 0.520, n = 10/group, Fig. 3B). In the test session, Aβ-treated mice

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showed significantly shorter latency to enter than vehicle-treated control mice.

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However, Aβ and TCS2002 (10 µM/3 µl)-cotreated mice showed latency to enter

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similar to those found for TCS2002-treated mice (two way ANOVA, TSC2002, F1, 36 =

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2.840, P = 0.101; Aβ, F1, 36 = 6.055, P = 0.019; interaction, F1, 36 = 11.07, P = 0.002,

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n = 10/group, Fig. 3A). In the object location recognition test, total exploration time

24

was not significantly different between groups (two way ANOVA, TSC2002, F1, 36 =

25

0.389, P = 0.537; Aβ, F1, 36 = 0.373, P = 0.545; interaction, F1, 36 = 0.012, P = 0.913,

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ACCEPTED MANUSCRIPT n = 10/group, Fig. 3D). However, Aβ-treated mice could not distinguish moved object

2

and fixed object (t18 = 0.4830, P > 0.05, n = 10, Fig. 3C), in the test session. Aβ and

3

TCS2002-cotreated mice could distinguish those objects (t18 = 1.947, P < 0.05, n =

4

10, Fig. 3C). Moreover, Aβ-treated mice showed a significantly lower number of

5

spontaneous alternations, while Aβ and z-DEVD-fmk-cotreated mice shower

6

significantly higher spontaneous alternations (two way ANOVA, TSC2002, F1,

7

3.733, P = 0.061; Aβ, F1, 36 = 3.877, P = 0.056; interaction, F1, 36 = 4.246, P = 0.047,

8

n = 10, Fig. 3E) in the Y-maze test, without total arm entries being affected (two way

9

ANOVA, TSC2002, F1, 36 = 0.582, P = 0.451; Aβ, F1, 36 = 0.009, P = 0.921; interaction,

36

=

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F1,

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pharmacological inhibition of GSK-3β rescued spatial fear memory and object

12

location recognition memory deficits induced by Aβ. Treatment of TCS2002 with Aβ

13

also attenuated the effect of Aβ on LTP and LTD (LTP: two way ANOVA, TSC2002,

14

F1, 24 = 2.234, P = 0.148; Aβ, F1, 24 = 2.006, P = 0.169; interaction, F1, 24 = 5.032, P =

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0.034, n = 7/group, Fig. 3G; LTD: two way ANOVA, TSC2002, F1, 324 = 5.642, P =

16

0.026; Aβ, F1,

17

10/group, Fig. 3H).

19

= 0.034, P = 0.854, n = 10/group, Fig. 3F). These results suggest that

= 1.520, P = 0.229; interaction, F1,

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= 4.271, P = 0.049, n =

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5. Akt activation restores Aβ-induced synaptic and memory dysfunction

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A previous report indicated that neurons injected with an Akt1 mutant mutated at

21

the caspase-3-cleavaging site show normal LTP in the presence of Aβ in an in vitro

22

system (Jo et al., 2011). Therefore, we tested the effect of pharmacological

23

activation of Akt in avoidance memory, working memory, recognition memory, and

24

long-term synaptic plasticity in the hippocampus. To activate Akt, we used SC79, an

25

activator of Akt; binds to the pleckstrin homology domain of Akt, which enhances Akt

12

ACCEPTED MANUSCRIPT phosphorylation by upstream protein kinases; also enables cytosolic activation of Akt.

2

In the inhibitory avoidance test, all groups showed similar latency to enter in the

3

training session (two way ANOVA, SC79, F1, 35 = 0.425, P = 0.519; Aβ, F1, 35 = 0.095,

4

P = 0.760; interaction, F1, 35 = 0.569, P = 0.456, n = 9 - 10/group, Fig. 4B). In the test

5

session, Aβ-treated mice showed significantly shorter latency to enter than vehicle-

6

treated control mice. However, Aβ and SC79 (100 µg/ 3 µl)-cotreated mice showed

7

latency to enter similar to those found for SC79-treated mice (two way ANOVA,

8

SC79, F1, 35 = 10.52, P = 0.003; Aβ, F1, 35 = 4.293, P = 0.046; interaction, F1, 35 =

9

4.153, P = 0.049, n = 9 - 10/group, Fig. 4A). In the object location recognition test,

