- Email: [email protected]
Estradiol alleviates the ischemic brain injury-induced decrease of neuronal calcium sensor protein hippocalcin
Neuroscience Letters 582 (2014) 32–37
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Estradiol alleviates the ischemic brain injury-induced decrease of neuronal calcium sensor protein hippocalcin Phil-Ok Koh ∗ Department of Anatomy, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, 501 Jinjudaero, Jinju 660-701, Gyeongnam, South Korea
h i g h l i g h t s • Estradiol plays a neuroprotective role against neuronal cell injury. • Estradiol prevents brain injury-induced decrease of in hippocalcin levels. • Estradiol attenuates the glutamate treatment-induced decrease in hippocalcin levels.
a r t i c l e
i n f o
Article history: Received 4 August 2014 Received in revised form 25 August 2014 Accepted 25 August 2014 Available online 3 September 2014 Keywords: Estradiol Hippocalcin Neuroprotection
a b s t r a c t Estradiol has protective and reparative effects in neurodegenerative diseases. Hippocalcin is a neuronal calcium-sensor protein that acts as a calcium buffer to regulate the intracellular concentration of Ca2+ . This study was investigated to elucidate whether estradiol regulates hippocalcin expression in a focal cerebral ischemia model and glutamate-treated neuronal cells. An ovariectomy was performed in adult female rats, and vehicle or estradiol was administered before middle cerebral artery occlusion (MCAO). Cerebral cortex tissues were collected at 24 h after MCAO. A proteomic approach revealed that hippocalcin expression decreased in vehicle-treated animals with combined MCAO, while estradiol treatment attenuated this decrease. Reverse transcription-PCR and Western blot analyses also showed that estradiol administration prevented the MCAO injury-induced decrease in hippocalcin expression. In cultured hippocampal cells, glutamate exposure increased the intracellular Ca2+ concentration, which was rescued by the presence of estradiol. Moreover, glutamate toxicity decreased hippocalcin expression, whereas estradiol attenuated this decrease. Together, these findings suggest that estradiol has a neuroprotective function by regulating hippocalcin expression and intracellular Ca2+ levels in ischemic brain injury. © 2014 Elsevier Ireland Ltd. All rights reserved.
Estradiol is a representative female sexual steroid hormone. In addition to its reproductive function, estradiol plays a neuroprotective role against neuronal cells injury [26,28]. Estradiol prevents neuronal cells death in response to oxidative stress and alleviates brain lesions in focal cerebral ischemia [5,7]. Clinical studies have demonstrated that estradiol reduces the progression of Alzheimer’s disease and the risk of stroke [7,19]. Consistent with this, the risk of neurological diseases increases in postmenopausal women compared to age-matched men [3,22]. However, premenopausal women suffer less from neurodegenerative diseases than adult men [22]. Moreover, estradiol replacement therapy reduces the mortality of stroke-related deaths [23].
∗ Tel.: +82 55 772 2354; fax: +82 55 772 2349. E-mail address: [email protected] http://dx.doi.org/10.1016/j.neulet.2014.08.045 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Intracellular Ca2+ is involved in neuronal cells growth, neuronal transmission, and synaptic plasticity [2]. However, an excessive increase in intracellular Ca2+ activates the caspase cascade, leading to cell death and neuronal disorder [9]. Ischemic brain injury increases intracellular Ca2+ levels and leads to apoptotic and necrotic cell death [25]. Hippocalcin is a calcium-binding protein that is expressed mainly in the brain. It belongs to the neuronal calcium-sensor protein family and acts as a calcium buffer because it binds to Ca2+ that is released in the cytoplasm [10,21]. Thus, regulation of hippocalcin expression is considered important for intracellular Ca2+ homeostasis. Moreover, it has been reported that estradiol reduces the increase in intracellular Ca2+ concentration that occurs following neuronal injury [14]. Although previous studies have demonstrated that estradiol regulates Ca2+ concentration, it is unclear whether hippocalcin is regulated by estradiol in ischemic brain injury. A proteomics technique was used to identify various proteins that are differentially expressed following
P.-O. Koh / Neuroscience Letters 582 (2014) 32–37
estradiol treatment during ischemic brain injury. Among these identified proteins, we focused on change of hippocalcin. In this study, we investigated whether estradiol regulates hippocalcin expression in a middle cerebral artery occlusion (MCAO) animal model and glutamate-exposed neuronal cells. Female Sprague-Dawley rats (220–230 g, n = 60) were purchased from Samtako Co. (Animal Breeding Center, Korea) and maintained under controlled temperature and lighting (12 h/12 h light/dark cycle) with free access to food and water. Animals were divided randomly into the following four groups: vehicle + sham, estradiol + sham, vehicle + MCAO, and estradiol + MCAO groups (n = 15 per group). Animals were subjected to an ovariectomy and were implanted with a silastic capsule containing sesame oil (Sigma, St. Louis, MO, USA. vehicle) or 17-estradiol (180 g/ml, Sigma). This capsule of 17-estradiol consistently produces estradiol levels equivalent to circulating physiological levels [5]. Animals were anesthetized with sodium pentobarbital (100 mg/kg) and MCAO was performed as described previously [15]. Briefly, the bifurcation of the right common carotid artery