Original Paper Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
Diseases
Received: September 23, 2013 Accepted after revision: January 7, 2014 Published online: April 30, 2014
Protective Effects of Bexarotene against Amyloid-β25–35-Induced Dysfunction in Hippocampal Neurons through the Insulin Signaling Pathway Weiwei Dai a Jiajia Yang b Ting Chen b Zhuo Yang a
College of Medicine, Nankai University, and b College of Life Sciences, Nankai University, Tianjin, China
Key Words Alzheimer’s disease · Bexarotene · Excitability · Potassium currents · Insulin signaling pathway
Abstract Background: Bexarotene, a retinoid X receptor agonist, has been shown to reverse neurodegeneration in mouse models of Alzheimer’s disease (AD), accompanied by a decreased level of amyloid-β (Aβ), which is a hallmark of AD. However, the mechanism underlying this therapeutic effect may involve enhancing the sensitivity to insulin. Objective: This study was to test the hypothesis that bexarotene would protect against Aβ25–35-induced dysfunction through the insulin signaling pathway. Methods: Using a whole-cell patch clamp technique, the excitability and voltage-gated potassium currents of hippocampal neurons were examined in four groups of cells (control, Aβ, Aβ + bexarotene and bexarotene). Results: It was found that insulin increased the excitability of neurons. Bexarotene could enhance this effect and reverse the Aβ25–35-induced decrease in the firing rate of the action potential (AP). In addition, the properties of the single AP (sAP) and voltage-gated outward K+ currents were recorded, which finally showed similar changes to those in the firing frequency. Conclusion: The effects of bexarotene on Aβimpaired excitability and sAP duration were mainly associ-
© 2014 S. Karger AG, Basel 1660–2854/14/0142–0077$39.50/0 E-Mail [email protected] www.karger.com/ndd
ated with K+ channels through insulin signaling pathway, which may be an additional mechanism underlying the protective effect of bexarotene on AD. © 2014 S. Karger AG, Basel
Introduction
As a primary candidate for initiation of neuronal dysfunction and deterioration of cognitive function in Alzheimer’s disease (AD), the levels of soluble amyloid-β (Aβ) in the brain are highly correlated with the severity of neurodegeneration and are a predictor of cognitive impairment in AD [1, 2]. A recent study reported by Cramer et al. [3] demonstrated that a retinoid X receptor (RXR) agonist, bexarotene, rapidly stimulated plaque and soluble Aβ clearance by enhancing apolipoprotein E (apo E) levels, thereby improving cognitive and behavioral deficits in mouse models of AD. RXR is a nuclear receptor and predominantly functions as a transcription factor upon dimerization of other nuclear receptors including the peroxisome proliferator-activated receptor (PPAR)-γ and liver X receptor [4]. RXR forms heterodimers with
W.D. and J.Y. contributed equally to this work.
Prof. Zhuo Yang College of Medicine Nankai University Tianjin 300071 (China) E-Mail zhuoyang @ nankai.edu.cn
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Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
Materials and Methods Preparation of Hippocampal Slices The experimental protocols were approved by the Institutional Animal Care Committee at Nankai University. Male Wistar rats were used on postnatal days 15–20. Hippocampal slices were prepared as previously described [20, 21]. Horizontal slices from the entire hippocampus (400 μm in thickness) were prepared with a vibratome (VT1000M/E, Leica, Germany), incubated with artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 1.25 KCl, 1.25 NaH2PO4, 25 NaHCO3, 1.5 MgCl2, 2.0 CaCl2 and 10 glucose, and aerated with 95% O2 and 5% CO2 at pH 7.4 for at least 1 h before electrophysiological recording. The slices were submerged in a chamber perfused with ACSF during data logging. All experiments were performed at room temperature (24–26 ° C).
