3. Lipid metabolism and cell cycle: anti-proliferative effects of PC-PLC inhibitor D609 in stroke
R. Adibhatla1,2,3,4, J. Hatcher1, R. Dempsey1 and H. Kalluri1
1Neurological Surgery; 2Neuroscience Training Program; 3Cardiovascular Research Center, University of Wisconsin School of Medicine and Public Health; 4Research, William S Middleton Veteran Affairs Hospital, Madison, Wisconsin, USA
Introduction: The pro-inflammatory response after stroke due to up-regulation of TNF-α and IL-1 can activate phospholipases and stimulate ROS production. Transient middle cerebral artery occlusion (tMCAO) in rat significantly increased (4-fold) PC-PLC activity, up-regulated sphingomyelinases (SMase, neutral- and acidic-) and down-regulated PC synthesis and sphingomyelin (SM) synthases (SMS-1 and 2) in the ipsi cortex, causing significant PC and SM loss.
1
Lipid second messengers 1,2-diacylglycerol (DAG) and ceramide, produced by hydrolysis of PC and SM, respectively, are key regulators of the cell cycle. The PC-PLC product DAG can stimulate cyclin D1 expression through pERK by activation of protein kinase C, either directly or through ASMase/ceramide pathway, and activation of AKT via ASMase/ceramide/PI3 kinase pathway. Up-regulation of cyclin D1 can trigger the cell cycle, inducing proliferation of astrocytes and microglia/macrophages. However, for post-mitotic neurons, cell cycle entry results in their death.
Results: In tMCAO (1 h by intraluminal suture method in SHR), PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609) attenuated infarction by 35±5%. D609 blocked proliferation (BrdU uptake) of rat neural progenitor cells by 75±5% and attenuated pERK, pAKT and cyclin D1 expression (Western blotting). D609 did not alter expression of pAKT, pERK and cyclin D1 after stroke. This may be due to heterogeneous populations of neuronal (<5%) and non-neuronal cells and changes might have been blunted, unlike in the homogeneous rat neural progenitor cells. However, treatment with D609 did increase ceramide levels and expression of endogenous cell cycle inhibitor, p21cip1, suggesting cell cycle arrest at G1-phase after stroke. SM synthase transfers the phosphocholine group of PC to ceramide to form SM and DAG. D609 also inhibits SM synthase, which could account for the increase in ceramide following D609 treatment. Ceramide can induce G0/G1 cell cycle arrest by up-regulation of CDK inhibitor p21Cip1.
2
Recent studies indicate that p21cip1 exerts neuroprotection via mechanisms independent of its nuclear role in cell cycle inhibition.
3
In addition to PC hydrolysis and impaired synthesis, oxidation of PC to oxidized-PC (OxPC, detected by EO6-monoclonal antibodies) due to ROS generation was also observed after tMCAO.
4
OxPC may serve as a marker of neuroinflammation and apoptosis and also enhance pro-inflammatory signals. D609 also attenuated formation of OxPC-modified proteins after tMCAO, which could be mediated by increased p21 expression or reduction in pro-inflammatory cytokine levels.
Conclusions: We hypothesize that PC-PLC regulates cell cycle through DAG in proliferating non-neuronal cells and mediates cell death in mature post-mitotic neurons. D609 prevented bFGF stimulated astrocyte proliferation in other studies.
5
D609 may inhibit proliferation of cytokine-expressing microglia/macrophages and simultaneously prevent mature neurons from entering into cell cycle and dying, thus providing protection after stroke. Funded by NIH, AHA and UW Med School.
65. The HMGB1 receptor rage mediates ischemic brain damage
S. Muhammad1, B. Waleed1, S. Murikinati1, S. Stoyanov2, H. Yang3, K.J. Tracey3, M. Bendszus4, P.P. Nawroth2, A. Bierhaus2, G. Rossetti5 and M. Schwaninger1
1Pharmacological Institute; 2Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; 3Feinstein Institute for Medical Research, Manhasset, New York, USA; 4Department of Neuroradiology, University of Heidelberg, Heidelberg, Germany; 5HMGBiotech, Milan, Italy
In ischemic stroke, the necrotic core is surrounded by a zone of inflammation, in which delayed cell death aggravates the initial insult. Here, we provide evidence that the receptor for advanced glycation end products (RAGE) functions as a sensor of necrotic cell death and contributes to inflammation and ischemic brain damage. The RAGE ligand high mobility group box 1 (HMGB1) was elevated in serum of stroke patients and was released from ischemic brain tissue in a mouse model of cerebral ischemia. A neutralizing anti-HMGB1 antibody and HMGB1 box A, an antagonist of HMGB1 at the receptor RAGE, ameliorated ischemic brain damage. Interestingly, genetic RAGE deficiency and the decoy receptor soluble RAGE (sRAGE) reduced the infarct size. In vitro, expression of RAGE in (micro)glial cells mediated the toxic effect of HMGB1. Addition of macrophages to neural cultures further enhanced the toxic effect of HMGB1. To test whether immigrant macrophages in the ischemic brain mediate the RAGE effect, we generated chimeric mice by transplanting RAGE−/− bone marrow to wild-type mice. RAGE deficiency in bone marrow-derived cells significantly reduced the infarct size. Thus, HMGB1-RAGE signaling links necrosis with macrophage activation and may provide a target for anti-inflammatory therapy in stroke.
