- Email: [email protected]
PS1dE9 Alzheimer's disease model mice
Accepted Manuscript PPARγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ deposition stage in APPswe/PS1dE9 Alzheimer's disease model mice Junya Toba, Miyu Nikkuni, Masato Ishizeki, Aya Yoshii, Naoto Watamura, Takafumi Inoue, Toshio Ohshima PII:
S0006-291X(16)30507-1
DOI:
10.1016/j.bbrc.2016.04.012
Reference:
YBBRC 35607
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 23 March 2016 Accepted Date: 4 April 2016
Please cite this article as: J. Toba, M. Nikkuni, M. Ishizeki, A. Yoshii, N. Watamura, T. Inoue, T. Ohshima, PPARγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ deposition stage in APPswe/PS1dE9 Alzheimer's disease model mice, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.04.012. 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.
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PPARγγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ β deposition stage in APPswe/PS1dE9 Alzheimer’s disease model mice Junya Toba1, Miyu Nikkuni1, Masato Ishizeki2, Aya Yoshii1, Naoto Watamura1, Takafumi
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Inoue2, Toshio Ohshima1,3
Laboratory for Molecular Brain Science and 2Laboratory for Neurophysiology, Department of
Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering,
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Waseda University, Tokyo, 162-8480 Japan
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Running title: Pioglitazone for cerebellar phenotype of AD mice
Corresponding author: Toshio Ohshima
Department of Life Science and Medical Bioscience, Waseda University,
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2-2 Wakamatsu-cho Shinjuku-ku, Tokyo 162-8480, Japan
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Tel: -81-3-5369-7321, Fax: -81-3-5369-7302, e-mail: [email protected]
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Abstract
Alzheimer’s disease (AD) is one of the best known neurodegenerative diseases; it causes
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dementia and its pathological features include accumulation of amyloid β (Aβ) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that Aβ
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accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD)
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induction, at the pre-Aβ accumulation stage.
In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor γ, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-Aβ accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and
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AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-Aβ accumulation stage in AD model mice.
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Highlights
・Phosphorylation level of CRMP2 and p35 protein levels increased in the cerebellum of APP/PS1 mice.
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・Pioglitazone treatment improved cerebellar dysfunction of APP/PS1 mice at the pre-Aβ accumulation stage.
Key words: Alzheimer’s disease; protein phosphorylation; CRMP; mouse, cerebellum
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Abbreviations APP, amyloid precursor protein BSA, bovine serum albumin
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Cdk5, cyclin dependent kinase 5 CRMP2, collapsin response mediator protein 2 GSK3β, glycogen synthase kinase 3β PS1, presenilin 1
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Tg, transgenic
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Introduction
Alzheimer’s disease (AD) is a well-known progressive neurodegenerative disorder and is the most common type of dementia among elderly people. Cognitive dysfunction and memory
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impairment are the main clinical symptoms present in AD patients. Accumulations of aberrant aggregates of amyloid β (Aβ) called senile plaques, and neurofibrillary tangles (NFTs), composed of phosphorylated-tau protein, are regarded as the major pathological features in AD [1]. Aβ becomes neurotoxic when it aggregates, and these aggregates lead to a synaptic
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dysfunction and neuronal cell death [2]. It is clear that accumulation of Aβ contributes to the onset of AD; thus, the amyloid hypothesis, which holds that much of AD pathology is driven by
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deposition of Aβ in the brain [3], is widely supported. However, there is no significant correlation between the severity of dementia and the deposition of senile plaques in some patients [4]; hence, the oligomer hypothesis, which regards the soluble oligomer of the Aβ peptide as the primary toxin in AD, has been suggested [5]. Some studies have shown that oligomeric Aβ is present in the brain, and impairs the synaptic plasticity mechanisms that are
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required for memory processing, and also induces synaptic dysfunction [2, 6].
CRMP2 is a member of CRMP protein familiy, which regulates axonal guidance, proper dendritic organization, and microtubule stability, by associating with tubulin [7, 8]. The interaction of CRMP2 with tubulin is disrupted due to its phosphorylation by Cdk5 and GSK3β
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[7]. In AD, it is phosphorylated by Cdk5 and GSK3β, and this phosphorylated form is a component of NFTs [9]. Furthermore, phosphorylation of CRMP2 is increased in the early
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stages of AD, i.e., before the accmulation of Aβ and phosphorylated tau [10], but the detail of its role in AD pathology remains unknown.
Peroxisome proliferator-activated receptor γ (PPARγ), which is a nuclear receptor that is highly expressed in adipose tissue, is involved in the metabolism of fatty acids and survival of adipocytes [11]. In the brain, PPARγ is predominantly present in microglia and astrocytes, and its expression has been studied in relation to inflammation and neurodegeneration [12]. One of the thiazolidinedione (TZD) drugs, pioglitazone, is a ligand of PPARγ, and is used clinically for treatment of type 2 diabetes, in order to increase insulin sensitivity [13]. However, it is has been
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shown that treatment with pioglitazone ameliorates impaired cognitive function and reduces Aβ deposition in an AD mouse model [14, 15].
We have previously reported the presence of cerebellar dysfunction, such as impairment of
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motor coordination ability and long-term depression (LTD) induction, at the pre-Aβ accumulation stage in APPswe/PS1dE9 (APP/PS1) mice [16]. In the present study, we report elevated phosphorylation of CRMP2 and the p35 protein levels in the cerebellum of APP/PS1 mice in which high soluble Aβ levels and Aβ plaque formation were found. We then treated mice with pioglitazone and found normalized p35 protein levels and CRMP2 phosphorylation in
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the cerebellum, as well as recovery of motor coordination ability and LTD induction at the
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pre-Aβ accumulation stage.
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Materials and Methods
Mice and pioglitazone treatment Mice used in the experiments were housed in accordance with protocols approved by the
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Institutional Animal Care and Use Committee of Waseda University. APPswe/PS1dE9 (APP/PS1) transgenic mice were generated as described [17]. Pioglitazone (TCI, Tokyo, Japan)
previously described [15].
