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Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E
Biochemical and Biophysical Research Communications 379 (2009) 949–953
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E Lili Cao, Jingjing Xu, Youting Lin, Xiuhe Zhao, Xuewu Liu, Zhaofu Chi * Department of Neurology, Qilu Hospital of Shandong University, Wenhua West Road 107, Jinan 250012, China
a r t i c l e
i n f o
Article history: Received 17 December 2008 Available online 10 January 2009
Keywords: Autophagy Light chain 3 Beclin 1 Oxidative stress Hippocampus
a b s t r a c t Autophagy, a process of bulk degradation of cellular constituents through autophagosome–lysosomal pathway, is enhanced during oxidative stress. Whether autophagy is induced during status epilepticus (SE), which induces an excess production of reactive oxygen species (ROS) and leads to oxidative stress, is not established. We also sought to determine if pretreatment with Vitamin E reduced autophagy. We used pilocarpine to elicit SE in rats. The ratio of LC3 II to LC3 I and beclin 1 were used to estimate autophagy. We found that ratio of LC3 II to LC3 I and beclin 1 increased significantly at 2, 8, 16, 24 and 72 h, peaking at 24 h after SE onset. Pretreatment with Vitamin E partially inhibited autophagy by reducing LC3 II formation and de novo synthesis of beclin 1 at 24 h after seizures. These data show that autophagy is increased in rats with pilocarpine-induced SE, and Vitamin E have a partial inhibition on autophagy. Ó 2009 Elsevier Inc. All rights reserved.
Autophagy is a process in which cytoplasmic components such as organelles and proteins are delivered to the lysosomal compartment for degradation, and plays an essential role in maintenance of cellular homeostasis [1,2]. Macroautophagy (hereafter referred to as autophagy), the major process of autophagy, involves the formation of double-membrane vesicles, named autophagosomes that engulf and deliver cytosolic material to the lysosomes [3,4]. In the most cells, autophagy is suppressed to a basal level. It is hypothesized that up-regulation of this system may play a significant role in adaptation to environments where oxidative stress is a major selection pressure by eliminating unwanted or unnecessary organelles and recycling cellular components for reuse [5,6]. Recent studies have proved that oxidative stress is implicated in the induction of autophagy and reactive oxygen species (ROS) serve as signaling molecules that initiate autophagy [7,8]. A role for autophagy has been uncovered in central nervous system disease [9–12]. Status epilepticus (SE) induced by systemic injections of pilocarpine provide a valuable animal model of temporal lobe epilepsy (TLE). Oxidative stress has been implicated in seizure, and ROS increase significantly in animal with pilocarpine-induced SE, which in turn enhance seizure activity [13,14]. Moreover, Glutamate excitotoxicity has been involved in the pathophysiology of epilepsy and related to neuronal death in pilocarpine-induced status epilepticus [15]. Seizure can result in ATP exhaust [16]. ATP exhaust, glutamate and oxidative stress are important inducer of autophagy [3].
* Corresponding author. Fax: +86 53186026772. E-mail address: [email protected] (Z. Chi). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.178
To date, a large group of autophagy-related proteins (ATGs) are known to directly regulate the formation of autophagosome [2]. Modification of light chain 3 (LC3) I (apparent mobility, 18 kd), (the mammalian homolog of yeast Atg8) with covalent attachment of phosphatidylethanolamine results in LC3 II (apparent mobility, 16 kd), a process essential for autophagy referred to as an ‘LC3 shift’ or ‘LC3 lipidation’ [17]. The amount of LC3 II or the LC3 II to LC3 I ratio correlated with the number of autophagosome [18]. The measurement of LC3 II by immunoblotting is a simple and quantitative method for determining autophagic activity of mammalian cells [4]. Beclin 1, a Bcl-2-interacting protein, is the mammalian homolog of yeast VPS30p/Apg6p, forms a complex with phoshatidylinositol-3-kinase (PI3K), and participates in the early stages of autophagosome formation, promoting the nucleation of autophagosome [19]. It has been shown that beclin 1 is essential for autophagy as well as for lysosomal enzyme transport [20,21]. In this study, pilocarpine was used to elicit SE. We sought to determine whether autophagy was activated after SE in rats in vivo. In addition, whether pretreatment with antioxidant Vitamin E influenced autophagy and cell injury after SE were determined. LC3 II/LC3 I ratio and beclin 1 were used to estimate autophagy. Materials and methods Adult male Wistar rats (220–250 g) were used. Animals were kept in standard conditions (12 h light and dark cycle) with free access to food and water during all experimental time period. Experiments were performed following the ‘‘Principles of Laboratory