85. HGTD-P plays a pro-apoptotic role in rat brain with hypoxia-ischemia
Y. Qu and D. Mu
Department of Pediatrics, Westchina Second University Hospital, Sichuan University, Chengdu, China
Background: HGTD-P is a newly founded pro-apoptotic protein and an effector of hypoxia-ischemia (HI) induced cell death. The function of HGTD-P has been investigated in human prostate cancer cells. However, whether HGTD-P is involved in regulating apoptosis of rat neurons is not clear.
Objective: To elucidate the roles of HGTD-P in cellular apoptosis both in vitro and in vivo following HI.
Methods: Samples from primary cultured neurons and postnatal day 10 rat brains with HI were collected. RT-PCR, Western blots, and immunocytochemistry were used to detect the expression and distribution of HGTD-P. MTT assay, DAPI, TUNEL and flowcytometry were used to detect cell viability and apoptosis.
Results: We found that HGTD-P mRNA and protein expression were upregulated in cultured neurons and rat brains following HI. Antisense oligonuecleotides (AS) targeted to HGTD-P could inhibit the expression of HGTD-P, and thus rescued cell viability and attenuated cell apoptosis. In addition, we found that HGTD-P might play a pro-apoptotic role through the activation of caspase 3 and inducing the translocation of apoptosis inducible factor (AIF) to the nucleus.
Conclusions: Our findings suggested that HGTD-P plays a pro-apoptotic role in the developing rat brain after HI and targeting HGTD-P may be a potential therapy for HI-induced neonatal brain damage.
114. Proteasome inhibition by Lactacystin in an optimal concentration protects neuronal cells from ischemia and reperfusion injury in vitro
R. Liu, T. Urabe, K. Yamashiro and N. Hattori
Neurology, Juntendo University School of Medicine, Tokyo, Japan
Objectives: We have reported the magnitude of an intrinsic neuroprotectant, erythropoirtin (EPO) and its transcription factor hypoxia inducible factor-1 (HIF-1) α. However, reoxygenation significantly degraded HIF-1α activation, reduced subsequent transcription of EPO. In the present study, we design to investigate the neuroprotective effect of proteasome inhibition by Lactacystin against ischemia/reperfusion injury.
Methods: Primary cultures of cerebral cortical cells were obtained from embryos (E 16 to 17) of Wistar rats. In vitro ischemia/reperfusion was induced by hypoxia (2%) and reoxygenation using primary cerebral cortical culture cells. Cortical cultures were treated with 0.1 to 60 μmol/L lactacystin (Calbiochem, CA), a specific inhibitor of the proteasome, for 24 h under normoxic or hypoxia/reoxygenation condition before hypoxia or after onset of reoxygenation. The neuronal survival was assessment by immunofluorescence staining of PI and FDA, and the expression of HIF-1a as well as EPO protein and was detected by Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis in culture cells under normoxia, 6 h hypoxia followed 24 h reoxygenation only without lactacystin treatment (H6R24 Lac−), and the same period of hypoxia/reoxygenation with lactacysticn (H6R24 Lac+).
Results: We found that lactacystin is contributed to block the degradation of HIF-1α and EPO during oxygenation by using immuoblot analysis and RT-PCR. We also determined that near-complete blockade of proteasome activity (<5%) by Lactacystin (≥20 μmol/L) during reoxygenation or throughout hypoxia/reoxygenation improved neuronal survival to a range to 61.2% or 47.9% respectively, compared with that of 6 h hypoxia followed 24 h oxygenation only without Lactacytin treatment (24.3%, P<0.001 to 0.0001). Our results indicated that application of Lactacystin in an optimal Aconcentration dose promote an neuronprotective effect against in vitro ischemia/reperfusion injury, and interfering with the progression of apoptosis might partially by preventing degradation of HIF-1α and EPO.