10

total exploration time was not significantly different between groups (two way

11

ANOVA, SC79, F1, 35 = 0.104, P = 0.749; Aβ, F1, 35 = 0.479, P = 0.494; interaction, F1,

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35

13

distinguish moved object and fix objects in the test session (t18 = 0.5181, P > 0.05, n

14

= 10, Fig. 4C), while Aβ and SC79-cotreated mice could distinguish those objects (t18

15

= 1.903, P < 0.05, n = 10, Fig. 4C). Moreover, Aβ-treated mice showed a significantly

16

lower number of spontaneous alternations and SC79 improved this (two way ANOVA,

17

SC79, F1, 35 = 3.263, P = 0.067; Aβ, F1, 35 = 4.133, P = 0.049; interaction, F1, 35 =

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4.722, P = 0.037, n = 9 - 10, Fig. 4E) in the Y-maze test, without total arm entries

19

being affected (two way ANOVA, SC79, F1, 35 = 0.105, P = 0.748; Aβ, F1, 35 = 0.001,

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P = 0.979; interaction, F1, 35 = 0.180, P = 0.674, n = 9 - 10/group, Fig. 4F). These

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results suggest that direct activation of Akt rescued spatial fear memory and object

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location recognition memory deficits induced by Aβ. Treatment of SC79 with Aβ also

23

attenuated the effect of Aβ on LTP (two way ANOVA, SC79, F1, 24 = 2.191, P = 0.152;

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Aβ, F1, 24 = 1.984, P = 0.172; interaction, F1, 24 = 5.252, P = 0.031, n = 7/group, Fig.

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= 0.014, P = 0.906, n = 9 - 10/group, Fig. 4D). However, Aβ-treated mice could not

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ACCEPTED MANUSCRIPT 1

4G) and LTD (two way ANOVA, SC79, F1, 24 = 3.844, P = 0.062; Aβ, F1, 24 = 4.170, P

2

= 0.052; interaction, F1, 24 = 4.659, P = 0.041, n = 7/group, Fig. 4H).

3 6. Akt activation ameliorates synaptic and memory dysfunction in 5XFAD mice

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Since Injection of synthetic Aβ into the ventricle causes reversible deficits in

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synaptic plasticity, learning, and memory, we next tested if direct Akt activation could

7

rescue memory and synaptic plasticity deficits in 6-month-old 5XFAD AD model mice.

8

SC79 was infused into the 3rd ventricle through cannulae once a day for 2 weeks

9

(Fig. 5A). Then, we conducted inhibitory avoidance and object location recognition

10

tests, and an LTP experiment. In the inhibitory avoidance test, all groups showed

11

similar latency to enter in the training trial (two way ANOVA, SC79, F1, 36 = 0.742, P

12

= 0.395; Tg, F1, 36 = 0.150, P = 0.701; interaction, F1,

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10/group, Fig. 5B). However, vehicle-treated 5XFAD group showed significantly

14

shorter latency to enter than wild type group and this effect was ameliorated by

15

SC79 infusion (two way ANOVA, SC79, F1, 36 = 5.633, P = 0.023; Tg, F1, 36 = 4.587,

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P = 0.039; interaction, F1, 36 = 4.138, P = 0.049, n = 10/group, Fig. 5C). Results from

17

the object location recognition test were similar to the data from the inhibitory

18

avoidance test. Vehicle-treated 5XFAD group showed a significantly lower

19

discrimination index than wild type group and this effect was ameliorated by SC79

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infusion (two way ANOVA, SC79, F1, 36 = 1.832, P = 0.188; Tg, F1, 36 = 5.089, P =

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0.033; interaction, F1,

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exploration time being affected (two way ANOVA, SC79, F1, 36 = 0.988, P = 0.327; Tg,

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F1, 36 = 0.469, P = 0.498; interaction, F1, 36 = 0.008, P = 0.927,n = 10/group, Fig. 5D).

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Treatment with SC79 also improved the lower LTP level, which was shown in the

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vehicle-treated 5XFAD group (two way ANOVA, SC79, F1, 28 = 2.921, P = 0.099; Tg,

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= 0.007, P = 0.934, n =

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= 4.434, P = 0.046, n = 10/group, Fig. 5E), without total

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ACCEPTED MANUSCRIPT 1

F1, 28 = 14.29, P = 0.001; interaction, F1, 28 = 6.681, P = 0.015, n = 7 - 9/group, Fig.