Biochemical analysis
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Enzyme-Linked ImmunoSorbent Assay (ELISA) for Aβ
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was administered orally to male 5- to 6-months-old (80 mg/body weight kg-1d-1) for 9 days, as
Brain tissues were homogenized in 10-fold volume to-weight ratio of Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA) containing a protease inhibitor cocktail (Roche Applied, Berkeley, CA, USA), and then centrifuged at 100,000 × g for 60 min. The supernatants were analyzed using the human β-amyloid (1−42) ELISA kit (Wako, Osaka, Japan)
Western blotting
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according to the manufacturer’s instruction.
Brain tissues or slices were homogenized in 10-fold volume-to-weight ratio of radio immunoprecipitation buffer (RIPA buffer: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40,
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1% sodium deoxycholate 0.1% sodium dodecyl sulfate), containing a protease and phosphatase inhibitor cocktail. Homogenized samples were centrifuged at 15,000 rpm for 15 min and the
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supernatants were subjected to western blot analysis as described previously [16]. The primary antibodies used in this study were as follows: anti-phosphorylated CRMP2 (Ser522, CP2191, ECM Biosciences, Versailles, KY, USA), anti-CRMP2 (9F, [8]) , anti-β-actin (A2103, Sigma, St Louis, MO, USA), anti-p35/25 (C19, sc-820, Santa Cruz Biotechnology, Dallas, TX, USA). Incubation of Aβ peptides and inhibitors Aβ25–35 peptide monomer (Peptide Research Inc., Osaka, Japan) dissolved in sterilized water to a concentration of 1mg/ml and stored in aliquots, was incubated at 37ºC for 4 days, to allow oligomerization [18, 19]. Aβ35–25 (Peptide Research Inc.), prepared in the same way, was used as
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a control. Aβ peptide and/or MK801 (Sigma) were added to the solution at the indicated final concentration.
Cerebellar slice preparation and electrophysiology
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250-µm-thick cerebellar slices were prepared and maintained according to a standard procedure [20]. In recordings artificial cerebrospinal fluid (ACSF) containing 100 µM picrotoxin was superfused at 31ºC. Membrane potential of Purkinje cells was held at -70 mV by the patch-clamp method with borosilicate glass pipettes (5−7 MΩ) filled with (in mM) 65
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K-gluconate, 65 Cs-methanesulphonate, 10 KCl, 1 MgCl2, 4 Na2-ATP, 1 Na-GTP, 5 sucrose, 0.4 EGTA, 20 K-HEPES; pH 7.3; 295 mOsm. A stimulation glass electrode was placed on the
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molecular layer, through which monopolar square pulses (200 µs) were applied. LTD experiments were performed with 6-month-old APPswe/PS1dE9 mice and age-matched non-Tg mice as described [16]. LTD was induced by pairing depolarization of Purkinje cells (200 ms, -70 to 0 mV) with a single PF stimulation 240 times at 1 Hz.
Statistical analysis
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All data are presented the mean ±SEM. The statistical analyses were performed using Student’s t-test or one-way and two-way ANOVA repeated-measures as described. P < 0.05 was
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considered statistically significant.
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Results
Upregulation of phosphorylated CRMP2 in APP/PS1 mouse brain and reversal effect of pioglitazone treatment
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Upregulation of the phosphorylation level of CRMP2 in the AD brain has been reported in the cerebral cortex and hippocampus [10]; however, it had not yet been investigated in the cerebellum. To examine whether phosphorylation of CRMP2 is altered in the cerebellum of APP/PS1 mice, we compared the phosphorylation levels of CRMP2 in the cerebellum and
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cerebral cortex of 6-months-old APP/PS1 mice with those in non-Tg controls. We performed western blot analysis with brain lysates from the cerebral cortex and cerebella from these mice.
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There was trend for an increase in levels of CRMP2 phosphorylated at Ser522 in APP/PS1 cerebella, as compared with the control, although the difference was not statistically significant (Fig. 1A).
We next investigated the effect of pioglitazone on phosphorylation of CRMP2 to assess the correlation between soluble amyloid oligomer and CRMP2 phosphorylation levels. We
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administered 80 mg/kg pioglitazone for 9 days to 6-months-old APP/PS1 mice and determined the concentration of the Aβ oligomer in the brain lysate of APP/PS1 mice by ELISA. There was a trend for a decrease in Aβ concentration in both the cerebral cortex and cerebellum (Supplementary Fig. 1). Western blotting analysis showed that phosphorylation of CRMP2 at
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Ser522 was significantly decreased in both the cerebral cortex and cerebellum of APP/PS1 mice treated with pioglitazone, as compared with non-treated mice of the same genotype (Fig. 1B, *p
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< 0.05, student’s t-test).
Increased p35 and p25 protein levels in the brain of APP/PS1 and reversal effect of pioglitazone treatment
Hyperactivation of Cdk5 occurs in the AD brain; this is thought to be involved in the progression of AD pathology via phosphorylation of several substrates, such as tau, leading to neurodegenerative pathology [22]. A previous study has reported that hyperactivity of Cdk5 in the AD brain was caused by an increase in p25, which was due to calpain-mediated cleavage of p35 [23]. We found that p35 levels tended to increase in APP/PS1 mice and that p25 levels
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increased significantly in the cerebral cortex of APP/PS1 mice (Fig. 2A, p < 0.05, Student’s t-test). The protein levels of p25 was markedly lower than those of p35; therefore, we used a longer exposure to analyze p25 ("p25 [long]") in these samples. Since we found that phosphorylation of CRMP2 at Ser522 was reduced by pioglitazone treatment, we investigated
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the protein levels of p35 and p25 in the brains of 6-months-old APP/PS1 mice treated with pioglitazone. This treatment significantly decreased both p35 and p25 protein levels in the cerebral cortex and tended to decrease those in the cerebellum (Fig. 2B, p < 0.01, Student’s t-test). These results suggest that activation of Cdk5 in 6-months-old APP/PS1 mice depended
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on the increase in the levels of p35 and p25; these noted elevations were reversed by
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pioglitazone treatment.