Conclusion: Our initial studies confirmed the observations that proteasome inhibition by lactacystin especially during reoxygenation leads to an up-regulation of HIF-1a and EPO and promote as a neuroprotectant against ischemia/reperfusion injury. Our study suggests that the proteasome inhibition strategies is a potential therapeutic target for acute stroke.
300. Expression of nucleoside transporters in rat cortical astrocytes in primary culture: effects of hypoxia and glucose deprivation
Z. Redzic, S. Malatiali and M. Al-Bader
Department of Physiology, Faculty of Medicine, Kuwait University, Kuwait, Kuwait
Objectives: The objectives of this study were to explore the amount of mRNA for rat equilibrative nucleoside transporters (rENT) 1 and 2 and rat concentrative nucleoside transporter (rCNT) 1, 2 and 3 in rat cortical astrocytes in primary culture under normal conditions, after 1 h exposure to hypoxia and inhibition of glycolysis (2% O2, 5% CO2 in N2, 10 mmol/L 2-deoxy-D-glucose (2-DG), 1 mmol/L D-glucose) (ischemia group) and after 1 h exposure to hypoxia and inhibition of glycolysis, followed by 1 h exposure to normal conditions (5% CO2 in the air, 5 mmol/L glucose, no 2-DG) (reperfusion group).
Methods: Primary cultures of cortical astrocytes were produced as explained.
1
Real time polymerase chain reaction (PCR) was used to explore the amount of mRNA; the threshold cycles for target amplification (Ct) values were estimated for genes of interest and the difference between their Ct values and Ct values for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt) was calculated. The quantification of gene expression and the significance of difference was estimated using the comparative ΔΔCt method, as described earlier.
2
Results: In rat cortical astrocytes, the Ct value of GAPDH did not differ between the control (20.34±0.51, n = 5), ischemia (20.4±0.41, n = 3, P>0.05) and reperfusion group (20.7±0.3, n = 3, P>0.05). Therefore, this gene was used as the endogenous control. The data for ENTs and CNTs are presented in the Table 1, signs * and ** indicate P<0.05 and P<0.01 versus control, respectively; according to the ΔCt values from the control group, mRNA for rENT1, rENT2 and rCNT2 was abundant, while the mRNA for rCNT1 was less abundant. The mRNA for rCNT3 was either absent or present at very low abundance, since the ΔCt values were close to the ΔCt values in negative controls (>20) in all experimental groups (not shown). One hour exposure to ischemic-like conditions leads to a significant increase in the amount of mRNA for rCNT1 and to a significant decrease in the amount of mRNA for rENT1. When exposure to ischemic-like conditions was followed by exposure to normal conditions (reperfusion group) there was an increase in the amount of mRNA for rENT2, a decrease in the amount of mRNA for rENT1 and further increase in amount of mRNA for rCNT1.
Conclusions: Exposure of rat astrocytes in primary culture to ischemia-like conditions and ischemia-reperfusion like conditions leads to a significant change in the amount of mRNA for nucleoside transporters.
327. Vesicular GABA transporter (VGAT) is cleaved by calpains under excitotoxic conditions, generating a non-synaptic protein
J Gomes1, C. Melo1, A. Inacio2, L. Almeida1, T. Wieloch2 and C. Duarte1
1Center for Neuroscience and Cell Biology and Department of Zoology, University of Coimbra, Coimbra, Portugal; 2Wallenberg Neuroscience Center, Lund University, Lund, Sweden
Aims: Although GABA is the major inhibitory neurotransmitter in the CNS, and may be neuroprotective under excitotoxic conditions,
1
it has been suggested that activation of GABAA receptors may contribute to cell damage due to chloride entry and consequent neuronal swelling.2–4 Recent findings showed that vesicular transporter expression directly regulates neurotransmitter release, controlling the quantal efficacy of synaptic transmission.
5
Therefore, putative changes in VGAT protein levels during ischemia may affect GABAergic transmission and cell death. In this work we investigated the fate of VGAT in cultured neurons, subjected to excitotoxic conditions and in the ischemic brain.
Methods: Cultured hippocampal neurons were subjected to excitotoxic insults with glutamate. VGAT protein levels were determined by western blot, and gene expression was analysed by Real Time PCR. The cellular distribution of VGAT was visualized with immunocytochemistry. In vivo models used were: intrahippocampal injection of Kainate and transient Middle Cerebral Artery Occlusion (MCAO).
