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Effects of ApoE on intracellular calcium levels and apoptosis of neurons after mechanical injury
Neuroscience 301 (2015) 375–383
EFFECTS OF APOE ON INTRACELLULAR CALCIUM LEVELS AND APOPTOSIS OF NEURONS AFTER MECHANICAL INJURY L. JIANG, a J. ZHONG, a X. DOU, b C. CHENG, a Z. HUANG a AND X. SUN a*
INTRODUCTION Traumatic brain injury (TBI) is one of the most common central nervous system disorders with high morbidity and mortality. It often brings disastrous consequences and heavy economic burden to patients’ families and society (Werner and Engelhard, 2007; Maas et al., 2008; Hiekkanen et al., 2009). Although the processes underlying TBI have been studied and are better understood over the last decade, improving the prognosis of TBI remains challenging (Loane and Faden, 2010). The TBI process is generally divided into two stages: the primary injury and the secondary injury (or delayed injury) (Maas et al., 2008; Loane and Faden, 2010). The primary injury may be prevented but cannot be therapeutically treated. The secondary injury, which consists of a series of complicated events and is influenced by many factors, may be limited by influencing the factors involved in the process (Maas et al., 2008; Loane and Faden, 2010). Calcium plays a vital role in regulating cellular functions and has long been implicated in the process after TBI (Tymianski and Tator, 1996; Weber et al., 2001; Werner and Engelhard, 2007). Variation of intracellular calcium concentration can dramatically influence the function and metabolism of cells, and calcium overload can directly induce cell apoptosis. Therefore, maintenance of intracellular calcium homeostasis is of critical importance for cells, especially after injury. Intracellular calcium can be derived from two sources: exogenous calcium, the influx of extracellular calcium through calcium channels such as N-methyl-D-aspartate receptor (NMDAR), or endogenous calcium, the release of calcium from intracellular calcium stores such as endoplasmic reticulum (ER) and mitochondria (Weber et al., 2001; Veinbergs et al., 2002). Intracellular calcium levels ([Ca2+]i) after TBI can be influenced by both exogenous and endogenous calcium. Several genes have been implicated in the process after TBI and many studies suggest that apolipoprotein e gene (APOE) polymorphisms may influence the outcome of TBI (Teasdale et al., 1997; Crawford et al., 2002, 2009). Apolipoprotein e (ApoE) is a multifunctional protein involved predominantly in the transportation of cholesterol, maintenance of microtubules, and neural transmission. It is the major apolipoprotein in the central nervous system (CNS), and participates in the process after TBI. Three ApoE isoforms have been identified in humans, ApoE2, ApoE3 and ApoE4, which are encoded by three different alleles (e2, e3, and e4, respectively). APOEe4 is believed to be a negative factor contributing
a
Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, PR China b
Chongqing Medical University, PR China
Abstract—Objective: The current study aimed to explore the effects of apolipoprotein e (ApoE) on intracellular calcium ([Ca2+]i) and apoptosis of neurons after mechanical injury in vitro. Methods: A neuron mechanical injury model was established after primary neurons obtained from APOE knockout and wild-type (WT) mice, and four experimental groups were generated: Group-ApoE4, Group-ApoE3, Group-ApoE( ) and Group-WT. Recombinant ApoE4 and ApoE3 were added to Group-ApoE4 and Group-ApoE3 respectively, and Group-ApoE( ) and Group-WT were control groups. Intracellular calcium was labeled by fluo-3/AM and examined using laser scanning confocal microscope and flow cytometry, and the apoptosis of neurons was also evaluated. Results: The intracellular calcium levels and apoptosis rates of mice neurons were significantly higher in Group-ApoE4 than in Group-ApoE3 and Group-WT after mechanical injury. However, without mechanical injury on neurons, no significant differences in intracellular calcium levels and apoptosis rates were found among all four experimental groups. The effects of ApoE4 on intracellular calcium levels and apoptosis rates of injured neurons were partly decreased by EGTA treatment. Conclusion: Compared with ApoE3-treatment and WT neurons, ApoE4 caused higher intracellular calcium levels and apoptosis rates of neurons after mechanical injury. This suggested APOE polymorphisms may affect neuron apoptosis after mechanical injury through different influences on intracellular calcium levels. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: TBI, intracellular calcium polymorphism, neuron, mechanical injury.
level,
APOE
*Corresponding author. Address: Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, No. 1, Youyi Road, Yuanjiagang, Yuzhong District, Chongqing 400016, PR China. Tel: +86-23-89011152; fax: +86-23-68734337. E-mail address: [email protected] (X. Sun). Li Jiang and Jianjun Zhong contributed equally to this work. Abbreviations: ApoE, apolipoprotein e; BBB, blood–brain barrier; EGTA, ethylene glycol tetraacetic acid; FCM, flow cytometry; FITC, fluorescein isothiocyanate; MK801, dizocilpine; NMDAR, N-methyl-Daspartate receptor; PBS, phosphate-buffered saline; PI, propidium iodide; TBI, traumatic brain injury; WT, wild-type. http://dx.doi.org/10.1016/j.neuroscience.2015.06.005 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 375
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to unfavorable outcomes after TBI, and ApoE4, encoded by APOEe4, is considered a dysfunctional or harmful protein. For example, compared with ApoE2 and ApoE3, ApoE4 may induce more Ab deposition and generate more neurotoxic segments after being cleaved by a protease (Yasuda et al., 1993; Teasdale et al., 1997; Friedman et al., 1999; Chang et al., 2005). However, the exact mechanism through which ApoE4 negatively affects the outcome of TBI is still unknown. To investigate ApoE involvement in the process after TBI, an in vitro mechanical injury model of neurons was generated, and the effects of different ApoE isoforms on [Ca2+]i and apoptosis of neurons were measured after mechanical injury.
