Peripheral Administration of GSK-3β Antisense Oligonucleotide Improves Learning and Memory in SAMP8 and Tg2576 Mouse Models of Alzheimer’s Disease

Abstract

Glycogen synthase kinase (GSK)-3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer’s disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ), and neurodegeneration. We have previously shown that an antisense oligonucleotide directed at the Tyr 216 site on GSK-3β (_GAO) when injected centrally can decrease GSK-3β levels, improve learning and memory, and decrease oxidative stress. In addition, we showed that _GAO can cross the blood-brain barrier. Herein the impact of peripherally administered _GAO in both the non-transgenic SAMP8 and transgenic Tg2576 (APPswe) models of AD were examined respective to learning and memory. Brain tissues were then evaluated for expression changes in the phosphorylated-Tyr 216 residue, which leads to GSK-3β activation, and the phosphorylated-Ser9 residue, which reduces GSK-3β activity. SAMP8 _GAO-treated mice showed improved acquisition and retention using aversive T-maze, and improved declarative memory as measured by the novel object recognition (NOR) test. Expression of the phosphorylated-Tyr 216 was decreased and the phosphorylated-Ser9 was increased in _GAO-treated SAMP8 mice. Tg2576 _GAO-treated mice improved acquisition and retention in both the T-maze and NOR tests, with an increased phosphorylated-Ser9 GSK-3β expression. Results demonstrate that peripheral administration of _GAO improves learning and memory, corresponding with alterations in GSK-3β phosphorylation state. This study supports peripherally administered _GAO as a viable means to mediate GSK-3β activity within the brain and a possible treatment for AD.

Keywords

Antisense GSK-3β learning memory SAMP8 tau

INTRODUCTION

Glycogen synthase kinase-3 (GSK-3) is a proline-directed serine/threonine kinase that has been implicated in the pathophysiology of various central nervous system disorders [1]. GSK-3 has been implicated as a central factor in Alzheimer’s disease (AD) due to its role in the phosphorylation the microtubule protein tau [2]. Recently, there has been interest in GSK-3 as a target for therapy for AD [3].

GSK-3 comes in two forms: GSK-3α and GSK-3β. They are ubiquitously expressed and involved in a variety of cellular processes [2]. GSK-3β is thought to be involved in multiple diseases such as cancer, diabetes, and neurological pathologies such as Parkinson’s disease, bipolar disorder, and AD [4 –7]. In AD, GSK-3β is involved in the regulation of microtubule-associated protein tau [8]. In addition, GSK-3β is thought to be the molecular link between Aβ and tau, by regulating the phosphorylation of tau, and Aβ through the α- and γ-secretases [9].

GSK-3β is phosphorylated at tyrosine 216 (GSK-3βTyr216) to make an active form and at serine 9 (GSK-3β) to make an inactive form [10]. Enhancing GSK-3βSer9 reduces the negative effects of GSK-3β had in the brain, such as increased phosphorylation of tau, elevated amyloid-beta (Aβ), and increased oxidative stress [9]. Decreasing GSK-3βTyr216 reduces the phosphorylation of tau [11].

The SAMP8 mouse is a model of sporadic AD that develops deficits in learning and memory starting at approximately 8 months of age [12, 13]. SAMP8 mice exhibit an age-related increase in Aβ, tau phosphorylation, and oxidative stress [14 –17]. The cognitive deficits can be reversed by lowering Aβ with antisense directed at APP [14, 18]. More recently, treatments that reduce GSK-3β have been found by us and others [19, 20] to improve learning and memory, and decrease oxidative stress in SAMP8 mice.

The Tg2576(APPswe) mouse is a transgenic mouse model of AD which is genetically altered to overexpress human Aβ [21]. The Tg2576 has also been found to have elevated tau [22]. Tau immunotherapy in the Tg2576 improved learning and memory and decreased Aβ oligomers [23]. The antihypertensive drug propranolol given to Tg2576 mice also decreased Aβ and phosphorylated tau as well as increasing active GSK-3β [22].

We recently published results showing the _GAO antisense could improve learning and memory when administered intracerebroventricularly (ICV) to the SAMP8 mouse, and lowered GSK-3β and decreased oxidative stress [19]. We also showed that the antisense can cross the blood-brain barrier. Here, we examined the effects of GSK antisense on learning and memory after peripheral administration in two mouse strains of AD. In addition, we verified that the antisense alters levels of active and inactive GSK-3β after peripheral administration. These studies demonstrated that lowering GSK-3β in the brain improves AD-like changes that occur with aging in the SAMP8 mouse.