2

5F and 5G). Activity of GSK-3β, a direct target of Akt, was also increased in both wild type and

4

5XFAD groups after SC79 administration (two way ANOVA, SC79, F1, 16 = 11.93, P =

5

0.003; Tg, F1, 16 = 0.602, P = 0.449; interaction, F1, 16 = 6.007, P = 0.026, n = 5/group,

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Fig. 5H and 5I). Akt1 activity was increased in 5XFAD group after SC79

7

administration (two way ANOVA, SC79, SC79, F1, 16 = 13.38, P = 0.002; Tg, F1, 16 =

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3.201, P = 0.092; interaction, F1, 16 = 21.60, P = 0.003, n = 5/group, Fig. 5H and 5J).

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Aberrant regulation of Akt is one of the key events in the AD brain. Since Akt is

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involved in various up-stream signaling pathways including mTORC2 and PDK1, and

13

in down-stream pathways including GSK-3β, CREB, and Tau, all known to be

14

aberrantly regulated in AD pathology, the regulation of Akt has been identified as a

15

candidate for AD treatment. However, the effect of direct regulation of Akt has not

16

been investigated before. In the present study, we found reduced Akt activity in Aβ-

17

injected or 5XFAD mouse AD models. Therefore, we tested the effect of direct

18

activation of Akt with SC79, an Akt activator, on AD-like memory impairments and

19

aberrant synaptic plasticity. We found that short-term treatment with SC79

20

ameliorated memory and LTP impairments and attenuated facilitated LTD in Aβ-

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injected as well as 5XFAD AD model mice.

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The Inhibitory avoidance task is notorious for its sensitivity to confounding factors.

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At least hyperactivity, reduced anxiety and increased pain threshold can yield a false

24

impression of impaired memory. Total arm visits in the spontaneous alternation in Y-

25

maze can be used as a control for activity. In the present study, because total arm

15

ACCEPTED MANUSCRIPT visits were not significantly different, the effect of Aβ infusion and the applied drugs

2

on animal activities could be rule out. We also could infer anxiety level from time

3

spent in exploring the object on the first day of object location recognition task.

4

Because this was not significantly different among groups, the effect of Aβ infusion

5

and the applied drugs on anxiety level could be rule out. To test the effect of Aβ

6

infusion and the applied drugs on sensitivity to foot shock, we measured responses

7

to foot shock in training trial (no response, 0; immobilization, 1; vocalization, 2;

8

jumping 3) (supplemental Fig. 4). In this test, all groups showed similar scores in

9

responses to food shock. Therefore, we demonstrate that the data from inhibitory

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avoidance task used in this study could be acceptable.

Caspase-3 is a molecule which is linked to Akt (Chu et al., 2017; Jahani-Asl et al.,

12

2007; Li et al., 2010). Caspase-3 activation is a cause of neuronal death in AD

13

pathology (Bredesen, 2009; D'Amelio et al., 2011). Growth factors and neurotropic

14

factors protect neurons from Aβ toxicities via inhibition of caspase-3 through Akt

15

activation (Schindowski et al., 2008; Zheng et al., 2000). However, since neuronal

16

loss is a late event in AD patients, its significance has been under-estimated. Recent

17

reports show that caspase-3 involved in early synaptic dysfunction induced by Aβ

18

and blocking caspase-3 is effective in preventing AD-like symptoms (D'Amelio et al.,

19

2011; Jo et al., 2011). Moreover, caspase-3 is involved in physiological synaptic

20

depression, which is related to brain development and memory flexibility (Li et al.,

21

2010). Recent studies also found that caspase-3 exists in the synapse as well as the

22

cell body, suggesting that local caspase-3 activation can regulate local physiological

23

and pathological events without affecting cell fate. The Aβ-injected AD model mice

24

that we used showed transient memory impairments and aberrant synaptic plasticity

25

in the hippocampus without evident neuronal loss suggesting that this model can be

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ACCEPTED MANUSCRIPT used to mimic early AD patients. In the present study, inhibition of caspase-3

2

blocked memory impairments and aberrant synaptic plasticity. These findings

3

suggest that if good pharmacological inhibitors are good drug candidates for early

4

phase of AD patients. However, since caspase-3 also has various physiological

5

roles, further research is required.