Defective cerebellar function in 6-months-old APP/PS1 mice and the effect of pioglitazone treatment
The cerebellum is a brain region that is important for controlling balance and motor coordination. To compare the cerebellar function of APP/PS1 mice with that of WT mice, we conducted a behavior test, the rotarod test, to assess the motor coordination ability of the mice.
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The 6-months-old control WT mice showed a gradual improvement in performance, i.e., increased retention time, through the consecutive 4-days trial period, but the APP/PS1 mice showed a short retention time, without improvement during the 4-day period (Fig. 3A, *p < 0.05, two-way repeated-measures ANOVA), which was consistent with the findings of our previous
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study (Kuwabara et al., 2014). Next, we assessed the effect of pioglitazone treatment on defective cerebellar motor coordination ability of APP/PS1 mice. APP/PS1 mice treated with
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pioglitazone showed gradual improvement in the score of the rotarod test (Fig. 3A, *p < 0.05, two-way repeated-measures ANOVA), which was similar to that in the control mice. This result indicated that the cerebellar dysfunction expressed as defective motor coordination ability in 6-months-old APP/PS1 mice was ameliorated by treatment with pioglitazone.
Additionally, impairment of synaptic plasticity in the hippocampus has been shown in an AD mouse model, and by application of Aβ [18, 24], which was thought to cause memory and cognitive dysfunction. Our previous study showed that cerebellar LTD was impaired in APP/PS1 mice [16]. To assess changes in cerebellar synaptic plasticity at the pre-Aβ
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accumulation stage, we compared cerebellar LTD in 6-months-old APP/PS1 mice and age-matched WT control mice. LTD was induced in control mice, and was expressed as a constant decrease in the amplitude of parallel fiber−Purkinje cell synapses. In contrast, impairment of LTD induction was seen in APP/PS1 mice (Fig. 3B, p < 0.001, one-way
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repeated-measures ANOVA), showing impairment in the synaptic function in the cerebellum of APP/PS1 mice. Cerebellar slices from APP/PS1 mice treated with pioglitazone showed normal LTD induction, expressed as a long-lasting decrease in the EPSC amplitude (Fig. 3B, p < 0.001, vs. non-treated APP/PS1 mice, one-way repeated-measures ANOVA). These results indicated
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that treatment with pioglitazone ameliorated cerebellar dysfunction in the cerebella of
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6-months-old APP/PS1 mice.
Aβ treatment of cerebellar slices tends to increase CRMP2 phosphorylation and p35 protein levels
Our previous study showed that phosphorylation of CRMP2 at Ser522 was increased by Aβ25-35 injection into the hippocampus [25]. To test whether Aβ25-35 also increases CRMP2 phosphorylation in the cerebellum, we incubated cerebellar slices with pre-oligomerized Aβ25-35
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or Aβ35-25 peptides for 2 h, and determined the levels of CRMP2 phosphorylated at Ser522 and of p35/p25 by western blot analysis. Aβ25-35 tended to increase CRMP2 Ser522 phosphorylation in cerebellar slices, as compared with incubation with Aβ35-25 (Fig. 4A). The levels of p35 were significantly increased in the cerebellar slices treated with Aβ25-35 as compared with Aβ35-25
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treatment of slices, while the levels of p25 were not altered (Fig. 4B, p < 0.05, Student’s t-test).
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Previous studies have suggested that Aβ increases the activity of NMDA receptors, which
increases Ca2+ influx into cells. Increased intracellular Ca2+ levels would activate calpain activity, which would cleave p35 to yield p25, resulting in increased phosphorylation of CRMP2 in cerebellar slices incubated with Aβ25-35. To test this hypothesis, we added 20 µM MK801, an NMDA receptor inhibitor, to the cerebellar slices incubated with Aβ25-35 peptide. After a 2-h co-incubation, slices were subjected to western blotting analysis. A decrease of CRMP2 phosphorylation at Ser522 was found in cerebellar slices treated with Aβ25-35 and MK801, as compared with slices treated with Aβ25-35 alone (Fig. 4C, p < 0.05, one-way repeated-measures ANOVA). However, MK801 had no effect on the levels of p35 and p25 (data not shown).
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Discussion
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In the present study, we demonstrated that phosphorylation of CRMP2 at Ser522 was increased in the cerebellum, as well as in the cerebral cortex of 6-months-old APP/PS1 mice (Fig. 1A). Furthermore, pioglitazone treatment of APP/PS1 mice resulted in decreased phosphorylation (Fig. 1B). Behavioral and electrophysiological impairment of the cerebellum was ameliorated
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by treatment with pioglitazone (Fig. 3), which accompanied the decrease in phosphorylated CRMP2. These results suggest that alteration of CRMP2 is involved in functional improvement
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of APP/PS1 mice. It is possible that increased phosphorylation of CRMP2 contributes to the impairment of cerebellar synaptic plasticity, because a previous study reported that impairment of hippocampal LTP by addition of Aβ was reversed by mutation of CRMP2S522A, in which phosphorylation of CRMP2S5522 was inhibited [25]. Furthermore, we demonstrated that elevation of phosphorylated CRMP2 was induced by addition of synthesized Aβ to cerebellar slices (Fig. 4A). Taken together with the evidence that application of Aβ to cerebellar slices
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impaired synaptic plasticity in the cerebellum [16], we concluded that there is close correlation between the phosphorylation of CRMP2 and functional impairment in AD.