MATERIALS AND METHODS

Animal subjects

The SAMP8 male mice were 12-month-old males from our breeding colony at the time of behavioral testing. The Tg2576 (APPswe) mice were 13-month-old males and their age equivalent wild type controls (WT) from Taconic (Germantown, NY). Mice were 2 months old when received from the vendor and housed 2 to 4 per cage until testing at 13 months of age. Mice were individually housed for 3 weeks during behavioral testing to ensure each mouse was equally aroused and equally exposed to external stimuli (T-maze buzzer) during testing. All studies were conducted with the approval of the Animal Care and Use Committee at the VA Medical Center, St. Louis, MO. Sentinels from the facility were tested regularly to ensure our facility is virus- and pathogen-free. Food (Richland 5001) and water were available on an ad libitum basis and the rooms had a 12-h light-dark cycle with lights on at 0600 h. Behavioral experiments were conducted between 0730 and1100 h.

Antisense

GSK antisense oligonucleotide (_GAO) [sequence: 5’ (P = S) GGTTACCTTGCTGCCATCTT-3’], or random antisense (_RAO) (sequence: 5’ (P = S) GATCACGTACACATCGACAC-3’) (Midland Certified Reagent Company, Midland, TX) were synthesized. Mice received one weekly intravenous (IV) injection for a total of 5 injections of antisense (6 μg/100 μl/IV) during the course of study. Behavioral testing began 3 days after the third injection. Mice were sacrificed 3 days after their 5th injection at the end of behavioral testing.

Chemicals and materials

All chemicals were of the highest purity and purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. Nitrocellulose membranes, polyacrylamide gels, XT MES electrophoresis running buffer, and Precision Plus Proteintrademark, and all Blue Standards were purchased from Biorad (Hercules, CA).

Behavioral testing

T-Maze training and testing procedures

Acquisition was tested 5 days after the third injection in an aversive T-maze. The T-maze is both a learning task based on working-memory and a reference-memory task. The T-maze consisted of a black plastic alley with a start box at one end and two goal boxes at the other. The start box was separated from the alley by a plastic guillotine door that prevented movement down the alley until raised at the onset of training. An electrifiable floor of stainless steel rods ran throughout the maze to deliver a mild scrambled foot-shock.

Mice were not permitted to explore the maze prior to training. A block of training trials began when a mouse was placed into the start box. The guillotine door was raised and a cue buzzer sounded simultaneously; 5 s later foot-shock was applied. The arm of the maze entered on the first trial was designated “incorrect” and the mild foot-shock was continued until the mouse entered the other goal box, which in all subsequent trials was designated as “correct” for the particular mouse. At the end of each trial, the mouse was returned to its home cage until the next trial.

Mice were trained until they made one avoidance. Training used an inter-trial interval of 35 s, the buzzer was of the door-bell type sounded at 55 dB, and shock was set at 0.35 mA (Coulbourn Instruments scrambled grid floor shocker model E13-08). Retention was tested one week later by continuing training until mice reached the criterion of 5 avoidances in 6 consecutive trials. The results were reported as the number of trials to first avoidance for acquisition and the number of trials to criterion for the retention test.

Novel object recognition

Novel object recognition (NOR) was tested the three days following T-maze retention testing. NOR is a declarative memory task that involves the hippocampus when, as performed here, the retention interval is 24 h after initial exposure to the objects [24]. Mice were habituated to an empty apparatus for 5 min a day for 3 days prior to entry of the objects. During the training session, the mouse was exposed to two similar objects (plastic frogs), which it was allowed to examine for 5 min. The apparatus and the objects were cleaned between each mouse. Twenty-four h later, the mouse was exposed to one of the original objects and a new novel object in a new location and the percent of time spent examining the new object was recorded. The novel object was made out of the same material as the original object and of the same size, but a different shape. This eliminated the possibility of smell associated with a particular object being a factor. The underlying concept of the task is based on the tendency of mice to spend more time exploring new, novel objects than familiar objects. Thus, the greater the retention/memory at 24 h, the more time spent with the new object. The results were reported as the percent time exploring the novel object of the total exploration time.