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L. Cao et al. / Biochemical and Biophysical Research Communications 379 (2009) 949–953
Animals Care” (NIH Publication No. 85-23, revised 1996) and according to the guidelines of the Commission of Shandong University for ethics of experiments on animals. Seizures were induced by pilocarpine injections as reported in previous study [14]. Rats were divided to four groups. The first group was pilocarpine-injected (340 mg/kg i.p.; Sigma) 30 min after an injection of atropine methyl nitrate (1 mg/kg i.p.) to reduce the number of peripheral cholinergic signs. Animals with 2 h SE, defined as a continuous motor seizure of stage 4 (rearing and falling), stage 5 (loss of balance, continuous rearing and falling), or stage 6 (severe tonic–clonic seizures) [22] were killed respectively at 2, 8,16, 24 and 72 h after seizure-onset. The control rats were injected with a physiological salt solution instead of pilocarpine. The third group was Vitamin E (200 mg/kg, Sigma) preinjected 30 min before pilocarpine injection, and were killed at 24 h after seizure-onset. The fourth group was injected with only Vitamin E as vehicle treatment group [23]. In order to standardize the different experimental groups, all animals were treated by diazepam administration (i.m. injection, at the dose of 4 mg/kg, Sigma) 2 h after pilocarpine (or saline) injection. The rats were sacrificed by decapitation and the brains were quickly removed and dissected. Hippocampus regions were maintained at 80 °C until determinations. Rats were anesthetized and intracardially perfusion with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) at different times after seizures. The brains were removed and kept in 4% paraformaldehyde for 12 h, then immersed in 25% sucrose for 3– 4 days at 4 °C. The paraffin-fixed brains were sectioned coronally in 0.5 mm thickness. Nissl staining with toluidine blue was first performed. The surviving cells were defined as round-shaped, cytoplasmic, membrane-intact cells, without any nuclear condensation or distorted aspect. Using high magnification (40), the number of surviving neurons/0.5mm length in hippocampus CA1 regions was blindly counted. Detailed procedure was carried out as described previously [24]. Total RNA was prepared from hippocampus with Trizol reagent (Invitrogen, USA) and reversely transcribed to cDNA using AMV First Strand DNA Synthesis Kit (Biotech Company, China). Briefly, a 1 lg of the isolated RNA was reversely transcribed to cDNA at 37 °C for 1 h in a 20 ll of reaction mixture containing 1 ll AMV reverse transcriptase, 1 ll random hexamer, 4 ll 5 AMV buffer, 1 ll RNase inhibitor (20 U/ll), 1 ll dNTP (10 mM). The PCR amplification mixture (25 ll) consisted of 1 ll cDNA mixture, 1 ll Taq DNA polymerase, 5 ll of 5 PCR buffer, 5 mM dNTP mixture, and 50 pM sense and antisense primers each. Beta-actin was used as an internal control. Beclin 1 was analyzed by PCR and the used oligonucleotide primers included: for beclin 1 (316 bp) forward primer 50 -AGGAGCAGTGGACAAAGG-30 and reverse primer 50 AGGGAAGAGGGAAAGGAC-30 , for beta-actin (142 bp) forward primer 50 -GACAGGATGCAGAAGGAGATTACT-30 and reverse primer 50 -TGATCCACATCTGCTGGAAGGT-30 . The PCR conditions were as follows: for initial denaturing at 94 °C for 5 min, followed by 30 PCR cycles with temperatures below: 94 °C for 40 s, for beclin 1 54 °C for 40 s, or for beta-actin 52 °C for 40 s each, and then 72 °C for 40 s; a final extension at 72 °C for 10 min. The amplified products were subjected to electrophoresis at 120 V for 30 min on 1.5% agarose gels containing 0.5 mg/ml ethidium bromide, and quantified by respectively comparing luminosity of beclin 1 to that of b-actin using AlphaEase FC (FluorChem 9900) (Alpha Innotech). Equal amounts of protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), and then transferred to 0.45 lm nitrocellulose membrane using a Trans-Blot semidry system (Bio-Rad). After blocking in 5% fat-free milk in Tris-buffered saline with Tween buffer for 2 h, the membranes were incubated with a primary antibody such as anti-LC3 (1:6000, Burlingame, Epitomics), anti-beclin 1 (1:500, Ab-