was exposed through a midline incision. The internal carotid artery and external carotid artery were dissected from the adjacent tissues. A 4/0 monofilament nylon with its tip rounded by heat was gently inserted from the external carotid artery into the internal carotid artery, and advanced up to the origin of the middle cerebral artery. Sham-operated animals were subjected to the same surgical process without arterial blockade. Hematoxylin and eosin staining was performed for the histopathological finding. Brain tissues were fixed in 4% phosphate buffered paraformaldehyde solution, embedded with paraffin, and cut into 4 m coronal section. The paraffin section were deparaffined in xylene and rehydrated in gradient ethanol from 100% to 70%. The sections were generally stained with hematoxylin and eosin solution. The stained sections were dehydrated using gradient ethanol, slipped with permount (Sigma), and observed under light microscope. A proteomic analysis was performed as previously described [11]. Right cerebral cortices were homogenized in lysis buffer (8 M urea, 4% CHAPS, ampholytes, and 40 mM Tris–HCl). Protein extracts were centrifuged at 16,000 × g for 20 min at 4 ◦ C. Bradford assay kit (Bio-Rad, Hercules, CA, USA) was used to determine total protein concentration. Total protein (100 g) was subjected to isoelectric focusing (IEF) on immobilized pH gradient (IPG) gel strips (pH 4–7 and pH 6–9, 17 cm, Bio-Rad). IEF was performed using a multistep protocol: 250 V (15 min), 10,000 V (3 h), and then 10,000 V to 50,000 V using a Protean IEF Cell (Bio-Rad). After equilibration of the IEF strips, strips were applied to gradient gels (7.5–17.5%) for second dimension electrophoresis. Gels were loaded on Protein-II XI electrophoresis equipment (Bio-Rad) at 5 mA for 2 h followed by 10 mA for 10 h at 10 ◦ C. Gels were fixed in 12% acetic acid and 50% methanol for 2 h, and stained with silver solution (0.2% silver nitrate, 0.75 ml/l formaldehyde) for 20 min. Gel images were recorded immediately using Agfar ARCUS 1200TM (Agfar-Gevaert, Mortsel, BEL). Spot analysis was performed using PDQuest 2-D analysis software (Bio-Rad). Gel pieces containing the desired protein spots were excised and destained for MALDI-TOF. Gel particles were digested in trypsin-containing buffer. Extract peptides were analyzed using a Voyager-DETM STR biospectrometry workstation (Applied Biosystem, Foster city, CA, USA) for MALDI-TOF mass spectrometry. Proteins were identified using the search programs MS-Fit and ProFound. SWISS-PROT and NCBI were used as the target protein sequence databases. The intensity of protein spots was measured using PDQuest software. The spot intensity is described as a ratio of the intensity of that spot in the experimental group relative to that of the corresponding spot in the vehicle + sham group.
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Fig. 1. Representative photos of H–E stain in the cerebral cortices from vehicle + middle cerebral artery occlusion (MCAO) (A), vehicle + sham (B), estradiol + MCAO (C), estradiol + sham (D) animals. Arrows indicate apoptotic bodies and arrowhead indicate necrotic changes with scalloped shrunken form in ischemic lesion. Scale bar = 100 m.
Right cerebral cortices were collected and frozen quickly. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA synthesis was performed with total RNA (1 g) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. The following primers were used: hippocalcin primer (forward primer, 5 -ACGCCAACTTCTTCCCCTATG-3 ; reverse primer, 5 -AGCCATCAGCGTCTTTGTTT-3 ) and actin primer (forward primer, 5 -GGGTCAGAAGGACTCCTACG-3 ; reverse primer, 5 TTTCACTGCGGCTGATGTAG-3 ). The amplification program consists of a denaturating at 94 ◦ C for 30 s, annealing at 54 ◦ C for 30 s, and extension at 72 ◦ C for 1 min. Samples were amplified for 30 cycles. PCR products were run on a 1% agarose gel and visualized under UV light. For Western blot analysis, equal amounts of protein (30 g) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with 5% skim milk solution for 1 h and then washed in Tris-buffered saline containing 0.1% Tween-20 (TBST). Membranes were probed with the following antibodies: anti-hippocalcin (diluted 1:1000, Abcam, Cambridge, UK) and anti-actin (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibodies at 4 ◦ C for 15 h. Membranes were then rinsed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Pierce, Rockford, IL, USA). Immunoreactivity was detected using enhanced chemiluminescence (ECL Western blotting detection kit, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Mouse hippocampal cells (HT22) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone Laboratory, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum
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Fig. 2. Proteomic analysis (A and B), reverse transcription-PCR analysis (C and D), and Western blot analysis (E and F) of hippocalcin in the cerebral cortices from vehicle + middle cerebral artery occlusion (MCAO), estradiol + MCAO, vehicle + sham, estradiol + sham animals. Circles indicate hippocalcin protein spots (A). Spot intensities were measured by PDQuest software. Spot intensities are reported as a ratio relative to vehicle + sham animals (B). Densitometric analyses from reverse transcription-PCR (D) and Western blot (F) are represented as a ratio of hippocalcin intensity to actin intensity. Each lane represents an individual animal. Data (n = 4) are shown as means ± S.E.M. * P < 0.05. Mw and pI indicate molecular weight and isoelectric point, respectively.