EXPERIMENTAL PROCEDURES Preparation of primary neuron culture This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chongqing Medical University. Neurons were harvested by a standard enzyme treatment protocol from APOE knockout (APOE-KO) mice (obtained from the Peking University Laboratory Animal Centre) and wildtype (WT) mice (obtained from the Laboratory Animal Centre of the Chongqing Medical University) within 24 h after birth. Neurons harvested from three mice were included in each group. Briefly, the cerebral cortex was minced into 2 mm3 pieces and dissociated with trypsin to obtain a cell suspension. After centrifugation, the resulting cell pellets were resuspended in DMEM/F12 containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, New York, USA) and added to culture flasks. Primary cultures were maintained in a humidified incubator with 5% CO2, 95% air at 37 °C. The culture medium was replaced with fresh medium every day, and neurons were cultured for 5 days. The neuron purity was determined by neuron specific enolase (NSE) positivity through immunocytochemistry.
recombinant human ApoE3 and ApoE4 were obtained from Peprotech (Peprotech, Rocky Hill, New Jersy, USA). The concentration of recombinant ApoE in the extracellular fluid was maintained at 10 lg/ml (Qiu et al., 2003), and neurons were treated with recombinant ApoE for 24 h (Veinbergs et al., 2002). Analysis of calcium levels [Ca2+]i in neurons was evaluated at 2, 12 and 24 h after injury by using the calcium indicator fluo-3/AM and by measuring the fluorescence intensity through laser scanning confocal microscopic imaging (Johnson et al., 2007). Briefly, neurons were loaded with fluo-3/AM (Biotium, Bay Area, San Francisco, USA) for 30 min at 37 °C in the dark, and then washed with D-Hanks medium. Fluo-3 was excited at 488 nm, and the fluorescence was detected by a (Rice et al., 1998) (Leica, Wetzlar, Hesse, Germany). In each microscopic field, the fluorescence intensity of approximately 5–10 neuron soma around the injured region was separately measured. The fluorescence intensity of selected microscopic fields was digitized. All data were analyzed by the accompanying software of the laser scanning confocal microscope system. The mean fluorescence intensity of [Ca2+]i in each group was also detected by flow cytometry (FCM) at 2, 12 and 24 h after injury. Neurons were harvested, washed twice with phosphate-buffered saline (PBS), resuspended and loaded with 5 lmol/l fluo-3/AM for 30 min at 37 °C in the dark. After washing with D-Hanks medium, neurons were immediately analyzed using a flow cytometer (Beckman Coulter, Epics XL) by counting 10,000 events per sample. To determine the source of calcium after mechanical injury, the calcium chelator EGTA (Sigma, Saint Louis, Missouri, USA) was applied to block the influx of extracellular calcium (Veinbergs et al., 2002). Meanwhile, MK801, an uncompetitive NMDAR antagonist, was also applied. Calcium levels were also measured by laser scanning confocal microscope. Apoptosis assay
Establishment of the neuronal mechanical injury model and ApoE administration The mechanical injury model was produced by scraping adherent neurons on a culture dish (Tecoma et al., 1989; Kumaria and Tolias, 2008). Briefly, a 10 ll pipette tip was drawn evenly across a 20-mm diameter culture dish under a dissecting microscope, producing an approximately 0.6 mm wide linear tear in the neuron cell layer down to the bottom of the plastic dish; five linear tears were generated in the neuron cell layer on each culture dish. The experimental cell groups, both with and without mechanical injury, were divided as follows: Group-ApoE4, Group-ApoE3, Group-ApoE( ) (all prepared from the APOE-KO mouse), and Group-WT (prepared from the WT mouse). Group-ApoE4 and Group-ApoE3 were respectively treated with recombinant human ApoE4 and ApoE3, and the controls Group-ApoE( ) and Group-WT were untreated. Full-length exogenous
The apoptosis rates of neurons were analyzed at 24 h after the mechanical injury by FCM with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Briefly, cells were harvested, washed twice with PBS, and resuspended in binding buffer containing 1:100 PI and Annexin V-FITC (Annexin V-FITC Apoptosis Detection Kit, Sigma, USA). After incubation at room temperature for 30 min in the dark, neurons were immediately analyzed using a flow cytometer by counting 10,000 events per sample. Apoptotic events were recorded as a combination of Annexin-V+/PI (early apoptotic) and Annexin-V+/PI+(late apoptotic/dead) events. Statistical analysis Statistical comparisons among the groups were conducted by a one-factor analysis of variance
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(ANOVA). All values were expressed as mean ± S.E.M, and P < 0.05 was considered statistically significant.
RESULTS [Ca2+]i of neurons under various ApoE conditions To investigate the effects of ApoE on [Ca2+]i in neurons after injury, an in vitro mechanical injury model was established. Intracellular calcium in neurons was labeled by fluo-3/AM, which indicates [Ca2+]i by fluorescence intensity using a laser scanning confocal microscope. In our model, the fluorescence intensity in neurons after injury was observed to be significantly increased compared with uninjured neurons. As shown in Fig. 1A, at 2 h after injury, the fluorescence intensity of GroupApoE4 neurons was significantly higher than both Group-ApoE3 and Group-WT, indicating that neurons treated with ApoE4 had higher [Ca2+]i than ApoE3treated neurons and ApoE-WT neurons (P < 0.05). However, the fluorescence intensity of neurons in Group-ApoE( ) was significantly higher than that in Group-ApoE4, indicating neurons without ApoE had higher [Ca2+]i than neurons treated only with ApoE4 (P < 0.05). Although the fluorescence intensity of Group-WT neurons was lower than that in Group-ApoE3 after injury, the difference was not statistically significant (P > 0.05). The fluorescence intensity in all four groups continued to increase from 12 to 24 h after injury (Fig. 1A), suggesting that [Ca2+]i of neurons around the injury region continued to increase at early stages of injury. The fluorescence intensity of neurons treated with ApoE4 remained higher than that of neurons treated with ApoE3 and WT neurons, but lower than that of Group-ApoE( ) (P < 0.05). No significant difference of fluorescence intensity was found between Group-ApoE3 and GroupWT (P > 0.05). The trend of rate of [Ca2+]i increase was Group-ApoE( ) > Group-ApoE4 > Group-ApoE3 and Group-WT (P < 0.05) (Fig. 1A). To further determine the mean [Ca2+]i in all four groups after mechanical injury, the fluorescence intensity was measured with fluo-3/AM by FCM. Consistent with the above results, the mean fluorescence intensity of neurons treated with ApoE4 was significantly higher than neurons treated with ApoE3 and WT neurons, but lower than Group-ApoE( ) at all measured time points after injury (P < 0.05) (Fig. 1B). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). Under normal conditions, both microscopy and FCM data showed no significant difference in fluorescence intensity of [Ca2+]i among all four groups in the absence of mechanical injury (P > 0.05). Apoptosis of neurons induced by mechanical injury under various ApoE conditions To analyze apoptosis in injured neurons in the experimental groups at the early stages after mechanical injury, apoptosis was evaluated by FCM
with Annexin V-FITC and PI. FCM data showed that 24 h after mechanical injury, the apoptosis rate of neurons in Group-ApoE4 was significantly higher than that in Group-ApoE3 and Group-WT (P < 0.05), but significantly lower than that in Group-ApoE( ) (P < 0.05) (Fig. 2). No significant difference in apoptosis rate was found between neurons in GroupWT and Group-ApoE3 after mechanical injury (P > 0.05). Also, no significant difference in apoptosis levels was found in uninjured neurons of all four groups (P > 0.05). Effects of ApoE4 on [Ca2+]i and neurons apoptosis were influenced by EGTA and MK801 To determine the calcium source involved in the ApoE4 induced high [Ca2+]i level, the calcium chelator, EGTA, was added into the culture medium after mechanical injury. After blocking the extracellular calcium by EGTA treatment, the fluorescence intensity of injured GroupApoE4 neurons was significantly decreased compared with untreated Group-ApoE4 neurons (P < 0.05) (Fig. 3). However, even though treated by EGTA, the fluorescence intensity of injured neurons in GroupApoE4 was still significantly higher than that of uninjured neurons. Meanwhile, an uncompetitive NMDAR antagonist, MK801, was also added after mechanical injury. After treatment with MK801, the fluorescence intensity of injured Group-ApoE4 neurons was significantly decreased compared with untreated GroupApoE4 neurons (P < 0.05), but still significantly higher than that of uninjured neurons (P < 0.05) (Fig. 5). Above data indicated that the influx of extracellular calcium was involved in the ApoE4 induced high level of [Ca2+]i, but it was not the only calcium source. The apoptosis rate of injured Group-ApoE4 neurons was also significantly decreased after EGTA treatment, but was still higher than that of uninjured Group-ApoE4 neurons (P < 0.05) (Fig. 4). These results suggested that extracellular calcium partly contributes to the impact of ApoE4 on apoptosis of injured neurons.