Western blot sample preparation

In study one, the cortex, hippocampus, septum, and amygdala of respective mice were flash frozen and stored at –80°C until use. Respective matched brain regions were pooled in twos. In the second study, brains were cut in ½ and flash frozen. The assays were performed on ½ brain. Based on the weight, cold RIPA buffer containing a mini-complete protease inhibitor (1 tablet/10 ml Roche, NJ) and phos-stop phosphatase inhibitor cocktail tablets(1 tablet/10 ml Roche, NJ) was added in a 1:10 volume (0.1 g = 1 ml) and then homogenized. The sample was then centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was taken and a BCA protein assay (Pierce/Thermo Fisher, IL) was run using prepared albumin standards (Pierce/Thermo Fisher, IL), and sample aliquots frozen at –80°C.

Western blot

Antibodies used in western blot analysis: pSer9 GSK-3β (Cell signaling), pTyr216 GSK-3β (BD Biosciences), total GSK-3β (BD biosciences), and actin (Sigma). The secondary antibodies were anti-rabbit IgG HRP (Cell signaling), anti-mouse IgG HRP (BD Biosciences), and anti-mouse IgG HRP (BD Biosciences).

Samples were heated at 95°C for 10 min in 2x SDS/20x reducing agent and PBS at a 1x concentration. They were then separated using an electrophoretic field using 10% Bis/Tris Criterion XT gels (Bio-Rad) at 200 V for 55 min using MOPS running buffer for GSK-3β analysis. The proteins were then transferred to nitrocellulose membranes at 240 mA for 45 min (4°C). After all transfers, gels were additionally evaluated with GelCode Blue Stain (Pierce, Rockford, IL).

The membranes were blocked overnight at 4°C using 5% BSA-Tris-buffered saline (20 mM Tris base, 137 mM NaCl, pH 7.6 with 0.1% Tween) for pSer9 GSK-3β and pTyr216 GSK-3β, or 5% nonfat milk-Tris buffered saline for total GSK-3β. Following overnight blocking, membranes were incubated with either pSer9 GSKβ (1:1000), pTyr216 GSK-3β (1:1000), or total GSK-3β (1:1000) at 4°C for 24 h. Following standard wash protocol and respective secondary incubation, anti-rabbit IgG HRP (1:2000), anti-mouse IgG HRP (1:1000), and anti-mouse IgG HRP (1:1000), respectively (60 min), blots were developed using the enhanced chemiluminescence method for pSer9 GSK-3β and pSer9 GSK-3β (SuperSignal West Dura, Pierce) or using Amersham ECL Western blotting detection for actin or total GSK-3β. Optical densities of expressed bands were measured by calibrated densitometer (ChemiDoc XRS, Biorad). The percent expression as compared to controls was calculated for each sample. pSer9 GSK-3β and pTyr216 GSK-3β expression were additionally normalized using total GSK-3β expression.

Statistical analysis

Results from the T-maze were analyzed by aT-test. Statistical significance for comparing changes in GSK-3β expression in different brain regions between treated and control SAMP8 mice were analyzed using independent samples t-test. Statistical significance for comparing changes in GSK-3β expression in transgenic control, transgenic treated, and wild-type control mice were analyzed using one-way ANOVA. Bonferroni post-hoc tests were used if the one-way ANOVA was significant. Expression statistical analyses were conducted using R studio. Significant differences were set at p < 0.05.

RESULTS

_GAO on learning and memory in SAMP8 mice

Twelve-month-old SAMP8 mice administer _GAO or _RAO were tested in T-maze foot shock avoidance acquisition. One week later, the mice were tested for retention. The T-test for trial to first avoidance during acquisition in the T-maze showed a significant effect T(17) = 3.21, p < 0.005. The mice that received _GAO took significantly fewer trials to reach their first avoidance (10.10±0.566) than the mice that received random antisense (12.56±0.503) (Fig. 1A). The T-test for trials to criterion on the retention test showed a significant effect T(17) = 3.427, p < 0.003 (Fig. 1B). The mice that received _GAO (8.50±0.76) took significantly fewer trials to reach criterion than the mice that received _RAO (15.88±2.224).