Results: We found that VGAT (58 KDa) is cleaved upon glutamate stimulation of hippocampal neurons, in a biphasic manner, giving rise to a product of about 46 KDa (tVGAT). About 40% of VGAT is cleaved within the first hour after glutamate stimulation, and 35% of the protein is cleaved 2 to 7 h later. Truncated VGAT is stable for more than 24 h. Under the same conditions, VGAT mRNA is downregulated, by a process of active mRNA degradation. Calpain I inhibition with ALLN or MDL28170 prevented the cleavage of VGAT in hippocampal neurons subjected to excitotoxicity. Inhibition of Caspases with Z-VAD-FMK did not affect glutamate-induced VGAT cleavage. VGAT cleavage was also observed in the cerebral cortex and striatum following transient Middle Cerebral Artery Occlusion (MCAO), a cerebral ischemia model. VGAT was also cleaved following intrahippocampal injection of kainate, a different model of in vivo excitotoxicity, but no effect was observed in transgenic mice overexpressing calpastatin, an endogenous calpain inhibitor. In vitro studies, using cerebrocortical synaptic vesicles and recombinant calpain I, also showed the cleavage of VGAT to the same product of about 46 KDa. Immunoblot experiments using different antibodies against VGAT showed that calpain cleaves VGAT in the N-terminal region of the protein, giving rise to two similar truncated forms, at amino acid 52 and amino acid 60. Imunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with VGAT or tVGAT proteins, showed that tVGAT immunoreactivity loses the synaptic localization, being homogenously distributed along the axons. This change in VGAT distribution was also observed with endogenous VGAT in glutamate stimulated hippocampal cultures.
Conclusions: Taken together, our results show that glutamate toxicity causes a down-regulation of VGAT, with a concomitant generation of a truncated VGAT, which is likely to affect GABAergic neurotransmission and may influence cell death, during ischemia.
510. Downregulation of Kv4.2 channels by NR2B-containing NMDA receptors in cultured hippocampal neurons
Z. Lei, P. Deng, Y. Li and Z. Xu
Indiana University School of Medicine, Indianapolis, Indiana, USA
Objective: Somatodendritic Kv4.2 channels mediate transient A-type potassium currents (IA), and play critical roles in controlling neuronal excitability and modulating synaptic plasticity.
1
Our previous studies have shown a downregulation of Kv4.2 and IA caused by activation of NMDA receptors.
2
Our previous studies have shown a NMDA-dependent downregulation of Kv4.2 and IA. NMDA receptors are wildly believed to be heteromeric complexes of NR1A combined with NR2A-NR2D.
3
NR2A- and NR2B-containing NMDA receptors have opposite role on excitotoxic neuronal injury.
4
Here, we investigate the involvement of NR2A- and NR2B-containing NMDA receptors in glutamate modulation of Kv4.2 and IA in cultured hippocampal neurons.
Methods: Primary neuronal cultures were prepared from the hippocampus of E18 embryos.
5
The neurons were treated on DIV18. The expression levels of Kv4.2 were analyzed by Western blotting, the cellular distributions of Kv4.2 were detected by immunostaining, and the properties of IA currents were measured by whole-cell voltage clamp recording.
Results: Bath application of glutamate caused a reduction in total Kv4.2 protein levels and Kv4.2 clusters, and produced a hyperpolarized shift in the inactivation curve of IA. The effect of glutamate on Kv4.2 and IA was inhibited by pretreatment of specific NR2B antagonists. In contrast to the predominant expression of NR2A-containing NMDA receptors at synapse, NR2B-containing NMDA receptors is wildly believed to be the predominant NMDA receptors expressed at extrasynaptic sites in mature hippocampal neurons in vitro.
6
We found that, like bath application of glutamate, selective activation of extrasynaptic NMDA receptors caused a reduction in total Kv4.2 protein levels and Kv4.2 clusters. In contrast, specific stimulation of synaptic NMDA receptors had no effect on Kv4.2. In addition, the influx of Ca2+ was essential for extrasynaptic modulation of Kv4.2.
Conclusions: These results demonstrate that the downregulation of Kv4.2 and IA induced by glutamate is mediated by the activation of NR2B-containing receptors, which might be associated with NR2B-mediated NMDA responses, such as excitotoxic neuronal death.
Supported by NIH grant NS38053 and AHA grant 0655747Z, AHA0526007Z, AHA0425689Z, AHA0630172N, AHA0710027Z, AHA0825810G.
727. Astrocyte survival to ischemic damage requires an intact mTOR/S6 kinase pathway
M.D. Pastor, N. Fradejas, P. Tranque and S. Calvo
School of Medicine University Castilla-La Macha Spain, Albacete, Spain
Astrocytes functions are crucial for CNS homeostasis, as they provide trophic, metabolic and antioxidant supports to neurons and play protective functions during brain damage.