Several studies have reported that aberrant activation of Cdk5 in AD is involved in the
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progression of pathology via production of Aβ aggregates and development of NFTs [22, 23]. It is generally considered that hyperactivation of Cdk5 in AD is dependent on production of p25,
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which is mediated by calpain, a calcium-dependent protease, induced by the toxicity of Aβ [23]. In this study, we detected an increase in p35 in the brain lysate; however, the p25 protein levels remained very low (Fig. 2A). Previous studies showed that the activity of calpastatin, an endogenous calpain inhibitor, was high in the cerebellum as compared with other brain regions, and little p25 was produced [26, 27]. In the present study, variation in the protein levels of p35 and p25 in the cerebellum of APP/PS1 mice was less than that in the cerebral cortex; thus, it is possible that other pathways deregulate Cdk5 activity, or other factors are involved in the cerebellar dysfunction. It has been reported that p35 is degraded by the ubiquitin-proteasome degradation pathway [28]. In addition, Aβ-induced inactivation of the proteasome in 3xTg-AD
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mice and enhanced accumulation of amyloid and tau by inhibition of the degradation of Aβ [29] suggest that hyperactivation of Cdk5 in AD is possibly induced by the increase in p35, by inhibiting its degradation via the proteasome. The present study showed that a decrease in p35 was induced by pioglitazone treatment in APP/PS1 mice. According to previous studies,
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degradation of p35 was promoted by treatment with TDZ drugs, including pioglitazone in a proteasome-dependent, but not PPARγ-dependent manner [30], which is consistent with the present study. Another study showed a reduction of p35 by treatment with pioglitazone in vitro; however, the elevation of p35 in AD confirmed in the present study was not investigated
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[31].The findings of the present study and or previous studies [31] indicate a correlation between the Cdk5 activity mediated by p35 regulation and pioglitazone treatment, but further
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investigation is required.
Our results showed the effects of pioglitazone treatment on cerebellar dysfunction in an AD mouse model. Further studies are required to determine the effect of pioglitazone on other brain function at the pre-Aβ-accumulation stage. In addition, the findings of the present study support
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the importance of Cdk5 and phosphorylated CRMP2 as therapeutic targets of AD.
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Conflict of Interest
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The authors declare no conflict of interest.
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Figure Legends
Fig. 1 Upregulation of phosphorylated CRMP2 in APP/PS1 mouse brain and its reversal by pioglitazone treatment
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A, Western blot analysis of brain lysates from the cerebral cortex (Cc) and cerebellum (Cb) in 6-months-old APP/PS1 mice, as compared with age-matched wild-type (WT) mice (n = 3 for each group), to examine levels of CRMP2 phosphorylated at Ser522. B, Western blot analysis
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shows a significant decrease in phosphorylated CRMP2S522 in 6-months-old APP/PS1 mice treated with pioglitazone (Pio) as compared with non-treated mice in both the cerebral cortex and cerebellum (n = 3 for each group, *p < 0.05, Student’s t-test).
Fig. 2 Increased p35 protein level in the brain of APP/PS1 and its reversal by pioglitazone
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treatment
A, Western blot analysis with brain lysates from the cerebral cortex (Cc) and cerebellum (Cb), to examine the protein level of p35 and p25 (n = 3 for each group, *p < 0.05, Student’s t-test). p25 had a much lower signal than p35; thus, a longer exposure is shown (long). B, Western blot
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analysis shows a decrease in p35 and p25 protein levels in both the cerebral cortex and cerebellum in 6-months-old APP/PS1 mice treated with pioglitazone (Pio), as compared with
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non-treated mice (n = 6 for each group, **p < 0.01, Student’s t-test).
Fig. 3 Defective cerebellar function in 6-months-old APP/PS1 mice and the effect of pioglitazone treatment
A, The motor coordination ability of 6-months-old APP/PS1 mice was assessed using the rotarod test. APP/PS1 mice showed defective motor coordination ability as compared with wile-type (WT) mice (n = 6 for each group, *p < 0.05, two-way repeated-measures ANOVA), and APP/PS1 mice treated with pioglitazone (Pio) showed an improved score, approaching that of WT mice. There was a significant difference between the scores of pioglitazone-treated APP/PS1 mice and non-treated mice (n = 6 for each group, *p < 0.05, two-way 17
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repeated-measures ANOVA). B, Long-term depression (LTD) induction at the PF-Purkinje cell synapse in cerebellar slices of 6-months-old APP/PS1 mice. Slices from WT mice (n = 6) showed LTD, which was significantly impaired in slices from APP/PS1 mice (n = 5, ***p < 0.001, one-way repeated-measures ANOVA). Slices from APP/PS1 mice treated with
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pioglitazone (Pio; n = 11) showed almost the same degree of reduction in EPSC amplitudes as WT mice, which was significant as compared with non-treated APP/PS1 mice (***p < 0.001, one-way repeated-measures ANOVA). Upper traces show the EPSCs averaged from 5
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consecutive recordings, before and 20 min after LTD induction. Scale bars: 200 pA and 20 ms.
Fig. 4 Aβ treatment tends to increase CRMP2 phosphorylation
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A, Western blot analysis of cerebellar slices treated with Aβ25-35 or Aβ35-25 to examine phosphorylation of CRMP2 at Ser522 (n = 4, Student’s t-test).
B, Western blot analysis showing a significant increase in p35 levels in the cerebellar slices treated with Aβ25-35 as compared with those treated with Aβ35-25 (n = 4, *p < 0.05, Student’s t-test).
C, Western blot analysis showing a significant decrease in CRMP2 phosphorylated at Ser522 in
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the cerebellar slices treated with Aβ25-35 and with MK801, as compared with those treated with
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Aβ25-35 alone (n = 3, *p < 0.05, one-way repeated-measures ANOVA).
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Legends for supplementary Figures
Supplementary Fig. 1 Biochemical analysis of level of soluble Aβ in 6-months-old APP/PS1 mice
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Detection of soluble Aβ1-42 by ELISA showing a decrease in its protein concentration in the brain lysates of the cerebral cortex (Cc) and cerebellum (Cb) of 6-months-old APP/PS1 mice (n
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Highlights ・Phosphorylation level of CRMP2 increased in the cerebellum of APP/PS1 mice. ・p35 protein levels increased in the cerebellum of APP/PS1 mice.