(Gibco BRL, Gaithersburg, MD, USA), penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C under an atmosphere of 95% air and 5% CO2 [17]. Cells were seeded onto 60-mm tissue culture dishes at a density of 100,000 cells per dish, and were cultured for 24 h. Glutamate (Sigma) was added to cells at a final concentration of 5 mM and cells were then incubated for 24 h. 17-Estradiol was added 15 min before the addition of glutamate (Sigma). 17Estradiol was dissolved in 95% ethanol at a concentration of 1 mM and diluted to the appropriate concentration (1 M or 10 M) in culture medium. This concentration of ethanol had no effect on cell viability or glutamate toxicity. Cell viability was determined by measuring metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT). MTT solution (5 mg/ml) was added to each well in serum-free medium and cells were incubated for 2 h. Medium was removed and formazan dye was trapped in the living cells. Absorbance was measured at a wavelength of 570 nm.
Cell survival was expressed as the percentage of neuroprotection vs. vehicle (set to100%). Cells were fixed in 4% paraformaldehyde for 20 min, washed with phosphate buffer saline (PBS), incubated with 5% normal goat serum (Jackson Immuno Research labs, West Grove, PA, USA) for 1 h, and then washed with PBS. Cells were reacted with antihippocalcin antibody (diluted 1:100, Cell Signaling) at 4 ◦ C for 15 h, followed by reaction with a fluorescein isothiocyanate (FITC)conjugated secondary antibody (1:200, Jackson Immuno Research labs). The positive cells for hippocalcin were observed under a fluorescence microscope (AXIO, Carl Zeiss Corporation, Thornwood, NY, USA). Intracellular Ca2+ concentration was measured with Fura2/AM [18]. Fura-2/AM, a calcium-sensitive fluorescent dye, is a fluorescent intracellular Ca2+ indicator. Fura-2/AM fluorescence dual-wavelength excitation was performed as described previously
P.-O. Koh / Neuroscience Letters 582 (2014) 32–37
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Fig. 3. Cell viability (A), intracellular calcium concentration (B), Western blot analysis (C), and immunocytochemical staining (D) of hippocalcin in hippocampal neuronal cells (HT22). HT22 cells were treated with glutamate (5 mM) for 24 h and estradiol (1 and 10 M) was added 15 min before glutamate exposure. Cell viability was assessed with the MTT assay (A). Cell survival was expressed as a percentage of vehicle-treated cells. Neurons were labeled with Fura-2/AM, and fluorescence spectra for calcium were measured using a luminescence spectrophotometer (B). Densitometric analysis results are presented as the ratio of hippocalcin intensity to actin intensity (C). Data (n = 3) are presented as means ± S.E.M. * P < 0.05. Estradiol treatment prevented the glutamate-induced decrease in number of hippocalcin-positive HT22 cells (D). Positive cells of hippocalcin fluoresced green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[6,11]. Cells were incubated in DMEM media containing 10 M fura-2/AM at 37 ◦ C for 1 h, then rinsed to remove fura-2/AM and centrifuged at 1000 × g for 5 min. Fura-2/AM fluorescence signals were measured with a luminescence spectrophotometer (LS50B, Perkin Elmer, Boston, MA, USA). Fura-2 fluorescence signals were analyzed by a Micro Vax II computer and software (Origin 7). All data are expressed as mean ± S.E.M. The results in each group were compared by two-way analysis of variance (ANOVA) followed by to post-hoc Scheffe’s test. A P < 0.05 was considered to represent statistical significance. Fig. 1 showed the histopathological changes in ischemic core area. Both necrotic cells and apoptotic cells were observed in ischemic lesion of vehicle + MCAO animals (Fig. 1A). Apoptotic cells typically appeared intensive dark apoptotic bodies and necrotic cells contained scalloped shrunken form. However, sham-operated animals had intact neurons (Fig. 1B and D). Moreover, estradiol treatment decreased the apoptotic cells and necrotic cells during MCAO (Fig. 1C). A proteomics approach revealed that hippocalcin protein expression was decreased in the cerebral cortices
of vehicle + MCAO animals compared to sham-operated animals. However, estradiol treatment restored the MCAO injury-induced decrease in hippocalcin expression (Fig. 2A). The peptide mass of hippocalcin was 10/102 and the sequence of this protein was 52%. Hippocalcin levels were 0.38 ± 0.03 and 1.03 ± 0.04 in vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2B). Reverse transcription-PCR and Western blot analyses showed that hippocalcin levels were decreased in vehicle + MCAO animals versus sham animals, whereas these decreases in hippocalcin were attenuated in estradiol + MCAO animals (Fig. 2C and E). Transcript levels of hippocalcin were 0.57 ± 0.03 and 0.73 ± 0.02 in the cerebral cortices of vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2D). Hippocalcin protein levels were 0.78 ± 0.02 and 1.05 ± 0.02 in the cerebral cortices of vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2F). Hippocalcin transcript and protein levels were similar in vehicle + sham and estradiol + sham animals. Fig. 3A showed the neuroprotective effect of estradiol against glutamate toxicity. Glutamate exposure dramatically decreased
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P.-O. Koh / Neuroscience Letters 582 (2014) 32–37