1
In order to identify genes involved in the astrocyte response to ischemia, we performed mRNA differential display using astrocytes subjected to oxygen and glucose deprivation (OGD). We detected a robust downregulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein level and activity. The main goal of this work was thus to analyze the functions of this kinase during brain ischemia.
Methods: To carry out this objective we used both in vitro and in vivo models of ischemia and S6K deficient mice. Cell viability and ROS production were measured by flow cytometry. Quantitative RT-PCR, Western blot and immunoprecipitation were used to analyze the expression levels of S6K and several pro- or antiapototic factors.
Results: Our data indicate that S6K protects astrocytes against in vitro ischemia, since OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes. Moreover, treatment with rapamycin, that inhibits S6K activity by acting on its upstream regulator mTOR, also incremented astrocyte injury caused by OGD. Among the mechanisms potentially involved in the increased damage observed in S6K−/− astrocytes, we have observed a defect in BAD inactivation by phosphorylation at its Ser136. In addition, the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL was significantly decreased in astrocytes lacking S6K, suggesting that this kinase is involved in maintaining the balance between pro- and antiapoptotic factors under stress conditions. Besides, ischemia-induced reactive oxygen species accumulation was much more prominent in S6K−/− astrocytes while pretreatment with the antioxidant N-acetyl-L-cysteyne completely abrogated OGD-induced S6K−/− astrocyte death, suggesting that S6K regulates oxidative metabolism. Finally, when the effects of S6K on in vivo ischemia were analyzed following permanent middle cerebral artery occlusion, we observed that both, infarct volume and mice mortality were increased in S6K−/− mice. In summary, our results indicate that S6K confers protection against ischemia through the regulation of astrocyte ROS and pro- and antiapoptotic factors.
731. Cell type specific effects of excitotoxic insult and energy limitation on cultured hippocampal neurons and astrocytes
S. Kahlert and G. Reiser
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke Universität, Magdeburg, Germany
Objectives: A characteristic of acute neurodegeneration after stroke is the massive increase in extracellular glutamate concentration. The ATP level gradually recovers with increasing distance from the core. In the penumbra, the region surrounding the core, the cellular energy is reduced due to reduced oxygen and glucose supply and increased extracellular glutamate concentration. The aim of the present study was to elucidate the question why neurons are much more affected by such adverse situations than the neighboring astrocytes.
Methods: To understand the mechanism of selective vulnerability of neurons we made a direct side by side comparison of rat hippocampal astrocytes and neurons in culture. We measured cytosolic Ca2+, mitochondrial potential, ROS generation, cytosolic ATP and NADH level.1,2 The cells, which were observed during kinetic measurements, were localized after immunofluorescence staining with NeuN and GFAP for a unequivocal identification of the cell types.
Results: Glutamate triggers massive Ca2+ influx in both neurons and astrocytes. Maximal Ca2+ loads were 5 to 6 times above basal level. However, both cell types could significantly remove the glutamate—triggered cytosolic Ca2+ load in the prolonged presence of glutamate. The Ca2+ response pattern was dramatically changed, when mitochondrial inhibitors were applied in combination with glutamate. Astrocyte-specific mechanisms are responsible for Ca2+ removal, which are largely not dependent on the mitochondrial polarization. Continuous detection of the MgGreen fluorescence allows an analysis of the kinetics of ATP changes. in neurons the application of glutamate induced a strong increase in MgGreen fluorescence, indicating a significant ATP decline. In neurons, removal of Ca2+ is linked to an increase of cytosolic Mg2+, released from Mg-ATP complex and mitochondrial activation, characterized by enhanced mitochondrial polarization and ROS generation. ROS increase was not due to inhibition of the respiratory chain, as NADH did not accumulate. Co-application of mitochondrial inhibitors of complex I or III, rotenone or antimycin A, with glutamate completely depolarized mitochondria, but did not further decrease the cellular ATP level. In these cases, the neuronal Ca2+ recovery was completely abrogated.
Conclusions: Hippocampal neurons are more susceptible than surrounding glial cells to ischemic insult with pathologic levels of the excitatory neurotransmitter glutamate. We conclude that inherent differences in energy supplementation of Ca2+ handling determine the sensitivity of neurons to oxygen limitation with mitochondrial impairment.
740. Kinase activities of JNK and ERK do not match their phosphorylation levels after preconditioning in focal ischemia in rats
T. Takahashi, X. Gao, G. Steinberg and H. Zhao
Stanford University, Stanford, California, USA
Objectives: Multiple cell signaling pathways, such as the Akt, JNK and ERK, are involved in neuronal injury induced by stroke.