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・Pioglitazone treatment improved cerebellar dysfunction of APP/PS1 mice.
S0006-291X(16)30507-1
DOI:
10.1016/j.bbrc.2016.04.012
Reference:
YBBRC 35607
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 23 March 2016 Accepted Date: 4 April 2016
Please cite this article as: J. Toba, M. Nikkuni, M. Ishizeki, A. Yoshii, N. Watamura, T. Inoue, T. Ohshima, PPARγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ deposition stage in APPswe/PS1dE9 Alzheimer's disease model mice, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.04.012. 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.
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PPARγγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ β deposition stage in APPswe/PS1dE9 Alzheimer’s disease model mice Junya Toba1, Miyu Nikkuni1, Masato Ishizeki2, Aya Yoshii1, Naoto Watamura1, Takafumi
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Inoue2, Toshio Ohshima1,3
Laboratory for Molecular Brain Science and 2Laboratory for Neurophysiology, Department of
Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering,
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Waseda University, Tokyo, 162-8480 Japan
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Running title: Pioglitazone for cerebellar phenotype of AD mice
Corresponding author: Toshio Ohshima
Department of Life Science and Medical Bioscience, Waseda University,
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2-2 Wakamatsu-cho Shinjuku-ku, Tokyo 162-8480, Japan
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Tel: -81-3-5369-7321, Fax: -81-3-5369-7302, e-mail: [email protected]
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Abstract
Alzheimer’s disease (AD) is one of the best known neurodegenerative diseases; it causes
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dementia and its pathological features include accumulation of amyloid β (Aβ) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that Aβ
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accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD)
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induction, at the pre-Aβ accumulation stage.
In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor γ, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-Aβ accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and
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AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-Aβ accumulation stage in AD model mice.
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Highlights
・Phosphorylation level of CRMP2 and p35 protein levels increased in the cerebellum of APP/PS1 mice.
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・Pioglitazone treatment improved cerebellar dysfunction of APP/PS1 mice at the pre-Aβ accumulation stage.
Key words: Alzheimer’s disease; protein phosphorylation; CRMP; mouse, cerebellum
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Abbreviations APP, amyloid precursor protein BSA, bovine serum albumin
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Cdk5, cyclin dependent kinase 5 CRMP2, collapsin response mediator protein 2 GSK3β, glycogen synthase kinase 3β PS1, presenilin 1
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Tg, transgenic
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Introduction
Alzheimer’s disease (AD) is a well-known progressive neurodegenerative disorder and is the most common type of dementia among elderly people. Cognitive dysfunction and memory
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impairment are the main clinical symptoms present in AD patients. Accumulations of aberrant aggregates of amyloid β (Aβ) called senile plaques, and neurofibrillary tangles (NFTs), composed of phosphorylated-tau protein, are regarded as the major pathological features in AD [1]. Aβ becomes neurotoxic when it aggregates, and these aggregates lead to a synaptic
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dysfunction and neuronal cell death [2]. It is clear that accumulation of Aβ contributes to the onset of AD; thus, the amyloid hypothesis, which holds that much of AD pathology is driven by
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deposition of Aβ in the brain [3], is widely supported. However, there is no significant correlation between the severity of dementia and the deposition of senile plaques in some patients [4]; hence, the oligomer hypothesis, which regards the soluble oligomer of the Aβ peptide as the primary toxin in AD, has been suggested [5]. Some studies have shown that oligomeric Aβ is present in the brain, and impairs the synaptic plasticity mechanisms that are
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required for memory processing, and also induces synaptic dysfunction [2, 6].
CRMP2 is a member of CRMP protein familiy, which regulates axonal guidance, proper dendritic organization, and microtubule stability, by associating with tubulin [7, 8]. The interaction of CRMP2 with tubulin is disrupted due to its phosphorylation by Cdk5 and GSK3β
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[7]. In AD, it is phosphorylated by Cdk5 and GSK3β, and this phosphorylated form is a component of NFTs [9]. Furthermore, phosphorylation of CRMP2 is increased in the early
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stages of AD, i.e., before the accmulation of Aβ and phosphorylated tau [10], but the detail of its role in AD pathology remains unknown.
Peroxisome proliferator-activated receptor γ (PPARγ), which is a nuclear receptor that is highly expressed in adipose tissue, is involved in the metabolism of fatty acids and survival of adipocytes [11]. In the brain, PPARγ is predominantly present in microglia and astrocytes, and its expression has been studied in relation to inflammation and neurodegeneration [12]. One of the thiazolidinedione (TZD) drugs, pioglitazone, is a ligand of PPARγ, and is used clinically for treatment of type 2 diabetes, in order to increase insulin sensitivity [13]. However, it is has been
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shown that treatment with pioglitazone ameliorates impaired cognitive function and reduces Aβ deposition in an AD mouse model [14, 15].
We have previously reported the presence of cerebellar dysfunction, such as impairment of
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motor coordination ability and long-term depression (LTD) induction, at the pre-Aβ accumulation stage in APPswe/PS1dE9 (APP/PS1) mice [16]. In the present study, we report elevated phosphorylation of CRMP2 and the p35 protein levels in the cerebellum of APP/PS1 mice in which high soluble Aβ levels and Aβ plaque formation were found. We then treated mice with pioglitazone and found normalized p35 protein levels and CRMP2 phosphorylation in
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the cerebellum, as well as recovery of motor coordination ability and LTD induction at the
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pre-Aβ accumulation stage.
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Materials and Methods
Mice and pioglitazone treatment Mice used in the experiments were housed in accordance with protocols approved by the
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Institutional Animal Care and Use Committee of Waseda University. APPswe/PS1dE9 (APP/PS1) transgenic mice were generated as described [17]. Pioglitazone (TCI, Tokyo, Japan)
previously described [15].