DISCUSSION Our results revealed that the [Ca2+]i was significantly increased in neurons in the early stages after mechanical injury. Furthermore, changes in [Ca2+]i in neurons after mechanical injury can be influenced by the presence of different ApoE isoforms. Both laser scanning confocal microscopy and FCM showed that 2 h after mechanical injury, [Ca2+]i of Group-ApoE4 neurons was significantly higher than that in GroupApoE3 and Group-WT. The [Ca2+]i in all four groups kept increasing at 12 and 24 h after injury, and notably, the [Ca2+]i of Group-ApoE4 neurons was still remarkably higher than that in Group-ApoE3 and GroupWT (P < 0.05). This result indicated that exogenous ApoE4 can induce higher [Ca2+]i in injured neurons compared with ApoE3 treatment and WT conditions. The process of secondary injury (or delayed injury) after TBI involves many factors, including calcium and APOE. Disruption of [Ca2+]i homeostasis is considered
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Fig. 1A. [Ca2+]i in the four experimental neuron groups after mechanical injury. The fluorescence intensities of neurons labeled with fluo-3/AM before and after mechanical injury were evaluated by laser scanning confocal microscopy. The fluorescence of neurons in Group-ApoE4 was significantly higher than that in neurons in Group-ApoE3 and Group-WT, but lower than that in Group-ApoE( ) (untreated APOE-KO neurons), suggesting that ApoE4 can lead to higher [Ca2+]i compared to WT and ApoE3-treated neurons at the early stages of mechanical injury (*P < 0.05). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). In addition, no significant difference of [Ca2+]i was found among all four groups in the absence of mechanical injury (NP > 0.05).
as a fundamental pathological mechanism underlying secondary injury in the CNS after TBI (Weber et al., 2001; Houlden and Greenwood, 2006; Jordan, 2007; Weber, 2012). Moreover, excessively high [Ca2+]i plays an important role in the apoptosis process after TBI. APOE polymorphism also influences the outcome of TBI, and our previous studies have already revealed that APOEe4 can deteriorate the outcome of patients at the early stage of TBI (Jiang et al., 2006, 2007, 2011), but the exact mechanism remains unclear. Furthermore, the
relationship between APOE and [Ca2+]i after mechanical injury has not been clarified. Therefore, in the present study, we hypothesized that APOE polymorphism might influence [Ca2+]i of neurons after mechanical injury. To address this hypothesis, an in vitro mechanical injury model of neurons was constructed, and the relationship between various ApoE isoforms and [Ca2+]i was investigated. Our results showed that ApoE4 significantly increased [Ca2+]i and the apoptosis rate of injured neurons, and that both [Ca2+]i and the apoptosis rate of
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Fig. 1B. Measurement of [Ca2+]i in the four experimental neuron groups after mechanical injury. Measurement of fluorescence intensity of calcium in neurons by FCM showed that the mean [Ca2+]i of injured neurons in Group-ApoE4 was significantly higher than that in Group-WT and GroupApoE3, but lower than that in Group-ApoE( ) at 2, 12 and 24 h (*P < 0.05). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). No significant difference of [Ca2+]i was found among all four groups in the absence of mechanical injury (NP > 0.05).
Fig. 2. Apoptosis rates in the four experimental groups after mechanical injury. At 24 h after mechanical injury, the apoptosis rate of neurons in Group-ApoE4 was significantly higher than that in Group-WT and Group-ApoE3 (P < 0.05), but significantly lower than that in Group-ApoE( ) (*P < 0.05). No significant difference of apoptosis rate was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). No significant difference was found among all four groups in the absence of mechanical injury (NP > 0.05).
neurons were lower in neurons treated with ApoE3 than those treated with ApoE4. This may be one possible mechanism through which APOEe4 deteriorates the outcome of patients at the early stages of TBI, indicating that ApoE4 may function as a negative factor. Increased intracellular Ca2+ after TBI could be derived from either an influx of Ca2+ from the extracellular space or release of Ca2+ from intracellular calcium stores. We investigated the source of ApoE4induced increased calcium after injury by using EGTA and MK801 to block the influx of extracellular calcium.