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GSK-3β is a well-studied target in AD pathology and a down-stream molecule of

7

Akt (Hooper et al., 2008; Reddy, 2013). GSK-3β is involved in Aβ-induced synaptic

8

pathology and memory impairments (Bradley et al., 2012; Peineau et al., 2008).

9

Moreover, GSK-3β inhibitors show neuroprotection and regulation of symptoms in

10

various AD models (Dominguez et al., 2012; Martinez et al., 2011). Lithium is a

11

representative GSK-3β inhibitor. Unfortunately, a clinical study failed to show the

12

effectiveness of lithium on moderate AD pathology (Hampel et al., 2009). However,

13

Nunes et al. (2013) showed a delay of progression of mild cognitive impairments to

14

AD suggesting that GSK-3β inhibition may be effective before onset or in the very

15

early phase of AD (Nunes et al., 2013). This may due to the fact that the time course

16

of AD onset is similar Tau hyperphosphorylation, rather than to Aβ deposition

17

(Selkoe and Hardy, 2016). Since GSK-3β is a Tau kinase, once Tau is

18

phosphorylated, GSK-3β inhibition cannot restore this event. In the present study, we

19

first examined the effect of TCS2002, a cell permeable GSK-3β inhibitor, in an Aβ-

20

injected AD mouse model. TCS2002 ameliorated memory impairments and aberrant

21

synaptic plasticity. These results suggest that TCS2002 may be a good candidate for

22

preventing the progression of MCI to AD.

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Akt is both a regulator and a substrate of caspase-3. Akt not only suppresses

24

caspase-3 activation but is also cleaved by activated caspase-3 (Chu et al., 2017).

17

ACCEPTED MANUSCRIPT This suggests that Aβ can suppress activity and reduce the protein level of Akt. In

2

the present study, we found that Akt phosphorylation was significantly lower in Aβ-

3

injected AD mouse model compared to a vehicle-injected control in the dendrite

4

area. However, levels of the Akt were slightly lower in AD group; the effect was

5

however not significant in the dendrite area. This suggests that changes of activity

6

rather than protein levels of Akt are evident in the early phase of AD. This was

7

confirmed in the 5XFAD model (supplemental Fig. S2A). Early-phase (6-month-old)

8

AD brains showed a reduction of pAkt but normal Akt levels (supplemental Fig. S2A).

9

However, late-phase (1-year-old) AD brains showed a reduction of Akt levels

10

(supplemental Fig. S2B). These results suggest that the Akt activator could be more

11

useful in the early rather than the late phase of AD. In the present study, we

12

confirmed that SC79 ameliorates memory impairments and aberrant synaptic deficits

13

in 3-month-old 5XFAD AD model mice but not in 1-year-old 5XFAD AD model mice

14

(supplemental Fig S3). Although we cannot rule out that prolonged administration or

15

an increase of the dose of SC79 may be effective in late-phase AD as well, it is

16

obvious that treatment protocols and agents should be different for early- and late-

17

phase AD.

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Akt activity is aberrantly regulated in AD brain (Lee et al., 2009; Sajan et al., 2016;

19

Talbot et al., 2012). Many researches showed controversial results in Akt activity in

20

brain of AD patients. Several findings showed increase of Akt activity in AD patients,

21

but others and we found opposite results in Aβ-treated neurons or mouse AD

22

models. In AD brain, there might be various pathological initiators beside Aβ. But, in

23

the Aβ-treated AD model system, Aβ is only involved in AD-like pathology. This might

24

be the reason of the controversy. Although Aβ-treated AD model system has long

25

been used to develop AD treatment, promising medicine for AD is still not developed.

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The controversial results may reflect the difficulty of developing AD treatment with

2

this system.

3 Funding and Disclosure

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This study was supported by the National Research Foundation of Korea (NRF)

6

grand funded by the Ministry of Education, Science and Technology (MEST)

7

(2016R1A5A2007009 and 2015R1A2A2A01007838).

8

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The authors declare no competing financial interests.

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Figure legends

2 Figure 1. Effect of Aβ on signaling molecules in the hippocampus. Aβ (10 µM/3 µl)

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was injected into third ventricle and hippocampal tissues were collected 3 days after

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the injection. a. Photograph of blots. b. Quantitative analysis of the expression level

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of cleaved caspase-3, pAkt and pGSK-3β, which were normalized to actin. c.