Patch Clamp Recording For whole-cell recording, patch electrodes were made from borosilicate glass using a vertical electrode puller (PIP5, HEKA, Germany) to yield tip openings of 1–2 μm. Using pipettes with 4–8 MΩ resistance after being filled with pipette solution containing (in mM) 140 KCl, 10 HEPES, 2 MgCl2, 10 EGTA and 2 ATP-Na2, pH 7.3, whole-cell voltage clamp or current clamp recordings were performed in hippocampal CA1 neurons. For recording of potassium currents, tetrodotoxin (TTX, 1 μM) was added into the ACSF to block Na+ currents. Slices were transferred into a glass-bottomed chamber with a volume of 1 ml and anchored by nylon threads and a U-shaped steel kit [21]. Pyramidal neurons in the hippocampal CA1 region were visually identified with infrared differential interference contrast optics (BX51WI, Olympus, Japan) and visualized on a television monitor connected to a low-lightsensitive CCD camera (710M, DVC). All cells were held at –70 mV when slow and fast capacitance compensation was automatically performed. Only when seal resistance was >500 MΩ and series resistance ( 0.05, one-way ANOVA), which indicated that Aβ25–35 did not impair the amplitude of sAP. In the control group, the amplitudes before (R1) and after (R2) insulin application were 103.8 ± 3.2 and 100.3 ± 2.7 mV, respectively (n = 7; p > 0.05, paired t test), which indicated that the amplitude was not sensitive to insulin. Thus, it is not necessary to investigate its change in IR sensitivity. In contrast, the half-width of sAP was more sensitive to insulin. As shown in figure 3b–e, in control group, bexaro-
Bexarotene Improves Aβ-Induced Dysfunction
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t test. Relative comparisons, i.e. (R2 – R1)/R1, between groups were made by one-way analysis of variance (ANOVA), followed by the least significant difference post hoc test with the p value set at 0.05.
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Fig. 2. Effect of insulin on the firing frequency of CA1 neurons. a A typical recording before (R1) and after (R2) application of in-
sulin in the control group. To examine the differences in insulin sensitivity between the four groups, embodied by AP firing frequency, a long-term depolarizing current (500 ms, 50 pA) was applied to the neurons. b Comparison of firing frequency before ap-
tene and Aβ + bexarotene groups, the half-width of sAP were significantly increased by insulin, to varying degrees, while in Aβ group, it was decreased. We next calculated the change in half-width in each group and found a significant difference between them (fig. 3f; p < 0.05, one-way ANOVA). The results illustrated that the half-width of sAP could be affected by insulin in the control group (increase of 24.6 ± 6.6%), and bexarotene significantly enhanced this effect (61.8 ± 15.5%). In Aβ group, the half-width of sAP was decreased (11.5 ± 3.7%), which was significantly dif80
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Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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plication of insulin (R1) among the four groups. c Comparison of firing frequency before and after application of insulin in each group. d The change in firing frequency induced by application of insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene.
ferent compared with that of the control group, Aβ + bexarotene group and bexarotene group, respectively (fig. 3f). Thus, we concluded that bexarotene alleviated the effect of Aβ25–35 on the half-width, which made it change to the direction of control group. Changes in the Properties of the AP between Groups May Involve Potassium Currents Studies from our laboratory have reported that voltage-dependent outward K+ currents played the major Dai/Yang/Chen/Yang
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Fig. 3. Effect of insulin on the half-width of the sAP. a sAPs were
elicited by 5-ms brief depolarizing current pulses before and after application of insulin in the control group. b–e Comparison of the half-width of the sAP before and after application of insulin in each group: control, from 3.02 ± 0.17 to 3.73 ± 0.23 ms; Aβ, from 2.55 ± 0.11 to 2.27 ± 0.18 ms; Aβ + bexarotene, from 3.04 ±
role in regulating neuronal excitability [20, 22, 23]. Thus, we examined the downstream mechanism of the protection of bexarotene. When recording potassium currents, the neurons were held at –70 mV and current traces were evoked by using an 80-ms constant depolarizing pulse from –50 to +90 mV in increments of 10 mV (fig. 4a). In the current-voltage curves shown in figure 4b–e, we observed that outward currents were reduced by the application of insulin from +60 mV, which was visible on current-voltage curves in control group (fig. 4b). However, after application of Aβ, the effect of insulin on K+ channels was abolished (fig. 4c). In contrast, bexarotene could enhance the effect of insulin (fig. 4d) and reverse the effect of Aβ in the Aβ + bexarotene group (fig. 4e). After normalization by the formula (R2 – R1)/R1, the changes in the amplitude of potassium currents at +90 mV in four groups were shown in figure 4f, and there were significant differences between them (p < 0.01, oneway ANOVA).