Our results showed that both EGTA and MK801 treatment decreased, but did not completely abolish the effects of ApoE4 on [Ca2+]i, indicating that the influx of calcium from the extracellular space was not the only contributing factor, and that the release of intracellular calcium may also be involved. We speculated that ApoE4 may increase [Ca2+]i of neurons by reinforcing both an influx of calcium from extracellular space and release of calcium from intracellular calcium store after mechanical injury. Besides neurons, other cell types including, astrocyte, microglia, endothelial cell (as well
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Fig. 3. Effect of EGTA on [Ca2+]i of injured Group-ApoE4 neurons. Laser scanning confocal microscopy revealed that [Ca2+]i of Group-ApoE4 neurons was significantly increased at 24 h after injury. However, after treatment with EGTA, [Ca2+]i in injured Group-ApoE4 neurons remarkably decreased. Notably, the [Ca2+]i of the injured Group-ApoE4 neurons treated with EGTA was still significantly higher than that of uninjured neurons. This indicated that extracellular calcium may not be the only source contributing to the effects of ApoE4 on [Ca2+]i (*P < 0.05).
as the blood–brain barrier (BBB) integrity) also involve in the process after TBI (Maas et al., 2008). In the process of secondary injury, inflammatory factors like cytokine, are released from activated microglia and astrocytes. The subsequent inflammatory reaction can influence structure and function of BBB, which may lead to cell death and brain edema. Meanwhile, excessive excitatory neurotransmitters and calcium over-load may also cause cell swelling and apoptosis, finally leading to brain swelling and raised intracranial pressure (ICP). Recently, Berislav V and his colleagues confirmed that expression of APOE4 but not APOE2 and APOE3 has direct toxic effects on the cerebrovascular system. ApoE4 can lead to BBB breakdown by activating a proinflammatory CypA–NF-kB–MMP9 pathway in pericytes (Bell et al., 2012; Halliday et al., 2013; Zlokovic, 2013). This, in turn, can lead to secondary neuronal injury, for instance, the exposure of neurons to multiple blood-derived neurotoxic proteins, such as thrombin, fibrin and hemosiderin. Both thrombin and fibrin are neurotoxic and hemosiderin generates reactive oxygen species which finally impose direct injury to the neuron membrane, implicating multiple potential BBB-derived neuronal injury (Paul et al., 2007; Zhong et al., 2009; Grammas, 2011). In our previous study, we have also found ApoE could influence permeability of BBB in mice after mechanical injury. Furthermore, we also revealed ApoE protected astrocytes from hypoxia-induced apoptosis in vitro (Zhou et al., 2013). This study showed that ApoE4 induced remarkably higher apoptosis rates in neurons after mechanical
injury than ApoE3-treated and WT neurons. The [Ca2+]i of each group exhibited a continuous increase, but at different rates. The [Ca2+]i of neurons treated with ApoE4 was higher than that with ApoE3 or WT neurons, with a similar trend observed in apoptosis rates, suggesting that higher [Ca2+]i leads to increased apoptosis in neurons. The calcium chelator, EGTA, which blocks the influx of extracellular calcium, could also partly decrease the apoptosis rate of injured neurons, but the apoptosis rate of injured neurons treated with a combination of ApoE4 and EGTA was still significantly higher than uninjured neurons. Furthermore, an increased apoptosis rate of neurons may not be the only consequence of increased [Ca2+]i, caused by ApoE4, and the function and metabolism of neurons may also be affected (Aono et al., 2002). Another important finding from this study is that no significant differences were found in [Ca2+]i and apoptosis rates among neurons in all four groups without mechanical injury. This indicated that without mechanical injury, ApoE4 treatment did not significantly increase [Ca2+]i and apoptosis of neurons as compared to ApoE3 treatment and WT condition, at least in a short period of time (24 h). However, after mechanical injury, the effects of ApoE4 on [Ca2+]i and apoptosis of neurons were evident. This result suggested that the effects of different ApoE on [Ca2+]i of neurons absent under normal conditions, but induced in response to mechanical injury. We speculate that, as a negative regulatory factor, ApoE4 can cause adverse effects on the physiological activity of the brain. However, under
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Fig. 4. Effect of EGTA on apoptosis of injured neurons in Group-ApoE4. The apoptosis rate of Group-ApoE4 neurons significantly increased at 24 h after injury, and EGTA treatment caused a significant reduction in apoptosis. Apoptosis of Group-ApoE4 neurons treated with EGTA was still significantly higher than that of uninjured neurons (*P < 0.05).
Fig. 5. Effect of MK801 on [Ca2+]i of injured Group-ApoE4 neurons. As revealed by laser scanning confocal microscopy, [Ca2+]i of Group-ApoE4 neurons was significantly increased at 24 h after injury. After treatment with MK801, [Ca2+]i of injured neurons in Group-ApoE4 remarkably decreased, but was still significantly higher than that of uninjured neurons (*P < 0.05).
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normal conditions, the adverse effects may be compensated for or blocked, and a dynamic balance is established. Once the balance is disrupted, under TBI for example, the adverse effects from ApoE4 appear. This may also be the reason why ApoE4 did not significantly increase [Ca2+]i of uninjured neurons in vitro in a short period of time. If the adverse effects of ApoE4 accumulate over a long period of time, the balance can be broken. This view is consistent with the results of our previous study and a clinical study of Alzheimer’s disease (Ponomareva et al., 2008; Jiang et al., 2011). In addition, [Ca2+]i of mechanically injured neurons without ApoE was even higher than that in injured neurons treated with ApoE4. Thus, by regarding neurons without ApoE as a control group, we found that ApoE4 may also decrease [Ca2+]i and the apoptosis rate of injured neurons, although its effect was much weaker as compared with ApoE3. This result is similar to the findings of Huang et al., who recently showed that ApoE4 also stimulated Ab clearance, although its effect was significantly weaker as compared with ApoE2 and ApoE3 (Huang and Mucke, 2012). We acknowledge that although the current used model can simulate mechanical injury to some extent (Tecoma et al., 1989; Kumaria and Tolias, 2008), our injury model has several disadvantages. First, it is difficult to estimate the force and strain of injury. Furthermore, the results only showed the influence of ApoE in vitro. So in our further studies, both in vivo and in vitro models (including neuron-astrocyte co-culture) will be founded. Meanwhile more calcium channels, cell membrane permeability and integrity, and IP3 receptor and RYR receptor, with more time points will be considered.
CONCLUSION In conclusion, after mechanical injury, significantly higher [Ca2+]i and apoptosis rate of neurons were caused by ApoE4 treatment, as compared with ApoE3 treatment and WT conditions, and this may be one of the possible mechanisms through which APOEe4 deteriorate the outcome of TBI. Moreover, the results of this study may also provide some argument for translational medicine and the development of individualized treatment according to genotype.
DISCLOSURE The authors declare that they have no conflict of interest. The study was supported by National Science Foundation of China (approval number: 30973087 and 81371378).