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Quantitative analysis of the expression level of caspase-3, Akt and GSK-3β, which

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were normalized to actin. Data are expressed as mean ± SEM. *P < 0.05 vs vehicle

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group.

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Figure 2. Effect of Z-DEVD-fmk on Aβ-induced memory impairments and aberrant

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synaptic plasticity. Aβ (10 µM/3 µl) and/or Z-DEVD-fmk (30 µM/3 µl) was injected

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into third ventricle and hippocampal tissues were collected 3 days after the injection.

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a. Latency to enter in retention trial of inhibitory avoidance test. b. Latency to enter in

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acquisition trial of inhibitory avoidance test. c. Preference ratio in test session of

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object location recognition test. d. Total arm entry in test session of object location

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recognition test. e. Spontaneous alternations in Y-maze test. f. Total arm entry in Y-

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maze test. g. Long-term potentiation (LTP) induced by theta burst stimulation (TBS).

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h. Long-term depression (LTD) induced by low frequency stimulation (1 Hz, 900

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pulses). Data are expressed as mean ± SEM. *P < 0.05 vs vehicle group.

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Figure 3. Effect of TCS2002 on Aβ-induced memory impairments and aberrant

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synaptic plasticity. Aβ (10 µM/3 µl) and/or TCS2002 (10 µM/3 µl) was injected into

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third ventricle and hippocampal tissues were collected 3 days after the injection. a.

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Latency to enter in retention trial of inhibitory avoidance test. b. Latency to enter in

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ACCEPTED MANUSCRIPT acquisition trial of inhibitory avoidance test. c. Preference ratio in test session of

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object location recognition test. d. Total arm entry in test session of object location

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recognition test. e. Spontaneous alternations in Y-maze test. f. Total arm entry in Y-

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maze test. g. Long-term potentiation (LTP) induced by theta burst stimulation (TBS).

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h. Long-term depression (LTD) induced by low frequency stimulation (1 Hz, 900

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pulses). Data are expressed as mean ± SEM. *P < 0.05 vs vehicle group.

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Figure 4. Effect of SC79 on Aβ-induced memory impairments and aberrant synaptic

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plasticity. Aβ (10 µM/3 µl) and/or SC79 (100 µg/3 µl) was injected into third ventricle

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and hippocampal tissues were collected 3 days after the injection. a. Latency to

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enter in retention trial of inhibitory avoidance test. b. Latency to enter in acquisition

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trial of inhibitory avoidance test. c. Preference ratio in test session of object

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recognition test. d. Total arm entry in test session of object recognition test. e.

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Spontaneous alternations in Y-maze test. f. Total arm entry in Y-maze test. g. Long-

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term potentiation (LTP) induced by theta burst stimulation (TBS). h. Long-term

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depression (LTD) induced by low frequency stimulation (1 Hz, 900 pulses). Data are

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expressed as mean ± SEM. *P < 0.05 vs vehicle group.

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Figure 5. Effect of SC79 on memory impairments and aberrant synaptic plasticity of

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5XFAD mice. a. Experimental schedule. SC79 (10 µg/3 µl) was once a day for 11

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days. Inhibitory avoidance test was conducted 8 and 9 d after the start of SC79

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injection. Object recognition test (ORM) was conducted 10 and 11 d after the start of

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SC79 injection. b. Latency to enter in acquisition test of inhibitory avoidance test. c.

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Latency to enter in retention trial of inhibitory avoidance test. d. Total exploration

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time in test session of object recognition test. e. Discrimination index in test session

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stimulation (TBS). g. Level of LTP at last 5 min. h. Western blot analysis of Akt1 and

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GSK-3β activity. I and J. Quantitative analysis of immunoreactivity of western blot of

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GSK-3β (I) and Akt1 (J). Data are expressed as mean ± SEM. *P < 0.05.

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ACCEPTED MANUSCRIPT Highlight

Oligomeric Aβ impaired learning and memory and synaptic plasticity

Akt activation ameliorated Aβ-induced deficits

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Caspase inhibition ameliorated Aβ-induced deficits

GSK-3β inhibition ameliorated Aβ-induced deficits

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Akt activation ameliorated learning and memory and synaptic deficits in

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