Bexarotene Improves Aβ-Induced Dysfunction
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0.19 to 3.29 ± 0.17 ms; bexarotene, from 2.77 ± 0.2 to 4.35 ± 0.24 ms. f The change in the half-width of sAP caused by insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene.
Discussion
AD is a chronic and devastating neurodegenerative disorder that impairs the patient’s memory, destroys the ability to reason, rational judgments and leads to myriad behavioral and psychiatric symptoms. The accumulation of Aβ in the brain is postulated to initiate a cascade of events leading to AD [24]. However, therapeutic approaches that aim to reduce Aβ production or aggregation have so far been unsuccessful. However, Cramer et al. [3] recently reported that the drug bexarotene (Targretin), already approved by the Food and Drug Administration for the treatment of cutaneous T cell lymphoma, showed promising efficacy in preclinical models of AD. In this study, we proposed an additional mechanism of action of bexarotene, whereby it alleviated Aβ-induced neuron dysfunction in insulin signaling pathway. Furthermore, despite the encouraging results reported by Cramer et al. [3], several groups have failed to replicate the effect of bexarotene on Aβ plaque burden in these and other related Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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Fig. 4. Effect of insulin on outward potassium currents. a Potassium currents were obtained by 80-ms depolarizing pluses from a command potential of –50 to +90 mV in increments of 10 mV, and the holding potential was –70 mV. The diagram shows the potassium current wave before and after application of insulin in the control group. b–e Comparison of current-voltage curves before and after
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Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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application of insulin in the control (b), Aβ (c), Aβ + bexarotene (d) and bexarotene (e) group. f The change in the amplitude of the potassium current at +90 mV induced by insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene; IK = delayed rectifying K+ channels. (For figure 4f see next page.) Dai/Yang/Chen/Yang
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mouse models [25–29], thus the effects of this compound remain controversial and need to be studied further. The results of our study supported the hypothesis in terms of the electrophysiological properties of rat hippocampal CA1 neurons. We demonstrated in this study that the protective effects of bexarotene against Aβ-induced impairment in neurons may involve the insulin signaling pathway. As we know, the AP is a fundamental property of excitable cells in the mammalian central nervous system and a dynamic reflection of changes in ion channels in the membrane. Either activation of signaling pathways or toxic impairment would modify the excitability of neurons. Consistent with this principle, our study did indicate that the application of insulin to normal neurons led to an increase in their excitability. Nonetheless, the direction of the response to insulin was reversed after the application of Aβ, which was represented by the decreased frequency of APs. Intriguingly, the deterioration of the response to insulin caused by Aβ was ameliorated by the application of bexarotene. In the bexarotene + Aβ group, the impact of insulin on neurons was restored. Moreover, bexarotene alone could enhance the response to insulin, which indicated an enhanced IR sensitivity. These results suggested that bexarotene had a protective effect against Aβ-induced impairment through the insulin signaling pathway. In order to explore the detailed mechanism, we focused on an important ion channel which plays an essential role in determining the excitability of neurons. It has been acknowledged that ion channels in the cell membrane are targets of many toxins and drugs, and in the nervous system, voltage-gated K+ currents are largely responsible for repolarization and hyperpolarization, which constitute the major part of the whole AP and therefore regulate a variety of neuronal properties, such as resting membrane potential, AP waveform and firing frequency. Thus, we tested whethBexarotene Improves Aβ-Induced Dysfunction