Biochemical analysis
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Enzyme-Linked ImmunoSorbent Assay (ELISA) for Aβ
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was administered orally to male 5- to 6-months-old (80 mg/body weight kg-1d-1) for 9 days, as
Brain tissues were homogenized in 10-fold volume to-weight ratio of Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA) containing a protease inhibitor cocktail (Roche Applied, Berkeley, CA, USA), and then centrifuged at 100,000 × g for 60 min. The supernatants were analyzed using the human β-amyloid (1−42) ELISA kit (Wako, Osaka, Japan)
Western blotting
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according to the manufacturer’s instruction.
Brain tissues or slices were homogenized in 10-fold volume-to-weight ratio of radio immunoprecipitation buffer (RIPA buffer: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40,
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1% sodium deoxycholate 0.1% sodium dodecyl sulfate), containing a protease and phosphatase inhibitor cocktail. Homogenized samples were centrifuged at 15,000 rpm for 15 min and the
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supernatants were subjected to western blot analysis as described previously [16]. The primary antibodies used in this study were as follows: anti-phosphorylated CRMP2 (Ser522, CP2191, ECM Biosciences, Versailles, KY, USA), anti-CRMP2 (9F, [8]) , anti-β-actin (A2103, Sigma, St Louis, MO, USA), anti-p35/25 (C19, sc-820, Santa Cruz Biotechnology, Dallas, TX, USA). Incubation of Aβ peptides and inhibitors Aβ25–35 peptide monomer (Peptide Research Inc., Osaka, Japan) dissolved in sterilized water to a concentration of 1mg/ml and stored in aliquots, was incubated at 37ºC for 4 days, to allow oligomerization [18, 19]. Aβ35–25 (Peptide Research Inc.), prepared in the same way, was used as
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a control. Aβ peptide and/or MK801 (Sigma) were added to the solution at the indicated final concentration.
Cerebellar slice preparation and electrophysiology
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250-µm-thick cerebellar slices were prepared and maintained according to a standard procedure [20]. In recordings artificial cerebrospinal fluid (ACSF) containing 100 µM picrotoxin was superfused at 31ºC. Membrane potential of Purkinje cells was held at -70 mV by the patch-clamp method with borosilicate glass pipettes (5−7 MΩ) filled with (in mM) 65
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K-gluconate, 65 Cs-methanesulphonate, 10 KCl, 1 MgCl2, 4 Na2-ATP, 1 Na-GTP, 5 sucrose, 0.4 EGTA, 20 K-HEPES; pH 7.3; 295 mOsm. A stimulation glass electrode was placed on the
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molecular layer, through which monopolar square pulses (200 µs) were applied. LTD experiments were performed with 6-month-old APPswe/PS1dE9 mice and age-matched non-Tg mice as described [16]. LTD was induced by pairing depolarization of Purkinje cells (200 ms, -70 to 0 mV) with a single PF stimulation 240 times at 1 Hz.
Statistical analysis
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All data are presented the mean ±SEM. The statistical analyses were performed using Student’s t-test or one-way and two-way ANOVA repeated-measures as described. P < 0.05 was
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Results
Upregulation of phosphorylated CRMP2 in APP/PS1 mouse brain and reversal effect of pioglitazone treatment
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Upregulation of the phosphorylation level of CRMP2 in the AD brain has been reported in the cerebral cortex and hippocampus [10]; however, it had not yet been investigated in the cerebellum. To examine whether phosphorylation of CRMP2 is altered in the cerebellum of APP/PS1 mice, we compared the phosphorylation levels of CRMP2 in the cerebellum and
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cerebral cortex of 6-months-old APP/PS1 mice with those in non-Tg controls. We performed western blot analysis with brain lysates from the cerebral cortex and cerebella from these mice.
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There was trend for an increase in levels of CRMP2 phosphorylated at Ser522 in APP/PS1 cerebella, as compared with the control, although the difference was not statistically significant (Fig. 1A).
We next investigated the effect of pioglitazone on phosphorylation of CRMP2 to assess the correlation between soluble amyloid oligomer and CRMP2 phosphorylation levels. We
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administered 80 mg/kg pioglitazone for 9 days to 6-months-old APP/PS1 mice and determined the concentration of the Aβ oligomer in the brain lysate of APP/PS1 mice by ELISA. There was a trend for a decrease in Aβ concentration in both the cerebral cortex and cerebellum (Supplementary Fig. 1). Western blotting analysis showed that phosphorylation of CRMP2 at
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Ser522 was significantly decreased in both the cerebral cortex and cerebellum of APP/PS1 mice treated with pioglitazone, as compared with non-treated mice of the same genotype (Fig. 1B, *p
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< 0.05, student’s t-test).
Increased p35 and p25 protein levels in the brain of APP/PS1 and reversal effect of pioglitazone treatment
Hyperactivation of Cdk5 occurs in the AD brain; this is thought to be involved in the progression of AD pathology via phosphorylation of several substrates, such as tau, leading to neurodegenerative pathology [22]. A previous study has reported that hyperactivity of Cdk5 in the AD brain was caused by an increase in p25, which was due to calpain-mediated cleavage of p35 [23]. We found that p35 levels tended to increase in APP/PS1 mice and that p25 levels
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increased significantly in the cerebral cortex of APP/PS1 mice (Fig. 2A, p < 0.05, Student’s t-test). The protein levels of p25 was markedly lower than those of p35; therefore, we used a longer exposure to analyze p25 ("p25 [long]") in these samples. Since we found that phosphorylation of CRMP2 at Ser522 was reduced by pioglitazone treatment, we investigated
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the protein levels of p35 and p25 in the brains of 6-months-old APP/PS1 mice treated with pioglitazone. This treatment significantly decreased both p35 and p25 protein levels in the cerebral cortex and tended to decrease those in the cerebellum (Fig. 2B, p < 0.01, Student’s t-test). These results suggest that activation of Cdk5 in 6-months-old APP/PS1 mice depended
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on the increase in the levels of p35 and p25; these noted elevations were reversed by
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pioglitazone treatment.