The T-test for time spent exploring the two objects was not significantly different between the two groups: T(16) = 0.18, p NS (1C); _GAO (13.60±2.62) and _RAO (13.00±1.79). The T-test for time exploring the novel object during the 24-h retention test was not significant: T(16) = 2.758, p < 0.01. The mice that received _GAO (63.60±2.20) spent a significantly greater amount of time exploring the novel object than the mice that received random antisense (52.86±3.45) (Fig. 1D).

_GAO on levels of total GSK-3, pSer9GSK-3, and pTyrGSK216 in SAMP8 mice

Western blot analysis was used to determine the effect of _GAO treatment on expression of pTyr 216 GSK-3β and pSer9 GSK-3β in different brain regions of SAMP8 mice. In mice treated with _GAO, there were no differences in total GSK-3; however, expression of pTyr 216 GSK-3β (active) in the amygdala significantly decreased by 21.57% compared to mice treated with _RAO (Fig. 2, p < 0.05, n = 6/group). Expression of pSer9 GSK-3β (inactive) in the hippocampus significantly increased by 18.40% in mice treated with _GAO compared to mice treated with _RAO (Fig. 2, p < 0.05, n = 6/group).

_GAO on learning and memory in Tg2576 mice

Tg2576 treated with _GAO or _RAO or WT control mice treated with _RAO were tested in T-maze foot shock avoidance acquisition. One week later, the mice were tested for retention. The ANOVA for trials to criterion during acquisition in the T-maze produced a significant effect F(2,24) = 5.53, p < 0.01. Tukey’s post hoc test indicated that the Tg2576 mice that received _GAO and the WT mice that receive _RAO took significantly fewer trials to reach first avoidance than the Tg2576 mice that received _RAO. There was no difference between the Tg2576 mice that received _GAO versus the WT mice that received _RAO (Fig. 3A). The ANOVA for trials to criterion on the retention test showed a significant effect: F(2, 24) = 12.46, p < 0.001 (Fig. 3B). The Tg2576 mice that received _GAO and the WT mice that received _RAO took significantly fewer trials to reach criterion than the mice that received _RAO. The Tg2576 mice that received _GAO were not significantly different from the WT mice that received _RAO.

The ANOVA for time exploring the novel object during the 24-h retention test showed a significant effect: F(2,27) = 6.93, p < 0.004. Tukey’s post hoc test indicate that the Tg2576 mice that received _GAO and the WT mice that received _RAO spent significantly more time exploring the novel object than the mice that received _RAO. There was no difference between the Tg2576 mice that received _GAO and the WT mice that received _RAO (Fig. 3D). The ANOVA for total exploration time on Day 1 was not significant: F(2,27) = 0.99, p NS.

_GAO on levels of total GSK-3, pSer9GSK-3 GSK, and pTyrGSK216 GSK in Tg2576 mice

Western blot was used to determine the effect of antisense treatment on expression of pTyr216 GSK-3β (active) and pSer9 GSK-3β in the brains of Tg2576 and WT mice (n = 5/group, Fig. 4). Treatment was found to have no effect on total GSK-3; however, there was a significant effect on expression of pSer9 GSK-3β (p < 0.05, one-way ANOVA). In transgenic mice treated with _GAO, pSer9 GSK-3β expression significantly increased by 21.1% compared to transgenic mice treated with _RAO (Bonferroni post-hoc, p < 0.05). Treatment was also found to have a significant effect on the expression of pTyr216 GSK-3β (p < 0.05, one-way ANOVA). In transgenic mice treated with _GAO, pTyr216 GSK-3β expression was decreased by 10.9% compared to transgenic mice treated with _RAO. However, the significant difference in pTyr216 expression was only found between transgenic mice treated with _GAO compared to WT mice treated with _RAO (Bonferonni post-hoc test, p < 0.05).

DISCUSSION

The regulatory kinase GSK-3β has received a lot of attention in the last 10 years as a possible target in AD [25 –29]. Evidence of the involvement of GSK-3β in AD continues to grow [30, 31]. We had previously shown that an antisense that targets GSK-3β improved cognition and decreased oxidative stress after central administration [19]. In the same set of studies, we demonstrated that this antisense can cross the blood-brain barrier. Here, we examined GSK-3β antisense after peripheral administration in both a spontaneous onset and transgenic mouse models of AD. Peripherally administered _GAO was able to improved learning and memory in both aged SAMP8 and Tg2576 mouse models of AD. This treatment resulted in increased inactive GSK-3βSer9 in both the SAMP8 and the Tg2576 mice and decreased active GSK-3βTyr9 in the SAMP8. The current results suggest that decreasing active and/or increasing inactive phosphorylated forms of GSK-3β are the important factors for improving memory in mouse models of AD.