cell viability, but estradiol treatment reduced neuronal cells death resulting from glutamate toxicity. Moreover, glutamate treatment increased intracellular Ca2+ levels, whereas estradiol treatment attenuated the glutamate toxicity-induced increase in intracellular Ca2+ (Fig. 3B). Western blot analysis also showed that hippocalcin expression decreased in the glutamate-treated group, whereas estradiol treatment restored the glutamate exposureinduced decrease (Fig. 3C). Hippocalcin levels were 0.57 ± 0.03 in the glutamate-treated group, and 0.94 ± 0.04 and 1.07 ± 0.02 in the estradiol-treated groups (1 M and 10 M, respectively) (Fig. 3D). Immunocytochemical staining clearly confirmed the hippocalcin-positive reaction in glutamate- and estradiol-treated HT22 cells. The number of hippocalcin-positive cells decreased in the glutamate-exposed group, whereas estradiol treatment attenuated this decrease (Fig. 3E). We previously demonstrated that estradiol protects neuronal cells from focal cerebral ischemia and glutamate toxicity-induced cell death [12]. Estradiol has been shown to significantly reduce infarct lesions following ischemic stroke and improve neuronal dysfunction [26,28]. Our previous study reported various proteins that are differentially expressed following estradiol treatment during cerebral ischemia in an animal model using a proteomics technique [27]. In this study, we additionally identified the change of hippocalcin protein following estradiol treatment during ischemic brain injury. Moreover, we demonstrated that estradiol regulated hippocalcin expression and modulated intracellular Ca2+ levels in MCAO-induced injury and glutamate-induced neuronal cells death. An increase in intracellular Ca2+ triggers cell death due to the activation of proteases and caspases [1,13]. Ischemic brain injury leads to oxygen deprivation and intracellular Ca2+ elevation, resulting in neuronal cells death [25]. Hippocalcin regulates calcium extrusion from neurons and protects neuronal cells against calcium-dependent excitotoxin damage [20]. Our proteomics study demonstrated that MCAO-induced injury caused a reduction in hippocalcin expression, while estradiol administration attenuated this reduction. Reverse transcription-PCR and Western blot analyses clearly confirmed that MCAO injury decreased hippocalcin expression and that estradiol prevented this decrease. These results demonstrated that estradiol regulated hippocalcin expression in MCAO animal model. A reduction in hippocalcin indicates an increase in intracellular Ca2+ levels and the initiation of neuronal cells death. Thus, maintenance of hippocalcin expression is important for regulating of intracellular Ca2+ homeostasis and preventing neuronal cells death in ischemic brain injury. MCAO induces a serious damage in cerebral cortex tissues and estradiol prevents cell death of cerebral cortex following MCAO. We observed the change of cerebral cortex from MCAO with estradiol treatment through the histopathological finding. Hippocalcin protein was detected as protein spots with different intensities between vehicle and estradiol-treated animals during MCAO injury. Hippocalcin is heavily expressed in the hippocampus. HT22 cells were used in vitro study to elucidate the relationship between estradiol and hippocalcin in ischemic injury. Glutamate toxicity is a major cause of neuronal cells death in neurodegenerative diseases [4]. Glutamate toxicity produces excess free radicals and leads to oxidative stress [4,17]. Glutamate activates glutamate receptors, prolongs neuronal depolarization, and triggers the deregulation of intracellular Ca2+ homeostasis [1]. Estradiol rapidly reduces glutamate-stimulated intracellular Ca2+ overload in primary hippocampal neuron cultures [8]. Furthermore, estradiol modulates the concentration of Ca2+ in the mitochondrial matrix and cytosol, consequently regulating energy metabolism, neurotransmission, and neuronal cells survival [24]. Estradiol protects neuronal cells against glutamate exposure and prevents the glutamate toxicity-induced increase in intracellular Ca2+ . Moreover,
glutamate toxicity decreased hippocalcin expression, whereas estradiol treatment attenuated this decrease. Immunocytochemical staining also demonstrated that glutamate toxicity decreased the number of hippocalcin-positive cells, and that this decrease was attenuated in the presence of estradiol. As a neuronal calcium sensor protein, hippocalcin plays a critical role in the modulation of intracellular Ca2+ concentration. A deficiency of hippocalcin destroys Ca2+ homeostasis and consequently leads to cells death. This study demonstrated that estradiol regulated intracellular Ca2+ concentration and hippocalcin expression in response to glutamate exposure. Both in vivo and in vitro studies showed that estradiol prevented a decrease in hippocalcin expression under ischemic conditions. Hippocalcin has been shown to interact with the neuronal apoptosis inhibitor protein (NAIP), which is a key regulator of apoptosis [16]. The hippocalcin-NAIP interaction protects neurons against calcium-induced cell death through caspase-3-dependent and independent pathways [21]. Although further studies are needed to explain the relationship between estradiol and hippocalcin in a stroke model, our results convincingly demonstrated that estradiol regulates hippocalcin expression and intracellular Ca2+ levels, thereby preventing neuronal cells death due to ischemic damage. Taken together, these results can suggest that estradiol prevents neuronal cells death in ischemic brain injury via modulation of hippocalcin expression and regulation of intracellular Ca2+ concentrations.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF2013R1A1A2007300).