Defective cerebellar function in 6-months-old APP/PS1 mice and the effect of pioglitazone treatment
The cerebellum is a brain region that is important for controlling balance and motor coordination. To compare the cerebellar function of APP/PS1 mice with that of WT mice, we conducted a behavior test, the rotarod test, to assess the motor coordination ability of the mice.
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The 6-months-old control WT mice showed a gradual improvement in performance, i.e., increased retention time, through the consecutive 4-days trial period, but the APP/PS1 mice showed a short retention time, without improvement during the 4-day period (Fig. 3A, *p < 0.05, two-way repeated-measures ANOVA), which was consistent with the findings of our previous
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study (Kuwabara et al., 2014). Next, we assessed the effect of pioglitazone treatment on defective cerebellar motor coordination ability of APP/PS1 mice. APP/PS1 mice treated with
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pioglitazone showed gradual improvement in the score of the rotarod test (Fig. 3A, *p < 0.05, two-way repeated-measures ANOVA), which was similar to that in the control mice. This result indicated that the cerebellar dysfunction expressed as defective motor coordination ability in 6-months-old APP/PS1 mice was ameliorated by treatment with pioglitazone.
Additionally, impairment of synaptic plasticity in the hippocampus has been shown in an AD mouse model, and by application of Aβ [18, 24], which was thought to cause memory and cognitive dysfunction. Our previous study showed that cerebellar LTD was impaired in APP/PS1 mice [16]. To assess changes in cerebellar synaptic plasticity at the pre-Aβ
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accumulation stage, we compared cerebellar LTD in 6-months-old APP/PS1 mice and age-matched WT control mice. LTD was induced in control mice, and was expressed as a constant decrease in the amplitude of parallel fiber−Purkinje cell synapses. In contrast, impairment of LTD induction was seen in APP/PS1 mice (Fig. 3B, p < 0.001, one-way
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repeated-measures ANOVA), showing impairment in the synaptic function in the cerebellum of APP/PS1 mice. Cerebellar slices from APP/PS1 mice treated with pioglitazone showed normal LTD induction, expressed as a long-lasting decrease in the EPSC amplitude (Fig. 3B, p < 0.001, vs. non-treated APP/PS1 mice, one-way repeated-measures ANOVA). These results indicated
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that treatment with pioglitazone ameliorated cerebellar dysfunction in the cerebella of
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6-months-old APP/PS1 mice.
Aβ treatment of cerebellar slices tends to increase CRMP2 phosphorylation and p35 protein levels
Our previous study showed that phosphorylation of CRMP2 at Ser522 was increased by Aβ25-35 injection into the hippocampus [25]. To test whether Aβ25-35 also increases CRMP2 phosphorylation in the cerebellum, we incubated cerebellar slices with pre-oligomerized Aβ25-35
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or Aβ35-25 peptides for 2 h, and determined the levels of CRMP2 phosphorylated at Ser522 and of p35/p25 by western blot analysis. Aβ25-35 tended to increase CRMP2 Ser522 phosphorylation in cerebellar slices, as compared with incubation with Aβ35-25 (Fig. 4A). The levels of p35 were significantly increased in the cerebellar slices treated with Aβ25-35 as compared with Aβ35-25
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treatment of slices, while the levels of p25 were not altered (Fig. 4B, p < 0.05, Student’s t-test).
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Previous studies have suggested that Aβ increases the activity of NMDA receptors, which
increases Ca2+ influx into cells. Increased intracellular Ca2+ levels would activate calpain activity, which would cleave p35 to yield p25, resulting in increased phosphorylation of CRMP2 in cerebellar slices incubated with Aβ25-35. To test this hypothesis, we added 20 µM MK801, an NMDA receptor inhibitor, to the cerebellar slices incubated with Aβ25-35 peptide. After a 2-h co-incubation, slices were subjected to western blotting analysis. A decrease of CRMP2 phosphorylation at Ser522 was found in cerebellar slices treated with Aβ25-35 and MK801, as compared with slices treated with Aβ25-35 alone (Fig. 4C, p < 0.05, one-way repeated-measures ANOVA). However, MK801 had no effect on the levels of p35 and p25 (data not shown).
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Discussion
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In the present study, we demonstrated that phosphorylation of CRMP2 at Ser522 was increased in the cerebellum, as well as in the cerebral cortex of 6-months-old APP/PS1 mice (Fig. 1A). Furthermore, pioglitazone treatment of APP/PS1 mice resulted in decreased phosphorylation (Fig. 1B). Behavioral and electrophysiological impairment of the cerebellum was ameliorated
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by treatment with pioglitazone (Fig. 3), which accompanied the decrease in phosphorylated CRMP2. These results suggest that alteration of CRMP2 is involved in functional improvement
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of APP/PS1 mice. It is possible that increased phosphorylation of CRMP2 contributes to the impairment of cerebellar synaptic plasticity, because a previous study reported that impairment of hippocampal LTP by addition of Aβ was reversed by mutation of CRMP2S522A, in which phosphorylation of CRMP2S5522 was inhibited [25]. Furthermore, we demonstrated that elevation of phosphorylated CRMP2 was induced by addition of synthesized Aβ to cerebellar slices (Fig. 4A). Taken together with the evidence that application of Aβ to cerebellar slices
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impaired synaptic plasticity in the cerebellum [16], we concluded that there is close correlation between the phosphorylation of CRMP2 and functional impairment in AD.