cam, UK), anti-actin (1:500, Zhongshan Goldenbriodge Biotechnology CO.LTD, China), which served as a loading control, overnight at 4 °C. Then the membranes were washed and incubated with horseradish peroxidaseconjugated secondary antibody (goat anti-rabbit IgG, 1:10000, Zhongshan Goldenbriodge Biotechnology CO.LTD, China) for 2 h under room temperature. Immunoreactivity was enhanced by chemiluminescence kit (Pierce, Rockford, Illinois, USA) and exposed to film. The bands on the film were scanned and analyzed with an image analyzer (Alpha Innotech, San Leandro, California, USA). Results in this study are presented as mean ± s.d. Two group comparisons were performed using Student’s t-test. Multiple group comparisons were performed by one-way analysis of variance, followed by the Newman–Keuls test. p < 0.05 was considered statistically significant. Results To semiquantify the amount of autophagosome formation after SE relative to naive controls, we examined hippocampi from rats with SE for evidence of an LC3 shift from an apparent B18 to B16 kd position [25]. By Western blot analysis, the time-course study of LC3 demonstrated that the ratio of LC3 II to LC3 I was significantly increased at 2, 8, 16, 24 and 72 h after SE, peaking at 24 h (Fig. 1A and B, p < 0.05). Since autophagy can be induced by mitochondrial oxidative stress and reactive oxygen species are essential for starvation-induced autophagy to proceed in vitro [8,12], we determined whether the antioxidant Vitamin E could reduce autophagy. Our data showed that pretreatment with Vitamin E reduced relative protein abundance of LC3 II to LC3 I at 24 h after SE versus vehicle-pretreated rats (Fig. 1C and D, p < 0.05), indicating that Vitamin E inhibited autophagy partly. Semiquatitive RT-PCR analysis showed that beclin 1 mRNA levels were significantly higher at 2 h after seizures onset compared with control group. A time-course of beclin 1 mRNA levels showed beclin 1 mRNA reached a peak at 24 h and remained at high levels at 72 h after SE (Fig. 2A and B). Western blot analysis showed that beclin 1 protein levels also began to increase at 2 h after SE, reached a maximum level at 24 h and remained significantly elevated until 72 h (Fig. 3A and B). Pretreatment with Vitamin E reduced both mRNA and protein levels of beclin 1 at 24 h after SE (Figs. 2C and D and 3C and D). To examine the extent of neuron loss in hippocampus after pilocarpine-induced seizures, toluidine blue was used for staining. Neurons in CA1 region are very vulnerable to SE stress. Our results showed that seizures led to severe cell death in CA1, which peaked at 24 h after seizure-onset. The surviving neuron numbers were sharply decreased in the pilocarpine-treated group compared with control group. Moreover, Vitamin E significantly attenuated the neuron loss induced by seizures (Table 1). Discussion The major finding of this study is that autophagy, detected biochemically, is induced in injured brain after SE in rats. An additional finding is that autophagosome formation, as detected by increases in LC3 II and beclin 1, is partially inhibited by the antioxidant Vitamin E. Treatment with Vitamin E partially reduces histological damage of SE in rat compared with vehicle treatment. These data represent the first evidence of autophagy activation and the first to suggest that reducing oxidative stress by Vitamin E reduces autophagy, after acute brain injury induced by pilocarpine-produced SE. Taken together, these data suggest that oxidative stress contributes to overall neuropathology after SE, at least in part by initiating or influencing autophagy. Similar to starvation-induced
L. Cao et al. / Biochemical and Biophysical Research Communications 379 (2009) 949–953
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Fig. 1. The effects of Vitamin E on hippocampus LC3 levels. (A) Western blot for analysis showing the time-course of LC3 II and LC3 I levels at various times after seizures. (B) Quantitative representation of LC3 II to LC3 I ratio at various times after seizures.(C) Western blot for analysis showing the effects of Vitamin E on LC3 II and LC3 I levels at 24 h after seizures. (D) Quantitative representation of effect of Vitamin E on LC3 II to LC3 I ratio at 24 h after seizures. Data are expressed as mean ± s.d. of at least four independent animals, *p < 0.05 vs. control, #p < 0.05 vs. 24 h, pilo = pilocarpine.
Fig. 2. The effects of Vitamin E on hippocampus beclin 1 mRNA levels after seizures. (A) Semi-quantitative RT-PCR analysis of time courses of beclin 1 mRNA in rat hippocampus after seizures. (B) Quantitative representation of the mRNA levels of beclin 1 at various times after seizures. (C) Semi-quantitative RT-PCR analysis of effects of Vitamin E on mRNA of beclin 1 at 24 h after SE onset. (D) Quantitative representation of the effects of Vitamin E on the beclin 1 mRNA. Data are expressed as mean ± s.d. of at least four independent animals. *p < 0.05 vs. control, #p < 0.05 vs. 24 h, pilo = pilocarpine.