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Estradiol alleviates the ischemic brain injury-induced decrease of neuronal calcium sensor protein hippocalcin Phil-Ok Koh ∗ Department of Anatomy, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, 501 Jinjudaero, Jinju 660-701, Gyeongnam, South Korea
h i g h l i g h t s • Estradiol plays a neuroprotective role against neuronal cell injury. • Estradiol prevents brain injury-induced decrease of in hippocalcin levels. • Estradiol attenuates the glutamate treatment-induced decrease in hippocalcin levels.
a r t i c l e
i n f o
Article history: Received 4 August 2014 Received in revised form 25 August 2014 Accepted 25 August 2014 Available online 3 September 2014 Keywords: Estradiol Hippocalcin Neuroprotection
a b s t r a c t Estradiol has protective and reparative effects in neurodegenerative diseases. Hippocalcin is a neuronal calcium-sensor protein that acts as a calcium buffer to regulate the intracellular concentration of Ca2+ . This study was investigated to elucidate whether estradiol regulates hippocalcin expression in a focal cerebral ischemia model and glutamate-treated neuronal cells. An ovariectomy was performed in adult female rats, and vehicle or estradiol was administered before middle cerebral artery occlusion (MCAO). Cerebral cortex tissues were collected at 24 h after MCAO. A proteomic approach revealed that hippocalcin expression decreased in vehicle-treated animals with combined MCAO, while estradiol treatment attenuated this decrease. Reverse transcription-PCR and Western blot analyses also showed that estradiol administration prevented the MCAO injury-induced decrease in hippocalcin expression. In cultured hippocampal cells, glutamate exposure increased the intracellular Ca2+ concentration, which was rescued by the presence of estradiol. Moreover, glutamate toxicity decreased hippocalcin expression, whereas estradiol attenuated this decrease. Together, these findings suggest that estradiol has a neuroprotective function by regulating hippocalcin expression and intracellular Ca2+ levels in ischemic brain injury. © 2014 Elsevier Ireland Ltd. All rights reserved.
Estradiol is a representative female sexual steroid hormone. In addition to its reproductive function, estradiol plays a neuroprotective role against neuronal cells injury [26,28]. Estradiol prevents neuronal cells death in response to oxidative stress and alleviates brain lesions in focal cerebral ischemia [5,7]. Clinical studies have demonstrated that estradiol reduces the progression of Alzheimer’s disease and the risk of stroke [7,19]. Consistent with this, the risk of neurological diseases increases in postmenopausal women compared to age-matched men [3,22]. However, premenopausal women suffer less from neurodegenerative diseases than adult men [22]. Moreover, estradiol replacement therapy reduces the mortality of stroke-related deaths [23].
∗ Tel.: +82 55 772 2354; fax: +82 55 772 2349. E-mail address: [email protected] http://dx.doi.org/10.1016/j.neulet.2014.08.045 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Intracellular Ca2+ is involved in neuronal cells growth, neuronal transmission, and synaptic plasticity [2]. However, an excessive increase in intracellular Ca2+ activates the caspase cascade, leading to cell death and neuronal disorder [9]. Ischemic brain injury increases intracellular Ca2+ levels and leads to apoptotic and necrotic cell death [25]. Hippocalcin is a calcium-binding protein that is expressed mainly in the brain. It belongs to the neuronal calcium-sensor protein family and acts as a calcium buffer because it binds to Ca2+ that is released in the cytoplasm [10,21]. Thus, regulation of hippocalcin expression is considered important for intracellular Ca2+ homeostasis. Moreover, it has been reported that estradiol reduces the increase in intracellular Ca2+ concentration that occurs following neuronal injury [14]. Although previous studies have demonstrated that estradiol regulates Ca2+ concentration, it is unclear whether hippocalcin is regulated by estradiol in ischemic brain injury. A proteomics technique was used to identify various proteins that are differentially expressed following
P.-O. Koh / Neuroscience Letters 582 (2014) 32–37
estradiol treatment during ischemic brain injury. Among these identified proteins, we focused on change of hippocalcin. In this study, we investigated whether estradiol regulates hippocalcin expression in a middle cerebral artery occlusion (MCAO) animal model and glutamate-exposed neuronal cells. Female Sprague-Dawley rats (220–230 g, n = 60) were purchased from Samtako Co. (Animal Breeding Center, Korea) and maintained under controlled temperature and lighting (12 h/12 h light/dark cycle) with free access to food and water. Animals were divided randomly into the following four groups: vehicle + sham, estradiol + sham, vehicle + MCAO, and estradiol + MCAO groups (n = 15 per group). Animals were subjected to an ovariectomy and were implanted with a silastic capsule containing sesame oil (Sigma, St. Louis, MO, USA. vehicle) or 17-estradiol (180 g/ml, Sigma). This capsule of 17-estradiol consistently produces estradiol levels equivalent to circulating physiological levels [5]. Animals were anesthetized with sodium pentobarbital (100 mg/kg) and MCAO was performed as described previously [15]. Briefly, the bifurcation of the right common carotid artery was exposed through a midline incision. The internal carotid artery and external carotid