Several studies have reported that aberrant activation of Cdk5 in AD is involved in the
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progression of pathology via production of Aβ aggregates and development of NFTs [22, 23]. It is generally considered that hyperactivation of Cdk5 in AD is dependent on production of p25,
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which is mediated by calpain, a calcium-dependent protease, induced by the toxicity of Aβ [23]. In this study, we detected an increase in p35 in the brain lysate; however, the p25 protein levels remained very low (Fig. 2A). Previous studies showed that the activity of calpastatin, an endogenous calpain inhibitor, was high in the cerebellum as compared with other brain regions, and little p25 was produced [26, 27]. In the present study, variation in the protein levels of p35 and p25 in the cerebellum of APP/PS1 mice was less than that in the cerebral cortex; thus, it is possible that other pathways deregulate Cdk5 activity, or other factors are involved in the cerebellar dysfunction. It has been reported that p35 is degraded by the ubiquitin-proteasome degradation pathway [28]. In addition, Aβ-induced inactivation of the proteasome in 3xTg-AD
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mice and enhanced accumulation of amyloid and tau by inhibition of the degradation of Aβ [29] suggest that hyperactivation of Cdk5 in AD is possibly induced by the increase in p35, by inhibiting its degradation via the proteasome. The present study showed that a decrease in p35 was induced by pioglitazone treatment in APP/PS1 mice. According to previous studies,
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degradation of p35 was promoted by treatment with TDZ drugs, including pioglitazone in a proteasome-dependent, but not PPARγ-dependent manner [30], which is consistent with the present study. Another study showed a reduction of p35 by treatment with pioglitazone in vitro; however, the elevation of p35 in AD confirmed in the present study was not investigated
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[31].The findings of the present study and or previous studies [31] indicate a correlation between the Cdk5 activity mediated by p35 regulation and pioglitazone treatment, but further
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investigation is required.
Our results showed the effects of pioglitazone treatment on cerebellar dysfunction in an AD mouse model. Further studies are required to determine the effect of pioglitazone on other brain function at the pre-Aβ-accumulation stage. In addition, the findings of the present study support
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the importance of Cdk5 and phosphorylated CRMP2 as therapeutic targets of AD.
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Conflict of Interest
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The authors declare no conflict of interest.
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Figure Legends
Fig. 1 Upregulation of phosphorylated CRMP2 in APP/PS1 mouse brain and its reversal by pioglitazone treatment
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A, Western blot analysis of brain lysates from the cerebral cortex (Cc) and cerebellum (Cb) in 6-months-old APP/PS1 mice, as compared with age-matched wild-type (WT) mice (n = 3 for each group), to examine levels of CRMP2 phosphorylated at Ser522. B, Western blot analysis
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shows a significant decrease in phosphorylated CRMP2S522 in 6-months-old APP/PS1 mice treated with pioglitazone (Pio) as compared with non-treated mice in both the cerebral cortex and cerebellum (n = 3 for each group, *p < 0.05, Student’s t-test).
Fig. 2 Increased p35 protein level in the brain of APP/PS1 and its reversal by pioglitazone
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A, Western blot analysis with brain lysates from the cerebral cortex (Cc) and cerebellum (Cb), to examine the protein level of p35 and p25 (n = 3 for each group, *p < 0.05, Student’s t-test). p25 had a much lower signal than p35; thus, a longer exposure is shown (long). B, Western blot
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analysis shows a decrease in p35 and p25 protein levels in both the cerebral cortex and cerebellum in 6-months-old APP/PS1 mice treated with pioglitazone (Pio), as compared with
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non-treated mice (n = 6 for each group, **p < 0.01, Student’s t-test).
Fig. 3 Defective cerebellar function in 6-months-old APP/PS1 mice and the effect of pioglitazone treatment
A, The motor coordination ability of 6-months-old APP/PS1 mice was assessed using the rotarod test. APP/PS1 mice showed defective motor coordination ability as compared with wile-type (WT) mice (n = 6 for each group, *p < 0.05, two-way repeated-measures ANOVA), and APP/PS1 mice treated with pioglitazone (Pio) showed an improved score, approaching that of WT mice. There was a significant difference between the scores of pioglitazone-treated APP/PS1 mice and non-treated mice (n = 6 for each group, *p < 0.05, two-way 17
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repeated-measures ANOVA). B, Long-term depression (LTD) induction at the PF-Purkinje cell synapse in cerebellar slices of 6-months-old APP/PS1 mice. Slices from WT mice (n = 6) showed LTD, which was significantly impaired in slices from APP/PS1 mice (n = 5, ***p < 0.001, one-way repeated-measures ANOVA). Slices from APP/PS1 mice treated with
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pioglitazone (Pio; n = 11) showed almost the same degree of reduction in EPSC amplitudes as WT mice, which was significant as compared with non-treated APP/PS1 mice (***p < 0.001, one-way repeated-measures ANOVA). Upper traces show the EPSCs averaged from 5
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Fig. 4 Aβ treatment tends to increase CRMP2 phosphorylation
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A, Western blot analysis of cerebellar slices treated with Aβ25-35 or Aβ35-25 to examine phosphorylation of CRMP2 at Ser522 (n = 4, Student’s t-test).
B, Western blot analysis showing a significant increase in p35 levels in the cerebellar slices treated with Aβ25-35 as compared with those treated with Aβ35-25 (n = 4, *p < 0.05, Student’s t-test).
C, Western blot analysis showing a significant decrease in CRMP2 phosphorylated at Ser522 in
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Aβ25-35 alone (n = 3, *p < 0.05, one-way repeated-measures ANOVA).
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Legends for supplementary Figures
Supplementary Fig. 1 Biochemical analysis of level of soluble Aβ in 6-months-old APP/PS1 mice
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Detection of soluble Aβ1-42 by ELISA showing a decrease in its protein concentration in the brain lysates of the cerebral cortex (Cc) and cerebellum (Cb) of 6-months-old APP/PS1 mice (n
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= 3).
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Highlights ・Phosphorylation level of CRMP2 increased in the cerebellum of APP/PS1 mice. ・p35 protein levels increased in the cerebellum of APP/PS1 mice.
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・Pioglitazone treatment improved cerebellar dysfunction of APP/PS1 mice.