Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia

Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia

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  Selective Over Expression Of EAAT2 In Astrocytes EnhancesNeuroprotection From Moderate But Not Severe Hypoxia-Ischemia Melodie L. Weller 1, Ida M. Stone , Amber Goss , Thomas Rau , Cherokee Rova , and David J.Poulsen *NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical andPharmaceutical Sciences, University of Montana, Missoula, MT 59812 Abstract Attempts have been made to elevate EAAT2 expression in effort to compensate for loss of functionand expression associated with disease or pathology. Increased EAAT2 expression has been notedfollowing treatment with β -lactam antibiotics, and during ischemic preconditioning (IPC). However,both of these conditions induce multiple changes in addition to alterations in EAAT2 expression thatcould potentially contribute to neuroprotection. Therefore, the aim of this study was to selectivelyoverexpress EAAT2 in astrocytes and characterize the cell type specific contribution of thistransporter to neuroprotection. To accomplish this we used a recombinant Adeno-associated virusvector, AAV1-GFAP-EAAT2, designed to selectively drive the overexpression of EAAT2 withinastrocytes. Both viral mediated gene delivery and β -lactam antibiotic (penicillin-G) treatment of rathippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, anddihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotectionin rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucosedeprivation (OGD). In contrast, the overexpression of EAAT2 in astrocytes provided enhancedneuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, thiscell type specific-increase in EAAT2 expression offers only limited protection compared to treatmentwith penicillin-G. INTRODUCTION Glutamate is the primary excitatory amino acid neurotransmitter in the mammalian centralnervous system. When released from presynaptic terminals, glutamate can activate ionotropicreceptors such as AMPA and KA to mediate standard fast excitatory signaling, or contributeto the higher order processing required in development, plasticity, learning and memory byactivating the NMDA and metabotropic glutamate receptors. Extracellular enzymes do notmetabolize free glutamate. Instead, high affinity, Na-dependant excitatory amino acid transportproteins (EAATs), located in the plasma membranes of both neurons and surrounding © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.* Corresponding Author:  David J. Poulsen, PhD, University of Montana, Dept Biomedical and Pharmaceutical Sciences,, 32 CampusDr., #1552, Missoula, MT 59812, 406-329-5702, current address:  NIH/NIDCR, Building 10 - Magnuson CC, 1N103, 10 Center Dr, Bethesda, MD, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript  Neuroscience . Author manuscript; available in PMC 2009 September 9. Published in final edited form as:  Neuroscience . 2008 September 9; 155(4): 12041211. doi:10.1016/j.neuroscience.2008.05.059. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    astrocytes, facilitate cellular uptake (for review see Billups et al., 1998, Bridges et al., 1999; Danbolt., 2001; Seal and Amara, 1999; Takahashi et al., 1997). The regulation of glutamate within the synaptic cleft is critical to limit the over stimulation of excitatory amino acidreceptors. Excitatory amino acid transporter 2 (EAAT2) is responsible for up to 90% of allglutamate uptake activity in the brain and is primarily localized on astrocytes (Chen, 2004;Maragakis, 2004).Given the extensive role of EAAT2 in regulating extracellular glutamate concentrations,alterations in EAAT2 expression and activity can have profound effects on neuroprotectionand neuropathology. Multiple neurodegenerative diseases have been associated with reducedEAAT2 expression and function (Boston-Howes, 2006; Cross, 1987; Guo, 2002; Li et al., 1997; Rao, 2001; Rothstein, 1996; Rothstein et al., 1996). In contrast, increased astrocytic EAAT2 expression appears to afford greater neuroprotection under excitotoxic conditions.Rosenberg and Aizenman (1989) demonstrated that cortical neuron cultures were significantlyless vulnerable to glutamate when cultured in an astrocyte-rich environment compared tocultures grown with few astrocytes. Recent studies have demonstrated that treatment of cultured neurons with β -lactam antibiotics results in increased expression and activity of EAAT2, which was hypothesized to enhance neuroprotection against hypoxia/ischemia(Rothstein et al., 2005; Lipski et al., 2007). However, an earlier study by Mitani and Tanaka (2003) reported higher extracellular concentrations of glutamate in the brains of wild type micecompared to EAAT2(GLT1) knock out mice following ischemia, suggesting that EAAT2 