artery were dissected from the adjacent tissues. A 4/0 monofilament nylon with its tip rounded by heat was gently inserted from the external carotid artery into the internal carotid artery, and advanced up to the origin of the middle cerebral artery. Sham-operated animals were subjected to the same surgical process without arterial blockade. Hematoxylin and eosin staining was performed for the histopathological finding. Brain tissues were fixed in 4% phosphate buffered paraformaldehyde solution, embedded with paraffin, and cut into 4 m coronal section. The paraffin section were deparaffined in xylene and rehydrated in gradient ethanol from 100% to 70%. The sections were generally stained with hematoxylin and eosin solution. The stained sections were dehydrated using gradient ethanol, slipped with permount (Sigma), and observed under light microscope. A proteomic analysis was performed as previously described [11]. Right cerebral cortices were homogenized in lysis buffer (8 M urea, 4% CHAPS, ampholytes, and 40 mM Tris–HCl). Protein extracts were centrifuged at 16,000 × g for 20 min at 4 ◦ C. Bradford assay kit (Bio-Rad, Hercules, CA, USA) was used to determine total protein concentration. Total protein (100 g) was subjected to isoelectric focusing (IEF) on immobilized pH gradient (IPG) gel strips (pH 4–7 and pH 6–9, 17 cm, Bio-Rad). IEF was performed using a multistep protocol: 250 V (15 min), 10,000 V (3 h), and then 10,000 V to 50,000 V using a Protean IEF Cell (Bio-Rad). After equilibration of the IEF strips, strips were applied to gradient gels (7.5–17.5%) for second dimension electrophoresis. Gels were loaded on Protein-II XI electrophoresis equipment (Bio-Rad) at 5 mA for 2 h followed by 10 mA for 10 h at 10 ◦ C. Gels were fixed in 12% acetic acid and 50% methanol for 2 h, and stained with silver solution (0.2% silver nitrate, 0.75 ml/l formaldehyde) for 20 min. Gel images were recorded immediately using Agfar ARCUS 1200TM (Agfar-Gevaert, Mortsel, BEL). Spot analysis was performed using PDQuest 2-D analysis software (Bio-Rad). Gel pieces containing the desired protein spots were excised and destained for MALDI-TOF. Gel particles were digested in trypsin-containing buffer. Extract peptides were analyzed using a Voyager-DETM STR biospectrometry workstation (Applied Biosystem, Foster city, CA, USA) for MALDI-TOF mass spectrometry. Proteins were identified using the search programs MS-Fit and ProFound. SWISS-PROT and NCBI were used as the target protein sequence databases. The intensity of protein spots was measured using PDQuest software. The spot intensity is described as a ratio of the intensity of that spot in the experimental group relative to that of the corresponding spot in the vehicle + sham group.
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Fig. 1. Representative photos of H–E stain in the cerebral cortices from vehicle + middle cerebral artery occlusion (MCAO) (A), vehicle + sham (B), estradiol + MCAO (C), estradiol + sham (D) animals. Arrows indicate apoptotic bodies and arrowhead indicate necrotic changes with scalloped shrunken form in ischemic lesion. Scale bar = 100 m.
Right cerebral cortices were collected and frozen quickly. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA synthesis was performed with total RNA (1 g) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. The following primers were used: hippocalcin primer (forward primer, 5 -ACGCCAACTTCTTCCCCTATG-3 ; reverse primer, 5 -AGCCATCAGCGTCTTTGTTT-3 ) and actin primer (forward primer, 5 -GGGTCAGAAGGACTCCTACG-3 ; reverse primer, 5 TTTCACTGCGGCTGATGTAG-3 ). The amplification program consists of a denaturating at 94 ◦ C for 30 s, annealing at 54 ◦ C for 30 s, and extension at 72 ◦ C for 1 min. Samples were amplified for 30 cycles. PCR products were run on a 1% agarose gel and visualized under UV light. For Western blot analysis, equal amounts of protein (30 g) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with 5% skim milk solution for 1 h and then washed in Tris-buffered saline containing 0.1% Tween-20 (TBST). Membranes were probed with the following antibodies: anti-hippocalcin (diluted 1:1000, Abcam, Cambridge, UK) and anti-actin (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibodies at 4 ◦ C for 15 h. Membranes were then rinsed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Pierce, Rockford, IL, USA). Immunoreactivity was detected using enhanced chemiluminescence (ECL Western blotting detection kit, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Mouse hippocampal cells (HT22) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone Laboratory, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum
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Fig. 2. Proteomic analysis (A and B), reverse transcription-PCR analysis (C and D), and Western blot analysis (E and F) of hippocalcin in the cerebral cortices from vehicle + middle cerebral artery occlusion (MCAO), estradiol + MCAO, vehicle + sham, estradiol + sham animals. Circles indicate hippocalcin protein spots (A). Spot intensities were measured by PDQuest software. Spot intensities are reported as a ratio relative to vehicle + sham animals (B). Densitometric analyses from reverse transcription-PCR (D) and Western blot (F) are represented as a ratio of hippocalcin intensity to actin intensity. Each lane represents an individual animal. Data (n = 4) are shown as means ± S.E.M. * P < 0.05. Mw and pI indicate molecular weight and isoelectric point, respectively.