1
These kinase activities have been thought to either protect (Akt), worsen (JNK), or protect or worsen (ERK) neuronal injury after stroke. Usually the activities of these kinases are represented by their phosphorylation levels;
2
however, we recently reported that changes in Akt phosphorylation levels do not match with the kinase activity assessed by in vitro kinase assay,
3
suggesting that using the phosphorylation level as a sole marker as kinase activity is unreliable. In this study, we further investigate whether changes in phosphorylation levels of ERK (P-ERK) and JNK (P-JNK) coincide with their kinase activities in a focal ischemia model and have also studied the protective effects of rapid ischemic preconditioning on these changes.
Methods: Focal cerebral ischemia was generated as previously described in male Sprague-Dawley rats.
3
Bilateral common carotid arteries (CCAs) were occluded for 30 mins after the distal portion of MCA was permanently cauterized. Ischemic preconditioning, which was induced by transient MCA occlusion for 15 mins, was performed 1 h prior to permanent MCA occlusion. Rats were sacrificed at 1, 5, 24 or 48 h after MCAo, and tissue samples corresponding to the ischemic core and penumbra was dissected and prepared for Western blot. ERK and JNK kinase assay was processed using Kinase Assay Kits (Cell Signaling Technology, MA).
Results: Rapid ischemic preconditioning significantly reduced infarction size (P<0.01). We first examined the effects of preconditioning without a test ischemia on P-ERK and P-JNK levelsand their kinase activities. Western blot results showed that both P-ERK and P-JNK levels were increased after preconditioning only, and in vitro kinase assay also showed that both of their activities were increased; suggesting that phosphorylation of ERK and JNK were consistent with their kinase activity. Next, we studied the effects of preconditioning after control ischemia on these pathways. As a result, both P-ERK and its activity were transiently increased at 1 and 5 h after control ischemia; preconditioning increased P-ERK levels while it blocked its kinase activity. Thus, P-ERK levels did not match its kinase activity when preconditioning was introduced. At last, P-JNK and its activity were investigated. P-JNK levels were reduced in rats with control ischemia compared with sham; preconditioning attenuated such reductions. To the opposite, JNK activities estimated by the in vitro kinase assay were increased after control ischemia, and preconditioning showed a trend to inhibit JNK activites.
Conclusions: Phosphorylation levels of JNK and ERK do not always match with their kinase activites. Kinase assay should be performed along with Western blot to detect phosphorylation levels to understand the roles of JNK and ERK in stroke.
838. Endoplasmic reticulum stress with increased XBP-1 activation leading to aggravated cerebral infarction in SOD1 knock-out mice
S. Nakajima1,2, M. Nibuya1 and K. Isoda2
1Neurology/Psychiatry; 21st Internal Medicine, NDMC, Tokorozawa, Japan
Objectives: Endoplasmic Reticulum (ER) plays important roles in maintaining intra-cellular Ca2+, folding proteins, and responding to various cellular stresses. Ischemia might be the maximum ER stress when combined with reactive oxygen species (ROS). Hypo-functions of CuZn superoxide disumutase (SOD1) are known to worsen tissue damages by ROS, but the grade of ER stress in this condition is not well analyzed compared to that of mitochondrial damage. We aimed to examine the possible overt ER stress in the stroke model induced by the singlet oxygen and ROS exposure.
Methods: SOD1 knockout (hetero n = 19, and homo n = 18) and C57BL/6 (wild n = 43) mice were anesthetized by the intra-peritoneal injection of 1% chloralose and 10% urethane mixture, keeping rectal temperatures at 37.5 degrees on the heating pad. Animals were fixed on the stereotactic apparatus and the non-invasive photothrombosis (PT) was created in the right distal-MCA by the transcranial irradiation of attenuated He-Ne laser beam (532 nm) guided through a single glass-fiber in the micromanipulator, after the injection of Rose-Bengal solution via a tail vein. Cerebral blood flow changes were recorded by a video-microscope and measured by a laser-Doppler flow (LDF) probe on the ischemic lesion. Ischemic changes were observed sequentially by the 9.4 T MRI (Bruker), and analyzed histologically up to the end-point of 1 month after loads. As the indicator of ER stress, spliced XBP-1 mRNA was measured by a RT-PCR method from samples of ischemic and contra-lateral hemispheres at 3, 12, and 24 h after ischemic insults, and effects of cilostazol (60 mg/kg, po) and edaravone (6 mg/kg, iv) combination therapy were also evaluated in each group of mice.