er the protective effect of bexarotene on excitability involved K+ channels, and the results offered a probable explanation for the alteration of neuron excitability. Previous studies have proved that the reduction of potassium currents will result in hyperexcitability of neurons [23, 30]. In addition, the sAP waveform was also altered in all four of our experimental groups, especially the half-width of the sAP, the change in which was similar to K+ currents. The half-width of sAP is most commonly measured as the width at half-maximal spike amplitude, which represents the duration of the AP. It has been reported that blocking of potassium currents would prolong the duration of the AP [31, 32]. The reason may be that the decreased outward K+ currents will lead to a longer time of repolarization for the AP. Thus, we speculated that the protective effect of bexarotene in Aβ-impaired excitability and sAP duration was mainly due to action on K+ channels through the insulin pathway. Although we proved that bexarotene could enhance IR sensitivity, the detailed mechanism was still unknown, because the decline in IR function can originate with aberrations in the expression or phosphorylation state of the receptor. For example, in certain familial forms of insulin resistance, the defective IR function is caused by the severe lesion of IR alleles and substantial decreases in IR levels [33]. Alternatively, the suppression of IR enzymatic activity has been linked to negative regulatory factors stimulated by IR signaling as well as other pathways. Additionally, IR activity can be inhibited by a downstream negative feedback pathway [34–36]. Due to all these possibilities, further studies need to be performed to explore the mechanism by which bexarotene enhances IR sensitivity.
Conclusion
This study demonstrated that the insulin signaling pathway may be an additional mechanism underlying the protective effect of bexarotene against Aβ25–35-induced dysfunction in hippocampal neurons, which was represented by neuronal AP properties, and the target might involve potassium channels. Therefore, the results provide further insight into the underlying mechanisms responsible for the effects of bexarotene on AD. Acknowledgements This work was partly supported by a grant from the National Basic Research Program of China (2011CB944003) and the National Natural Science Foundation of China (31271074 and 31300923).
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Diseases
Received: September 23, 2013 Accepted after revision: January 7, 2014 Published online: April 30, 2014
Protective Effects of Bexarotene against Amyloid-β25–35-Induced Dysfunction in Hippocampal Neurons through the Insulin Signaling Pathway Weiwei Dai a Jiajia Yang b Ting Chen b Zhuo Yang a
College of Medicine, Nankai University, and b College of Life Sciences, Nankai University, Tianjin, China
Key Words Alzheimer’s disease · Bexarotene · Excitability · Potassium currents · Insulin signaling pathway
Abstract Background: Bexarotene, a retinoid X receptor agonist, has been shown to reverse neurodegeneration in mouse models of Alzheimer’s disease (AD), accompanied by a decreased level of amyloid-β (Aβ), which is a hallmark of AD. However, the mechanism underlying this therapeutic effect may involve enhancing the sensitivity to insulin. Objective: This study was to test the hypothesis that bexarotene would protect against Aβ25–35-induced dysfunction through the insulin signaling pathway. Methods: Using a whole-cell patch clamp technique, the excitability and voltage-gated potassium currents of hippocampal neurons were examined in four groups of cells (control, Aβ, Aβ + bexarotene and bexarotene). Results: It was found that insulin increased the excitability of neurons. Bexarotene could enhance this effect and reverse the Aβ25–35-induced decrease in the firing rate of the action potential (AP). In addition, the properties of the single AP (sAP) and voltage-gated outward K+ currents were recorded, which finally showed similar changes to those in the firing frequency. Conclusion: The effects of bexarotene on Aβimpaired excitability and sAP duration were mainly associ-
© 2014 S. Karger AG, Basel 1660–2854/14/0142–0077$39.50/0 E-Mail [email protected] www.karger.com/ndd
ated with K+ channels through insulin signaling pathway, which may be an additional mechanism underlying the protective effect of bexarotene on AD. © 2014 S. Karger AG, Basel
Introduction
As a primary candidate for initiation of neuronal dysfunction and deterioration of cognitive function in Alzheimer’s disease (AD), the levels of soluble amyloid-β (Aβ) in the brain are highly correlated with the severity of neurodegeneration and are a predictor of cognitive impairment in AD [1, 2]. A recent study reported by Cramer et al. [3] demonstrated that a retinoid X receptor (RXR) agonist, bexarotene, rapidly stimulated plaque and soluble Aβ clearance by enhancing apolipoprotein E (apo E) levels, thereby improving cognitive and behavioral deficits in mouse models of AD. RXR is a nuclear receptor and predominantly functions as a transcription factor upon dimerization of other nuclear receptors including the peroxisome proliferator-activated receptor (PPAR)-γ and liver X receptor [4]. RXR forms heterodimers with
W.D. and J.Y. contributed equally to this work.