(Gibco BRL, Gaithersburg, MD, USA), penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C under an atmosphere of 95% air and 5% CO2 [17]. Cells were seeded onto 60-mm tissue culture dishes at a density of 100,000 cells per dish, and were cultured for 24 h. Glutamate (Sigma) was added to cells at a final concentration of 5 mM and cells were then incubated for 24 h. 17-Estradiol was added 15 min before the addition of glutamate (Sigma). 17Estradiol was dissolved in 95% ethanol at a concentration of 1 mM and diluted to the appropriate concentration (1 M or 10 M) in culture medium. This concentration of ethanol had no effect on cell viability or glutamate toxicity. Cell viability was determined by measuring metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT). MTT solution (5 mg/ml) was added to each well in serum-free medium and cells were incubated for 2 h. Medium was removed and formazan dye was trapped in the living cells. Absorbance was measured at a wavelength of 570 nm.
Cell survival was expressed as the percentage of neuroprotection vs. vehicle (set to100%). Cells were fixed in 4% paraformaldehyde for 20 min, washed with phosphate buffer saline (PBS), incubated with 5% normal goat serum (Jackson Immuno Research labs, West Grove, PA, USA) for 1 h, and then washed with PBS. Cells were reacted with antihippocalcin antibody (diluted 1:100, Cell Signaling) at 4 ◦ C for 15 h, followed by reaction with a fluorescein isothiocyanate (FITC)conjugated secondary antibody (1:200, Jackson Immuno Research labs). The positive cells for hippocalcin were observed under a fluorescence microscope (AXIO, Carl Zeiss Corporation, Thornwood, NY, USA). Intracellular Ca2+ concentration was measured with Fura2/AM [18]. Fura-2/AM, a calcium-sensitive fluorescent dye, is a fluorescent intracellular Ca2+ indicator. Fura-2/AM fluorescence dual-wavelength excitation was performed as described previously
P.-O. Koh / Neuroscience Letters 582 (2014) 32–37
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Fig. 3. Cell viability (A), intracellular calcium concentration (B), Western blot analysis (C), and immunocytochemical staining (D) of hippocalcin in hippocampal neuronal cells (HT22). HT22 cells were treated with glutamate (5 mM) for 24 h and estradiol (1 and 10 M) was added 15 min before glutamate exposure. Cell viability was assessed with the MTT assay (A). Cell survival was expressed as a percentage of vehicle-treated cells. Neurons were labeled with Fura-2/AM, and fluorescence spectra for calcium were measured using a luminescence spectrophotometer (B). Densitometric analysis results are presented as the ratio of hippocalcin intensity to actin intensity (C). Data (n = 3) are presented as means ± S.E.M. * P < 0.05. Estradiol treatment prevented the glutamate-induced decrease in number of hippocalcin-positive HT22 cells (D). Positive cells of hippocalcin fluoresced green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[6,11]. Cells were incubated in DMEM media containing 10 M fura-2/AM at 37 ◦ C for 1 h, then rinsed to remove fura-2/AM and centrifuged at 1000 × g for 5 min. Fura-2/AM fluorescence signals were measured with a luminescence spectrophotometer (LS50B, Perkin Elmer, Boston, MA, USA). Fura-2 fluorescence signals were analyzed by a Micro Vax II computer and software (Origin 7). All data are expressed as mean ± S.E.M. The results in each group were compared by two-way analysis of variance (ANOVA) followed by to post-hoc Scheffe’s test. A P < 0.05 was considered to represent statistical significance. Fig. 1 showed the histopathological changes in ischemic core area. Both necrotic cells and apoptotic cells were observed in ischemic lesion of vehicle + MCAO animals (Fig. 1A). Apoptotic cells typically appeared intensive dark apoptotic bodies and necrotic cells contained scalloped shrunken form. However, sham-operated animals had intact neurons (Fig. 1B and D). Moreover, estradiol treatment decreased the apoptotic cells and necrotic cells during MCAO (Fig. 1C). A proteomics approach revealed that hippocalcin protein expression was decreased in the cerebral cortices
of vehicle + MCAO animals compared to sham-operated animals. However, estradiol treatment restored the MCAO injury-induced decrease in hippocalcin expression (Fig. 2A). The peptide mass of hippocalcin was 10/102 and the sequence of this protein was 52%. Hippocalcin levels were 0.38 ± 0.03 and 1.03 ± 0.04 in vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2B). Reverse transcription-PCR and Western blot analyses showed that hippocalcin levels were decreased in vehicle + MCAO animals versus sham animals, whereas these decreases in hippocalcin were attenuated in estradiol + MCAO animals (Fig. 2C and E). Transcript levels of hippocalcin were 0.57 ± 0.03 and 0.73 ± 0.02 in the cerebral cortices of vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2D). Hippocalcin protein levels were 0.78 ± 0.02 and 1.05 ± 0.02 in the cerebral cortices of vehicle + MCAO and estradiol + MCAO animals, respectively (Fig. 2F). Hippocalcin transcript and protein levels were similar in vehicle + sham and estradiol + sham animals. Fig. 3A showed the neuroprotective effect of estradiol against glutamate toxicity. Glutamate exposure dramatically decreased