Results: Microscopic observation of distal-MCA demonstrated recurrent occlusion/recanalization by micro-emboli flown from the laser-irradiated locus, showing cyclic hyperemias in LDF before the complete occlusion. Time to occlusions were shorter in SOD1 knockout mice, and elongated in the treated groups. MRI disclosed superficial spindle-shaped ischemic changes within 1 h after distal-MCA occlusion, and enlarged gradually with surrounding edema. Ischemic T2WI area after 1 day in the wild group was 24.5±3.9% (M±SEM) of the ipsi-lesional hemisphere in the untreated group, and reduced to 10.1±3.7% in the therapy group (P<0.05). SOD1 knockout groups demonstrated broader edematous lesions, which subsided after 1 week revealing larger infractions. Spliced XBP-1 transcription in the ischemic hemisphere showed the highest increase in SOD1 homo-knockout mice at each time spans, and significantly reduced by the combined therapy.
Conclusions: During the acute stroke stage, SOD1 hypo-function elicited the marked ER stress response with exaggerated XBP1 splicing, and induced the larger cerebral infarction. Excessive and sustained ER stress may trigger the apoptotic cell death around necrotic foci, and the early treatment with potent free radical scavenger and anti-thrombotic drugs will salvage this vicious process, as shown in the present study. Non-invasive PT stroke model in this experiment enabled the individual analysis of ischemic lesions using the MRI, and might be suitable for the long-term study of SOD1 hypo- or hyper-function in combination with free radical inducers and scavengers.
843. Induction of autophagy in the rat hippocampus and cultured neurons by iron
Y. He, Y. Hua, S. Song, W. Liu, R.F. Keep and G. Xi
Department of Neurosurgery, University of Michigan, Ann Arbor, Michigan, USA
Objectives: Autophagy occurs in the brain after intracerebral hemorrhage (ICH).
1
Iron is an important factor causing neuronal death and brain atrophy after ICH.2,3 In this study, we examined whether iron can induce autophagy in the hippocampus and in cultured neurons.
Methods: For in vivo studies, rats received an infusion of either saline or ferrous iron into the right hippocampus and were killed at 1, 3, or 7 days later for Western blot analysis of microtubule—associated protein light chain-3 (LC3). For in vitro studies, primary cultured cortex neurons from rat embryos were exposed to ferrous iron. Cells were used for Western blot analysis of LC3 and monodansylcadaverine (MDC) staining 24 h later.
Results: Physiological variables including mean arterial blood pressure, blood pH, PaO2, PaCO2, hematocrit, and blood glucose level were controlled within normal ranges.
Using Western blot analysis, a time course study of LC3 showed that the ratio of LC3-II to LC3-I in the ipsilateral hippocampus was significantly increased at day 3 and remained at high levels at day 7 after intrahippocampal injection of ferrous iron. The ratio of LC3-II to LC3-I in the ipsilateral hippocampus at day 3 after ferrous iron injection was markedly higher than that in the ipsilateral hippocampus after saline injection (2.48±0.57 versus 0.42±0.08; P<0.01).
To test whether autophagy also happens after ferrous iron treatment in cultured neurons, cells were exposed to ferrous iron or control medium. Western blotting showed that cells treated with ferrous iron had a higher ratio of LC3-II to LC3-I compared with vehicle control (0.40±0.15 versus 0.13±0.05; P<0.01). MDC labeling showed an increase in the number of vacuoles and their size in cells treated with ferrous iron.
Conclusions: These results indicate that autophagy is induced by iron in neurons and that iron-induced autophagy may contribute to brain injury after ICH.
913. Protection of astrocytes from ischemia-like injury by endoplasmic reticulum chaperone protein Grp78
R.G. Giffard1, J.F. Emery1, L.-J. Xu1, A.S. Lee2, and Y.-B. Ouyang1
1Anesthesia, Stanford University, Stanford; 2Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California, USA
Objectives: Grp78 (BiP, Hsp78) is an endoplasmic reticulum (ER)-localized member of the 70 kD heat shock protein (HSP70) family. Many studies of animal and cell-culture models of stroke have shown the neuroprotective properties of other members of the HSP70 family,1,2 but little has been done with Grp78. In this study, we investigate the ability of Grp78 over-expression to protect primary astrocyte cultures under ischemic conditions.
Methods: Primary astrocyte cultures were prepared from postnatal (days 1 to 3) Swiss Webster mice (Charles River, Hollister, CA) as previously described.
3
After three days, cultures were co-transfected with equimolar amounts of pcDNA-Grp78
4
and pAcGFP1-Mito (BD Biosciences) using FuGene6 (Roche).
Cultures were subjected to ischemic conditions by either glucose deprivation (GD), or oxygen-glucose deprivation (OGD) as previously described.