Improvement of memory after _GAO administration and an associated increase in GSK-3βSer9 is similar to studies using lithium [32]. Lithium has been shown to increase GSK-3βSer9 and improve memory in transgenic mice [33]. In clinical studies, small doses of lithium had no effect on patients with moderate AD [7]. Lithium was able to stabilize patients with mild cognitive impairment, possibly delaying the progression of the disease [34].

GSK-3β antisense also reduced the active form GSK-3βTyr216 in SAMP8 mice. Increase GSK-3βTyr216 has been found in pretangle changes in AD [35] and upregulated in the prefrontal cortex and hippocampus of AD patients [31, 36]. Decreasing GSK-3βTyr216 has been found to improve memory in mice with streptozotocin-induce tau hyperphosphorylation [37]. Here, we found that in the SAMP8 decreasing active and/or increasing inactive around 20% in the hippocampus and amygdala had a positive effect on memory. In the transgenics, the change was around 10%; however, this may have been due to the fact that we only measured ½ brains as a verification we were changing brain GSK-3β phosphorylated forms.

GSK-3β has an active role in memory through the involvement in the phosphatidylinositol 3-kinase (PI3K) signaling [38]. Inhibition of PI3K leads to overactivation of GSK-3 resulting impaired memory [39]. In the SAMP8 with age-related memory impairment, we found that gene expression in the PI3K pathway was altered with age [40]. Impairment in PI3K pathway has been associated with impaired long-term potentiation and important factor in memory [38] as does overexpression of active GSK-3β [2]. Taken together these findings suggest that _GAO improvement in memory may be working through the PI3K pathway.

The SAMP8 and Tg2576 mice used in this study were treated with antisense oligonucleotide directed at GSK-3β and random antisense oligonucleotide, the latter serving as the control. The GSK-3β antisense has a sequence that corresponds to 94–113 nucleotides downstream from the initiation codon of GSK-mRNA. This is an internal sequence with high probability of being located away from any loop formation in the mRNA. As an internal site, it should not block 100% of GSK mRNA. This is important as GSK-3β is essential for intracellular signaling pathways such as cell proliferation, cellular migration, glucose regulation, inflammatory responses, and apoptosis [41]. Therefore, given the critical cellular functions of GSK-3β, antisense treatment may be an effective way to control the over-activity of the kinase without completely blocking its functions. Antisenses are currently in various stages of testing for such conditions as cancer, hypercholesterolemia, Ebola virus infection, type 2 diabetes, HIV infection, and ocular disease, and may be a feasible treatment for AD as well [42 –45].

In conclusion, this paper provides evidence that the reduction of GSK-3β with antisense improves cognition by altering levels of active and inactive GSK-3β in two mouse models of AD. The ability of peripherally administered _GAO to reduce active GSK-3β suggests that _GAO should be investigated further as a potential treatment for AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by a VA Merit Review S.A.F.

Authors’ disclosures available online ().

References

Kaidanovich-Beilin

, Woodgett

(2011) GSK-3: Functional insights from cell biology and animal models. Front Mol Neurosci 4, 40.

Hooper

, Killick

, Lovestone

(2008) The GSK3 hypothesis of Alzheimer’s disease. J Neurochem 104, 1433–1439.

Martinez

, Gil

, Perez

(2011) Glycogen synthase kinase 3 inhibitors in the next horizon for Alzheimer’s disease treatment. Int J Alzheimers Dis 2011, 1–7.

, Li

, Chen

, Liu

, Lv

, Dong

, Luo

, Gu

, Liu

(2016) Baicalin attenuates high fat diet-induced insulin resistance and ectopic fat storage in skeletal muscle, through modulating the protein kinase B/Glycogen synthase kinase 3 beta pathway. Chin J Nat Med 14, 48–55.

, Thrasher

, Terranova

(2015) Glycogen synthase kinase-3: A potential preventive target for prostate cancer management. Urol Oncol 33, 456–463.