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cell viability, but estradiol treatment reduced neuronal cells death resulting from glutamate toxicity. Moreover, glutamate treatment increased intracellular Ca2+ levels, whereas estradiol treatment attenuated the glutamate toxicity-induced increase in intracellular Ca2+ (Fig. 3B). Western blot analysis also showed that hippocalcin expression decreased in the glutamate-treated group, whereas estradiol treatment restored the glutamate exposureinduced decrease (Fig. 3C). Hippocalcin levels were 0.57 ± 0.03 in the glutamate-treated group, and 0.94 ± 0.04 and 1.07 ± 0.02 in the estradiol-treated groups (1 M and 10 M, respectively) (Fig. 3D). Immunocytochemical staining clearly confirmed the hippocalcin-positive reaction in glutamate- and estradiol-treated HT22 cells. The number of hippocalcin-positive cells decreased in the glutamate-exposed group, whereas estradiol treatment attenuated this decrease (Fig. 3E). We previously demonstrated that estradiol protects neuronal cells from focal cerebral ischemia and glutamate toxicity-induced cell death [12]. Estradiol has been shown to significantly reduce infarct lesions following ischemic stroke and improve neuronal dysfunction [26,28]. Our previous study reported various proteins that are differentially expressed following estradiol treatment during cerebral ischemia in an animal model using a proteomics technique [27]. In this study, we additionally identified the change of hippocalcin protein following estradiol treatment during ischemic brain injury. Moreover, we demonstrated that estradiol regulated hippocalcin expression and modulated intracellular Ca2+ levels in MCAO-induced injury and glutamate-induced neuronal cells death. An increase in intracellular Ca2+ triggers cell death due to the activation of proteases and caspases [1,13]. Ischemic brain injury leads to oxygen deprivation and intracellular Ca2+ elevation, resulting in neuronal cells death [25]. Hippocalcin regulates calcium extrusion from neurons and protects neuronal cells against calcium-dependent excitotoxin damage [20]. Our proteomics study demonstrated that MCAO-induced injury caused a reduction in hippocalcin expression, while estradiol administration attenuated this reduction. Reverse transcription-PCR and Western blot analyses clearly confirmed that MCAO injury decreased hippocalcin expression and that estradiol prevented this decrease. These results demonstrated that estradiol regulated hippocalcin expression in MCAO animal model. A reduction in hippocalcin indicates an increase in intracellular Ca2+ levels and the initiation of neuronal cells death. Thus, maintenance of hippocalcin expression is important for regulating of intracellular Ca2+ homeostasis and preventing neuronal cells death in ischemic brain injury. MCAO induces a serious damage in cerebral cortex tissues and estradiol prevents cell death of cerebral cortex following MCAO. We observed the change of cerebral cortex from MCAO with estradiol treatment through the histopathological finding. Hippocalcin protein was detected as protein spots with different intensities between vehicle and estradiol-treated animals during MCAO injury. Hippocalcin is heavily expressed in the hippocampus. HT22 cells were used in vitro study to elucidate the relationship between estradiol and hippocalcin in ischemic injury. Glutamate toxicity is a major cause of neuronal cells death in neurodegenerative diseases [4]. Glutamate toxicity produces excess free radicals and leads to oxidative stress [4,17]. Glutamate activates glutamate receptors, prolongs neuronal depolarization, and triggers the deregulation of intracellular Ca2+ homeostasis [1]. Estradiol rapidly reduces glutamate-stimulated intracellular Ca2+ overload in primary hippocampal neuron cultures [8]. Furthermore, estradiol modulates the concentration of Ca2+ in the mitochondrial matrix and cytosol, consequently regulating energy metabolism, neurotransmission, and neuronal cells survival [24]. Estradiol protects neuronal cells against glutamate exposure and prevents the glutamate toxicity-induced increase in intracellular Ca2+ . Moreover,
glutamate toxicity decreased hippocalcin expression, whereas estradiol treatment attenuated this decrease. Immunocytochemical staining also demonstrated that glutamate toxicity decreased the number of hippocalcin-positive cells, and that this decrease was attenuated in the presence of estradiol. As a neuronal calcium sensor protein, hippocalcin plays a critical role in the modulation of intracellular Ca2+ concentration. A deficiency of hippocalcin destroys Ca2+ homeostasis and consequently leads to cells death. This study demonstrated that estradiol regulated intracellular Ca2+ concentration and hippocalcin expression in response to glutamate exposure. Both in vivo and in vitro studies showed that estradiol prevented a decrease in hippocalcin expression under ischemic conditions. Hippocalcin has been shown to interact with the neuronal apoptosis inhibitor protein (NAIP), which is a key regulator of apoptosis [16]. The hippocalcin-NAIP interaction protects neurons against calcium-induced cell death through caspase-3-dependent and independent pathways [21]. Although further studies are needed to explain the relationship between estradiol and hippocalcin in a stroke model, our results convincingly demonstrated that estradiol regulates hippocalcin expression and intracellular Ca2+ levels, thereby preventing neuronal cells death due to ischemic damage. Taken together, these results can suggest that estradiol prevents neuronal cells death in ischemic brain injury via modulation of hippocalcin expression and regulation of intracellular Ca2+ concentrations.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF2013R1A1A2007300).
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