mayactually contribute to neuropathology following ischemia. In addition, Bonde et al (2003)observed a 181% increase in EAAT2(GLT1) expression following the treatment of hippocampal slice cultures with GDNF. This increase in EAAT2 was not associated withgreater neuronal survival, but rather with increased neuronal loss following hypoxia/ischemia.The picture is further complicated by the observation that EAAT2(GLT1) expression may shiftfrom astrocytes to neurons following ischemic insult (Danbolt, 2001; Martin et al., 1997, Bondeet al., 2003; Rao et al., 2001a; Fukamachi et al., 2001, Xu et al., 2003), and we have previously shown that over expression of EAAT2 in neurons increases neuronal sensitivity to glutamatemediated excitotoxicity (Selkirk, et al., 2005).Clearly EAAT2 activity has a profound impact on the regulation of normal glutamatergicneurotransmission, and can profoundly influence neuroprotection or neurodegeneration.However, the inability to selectively study the expression and function of EAAT2 in a cell typespecific manner has made it difficult to clearly determine the exact contribution of astrocyticEAAT2 towards neuroprotection or neurodegeneration. Therefore, we used recombinantAdeno-associated viral vectors to transduce rat hippocampal slice cultures and limit theoverexpression of human EAAT2 to astrocytes using the GFAP promoter. Under theseconditions we demonstrated that functional EAAT2 expression and activity could beselectively increased in astrocytes, leading to a significant increase in neuroprotectionfollowing moderate hypoxia/ischemia but not following a more sever insult. Methods Virus Preparation The AAV1-GFAP-hrGRP and AAV1-GFAP-EAAT2 viruses were packaged in HEK293Tcells cultures grown in standard growth media (DMEM, 10% heat inactivated FBS, 0.05%penicillin/streptomycin (5000U/ml), 0.1 mM MEM nonessential amino acids, 1 mM MEMsodium pyruvate, and gentamicin (25 mg/ml). Cells were transfected with three plasmids usingPolyfect Transfection Reagent (Qiagen, Valencia, CA). The three plasmids used in thetransfection were: 1) adeno helper plasmid (pFD6), AAV helper (H21) and the AAV packagingvector containing the glial fibrillary acidic protein (GFAP) promoter followed by either theenhanced green fluorescent protein (eGFP) gene or the human EAAT2 (GLT1a) gene sequence Weller et al.Page 2  Neuroscience . Author manuscript; available in PMC 2009 September 9. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    (obtained from J. Rothstein, Johns Hopkins), flanked by AAV2 inverted terminal repeats. Viruswas isolated from HEK293T cells through repeated freeze-thaw cycles, incubation for 30minutes at 37°C with 50U benzonase (Novagen, Madison, WI) and 0.5% sodium deoxycholate,briefly sonicated and further purified by iodixonol density gradient centrifugation as previouslyreported (Zolotukhin, 1999). The titer (genomic particles/ml (gp/ml)) of final virus isolate wasdetermined by quantitative real time-polymerase chain reaction (RT-PCR) using an ABI Prism7700 with primer and probe sets specific for the EAAT2 sequence or the WPRE sequence. Rat hippocampal slice cultures (RHSC) RHSC were prepared using a modified method of Noraberg (1999). Hippocampal tissue wasisolated from 7-day-old Sprague-Dawley rats. Tissue was cut into 400µm slices using aMcIlwain tissue chopper and transferred to ice-cold dissection media (Hanks balanced saltsolution, 20mM HEPES, 25mM D-glucose, pH 7.3, filter sterilized) and incubated for 30minutes on ice. Slices presenting clear hippocampal architecture were transferred to dissectionmedia alone or media containing 1 × 10 11  gp/ml of AAV1-GFAP-EAAT2, AAV1-GFAP-nullor AAV1-GFAP-hrGFP, oxygenated for 30 minutes and placed on inserts in 6-well platescontaining 1 ml of primary RHSC media (50% Optimem (Invitrogen, Carlsbad, CA), 25%HBSS, 25% heat inactivated horse serum, 25mM D-Glucose, (+/  − ) 100µM penicillin G, pH7.3, filter sterilized). On day three, media was changed to a secondary culture media(Neurobasal-A media, B-27 supplement, 1mM Gluta-max (Invitrogen, Carlsbad, CA), 25mMD-glucose, 2.7 +/  −  100µM penicillin G, pH 7.3, filter sterilized). Half of the secondary mediawas changed every other day. Cultures were maintained at 37°C, 5% CO 2  for 10 days. Oxygen and glucose deprivation (OGD) OGD  studies were performed using a modified method of Bonde, et al. (2003). Propidiumiodide (PI) is a polar compound that gains entry into dead and dying neurons and binds tonucleic acid. Binding of PI to DNA results in a red maximum fluorescence emission at 630nmupon excitation at 495nm. At least 6 hours prior to OGD, PI (Molecular Probes, Eugene, OR)was added to the media at a concentration of 2µM (Noraberg, 1999). At this concentration,staining is specific for damaged neurons. OGD was established by transferring