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(Accepted 3 June 2015) (Available online 11 June 2015)
EFFECTS OF APOE ON INTRACELLULAR CALCIUM LEVELS AND APOPTOSIS OF NEURONS AFTER MECHANICAL INJURY L. JIANG, a J. ZHONG, a X. DOU, b C. CHENG, a Z. HUANG a AND X. SUN a*
INTRODUCTION Traumatic brain injury (TBI) is one of the most common central nervous system disorders with high morbidity and mortality. It often brings disastrous consequences and heavy economic burden to patients’ families and society (Werner and Engelhard, 2007; Maas et al., 2008; Hiekkanen et al., 2009). Although the processes underlying TBI have been studied and are better understood over the last decade, improving the prognosis of TBI remains challenging (Loane and Faden, 2010). The TBI process is generally divided into two stages: the primary injury and the secondary injury (or delayed injury) (Maas et al., 2008; Loane and Faden, 2010). The primary injury may be prevented but cannot be therapeutically treated. The secondary injury, which consists of a series of complicated events and is influenced by many factors, may be limited by influencing the factors involved in the process (Maas et al., 2008; Loane and Faden, 2010). Calcium plays a vital role in regulating cellular functions and has long been implicated in the process after TBI (Tymianski and Tator, 1996; Weber et al., 2001; Werner and Engelhard, 2007). Variation of intracellular calcium concentration can dramatically influence the function and metabolism of cells, and calcium overload can directly induce cell apoptosis. Therefore, maintenance of intracellular calcium homeostasis is of critical importance for cells, especially after injury. Intracellular calcium can be derived from two sources: exogenous calcium, the influx of extracellular calcium through calcium channels such as N-methyl-D-aspartate receptor (NMDAR), or endogenous calcium, the release of calcium from intracellular calcium stores such as endoplasmic reticulum (ER) and mitochondria (Weber et al., 2001; Veinbergs et al., 2002). Intracellular calcium levels ([Ca2+]i) after TBI can be influenced by both exogenous and endogenous calcium. Several genes have been implicated in the process after TBI and many studies suggest that apolipoprotein e gene (APOE) polymorphisms may influence the outcome of TBI (Teasdale et al., 1997; Crawford et al., 2002, 2009). Apolipoprotein e (ApoE) is a multifunctional protein involved predominantly in the transportation of cholesterol, maintenance of microtubules, and neural transmission. It is the major apolipoprotein in the central nervous system (CNS), and participates in the process after TBI. Three ApoE isoforms have been identified in humans, ApoE2, ApoE3 and ApoE4, which are encoded by three different alleles (e2, e3, and e4, respectively). APOEe4 is believed to be a negative factor contributing
a
Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, PR China b
Chongqing Medical University, PR China
Abstract—Objective: The current study aimed to explore the effects of apolipoprotein e (ApoE) on intracellular calcium ([Ca2+]i) and apoptosis of neurons after mechanical injury in vitro. Methods: A neuron mechanical injury model was established after primary neurons obtained from APOE knockout and wild-type (WT) mice, and four experimental groups were generated: Group-ApoE4, Group-ApoE3, Group-ApoE( ) and Group-WT. Recombinant ApoE4 and ApoE3 were added to Group-ApoE4 and Group-ApoE3 respectively, and Group-ApoE( ) and Group-WT were control groups. Intracellular calcium was labeled by fluo-3/AM and examined using laser scanning confocal microscope and flow cytometry, and the apoptosis of neurons was also evaluated. Results: The intracellular calcium levels and apoptosis rates of mice neurons were significantly higher in Group-ApoE4 than in Group-ApoE3 and Group-WT after mechanical injury. However, without mechanical injury on neurons, no significant differences in intracellular calcium levels and apoptosis rates were found among all four experimental groups. The effects of ApoE4 on intracellular calcium levels and apoptosis rates of injured neurons were partly decreased by EGTA treatment. Conclusion: Compared with ApoE3-treatment and WT neurons, ApoE4 caused higher intracellular calcium levels and apoptosis rates of neurons after mechanical injury. This suggested APOE polymorphisms may affect neuron apoptosis after mechanical injury through different influences on intracellular calcium levels. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: TBI, intracellular calcium polymorphism, neuron, mechanical injury.
level,
APOE
*Corresponding author. Address: Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, No. 1, Youyi Road, Yuanjiagang, Yuzhong District, Chongqing 400016, PR China. Tel: +86-23-89011152; fax: +86-23-68734337. E-mail address: [email protected] (X. Sun). Li Jiang and Jianjun Zhong contributed equally to this work. Abbreviations: ApoE, apolipoprotein e; BBB, blood–brain barrier; EGTA, ethylene glycol tetraacetic acid; FCM, flow cytometry; FITC, fluorescein isothiocyanate; MK801, dizocilpine; NMDAR, N-methyl-Daspartate receptor; PBS, phosphate-buffered saline; PI, propidium iodide; TBI, traumatic brain injury; WT, wild-type. http://dx.doi.org/10.1016/j.neuroscience.2015.06.005 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 375
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to unfavorable outcomes after TBI, and ApoE4, encoded by APOEe4, is considered a dysfunctional or harmful protein. For example, compared with ApoE2 and ApoE3, ApoE4 may induce more Ab deposition and generate more neurotoxic segments after being cleaved by a protease (Yasuda et al., 1993; Teasdale et al., 1997; Friedman et al., 1999; Chang et al., 2005). However, the exact mechanism through which ApoE4 negatively affects the outcome of TBI is still unknown. To investigate ApoE involvement in the process after TBI, an in vitro mechanical injury model of neurons was generated, and the effects of different ApoE isoforms on [Ca2+]i and apoptosis of neurons were measured after mechanical injury.