Lazzara

, Kim

(2015) Potential application of lithium in Parkinson’s and other neurodegenerative diseases. Front Neurosci 9, 403.

Forlenza

, De-Paula

, Diniz

(2014) Neuroprotective effects of lithium: Implications for the treatment of Alzheimer’s disease and related neurodegenerative disorders. ACS Chem Neurosci 5, 443–450.

Medina

, Avila

(2013) Understanding the relationship between GSK-3 and Alzheimer’s disease: A focus on how GSK-3 can modulate synaptic plasticity processes. Expert Rev Neurother 13, 495–503.

Llorens-Martin

, Jurado

, Hernandez

, Avila

(2014) GSK-3beta, a pivotal kinase in Alzheimer disease. Front Mol Neurosci 7, 46.

10.

Qian

, Shi

, Yin

, Iqbal

, Grundke-Iqbal

, Gong

, Liu

(2010) PP2A regulates tau phosphorylation directly and also indirectly via activating GSK-3beta. J Alzheimers Dis 19, 1221–1229.

11.

Hanger

, Noble

(2011) Functional implications of glycogen synthase kinase-3-mediated tau phosphorylation. Int J Alzheimers Dis 2011, 352805.

12.

Yagi

, Katoh

, Akiguchi

, Takeda

(1988) Age-related deterioration of ability of acquisition in memory and learning in senescence accelerated mouse: SAM-P/8 as an animal model of disturbances in recent memory. Neurosci Biobehav Rev 474, 86–93.

13.

Flood

, Morley

(1998) Learning and memory in the SAMP8 mouse. Neurosci Biobehav Rev 22, 1–20.

14.

Morley

, Kumar

, Bernardo

, Farr

, Uezu

, Tumosa

, Flood

(2000) Beta-amyloid precursor polypeptide in SAMP8 mice affects learning and memory. Peptides 21, 1761–1767.

15.

Pallas

, Camins

, Smith

, Perry

, Lee

, Casadesus

(2008) From aging to Alzheimer’s disease: Unveiling “the switch” with the senescence-accelerated mouse model (SAMP8). J Alzheimers Dis 15, 615–624.

16.

Butterfield

, Stadtman

(1997) Protein oxidation processes in aging brain. Adv Cell Aging Gerontol 2, 161–191.

17.

Poon

, Castegna

, Farr

, Thongbookerd

, Lynn

, Banks

, Morley

, Klein

, Butterfield

(2004) Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mouse brain. Neuroscience 126, 915–926.

18.

Erickson

, Niehoff

, Farr

, Morley

, Dillman

, Lynch

, Banks

(2012) Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses ABPP and LRP-1 overexpression in the aged SAMP8 mouse brain. J Alzheimers Dis 28, 951–960.

19.

Farr

, Ripley

, Sultana

, Zhang

, Niehoff

, Platt

, Murphy

, Morley

, Kumar

, Butterfield

(2014) Antisense oligonucleotide against GSK-3beta in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease. Free Radic Biol Med 67, 387–395.

20.

Tajes

, Yeste-Velasco

, Zhu

, Chou

, Smith

, Pallas

, Camins

, Casadesus

(2009) Activation of Akt by lithium: Pro-survival pathways in aging. Mech Ageing Dev 130, 253–261.

21.

Hsaio

, Chapman

, Nilsen

, Erkman

, Harigaya

, Yang

, Cole

(1996) Correlative memory deficits, AB elevation, and amyloid plaques in transgenic mice. Science 274, 99–102.

22.

Dobarro

, Gerenu

, Ramierz

(2013) Propranolol reduces cognitive deficits, amyloid and tau pathology in Alzheimer’s transgenic mice. Int J Neuropsychopharmacol 16, 2245–2257.

23.

Castillo-Carranza

, Guerrero-Munoz

, Sengupta

, Hernandez

, Barrett

, Dineley

, Kayed

(2015) Tau immunotherapy modulates both pathological tau and upstream amyloid pathology in an Alzheimer’s disease mouse model. J Neurosci 35, 4857–4868.

24.

Hammond

, Tull

, Stackman

(2004) On the delay-dependent involvement of the hippocampus in object recognition memory. Neurobiol Learn Mem 82, 26–34.

25.

Lovestone

, Reynolds

, Latimer

, Davis

, Anderton

, Gallo

, Hanger

, Mulot

, Marquardt

, Stabel

, Woodgett

, Miller

CCJ

(1994) Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr Biol 4, 1077–1086.