2
Cell viability was quantified after GD or OGD by microscopic evaluation of Hoechst 33342 (5 μmol/L) and propidium iodide (PI, 5 μmol/L) labeled cells.
Fluorescence immunocytochemistry and immunoblotting were performed as previously described
2
with anti-Grp78 (Stressgen, Ann Arbor, MI) or anti-activating transcription factor (ATF)-4 primary antibody.
Mitochondria were isolated by percoll gradient centrifugation. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured over a three hour time course as described previously.
2
Mitochondrial complex I activity was measured using coenzyme Q0 as acceptor and NADH as donor, complex IV activity was measured using a CYTOX-OX1 kit (Sigma).
Results: The percentage of GFP-expressing cells after co-transfection was as high as 78% and the GFP signal was detectable through 25 days. The great majority of GFP positive cells also over-expressed Grp78 by immunostaining.
Over-expression of Grp78 protected primary cultured astrocytes against ischemic injury. Cultures over-expressing Grp78 demonstrated significantly reduced cell damage after both GD and OGD. ATF-4, a marker of ER stress, showed a smaller increase in Grp78 transfected cultures after GD, compared to controls. Grp78 immunostaining in unstressed cells demonstrated perinuclear distribution, consistent with ER localization. After GD, immunoblots from isolated mitochondria clearly showed that Grp78 was present in the mitochondria of Grp78-transfected cells. ROS production was reduced, and mitochondrial membrane potential and complex I and IV activities were better preserved in Grp78 transfected cells.
Conclusions: The ER-resident chaperone Grp78 can protect astrocytes from ischemic injury, and this is associated with reduced ER stress response and reduced ROS production. Under ischemic stress Grp78 retargets to mitochondria in over-expressing astrocytes. Mitochondrial functions are better preserved in these cells, indicating that part of the protection observed may be mediated by direct effects on mitochondria by Grp78.
942. Activation of protein kinase C delta following oxygen glucose deprivation leads to release of cytochrome c from mitochondria
M Perez-Pinzon, K. Dave, A. Raval, R.A. DeFazio and I. Saul
Neurology, University of Miami, Miami, Florida, USA
Objectives: Mitochondrial damage including release of pro-apoptotic factors such as cytochrome c from mitochondria following cerebral ischemia is a key event leading to cell death. In a previous study we observed release of cytochrome c and activation of δPKC following cerebral ischemia occurred closed to each other.1,2 However, the mechanism by which early activation of δPKC leads to cytochrome c release is not understood. In the present study we tested hypothesis that early activation of δPKC following cerebral ischemia phosphorylates protein phosphatase 2A (PP2A) leading to release of cytochrome c from mitochondria via activation of pro-apoptotic factor BAD.
Methods: To test this hypotheses, we used an asphyxial cardiac arrest (CA) model of cerebral ischemia in rats where we studied translocation of δPKC from cytosol to mitochondria, release of cytochrome c from mitochondria, and the extent of BAD (ser 136) phosphorylation by Western blotting at 1 h of reperfusion following 8 mins of CA. Mechanistic studies were performed using synaptosomes as a model system. Synaptosomes were subjected to 60 mins of oxygen glucose deprivation (OGD) in presence of δPKC inhibitor peptide (Tat- δV1–1) or carrier peptide (Tat) alone.
Results: Levels of δPKC increased in the mitochondrial fraction by 99±19% (P<0.02, n = 4) and cytochrome c increased in cytosolic fraction by 76±14% (P<0.05, n = 4) in CA group when compared to sham CA group. BAD phosphorylation decreased by 46±6% (P<0.005, n = 4) in CA animals versus Sham treatment. These results suggest that early activation of δPKC initiates the release of cytochrome c from mitochondria by decreasing phosphorylation of BAD via activation of PP2A. To confirm the role of δPKC following cerebral ischemia, we used synaptosomes as a model system. OGD increased levels of δPKC in mitochondria by 72% (P<0.05, n = 4). OGD in presence of δPKC inhibitor peptide (Tat-δV1–1) decreased cytochrome c release by 71% (n = 4, P<0.004) and increased BAD phosphorylation by 312% (n = 4, P<0.01) as compared to the Tat control group. Similarly, OGD in presence of PP2A inhibitor okadaic acid, prevented OGD induced cytochrome c release by 52% (n = 4, P<0.05) and BAD dephosphorylation by 144% (n = 4), respectively. The protein phosphatase 1 inhibitor calyculin had no effect.
Conclusion: These results suggest that early activation of δPKC following OGD initiates the release of cytochrome c from mitochondria by decreasing phosphorylation of BAD via activation of PP2A.
Support: PHS grants NS34773, NS05820, NS045676 and NS054147.