EXPERIMENTAL PROCEDURES Preparation of primary neuron culture This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chongqing Medical University. Neurons were harvested by a standard enzyme treatment protocol from APOE knockout (APOE-KO) mice (obtained from the Peking University Laboratory Animal Centre) and wildtype (WT) mice (obtained from the Laboratory Animal Centre of the Chongqing Medical University) within 24 h after birth. Neurons harvested from three mice were included in each group. Briefly, the cerebral cortex was minced into 2 mm3 pieces and dissociated with trypsin to obtain a cell suspension. After centrifugation, the resulting cell pellets were resuspended in DMEM/F12 containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, New York, USA) and added to culture flasks. Primary cultures were maintained in a humidified incubator with 5% CO2, 95% air at 37 °C. The culture medium was replaced with fresh medium every day, and neurons were cultured for 5 days. The neuron purity was determined by neuron specific enolase (NSE) positivity through immunocytochemistry.
recombinant human ApoE3 and ApoE4 were obtained from Peprotech (Peprotech, Rocky Hill, New Jersy, USA). The concentration of recombinant ApoE in the extracellular fluid was maintained at 10 lg/ml (Qiu et al., 2003), and neurons were treated with recombinant ApoE for 24 h (Veinbergs et al., 2002). Analysis of calcium levels [Ca2+]i in neurons was evaluated at 2, 12 and 24 h after injury by using the calcium indicator fluo-3/AM and by measuring the fluorescence intensity through laser scanning confocal microscopic imaging (Johnson et al., 2007). Briefly, neurons were loaded with fluo-3/AM (Biotium, Bay Area, San Francisco, USA) for 30 min at 37 °C in the dark, and then washed with D-Hanks medium. Fluo-3 was excited at 488 nm, and the fluorescence was detected by a (Rice et al., 1998) (Leica, Wetzlar, Hesse, Germany). In each microscopic field, the fluorescence intensity of approximately 5–10 neuron soma around the injured region was separately measured. The fluorescence intensity of selected microscopic fields was digitized. All data were analyzed by the accompanying software of the laser scanning confocal microscope system. The mean fluorescence intensity of [Ca2+]i in each group was also detected by flow cytometry (FCM) at 2, 12 and 24 h after injury. Neurons were harvested, washed twice with phosphate-buffered saline (PBS), resuspended and loaded with 5 lmol/l fluo-3/AM for 30 min at 37 °C in the dark. After washing with D-Hanks medium, neurons were immediately analyzed using a flow cytometer (Beckman Coulter, Epics XL) by counting 10,000 events per sample. To determine the source of calcium after mechanical injury, the calcium chelator EGTA (Sigma, Saint Louis, Missouri, USA) was applied to block the influx of extracellular calcium (Veinbergs et al., 2002). Meanwhile, MK801, an uncompetitive NMDAR antagonist, was also applied. Calcium levels were also measured by laser scanning confocal microscope. Apoptosis assay
Establishment of the neuronal mechanical injury model and ApoE administration The mechanical injury model was produced by scraping adherent neurons on a culture dish (Tecoma et al., 1989; Kumaria and Tolias, 2008). Briefly, a 10 ll pipette tip was drawn evenly across a 20-mm diameter culture dish under a dissecting microscope, producing an approximately 0.6 mm wide linear tear in the neuron cell layer down to the bottom of the plastic dish; five linear tears were generated in the neuron cell layer on each culture dish. The experimental cell groups, both with and without mechanical injury, were divided as follows: Group-ApoE4, Group-ApoE3, Group-ApoE( ) (all prepared from the APOE-KO mouse), and Group-WT (prepared from the WT mouse). Group-ApoE4 and Group-ApoE3 were respectively treated with recombinant human ApoE4 and ApoE3, and the controls Group-ApoE( ) and Group-WT were untreated. Full-length exogenous
The apoptosis rates of neurons were analyzed at 24 h after the mechanical injury by FCM with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Briefly, cells were harvested, washed twice with PBS, and resuspended in binding buffer containing 1:100 PI and Annexin V-FITC (Annexin V-FITC Apoptosis Detection Kit, Sigma, USA). After incubation at room temperature for 30 min in the dark, neurons were immediately analyzed using a flow cytometer by counting 10,000 events per sample. Apoptotic events were recorded as a combination of Annexin-V+/PI (early apoptotic) and Annexin-V+/PI+(late apoptotic/dead) events. Statistical analysis Statistical comparisons among the groups were conducted by a one-factor analysis of variance
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(ANOVA). All values were expressed as mean ± S.E.M, and P < 0.05 was considered statistically significant.
RESULTS [Ca2+]i of neurons under various ApoE conditions To investigate the effects of ApoE on [Ca2+]i in neurons after injury, an in vitro mechanical injury model was established. Intracellular calcium in neurons was labeled by fluo-3/AM, which indicates [Ca2+]i by fluorescence intensity using a laser scanning confocal microscope. In our model, the fluorescence intensity in neurons after injury was observed to be significantly increased compared with uninjured neurons. As shown in Fig. 1A, at 2 h after injury, the fluorescence intensity of GroupApoE4 neurons was significantly higher than both Group-ApoE3 and Group-WT, indicating that neurons treated with ApoE4 had higher [Ca2+]i than ApoE3treated neurons and ApoE-WT neurons (P < 0.05). However, the fluorescence intensity of neurons in Group-ApoE( ) was significantly higher than that in Group-ApoE4, indicating neurons without ApoE had higher [Ca2+]i than neurons treated only with ApoE4 (P < 0.05). Although the fluorescence intensity of Group-WT neurons was lower than that in Group-ApoE3 after injury, the difference was not statistically significant (P > 0.05). The fluorescence intensity in all four groups continued to increase from 12 to 24 h after injury (Fig. 1A), suggesting that [Ca2+]i of neurons around the injury region continued to increase at early stages of injury. The fluorescence intensity of neurons treated with ApoE4 remained higher than that of neurons treated with ApoE3 and WT neurons, but lower than that of Group-ApoE( ) (P < 0.05). No significant difference of fluorescence intensity was found between Group-ApoE3 and GroupWT (P > 0.05). The trend of rate of [Ca2+]i increase was Group-ApoE( ) > Group-ApoE4 > Group-ApoE3 and Group-WT (P < 0.05) (Fig. 1A). To further determine the mean [Ca2+]i in all four groups after mechanical injury, the fluorescence intensity was measured with fluo-3/AM by FCM. Consistent with the above results, the mean fluorescence intensity of neurons treated with ApoE4 was significantly higher than neurons treated with ApoE3 and WT neurons, but lower than Group-ApoE( ) at all measured time points after injury (P < 0.05) (Fig. 1B). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). Under normal conditions, both microscopy and FCM data showed no significant difference in fluorescence intensity of [Ca2+]i among all four groups in the absence of mechanical injury (P > 0.05). Apoptosis of neurons induced by mechanical injury under various ApoE conditions To analyze apoptosis in injured neurons in the experimental groups at the early stages after mechanical injury, apoptosis was evaluated by FCM
with Annexin V-FITC and PI. FCM data showed that 24 h after mechanical injury, the apoptosis rate of neurons in Group-ApoE4 was significantly higher than that in Group-ApoE3 and Group-WT (P < 0.05), but significantly lower than that in Group-ApoE( ) (P < 0.05) (Fig. 2). No significant difference in apoptosis rate was found between neurons in GroupWT and Group-ApoE3 after mechanical injury (P > 0.05). Also, no significant difference in apoptosis levels was found in uninjured neurons of all four groups (P > 0.05). Effects of ApoE4 on [Ca2+]i and neurons apoptosis were influenced by EGTA and MK801 To determine the calcium source involved in the ApoE4 induced high [Ca2+]i level, the calcium chelator, EGTA, was added into the culture medium after mechanical injury. After blocking the extracellular calcium by EGTA treatment, the fluorescence intensity of injured GroupApoE4 neurons was significantly decreased compared with untreated Group-ApoE4 neurons (P < 0.05) (Fig. 3). However, even though treated by EGTA, the fluorescence intensity of injured neurons in GroupApoE4 was still significantly higher than that of uninjured neurons. Meanwhile, an uncompetitive NMDAR antagonist, MK801, was also added after mechanical injury. After treatment with MK801, the fluorescence intensity of injured Group-ApoE4 neurons was significantly decreased compared with untreated GroupApoE4 neurons (P < 0.05), but still significantly higher than that of uninjured neurons (P < 0.05) (Fig. 5). Above data indicated that the influx of extracellular calcium was involved in the ApoE4 induced high level of [Ca2+]i, but it was not the only calcium source. The apoptosis rate of injured Group-ApoE4 neurons was also significantly decreased after EGTA treatment, but was still higher than that of uninjured Group-ApoE4 neurons (P < 0.05) (Fig. 4). These results suggested that extracellular calcium partly contributes to the impact of ApoE4 on apoptosis of injured neurons.