autophagy, a role for oxidative stress in SE-induced autophagy is also implicated. LC3 was the first identified mammalian protein localized in autophagosome membranes. LC3 has two forms: LC3 I is cytosolic form, and is activated and modified to a membrane-bound form, LC3 II. Thus, the ratio of LC3 II to LC3 I is used to semiquantify the amount of autophagosome formation [18,26]. In this study, the ration of LC3 II to LC3 I after SE was markedly increased at 2,
8, 16, 24 and 72 h, with peak relative abundance at 24 h (Fig. 1), although time point after 72 h are not evaluated and such a later peak cannot be ruled out. These data indicate the activation of autophagy in rat hippocampus after SE. In rat brain, beclin 1 is expressed in many areas including cerebral cortex, hippocampus and cerebellum [27,28]. Beclin 1 is part of a Class III PI3K complex that locates at trans-Golgi network and participates in autophagosome formation, mediating localiza-
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Fig. 3. The effects of Vitamin E on hippocampus beclin 1 levels after seizures. (A) Western blot analysis of time-course of beclin 1 in rat hippocampus after seizures. (B) Quantitative representation of beclin 1 at various times after seizures. (C) Western blot analysis of effects of Vtamin E on beclin 1 at 24 h after SE onset. (D) Quantitative representation of effects of Vitamin E on beclin 1 at 24 h after SE onset. Data are expressed are mean ± s.d. of at least four independent animals.*p < 0.05 vs. control, #p < 0.05 vs. 24 h group, pilo = pilocarpine.
Table 1 Protective effects of Vitamin E on neuron death in hippocampus CA1 after seizures. Group
Neuron numbers (mean ± s.d.)
Control 2h 8h 16 h 24 h 72 h Vitamin E + pilo
110.67 ± 18.56 98.17 ± 15.22 87.5 ± 17.38 79.83 ± 19.49* 75.5 ± 15.924 82.83 ± 19.59* 94.67 ± 13.02#
Neuron number is expressed as the number of surviving pyramidal cells/0.5mm length of the CA1 subfield of the hippocampus counted under light microscopy. Data are mean ± s.d. (n = 6). 4 p < 0.01 vs. control group. * p < 0.05 vs. control group. # p < 0.05 vs. 24 h group. Pilo = pilocarpine.
tion of other autophagy proteins to the preautophagosome membrane [29,30]. In rats challenged by SE, elevations of beclin 1 levels were detected at different postseizure time points in hippocampus which is susceptible to neurodegeneration after stress. The earliest alterations in beclin 1 levels occurred at 2 h, peaked at 24 h and lasted for at least 3 days post-SE (Fig. 3). An increase in synthesis of beclin 1 in vivo was also seen, highly correlated with changes of beclin 1 protein. Thus, the up-regulation of beclin 1 appears to be a response to the seizure insult. Under nutrient-starvation conditions, ROS is increased, contributing to autophagy [8]. Oxidative stress is also believed to be one mechanism by which nonstarvation-induced autophagy can be initiated [12]. ROS generation occurs upstream of autophagy [31,32] and induce an increase in beclin 1 expression [33]. ROS specifically regulate the activity of Atg4, the cysteine protease responsible for the cleavage of the C terminus of LC3 I (Atg8), a reaction essential for its lipidation during the formation of autophagosome in vitro [26,34]. For these reasons, we tested the antioxidant Vitamin E for its capacity to reduce oxidative stress and autophagosome formation. Consistent with either upstream initiation or regulation of
autophagy by oxidative stress, Vitamin E treatment resulted in a reduction in autophagy. By biochemical evidence, Vitamin E treatment reduced the shift of LC3 II to LC3 I (Fig. 1), and the levels of beclin 1 by inhibiting its synthesis (Figs. 2 and 3). Pretreatment with Vitamin E not only produced salient biochemical effects, but also reduced histological damages and partly improved histological outcome (Table 1). What remains unclear is whether postseizure alterations in food intake contributed to the observed increase in autophagy. The nutritional status of these rats was not ascertained, thus a contribution of nutrient deprivation-induced autophagy, cannot be completely ruled out. Arguing against this, starvation alone up to duration of 48 h does not typically produce autophagy in brain-traditionally considered protected from nutritional starvation—in vivo [4]. Autophagy is important in maintaining intracellular homeostasis and keeping the cell healthy, and is often considered to be primarily a survival strategy in multicellular organisms, which either is initiated by stressor. However, autophagy may also promote cell death (referred to as type II cell death) through excessive self-digestion and degradation of essential cellular constituent [2,3]. Up-regulation of LC3 II and beclin 1 is consistent with degree of cell death after SE, and pretreated Vitamin E reduced both auotphagy and cell death. All these data showed that autophagic cell