26.

Yamaguchi

, Ishiguro

, Uchida

, Takashima

, Lemere

, Imahori

(1996) Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3 beta and cyclin-dependent kinase 5, a component of TPK II. Acta Neuropathol 92, 232–241.

27.

Hong

, Chen

, Klein

, Lee

(1997) Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3. J Biol Chem 272, 25326–25332.

28.

Engel

, Goni-Oliver

, Lucas

, Avila

, Hernandez

(2006) Chronic lithium administration of FTDP-17 tau and GSK-3beta overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert. J Neurochem 99, 1445–1455.

29.

Lucas

, Hernandez

, Gomez-Ramos

, Moran

, Hen

, Avila

(2001) Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J 20, 27–39.

30.

Pei

, Tanaka

, Tung

, Braak

, Iqbal

, Grundke-Iqbal

(1997) Distribution, levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain. J Neuropathol Exp Neurol 56, 70–78.

31.

Leroy

, Yilmaz

, Brion

(2007) Increased level of active GSK-3beta in Alzheimer’s disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration. Neuropathol Applied Neurobiol 33, 43–55.

32.

, Yang

, Song

, Jiang

, Lin

, Wang

, Zhu

, Wang

, Tian

(2010) LiCl attenuates thapsigargin-induced tau hyperphosphorylation by inhibiting GSK-3beta in vivo and in vitro. J Alzheimers Dis 21, 1107–1117.

33.

Fiorentini

, Rosi

, Grossi

, Luccarini

, Casamenti

(2010) Lithium improves hippocampal neurogenesis, neuropathology and cognitive functions in APP mutant mice. PLoS One 5, e14382.

34.

Forlenza

, Diniz

, Radanovic

, Santos

, Talib

, Gattaz

(2011) Disease-modifying properties of long-term lithium treatment for amnestic mild cognitive impairment: Randomised controlled trial. Br J Psychiatry 198, 351–356.

35.

Pei

, Braak

, Grundke-Iqbal

, Iqbal

, Winblad

, Cowburn

(1999) Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes. J Neuropathol Exp Neurol 58, 1010–1019.

36.

Blalock

, Geddes

, Chen

, Porter

, Markesbery

, Landfield

(2004) Incipient Alzheimer’s disease: Microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc Natl Acad Sci U S A 101, 2173–2178.

37.

Kanno

, Tsuchiya

, Nishizaki

(2014) Hyperphosphorylation of Tau at Ser396 occurs in the much earlier stage than appearance of learning and memory disorders in 5XFAD mice. Behav Brain Res 274, 302–306.

38.

Sanna

, Cammalleri

, Berton

, Simpson

, Lutjens

, Bloom

, Francesconi

(2002) Phosphatidylinositol 3-kinase is required for the expression but not for the induction or the maintenance of long-term potentiation in the hippocampal CA1 region. J Neurosci 22, 3359–3365.

39.

Liu

, Zhang

, Li

, Wang

, Deng

, Netzer

, Xu

, Wang

(2003) Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory. J Neurochem 87, 1333–1344.

40.

Armbrecht

, Siddiqui

, Green

, Farr

, Kumar

, Banks

, Patrick

, Shah

, Morley

(2014) SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways. Neurobiol Aging 35, 159–168.

41.

Jope

, Yukaitis

, Beurel

(2007) Glycogen synthase kinase-3 (GSK3): Inflammation, diseases, and therapeutics. Neurochem Res 32, 577–595.

42.

Tamm

(2008) AEG-35156, an antisense oligonucleotide against X-linked inhibitor of apoptosis for potential tretment of cancer. Curr Opin Investig Drugs 9, 638–646.

43.

Evers

, Toonen

, van Roon-Mom

(2015) Antisense oligonucleotides in therapy for neurodegenerative disorders. Adv Drug Deliv Rev 87, 90–103.

44.

Lippi

, Harenberg

, Mattiuzzi

, Favaloro

(2015) Next generation antithrombotic therapy: Focus on antisense therapy against coagulation factor XI. Semin Thromb Hemost 41, 255–262.

45.

Davalos

, Chroni

(2015) Antisense oligonucleotides, microRNAs, and antibodies. Handb Exp Pharmacol 224, 649–689.