DISCUSSION Our results revealed that the [Ca2+]i was significantly increased in neurons in the early stages after mechanical injury. Furthermore, changes in [Ca2+]i in neurons after mechanical injury can be influenced by the presence of different ApoE isoforms. Both laser scanning confocal microscopy and FCM showed that 2 h after mechanical injury, [Ca2+]i of Group-ApoE4 neurons was significantly higher than that in GroupApoE3 and Group-WT. The [Ca2+]i in all four groups kept increasing at 12 and 24 h after injury, and notably, the [Ca2+]i of Group-ApoE4 neurons was still remarkably higher than that in Group-ApoE3 and GroupWT (P < 0.05). This result indicated that exogenous ApoE4 can induce higher [Ca2+]i in injured neurons compared with ApoE3 treatment and WT conditions. The process of secondary injury (or delayed injury) after TBI involves many factors, including calcium and APOE. Disruption of [Ca2+]i homeostasis is considered
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Fig. 1A. [Ca2+]i in the four experimental neuron groups after mechanical injury. The fluorescence intensities of neurons labeled with fluo-3/AM before and after mechanical injury were evaluated by laser scanning confocal microscopy. The fluorescence of neurons in Group-ApoE4 was significantly higher than that in neurons in Group-ApoE3 and Group-WT, but lower than that in Group-ApoE( ) (untreated APOE-KO neurons), suggesting that ApoE4 can lead to higher [Ca2+]i compared to WT and ApoE3-treated neurons at the early stages of mechanical injury (*P < 0.05). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). In addition, no significant difference of [Ca2+]i was found among all four groups in the absence of mechanical injury (NP > 0.05).
as a fundamental pathological mechanism underlying secondary injury in the CNS after TBI (Weber et al., 2001; Houlden and Greenwood, 2006; Jordan, 2007; Weber, 2012). Moreover, excessively high [Ca2+]i plays an important role in the apoptosis process after TBI. APOE polymorphism also influences the outcome of TBI, and our previous studies have already revealed that APOEe4 can deteriorate the outcome of patients at the early stage of TBI (Jiang et al., 2006, 2007, 2011), but the exact mechanism remains unclear. Furthermore, the
relationship between APOE and [Ca2+]i after mechanical injury has not been clarified. Therefore, in the present study, we hypothesized that APOE polymorphism might influence [Ca2+]i of neurons after mechanical injury. To address this hypothesis, an in vitro mechanical injury model of neurons was constructed, and the relationship between various ApoE isoforms and [Ca2+]i was investigated. Our results showed that ApoE4 significantly increased [Ca2+]i and the apoptosis rate of injured neurons, and that both [Ca2+]i and the apoptosis rate of
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Fig. 1B. Measurement of [Ca2+]i in the four experimental neuron groups after mechanical injury. Measurement of fluorescence intensity of calcium in neurons by FCM showed that the mean [Ca2+]i of injured neurons in Group-ApoE4 was significantly higher than that in Group-WT and GroupApoE3, but lower than that in Group-ApoE( ) at 2, 12 and 24 h (*P < 0.05). No significant difference in [Ca2+]i was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). No significant difference of [Ca2+]i was found among all four groups in the absence of mechanical injury (NP > 0.05).
Fig. 2. Apoptosis rates in the four experimental groups after mechanical injury. At 24 h after mechanical injury, the apoptosis rate of neurons in Group-ApoE4 was significantly higher than that in Group-WT and Group-ApoE3 (P < 0.05), but significantly lower than that in Group-ApoE( ) (*P < 0.05). No significant difference of apoptosis rate was found between neurons in Group-WT and Group-ApoE3 after mechanical injury (P > 0.05). No significant difference was found among all four groups in the absence of mechanical injury (NP > 0.05).
neurons were lower in neurons treated with ApoE3 than those treated with ApoE4. This may be one possible mechanism through which APOEe4 deteriorates the outcome of patients at the early stages of TBI, indicating that ApoE4 may function as a negative factor. Increased intracellular Ca2+ after TBI could be derived from either an influx of Ca2+ from the extracellular space or release of Ca2+ from intracellular calcium stores. We investigated the source of ApoE4induced increased calcium after injury by using EGTA and MK801 to block the influx of extracellular calcium.