death also occurred after SE. Whether autophagy contributes to delayed neurodegeneration induced by SE needs further study. The question that constantly arises, however, is whether autophagic activity is the cause of death or is actually an attempt to prevent it as a part of an endogenous neuroprotective response. Regardless, it is clear that autophagy and oxidative stress are connected during SE, and that one effect of antioxidant therapy is a reduction in autophagy. In summary, this study identified a role of autophagy as part of the seizure stress response. Autophagy is increased in pilocarpineinduced SE in rats. Antioxidant Vitamin E inhibited autophagy and may represent a therapeutic target. As in other neurological diseases, more work is needed to determine whether autophagy is
L. Cao et al. / Biochemical and Biophysical Research Communications 379 (2009) 949–953
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E Lili Cao, Jingjing Xu, Youting Lin, Xiuhe Zhao, Xuewu Liu, Zhaofu Chi * Department of Neurology, Qilu Hospital of Shandong University, Wenhua West Road 107, Jinan 250012, China
a r t i c l e
i n f o
Article history: Received 17 December 2008 Available online 10 January 2009
Keywords: Autophagy Light chain 3 Beclin 1 Oxidative stress Hippocampus
a b s t r a c t Autophagy, a process of bulk degradation of cellular constituents through autophagosome–lysosomal pathway, is enhanced during oxidative stress. Whether autophagy is induced during status epilepticus (SE), which induces an excess production of reactive oxygen species (ROS) and leads to oxidative stress, is not established. We also sought to determine if pretreatment with Vitamin E reduced autophagy. We used pilocarpine to elicit SE in rats. The ratio of LC3 II to LC3 I and beclin 1 were used to estimate autophagy. We found that ratio of LC3 II to LC3 I and beclin 1 increased significantly at 2, 8, 16, 24 and 72 h, peaking at 24 h after SE onset. Pretreatment with Vitamin E partially inhibited autophagy by reducing LC3 II formation and de novo synthesis of beclin 1 at 24 h after seizures. These data show that autophagy is increased in rats with pilocarpine-induced SE, and Vitamin E have a partial inhibition on autophagy. Ó 2009 Elsevier Inc. All rights reserved.
Autophagy is a process in which cytoplasmic components such as organelles and proteins are delivered to the lysosomal compartment for degradation, and plays an essential role in maintenance of cellular homeostasis [1,2]. Macroautophagy (hereafter referred to as autophagy), the major process of autophagy, involves the formation of double-membrane vesicles, named autophagosomes that engulf and deliver cytosolic material to the lysosomes [3,4]. In the most cells, autophagy is suppressed to a basal level. It is hypothesized that up-regulation of this system may play a significant role in adaptation to environments where oxidative stress is a major selection pressure by eliminating unwanted or unnecessary organelles and recycling cellular components for reuse [5,6]. Recent studies have proved that oxidative stress is implicated in the induction of autophagy and reactive oxygen species (ROS) serve as signaling molecules that initiate autophagy [7,8]. A role for autophagy has been uncovered in central nervous system disease [9–12]. Status epilepticus (SE) induced by systemic injections of pilocarpine provide a valuable animal model of temporal lobe epilepsy (TLE). Oxidative stress has been implicated in seizure, and ROS increase significantly in animal with pilocarpine-induced SE, which in turn enhance seizure activity [13,14]. Moreover, Glutamate excitotoxicity has been involved in the pathophysiology of epilepsy and related to neuronal death in pilocarpine-induced status epilepticus [15]. Seizure can result in ATP exhaust [16]. ATP exhaust, glutamate and oxidative stress are important inducer of autophagy [3].
* Corresponding author. Fax: +86 53186026772. E-mail address: [email protected] (Z. Chi). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.178
To date, a large group of autophagy-related proteins (ATGs) are known to directly regulate the formation of autophagosome [2]. Modification of light chain 3 (LC3) I (apparent mobility, 18 kd), (the mammalian homolog of yeast Atg8) with covalent attachment of phosphatidylethanolamine results in LC3 II (apparent mobility, 16 kd), a process essential for autophagy referred to as an ‘LC3 shift’ or ‘LC3 lipidation’ [17]. The amount of LC3 II or the LC3 II to LC3 I ratio correlated with the number of autophagosome [18]. The measurement of LC3 II by immunoblotting is a simple and quantitative method for determining autophagic activity of mammalian cells [4]. Beclin 1, a Bcl-2-interacting protein, is the mammalian homolog of yeast VPS30p/Apg6p, forms a complex with phoshatidylinositol-3-kinase (PI3K), and participates in the early stages of autophagosome