Prof. Zhuo Yang College of Medicine Nankai University Tianjin 300071 (China) E-Mail zhuoyang @ nankai.edu.cn
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Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
Materials and Methods Preparation of Hippocampal Slices The experimental protocols were approved by the Institutional Animal Care Committee at Nankai University. Male Wistar rats were used on postnatal days 15–20. Hippocampal slices were prepared as previously described [20, 21]. Horizontal slices from the entire hippocampus (400 μm in thickness) were prepared with a vibratome (VT1000M/E, Leica, Germany), incubated with artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 1.25 KCl, 1.25 NaH2PO4, 25 NaHCO3, 1.5 MgCl2, 2.0 CaCl2 and 10 glucose, and aerated with 95% O2 and 5% CO2 at pH 7.4 for at least 1 h before electrophysiological recording. The slices were submerged in a chamber perfused with ACSF during data logging. All experiments were performed at room temperature (24–26 ° C).
Patch Clamp Recording For whole-cell recording, patch electrodes were made from borosilicate glass using a vertical electrode puller (PIP5, HEKA, Germany) to yield tip openings of 1–2 μm. Using pipettes with 4–8 MΩ resistance after being filled with pipette solution containing (in mM) 140 KCl, 10 HEPES, 2 MgCl2, 10 EGTA and 2 ATP-Na2, pH 7.3, whole-cell voltage clamp or current clamp recordings were performed in hippocampal CA1 neurons. For recording of potassium currents, tetrodotoxin (TTX, 1 μM) was added into the ACSF to block Na+ currents. Slices were transferred into a glass-bottomed chamber with a volume of 1 ml and anchored by nylon threads and a U-shaped steel kit [21]. Pyramidal neurons in the hippocampal CA1 region were visually identified with infrared differential interference contrast optics (BX51WI, Olympus, Japan) and visualized on a television monitor connected to a low-lightsensitive CCD camera (710M, DVC). All cells were held at –70 mV when slow and fast capacitance compensation was automatically performed. Only when seal resistance was >500 MΩ and series resistance ( 0.05, one-way ANOVA), which indicated that Aβ25–35 did not impair the amplitude of sAP. In the control group, the amplitudes before (R1) and after (R2) insulin application were 103.8 ± 3.2 and 100.3 ± 2.7 mV, respectively (n = 7; p > 0.05, paired t test), which indicated that the amplitude was not sensitive to insulin. Thus, it is not necessary to investigate its change in IR sensitivity. In contrast, the half-width of sAP was more sensitive to insulin. As shown in figure 3b–e, in control group, bexaro-
Bexarotene Improves Aβ-Induced Dysfunction
Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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t test. Relative comparisons, i.e. (R2 – R1)/R1, between groups were made by one-way analysis of variance (ANOVA), followed by the least significant difference post hoc test with the p value set at 0.05.