inserts todeoxygenated, glucose-free balanced salt solution (BSS) (120 mM NaCl, 5mM KCl, 1.25 mMNa2HPO4, 2mM CaCl2, 25mM NaHCO3, 20mM HEPES, 25mM Sucrose, pH 7.3, filtersterilized). Cultures were placed directly into an oxygen deprivation chamber (37°C, 5%CO 2  and 95% N 2 , Biospheric, PRO-OX 110) in glucose free media for 60 minutes to establisha moderate insult, or incubated under normal oxygen conditions in glucose-free buffer for 15minutes then placed into the oxygen and glucose free conditions for 60 minutes to establish asevere insult. Non-OGD control slices were transferred to BSS with glucose and incubated innormal O 2  for 1 hour. After OGD, inserts were transferred back into wells with 1ml secondarymedia containing 25 mM D-glucose, B-27 without antioxidants and with 2µM PI then returnedto normal oxygen conditions. Fluorescent images were taken of the hippocampal slices priorto OGD and at 18, and 24 hours post-OGD on an Olympus IX71 inverted fluorescentmicroscope (Melville, NY) attached to an Hamamatsu ORCA-ER digital camera using Image-Pro Plus software package (Media Cybernetics, Bethesda, MD). Four independent experimentswere conducted with 8–12 slices/experiment. Western Blot Analysis Western blot analysis of rat hippocampal slice cultures were performed to evaluate EAAT2expression levels in control slices, penicillin G treated slices and cultures transduced withAAV1-GFAP-EAAT2. The RHSC were homogenized in lysis solution (2.5% sodiumdeoxycholate, 0.1% protease inhibitor cocktail set III (Calbiochem, San Diego, CA), and 0.05%benzonase (EMD Biosciences, San Diego, CA) in PBS). Protein concentrations of lysate Weller et al.Page 3  Neuroscience . Author manuscript; available in PMC 2009 September 9. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    samples were determined using the Bio-Rad DC protein assay (Hercules, CA). Aliquots of homogenized RHSC (30µg) were loaded onto NuPAGE 4–12% Bis-Tris gels (Invitrogen,Carlsbad, CA). Proteins were transferred to Immuno-Blot PVDF membrane (Bio-Rad,Hercules, CA) and blocked in Tris buffer containing Tween-20 and 0.5% non-fat milk.Membranes were probed with antibodies to GLT-1 to label EAAT2 (1:500, ABR) and actin(1:1000, Sigma, St. Louis, MO). Proteins were visualized using ECL (Pierce, Rockford, IL)with species-specific HRP conjugated secondary antibodies. Blots were imaged on a Kodak Image Station 440 CF. Dosimetry analysis of regions of interest were used to calculate foldchange in EAAT2 protein expression corrected to actin controls. Similar experiments wereperformed using the anti-EAAC1 (1:100 kindly provided by Dr Jeffery Rothstein) and anti-GLAST (1:100, ABR) to label EAAT3 and EAAT1 respectively. Immunohistochemistry Cell type specific expression obtained with the AAV1-GFAP vector was evaluated in RHSCtransduced with AAV1-GFAP-GFP. Slice cultures were transduced with AAV1-GFAP-GFPand fixed after 10 days in culture. Slices were fixed with 4% paraformaldehyde for 30 minutesat room temperature, and stored at 4°C in PBS. Slices were cryo-protected by incubation in asucrose solution at 4°C and cryo-sectioned into 10µM thick sections. Slices were thenincubated in blocking buffer (1% normal goat serum, 0.3% Triton-X 100) for 1 hour at roomtemperature. After initial blocking, neurons were stained with the fluorescent Nissl stain,NeuroTrace Blue (1:400; Invitrogen). Astrocytes were stained with a primary rabbit anti-GFAPantibody (1:500, Chemicon, Temecula, CA) overnight at 4°C, then rinsed in blocking buffer(1% normal goat serum) and incubated for 1 hour in secondary anti-rabbit antibody conjugatedto Alexa 546 (1:500, Molecular Probes, Eugene, OR). Fluorescent images were obtained on aBio-Rad Radiance 2000 MP laser scanning confocal microscope (Molecular Histology Core-University of Montana) and an Olympus IX71 inverted fluorescent microscope attached to aHamamatsu ORCA-ER camera using Image-Pro plus software. Glutamate uptake assays Uptake assays were performed using a modified method of Selkirk (Selkirk, 2005). Briefly,whole slices were homogenized on ice in tissue buffer (50mM Tris, 0.3M sucrose, pH 7.3) andcentrifuged at 14,000 × g for 10 minutes at 4°C. The pellet was resuspended in 250µL of eithersodium containing Krebs buffer or sodium-free Krebs containing equimolar amount of choline.Hippocampal homogenates were incubated with aspartic acid solution (200nM [ 3 H] D-asparticacid (PerkinElmer, Boston, MA) and 2.0µM cold D-aspartic acid (Novabiochem, San Diego,CA) in the presence and absence of 500µM dihydrokainic acid (DHK), (Tocris, Ellisville, MO).DHK is an EAAT2 selective uptake inhibitor and allows for assessment of non-EAAT2mediated aspartic acid uptake. Reactions were incubated at 37°C for 4 minutes and promptlyterminated by filtration through Whatman GC/F filter paper (Whatman, Brentford, Middlesex,UK) via a Brandel Cell Harvester (Brandel, Gaithersburg, MD). Filtrate was incubatedovernight in scintillation cocktail and counted on a Beckman LS 6500 Scintillation System.Specific EAAT2 mediated uptake of [ 3 H]-D-aspartic acid was calculated as total sodium-dependent uptake (pmol Asp/mg protein/min) less the uptake in the presence of DHK. Data Analysis One-way ANOVA was performed to determine statistical significance of western blotdosimetry analysis, functional [ 3 H]D-aspartic acid uptake and level of neuroprotection offeredbetween the control tissue and those treated with either penicillin G or transduced with AAV-GFAP-EAAT2. Statistical analysis was performed using Prism Software (GraphPad Software,Inc., San Diego, CA). Weller et al.Page 4  Neuroscience . Author manuscript; available in PMC 2009 September 9. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Results Astrocyte targeted transgene expression following AAV-mediated gene delivery The GFAP promoter sequence identified by Brenner et. al. (1994) has been used in multiplestudies to selectively drive protein expression in astrocytes. Feng et. al. (2004) demonstratedstable, long-term astrocyte specific expression of apolipoprotein E (ApoE) using rAAVcontaining the GFAP promoter. Guo et. al. (2003) utilized the GFAP promoter sequence todrive EAAT2 expression in a transgenic mouse model and noted a high degree of astrocytespecific transgene expression.We validated the astrocyte specificity of the AAV1-GFAP viral construct by transducing rathippocampal slice cultures (RHSC) with AAV1-GFAP-GFP. Extensive GFP expression wasobserved throughout the slices, with a greater density of GFP positive cells present within thestratum radiatum, stratum oriens, hilus and dentate (Figure 1B & C). Neurons within transducedslices were labeled with NeuroTrace Blue and showed a distinct pattern separate from GFPpositive cells (Figure 1A & C). Immunohistochemical analysis was performed on cryosectionsprepared from RHSCs to confirm astrocyte specificity of transgene expression and to establishthe extent to which AAV could penetrate slices and transduce cells within the core of thecultures. Neurons were labeled with NeuroTrace Blue (Figure 1F & G) and astrocytes werelabeled with anti-GFAP antibody (Figure 1E & G). Co-labeling of green GFP positive cellswith anti-GFAP antibody (Figure 1 G & J) indicated that transgene expression was effectivelytargeted to astrocytes, with no GFP expression observed in neurons (Figure 1G). Extensiveastrocytes-specific GFP expression was seen within all cryosections examined indicating thatAAV was capable of efficient astrocytes transduction throughout the entire slice culture. AAV-mediated delivery of the EAAT2 gene results in increased expression of functionalEAAT2 within astrocytes Western blot analysis was performed to determine the level of EAAT2 overexpression thatcould be achieved within astrocytes of AAV transduced RHSC compared to cultures treatedwith penicillin-G. Immunoblot analysis indicated that EAAT2 expression was significantlyincreased to 229% of controls following transduction with AAV1-GFAP-EAAT2 (Figure 2A&B). In comparison, treatment of RHSC with 100µM penicillin-G rendered a somewhat lower,but still statistically significant increase of 163% in EAAT2 expression over that of controls.Total intensity of all three bands characteristic of EAAT2, consisting of a monomer, dimer andmultimeric aggregated proteins, were used to calculate the total level of EAAT2 expressionnormalized against β  actin.We further wanted to determine if the overexpression of exogenous EAAT2 caused alterationsin the expression of other glutamate transporters. EAAC1(EAAT3) and GLAST(EAAT1) areglutamate transporters also expressed with in the hippocampus. EAAC1 is predominantlyexpressed in neurons and GLAST is expressed in astrocytes. Western blot analysis indicatedno changes in the expression of either of these transporters under any of the conditionsexamined (Figure 2C & D).To determine if exogenous EAAT2 transporters were trafficked to the cell surface andcontributed to functional uptake activity, we measured [ 3 H]D-aspartate uptake in control,penicillin-G treated and AAV1-GFAP-EAAT2 transduced RHSCs. Crude homogenatepreparations from AAV1-GFAP-EAAT2 and penicillin-G treated slices showed a 162% and128% increase in DHK sensitive glutamate uptake respectively, over that of controls (Figure3). The disparity observed between the level of EAAT2 expression in western blots to thatnoted in EAAT2 specific uptake activity could be attributed to the fact that western blot analysis Weller et al.Page 5  Neuroscience . Author manuscript; available in PMC 2009 September 9. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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