formation, promoting the nucleation of autophagosome [19]. It has been shown that beclin 1 is essential for autophagy as well as for lysosomal enzyme transport [20,21]. In this study, pilocarpine was used to elicit SE. We sought to determine whether autophagy was activated after SE in rats in vivo. In addition, whether pretreatment with antioxidant Vitamin E influenced autophagy and cell injury after SE were determined. LC3 II/LC3 I ratio and beclin 1 were used to estimate autophagy. Materials and methods Adult male Wistar rats (220–250 g) were used. Animals were kept in standard conditions (12 h light and dark cycle) with free access to food and water during all experimental time period. Experiments were performed following the ‘‘Principles of Laboratory
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Animals Care” (NIH Publication No. 85-23, revised 1996) and according to the guidelines of the Commission of Shandong University for ethics of experiments on animals. Seizures were induced by pilocarpine injections as reported in previous study [14]. Rats were divided to four groups. The first group was pilocarpine-injected (340 mg/kg i.p.; Sigma) 30 min after an injection of atropine methyl nitrate (1 mg/kg i.p.) to reduce the number of peripheral cholinergic signs. Animals with 2 h SE, defined as a continuous motor seizure of stage 4 (rearing and falling), stage 5 (loss of balance, continuous rearing and falling), or stage 6 (severe tonic–clonic seizures) [22] were killed respectively at 2, 8,16, 24 and 72 h after seizure-onset. The control rats were injected with a physiological salt solution instead of pilocarpine. The third group was Vitamin E (200 mg/kg, Sigma) preinjected 30 min before pilocarpine injection, and were killed at 24 h after seizure-onset. The fourth group was injected with only Vitamin E as vehicle treatment group [23]. In order to standardize the different experimental groups, all animals were treated by diazepam administration (i.m. injection, at the dose of 4 mg/kg, Sigma) 2 h after pilocarpine (or saline) injection. The rats were sacrificed by decapitation and the brains were quickly removed and dissected. Hippocampus regions were maintained at 80 °C until determinations. Rats were anesthetized and intracardially perfusion with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) at different times after seizures. The brains were removed and kept in 4% paraformaldehyde for 12 h, then immersed in 25% sucrose for 3– 4 days at 4 °C. The paraffin-fixed brains were sectioned coronally in 0.5 mm thickness. Nissl staining with toluidine blue was first performed. The surviving cells were defined as round-shaped, cytoplasmic, membrane-intact cells, without any nuclear condensation or distorted aspect. Using high magnification (40), the number of surviving neurons/0.5mm length in hippocampus CA1 regions was blindly counted. Detailed procedure was carried out as described previously [24]. Total RNA was prepared from hippocampus with Trizol reagent (Invitrogen, USA) and reversely transcribed to cDNA using AMV First Strand DNA Synthesis Kit (Biotech Company, China). Briefly, a 1 lg of the isolated RNA was reversely transcribed to cDNA at 37 °C for 1 h in a 20 ll of reaction mixture containing 1 ll AMV reverse transcriptase, 1 ll random hexamer, 4 ll 5 AMV buffer, 1 ll RNase inhibitor (20 U/ll), 1 ll dNTP (10 mM). The PCR amplification mixture (25 ll) consisted of 1 ll cDNA mixture, 1 ll Taq DNA polymerase, 5 ll of 5 PCR buffer, 5 mM dNTP mixture, and 50 pM sense and antisense primers each. Beta-actin was used as an internal control. Beclin 1 was analyzed by PCR and the used oligonucleotide primers included: for beclin 1 (316 bp) forward primer 50 -AGGAGCAGTGGACAAAGG-30 and reverse primer 50 AGGGAAGAGGGAAAGGAC-30 , for beta-actin (142 bp) forward primer 50 -GACAGGATGCAGAAGGAGATTACT-30 and reverse primer 50 -TGATCCACATCTGCTGGAAGGT-30 . The PCR conditions were as follows: for initial denaturing at 94 °C for 5 min, followed by 30 PCR cycles with temperatures below: 94 °C for 40 s, for beclin 1 54 °C for 40 s, or for beta-actin 52 °C for 40 s each, and then 72 °C for 40 s; a final extension at 72 °C for 10 min. The amplified products were subjected to electrophoresis at 120 V for 30 min on 1.5% agarose gels containing 0.5 mg/ml ethidium bromide, and quantified by respectively comparing luminosity of beclin 1 to that of b-actin using AlphaEase FC (FluorChem 9900) (Alpha Innotech). Equal amounts of protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), and then transferred to 0.45 lm nitrocellulose membrane using a Trans-Blot semidry system (Bio-Rad). After blocking in 5% fat-free milk in Tris-buffered saline with Tween buffer for 2 h, the membranes were incubated with a primary antibody such as anti-LC3 (1:6000, Burlingame, Epitomics), anti-beclin 1 (1:500, Ab-