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Fig. 2. Effect of insulin on the firing frequency of CA1 neurons. a A typical recording before (R1) and after (R2) application of in-
sulin in the control group. To examine the differences in insulin sensitivity between the four groups, embodied by AP firing frequency, a long-term depolarizing current (500 ms, 50 pA) was applied to the neurons. b Comparison of firing frequency before ap-
tene and Aβ + bexarotene groups, the half-width of sAP were significantly increased by insulin, to varying degrees, while in Aβ group, it was decreased. We next calculated the change in half-width in each group and found a significant difference between them (fig. 3f; p < 0.05, one-way ANOVA). The results illustrated that the half-width of sAP could be affected by insulin in the control group (increase of 24.6 ± 6.6%), and bexarotene significantly enhanced this effect (61.8 ± 15.5%). In Aβ group, the half-width of sAP was decreased (11.5 ± 3.7%), which was significantly dif80
* *
Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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plication of insulin (R1) among the four groups. c Comparison of firing frequency before and after application of insulin in each group. d The change in firing frequency induced by application of insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene.
ferent compared with that of the control group, Aβ + bexarotene group and bexarotene group, respectively (fig. 3f). Thus, we concluded that bexarotene alleviated the effect of Aβ25–35 on the half-width, which made it change to the direction of control group. Changes in the Properties of the AP between Groups May Involve Potassium Currents Studies from our laboratory have reported that voltage-dependent outward K+ currents played the major Dai/Yang/Chen/Yang
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Fig. 3. Effect of insulin on the half-width of the sAP. a sAPs were
elicited by 5-ms brief depolarizing current pulses before and after application of insulin in the control group. b–e Comparison of the half-width of the sAP before and after application of insulin in each group: control, from 3.02 ± 0.17 to 3.73 ± 0.23 ms; Aβ, from 2.55 ± 0.11 to 2.27 ± 0.18 ms; Aβ + bexarotene, from 3.04 ±
role in regulating neuronal excitability [20, 22, 23]. Thus, we examined the downstream mechanism of the protection of bexarotene. When recording potassium currents, the neurons were held at –70 mV and current traces were evoked by using an 80-ms constant depolarizing pulse from –50 to +90 mV in increments of 10 mV (fig. 4a). In the current-voltage curves shown in figure 4b–e, we observed that outward currents were reduced by the application of insulin from +60 mV, which was visible on current-voltage curves in control group (fig. 4b). However, after application of Aβ, the effect of insulin on K+ channels was abolished (fig. 4c). In contrast, bexarotene could enhance the effect of insulin (fig. 4d) and reverse the effect of Aβ in the Aβ + bexarotene group (fig. 4e). After normalization by the formula (R2 – R1)/R1, the changes in the amplitude of potassium currents at +90 mV in four groups were shown in figure 4f, and there were significant differences between them (p < 0.01, oneway ANOVA).
Bexarotene Improves Aβ-Induced Dysfunction
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0.19 to 3.29 ± 0.17 ms; bexarotene, from 2.77 ± 0.2 to 4.35 ± 0.24 ms. f The change in the half-width of sAP caused by insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene.
Discussion
AD is a chronic and devastating neurodegenerative disorder that impairs the patient’s memory, destroys the ability to reason, rational judgments and leads to myriad behavioral and psychiatric symptoms. The accumulation of Aβ in the brain is postulated to initiate a cascade of events leading to AD [24]. However, therapeutic approaches that aim to reduce Aβ production or aggregation have so far been unsuccessful. However, Cramer et al. [3] recently reported that the drug bexarotene (Targretin), already approved by the Food and Drug Administration for the treatment of cutaneous T cell lymphoma, showed promising efficacy in preclinical models of AD. In this study, we proposed an additional mechanism of action of bexarotene, whereby it alleviated Aβ-induced neuron dysfunction in insulin signaling pathway. Furthermore, despite the encouraging results reported by Cramer et al. [3], several groups have failed to replicate the effect of bexarotene on Aβ plaque burden in these and other related Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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20 mV
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Fig. 4. Effect of insulin on outward potassium currents. a Potassium currents were obtained by 80-ms depolarizing pluses from a command potential of –50 to +90 mV in increments of 10 mV, and the holding potential was –70 mV. The diagram shows the potassium current wave before and after application of insulin in the control group. b–e Comparison of current-voltage curves before and after