Our results showed that both EGTA and MK801 treatment decreased, but did not completely abolish the effects of ApoE4 on [Ca2+]i, indicating that the influx of calcium from the extracellular space was not the only contributing factor, and that the release of intracellular calcium may also be involved. We speculated that ApoE4 may increase [Ca2+]i of neurons by reinforcing both an influx of calcium from extracellular space and release of calcium from intracellular calcium store after mechanical injury. Besides neurons, other cell types including, astrocyte, microglia, endothelial cell (as well
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Fig. 3. Effect of EGTA on [Ca2+]i of injured Group-ApoE4 neurons. Laser scanning confocal microscopy revealed that [Ca2+]i of Group-ApoE4 neurons was significantly increased at 24 h after injury. However, after treatment with EGTA, [Ca2+]i in injured Group-ApoE4 neurons remarkably decreased. Notably, the [Ca2+]i of the injured Group-ApoE4 neurons treated with EGTA was still significantly higher than that of uninjured neurons. This indicated that extracellular calcium may not be the only source contributing to the effects of ApoE4 on [Ca2+]i (*P < 0.05).
as the blood–brain barrier (BBB) integrity) also involve in the process after TBI (Maas et al., 2008). In the process of secondary injury, inflammatory factors like cytokine, are released from activated microglia and astrocytes. The subsequent inflammatory reaction can influence structure and function of BBB, which may lead to cell death and brain edema. Meanwhile, excessive excitatory neurotransmitters and calcium over-load may also cause cell swelling and apoptosis, finally leading to brain swelling and raised intracranial pressure (ICP). Recently, Berislav V and his colleagues confirmed that expression of APOE4 but not APOE2 and APOE3 has direct toxic effects on the cerebrovascular system. ApoE4 can lead to BBB breakdown by activating a proinflammatory CypA–NF-kB–MMP9 pathway in pericytes (Bell et al., 2012; Halliday et al., 2013; Zlokovic, 2013). This, in turn, can lead to secondary neuronal injury, for instance, the exposure of neurons to multiple blood-derived neurotoxic proteins, such as thrombin, fibrin and hemosiderin. Both thrombin and fibrin are neurotoxic and hemosiderin generates reactive oxygen species which finally impose direct injury to the neuron membrane, implicating multiple potential BBB-derived neuronal injury (Paul et al., 2007; Zhong et al., 2009; Grammas, 2011). In our previous study, we have also found ApoE could influence permeability of BBB in mice after mechanical injury. Furthermore, we also revealed ApoE protected astrocytes from hypoxia-induced apoptosis in vitro (Zhou et al., 2013). This study showed that ApoE4 induced remarkably higher apoptosis rates in neurons after mechanical
injury than ApoE3-treated and WT neurons. The [Ca2+]i of each group exhibited a continuous increase, but at different rates. The [Ca2+]i of neurons treated with ApoE4 was higher than that with ApoE3 or WT neurons, with a similar trend observed in apoptosis rates, suggesting that higher [Ca2+]i leads to increased apoptosis in neurons. The calcium chelator, EGTA, which blocks the influx of extracellular calcium, could also partly decrease the apoptosis rate of injured neurons, but the apoptosis rate of injured neurons treated with a combination of ApoE4 and EGTA was still significantly higher than uninjured neurons. Furthermore, an increased apoptosis rate of neurons may not be the only consequence of increased [Ca2+]i, caused by ApoE4, and the function and metabolism of neurons may also be affected (Aono et al., 2002). Another important finding from this study is that no significant differences were found in [Ca2+]i and apoptosis rates among neurons in all four groups without mechanical injury. This indicated that without mechanical injury, ApoE4 treatment did not significantly increase [Ca2+]i and apoptosis of neurons as compared to ApoE3 treatment and WT condition, at least in a short period of time (24 h). However, after mechanical injury, the effects of ApoE4 on [Ca2+]i and apoptosis of neurons were evident. This result suggested that the effects of different ApoE on [Ca2+]i of neurons absent under normal conditions, but induced in response to mechanical injury. We speculate that, as a negative regulatory factor, ApoE4 can cause adverse effects on the physiological activity of the brain. However, under
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Fig. 4. Effect of EGTA on apoptosis of injured neurons in Group-ApoE4. The apoptosis rate of Group-ApoE4 neurons significantly increased at 24 h after injury, and EGTA treatment caused a significant reduction in apoptosis. Apoptosis of Group-ApoE4 neurons treated with EGTA was still significantly higher than that of uninjured neurons (*P < 0.05).
Fig. 5. Effect of MK801 on [Ca2+]i of injured Group-ApoE4 neurons. As revealed by laser scanning confocal microscopy, [Ca2+]i of Group-ApoE4 neurons was significantly increased at 24 h after injury. After treatment with MK801, [Ca2+]i of injured neurons in Group-ApoE4 remarkably decreased, but was still significantly higher than that of uninjured neurons (*P < 0.05).
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normal conditions, the adverse effects may be compensated for or blocked, and a dynamic balance is established. Once the balance is disrupted, under TBI for example, the adverse effects from ApoE4 appear. This may also be the reason why ApoE4 did not significantly increase [Ca2+]i of uninjured neurons in vitro in a short period of time. If the adverse effects of ApoE4 accumulate over a long period of time, the balance can be broken. This view is consistent with the results of our previous study and a clinical study of Alzheimer’s disease (Ponomareva et al., 2008; Jiang et al., 2011). In addition, [Ca2+]i of mechanically injured neurons without ApoE was even higher than that in injured neurons treated with ApoE4. Thus, by regarding neurons without ApoE as a control group, we found that ApoE4 may also decrease [Ca2+]i and the apoptosis rate of injured neurons, although its effect was much weaker as compared with ApoE3. This result is similar to the findings of Huang et al., who recently showed that ApoE4 also stimulated Ab clearance, although its effect was significantly weaker as compared with ApoE2 and ApoE3 (Huang and Mucke, 2012). We acknowledge that although the current used model can simulate mechanical injury to some extent (Tecoma et al., 1989; Kumaria and Tolias, 2008), our injury model has several disadvantages. First, it is difficult to estimate the force and strain of injury. Furthermore, the results only showed the influence of ApoE in vitro. So in our further studies, both in vivo and in vitro models (including neuron-astrocyte co-culture) will be founded. Meanwhile more calcium channels, cell membrane permeability and integrity, and IP3 receptor and RYR receptor, with more time points will be considered.
CONCLUSION In conclusion, after mechanical injury, significantly higher [Ca2+]i and apoptosis rate of neurons were caused by ApoE4 treatment, as compared with ApoE3 treatment and WT conditions, and this may be one of the possible mechanisms through which APOEe4 deteriorate the outcome of TBI. Moreover, the results of this study may also provide some argument for translational medicine and the development of individualized treatment according to genotype.
DISCLOSURE The authors declare that they have no conflict of interest. The study was supported by National Science Foundation of China (approval number: 30973087 and 81371378).
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(Accepted 3 June 2015) (Available online 11 June 2015)