cam, UK), anti-actin (1:500, Zhongshan Goldenbriodge Biotechnology CO.LTD, China), which served as a loading control, overnight at 4 °C. Then the membranes were washed and incubated with horseradish peroxidaseconjugated secondary antibody (goat anti-rabbit IgG, 1:10000, Zhongshan Goldenbriodge Biotechnology CO.LTD, China) for 2 h under room temperature. Immunoreactivity was enhanced by chemiluminescence kit (Pierce, Rockford, Illinois, USA) and exposed to film. The bands on the film were scanned and analyzed with an image analyzer (Alpha Innotech, San Leandro, California, USA). Results in this study are presented as mean ± s.d. Two group comparisons were performed using Student’s t-test. Multiple group comparisons were performed by one-way analysis of variance, followed by the Newman–Keuls test. p < 0.05 was considered statistically significant. Results To semiquantify the amount of autophagosome formation after SE relative to naive controls, we examined hippocampi from rats with SE for evidence of an LC3 shift from an apparent B18 to B16 kd position [25]. By Western blot analysis, the time-course study of LC3 demonstrated that the ratio of LC3 II to LC3 I was significantly increased at 2, 8, 16, 24 and 72 h after SE, peaking at 24 h (Fig. 1A and B, p < 0.05). Since autophagy can be induced by mitochondrial oxidative stress and reactive oxygen species are essential for starvation-induced autophagy to proceed in vitro [8,12], we determined whether the antioxidant Vitamin E could reduce autophagy. Our data showed that pretreatment with Vitamin E reduced relative protein abundance of LC3 II to LC3 I at 24 h after SE versus vehicle-pretreated rats (Fig. 1C and D, p < 0.05), indicating that Vitamin E inhibited autophagy partly. Semiquatitive RT-PCR analysis showed that beclin 1 mRNA levels were significantly higher at 2 h after seizures onset compared with control group. A time-course of beclin 1 mRNA levels showed beclin 1 mRNA reached a peak at 24 h and remained at high levels at 72 h after SE (Fig. 2A and B). Western blot analysis showed that beclin 1 protein levels also began to increase at 2 h after SE, reached a maximum level at 24 h and remained significantly elevated until 72 h (Fig. 3A and B). Pretreatment with Vitamin E reduced both mRNA and protein levels of beclin 1 at 24 h after SE (Figs. 2C and D and 3C and D). To examine the extent of neuron loss in hippocampus after pilocarpine-induced seizures, toluidine blue was used for staining. Neurons in CA1 region are very vulnerable to SE stress. Our results showed that seizures led to severe cell death in CA1, which peaked at 24 h after seizure-onset. The surviving neuron numbers were sharply decreased in the pilocarpine-treated group compared with control group. Moreover, Vitamin E significantly attenuated the neuron loss induced by seizures (Table 1). Discussion The major finding of this study is that autophagy, detected biochemically, is induced in injured brain after SE in rats. An additional finding is that autophagosome formation, as detected by increases in LC3 II and beclin 1, is partially inhibited by the antioxidant Vitamin E. Treatment with Vitamin E partially reduces histological damage of SE in rat compared with vehicle treatment. These data represent the first evidence of autophagy activation and the first to suggest that reducing oxidative stress by Vitamin E reduces autophagy, after acute brain injury induced by pilocarpine-produced SE. Taken together, these data suggest that oxidative stress contributes to overall neuropathology after SE, at least in part by initiating or influencing autophagy. Similar to starvation-induced
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Fig. 1. The effects of Vitamin E on hippocampus LC3 levels. (A) Western blot for analysis showing the time-course of LC3 II and LC3 I levels at various times after seizures. (B) Quantitative representation of LC3 II to LC3 I ratio at various times after seizures.(C) Western blot for analysis showing the effects of Vitamin E on LC3 II and LC3 I levels at 24 h after seizures. (D) Quantitative representation of effect of Vitamin E on LC3 II to LC3 I ratio at 24 h after seizures. Data are expressed as mean ± s.d. of at least four independent animals, *p < 0.05 vs. control, #p < 0.05 vs. 24 h, pilo = pilocarpine.
Fig. 2. The effects of Vitamin E on hippocampus beclin 1 mRNA levels after seizures. (A) Semi-quantitative RT-PCR analysis of time courses of beclin 1 mRNA in rat hippocampus after seizures. (B) Quantitative representation of the mRNA levels of beclin 1 at various times after seizures. (C) Semi-quantitative RT-PCR analysis of effects of Vitamin E on mRNA of beclin 1 at 24 h after SE onset. (D) Quantitative representation of the effects of Vitamin E on the beclin 1 mRNA. Data are expressed as mean ± s.d. of at least four independent animals. *p < 0.05 vs. control, #p < 0.05 vs. 24 h, pilo = pilocarpine.