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Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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application of insulin in the control (b), Aβ (c), Aβ + bexarotene (d) and bexarotene (e) group. f The change in the amplitude of the potassium current at +90 mV induced by insulin among the four groups, obtained by the following formula: (R2 – R1)/R1. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01. con = Control; bex = bexarotene; IK = delayed rectifying K+ channels. (For figure 4f see next page.) Dai/Yang/Chen/Yang
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mouse models [25–29], thus the effects of this compound remain controversial and need to be studied further. The results of our study supported the hypothesis in terms of the electrophysiological properties of rat hippocampal CA1 neurons. We demonstrated in this study that the protective effects of bexarotene against Aβ-induced impairment in neurons may involve the insulin signaling pathway. As we know, the AP is a fundamental property of excitable cells in the mammalian central nervous system and a dynamic reflection of changes in ion channels in the membrane. Either activation of signaling pathways or toxic impairment would modify the excitability of neurons. Consistent with this principle, our study did indicate that the application of insulin to normal neurons led to an increase in their excitability. Nonetheless, the direction of the response to insulin was reversed after the application of Aβ, which was represented by the decreased frequency of APs. Intriguingly, the deterioration of the response to insulin caused by Aβ was ameliorated by the application of bexarotene. In the bexarotene + Aβ group, the impact of insulin on neurons was restored. Moreover, bexarotene alone could enhance the response to insulin, which indicated an enhanced IR sensitivity. These results suggested that bexarotene had a protective effect against Aβ-induced impairment through the insulin signaling pathway. In order to explore the detailed mechanism, we focused on an important ion channel which plays an essential role in determining the excitability of neurons. It has been acknowledged that ion channels in the cell membrane are targets of many toxins and drugs, and in the nervous system, voltage-gated K+ currents are largely responsible for repolarization and hyperpolarization, which constitute the major part of the whole AP and therefore regulate a variety of neuronal properties, such as resting membrane potential, AP waveform and firing frequency. Thus, we tested whethBexarotene Improves Aβ-Induced Dysfunction
er the protective effect of bexarotene on excitability involved K+ channels, and the results offered a probable explanation for the alteration of neuron excitability. Previous studies have proved that the reduction of potassium currents will result in hyperexcitability of neurons [23, 30]. In addition, the sAP waveform was also altered in all four of our experimental groups, especially the half-width of the sAP, the change in which was similar to K+ currents. The half-width of sAP is most commonly measured as the width at half-maximal spike amplitude, which represents the duration of the AP. It has been reported that blocking of potassium currents would prolong the duration of the AP [31, 32]. The reason may be that the decreased outward K+ currents will lead to a longer time of repolarization for the AP. Thus, we speculated that the protective effect of bexarotene in Aβ-impaired excitability and sAP duration was mainly due to action on K+ channels through the insulin pathway. Although we proved that bexarotene could enhance IR sensitivity, the detailed mechanism was still unknown, because the decline in IR function can originate with aberrations in the expression or phosphorylation state of the receptor. For example, in certain familial forms of insulin resistance, the defective IR function is caused by the severe lesion of IR alleles and substantial decreases in IR levels [33]. Alternatively, the suppression of IR enzymatic activity has been linked to negative regulatory factors stimulated by IR signaling as well as other pathways. Additionally, IR activity can be inhibited by a downstream negative feedback pathway [34–36]. Due to all these possibilities, further studies need to be performed to explore the mechanism by which bexarotene enhances IR sensitivity.
Conclusion
This study demonstrated that the insulin signaling pathway may be an additional mechanism underlying the protective effect of bexarotene against Aβ25–35-induced dysfunction in hippocampal neurons, which was represented by neuronal AP properties, and the target might involve potassium channels. Therefore, the results provide further insight into the underlying mechanisms responsible for the effects of bexarotene on AD. Acknowledgements This work was partly supported by a grant from the National Basic Research Program of China (2011CB944003) and the National Natural Science Foundation of China (31271074 and 31300923).
Neurodegener Dis 2014;14:77–84 DOI: 10.1159/000358397
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