autophagy, a role for oxidative stress in SE-induced autophagy is also implicated. LC3 was the first identified mammalian protein localized in autophagosome membranes. LC3 has two forms: LC3 I is cytosolic form, and is activated and modified to a membrane-bound form, LC3 II. Thus, the ratio of LC3 II to LC3 I is used to semiquantify the amount of autophagosome formation [18,26]. In this study, the ration of LC3 II to LC3 I after SE was markedly increased at 2,
8, 16, 24 and 72 h, with peak relative abundance at 24 h (Fig. 1), although time point after 72 h are not evaluated and such a later peak cannot be ruled out. These data indicate the activation of autophagy in rat hippocampus after SE. In rat brain, beclin 1 is expressed in many areas including cerebral cortex, hippocampus and cerebellum [27,28]. Beclin 1 is part of a Class III PI3K complex that locates at trans-Golgi network and participates in autophagosome formation, mediating localiza-
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Fig. 3. The effects of Vitamin E on hippocampus beclin 1 levels after seizures. (A) Western blot analysis of time-course of beclin 1 in rat hippocampus after seizures. (B) Quantitative representation of beclin 1 at various times after seizures. (C) Western blot analysis of effects of Vtamin E on beclin 1 at 24 h after SE onset. (D) Quantitative representation of effects of Vitamin E on beclin 1 at 24 h after SE onset. Data are expressed are mean ± s.d. of at least four independent animals.*p < 0.05 vs. control, #p < 0.05 vs. 24 h group, pilo = pilocarpine.
Table 1 Protective effects of Vitamin E on neuron death in hippocampus CA1 after seizures. Group
Neuron numbers (mean ± s.d.)
Control 2h 8h 16 h 24 h 72 h Vitamin E + pilo
110.67 ± 18.56 98.17 ± 15.22 87.5 ± 17.38 79.83 ± 19.49* 75.5 ± 15.924 82.83 ± 19.59* 94.67 ± 13.02#
Neuron number is expressed as the number of surviving pyramidal cells/0.5mm length of the CA1 subfield of the hippocampus counted under light microscopy. Data are mean ± s.d. (n = 6). 4 p < 0.01 vs. control group. * p < 0.05 vs. control group. # p < 0.05 vs. 24 h group. Pilo = pilocarpine.
tion of other autophagy proteins to the preautophagosome membrane [29,30]. In rats challenged by SE, elevations of beclin 1 levels were detected at different postseizure time points in hippocampus which is susceptible to neurodegeneration after stress. The earliest alterations in beclin 1 levels occurred at 2 h, peaked at 24 h and lasted for at least 3 days post-SE (Fig. 3). An increase in synthesis of beclin 1 in vivo was also seen, highly correlated with changes of beclin 1 protein. Thus, the up-regulation of beclin 1 appears to be a response to the seizure insult. Under nutrient-starvation conditions, ROS is increased, contributing to autophagy [8]. Oxidative stress is also believed to be one mechanism by which nonstarvation-induced autophagy can be initiated [12]. ROS generation occurs upstream of autophagy [31,32] and induce an increase in beclin 1 expression [33]. ROS specifically regulate the activity of Atg4, the cysteine protease responsible for the cleavage of the C terminus of LC3 I (Atg8), a reaction essential for its lipidation during the formation of autophagosome in vitro [26,34]. For these reasons, we tested the antioxidant Vitamin E for its capacity to reduce oxidative stress and autophagosome formation. Consistent with either upstream initiation or regulation of
autophagy by oxidative stress, Vitamin E treatment resulted in a reduction in autophagy. By biochemical evidence, Vitamin E treatment reduced the shift of LC3 II to LC3 I (Fig. 1), and the levels of beclin 1 by inhibiting its synthesis (Figs. 2 and 3). Pretreatment with Vitamin E not only produced salient biochemical effects, but also reduced histological damages and partly improved histological outcome (Table 1). What remains unclear is whether postseizure alterations in food intake contributed to the observed increase in autophagy. The nutritional status of these rats was not ascertained, thus a contribution of nutrient deprivation-induced autophagy, cannot be completely ruled out. Arguing against this, starvation alone up to duration of 48 h does not typically produce autophagy in brain-traditionally considered protected from nutritional starvation—in vivo [4]. Autophagy is important in maintaining intracellular homeostasis and keeping the cell healthy, and is often considered to be primarily a survival strategy in multicellular organisms, which either is initiated by stressor. However, autophagy may also promote cell death (referred to as type II cell death) through excessive self-digestion and degradation of essential cellular constituent [2,3]. Up-regulation of LC3 II and beclin 1 is consistent with degree of cell death after SE, and pretreated Vitamin E reduced both auotphagy and cell death. All these data showed that autophagic cell death also occurred after SE. Whether autophagy contributes to delayed neurodegeneration induced by SE needs further study. The question that constantly arises, however, is whether autophagic activity is the cause of death or is actually an attempt to prevent it as a part of an endogenous neuroprotective response. Regardless, it is clear that autophagy and oxidative stress are connected during SE, and that one effect of antioxidant therapy is a reduction in autophagy. In summary, this study identified a role of autophagy as part of the seizure stress response. Autophagy is increased in pilocarpineinduced SE in rats. Antioxidant Vitamin E inhibited autophagy and may represent a therapeutic target. As in other neurological diseases, more work is needed to determine whether autophagy is
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