Time course of protein changes following in vitro ischemia in the rat hippocampal slice

BRAIN RESEARCH Brain Research 694 (1995) 94-102

ELSEVIER

Research report

Time course of protein changes following in vitro ischemia in the rat hippocampal slice Kathleen M. Raley-Susman *, Jennifer Murata Vassar College, Department of Biology, Poughkeepsie, NY 12601, USA Received 31 May 1995; accepted 7 June 1995

Abstract

Following 5 min in vitro ischemia, total protein synthesis is dramatically and persistently inhibited in neurons in the rat hippocampal slice. This model system was used to explore the responses of individual proteins to this irreversible insult. In vitro ischemia inhibited new protein synthesis of most proteins analyzed; however, the synthesis of a 68/70 kDa protein was substantially stimulated for the first hour after ischemia. By 3 h postischemia, its synthesis rates were depressed to 60% of control rates. Although the total amounts of most proteins were not significantly depleted for the first few hours after an ischemic episode, there were several notable exceptions. The levels of HSC73, a constitutively expressed member of the 70 kDa stress protein family, were reduced after in vitro ischemia. In addition, MAP-2 (microtubule-associated protein-2) and ot-tubulin were depleted in the early hours after the insult, with MAP-2 exhibiting a detectable depletion earlier than tubulin. In contrast, the levels and distribution of a 68 kDa neurofilament protein localized to CA3 pyramidal neurons in the slice, apparently distinct from the band whose new synthesis was stimulated, were not affected by the 5 min in vitro ischemia insult. Thus, the responses of individual proteins to ischemia varied considerably. These individual responses could play an important role in the damage mechanism that is initiated in response to in vitro ischemia. Keywords: Hippocampus; lschemia, in vitro; MAP-2; Tubulin; Heat shock protein

I. Introduction

Brief episodes of ischemia suppress protein synthesis in many regions of the brain; the inhibition is persistent and in some cases irreversible in vulnerable neurons within the hippocampus, most notably the CA1 pyramidal neurons [6,16,28]. The inhibition of synthesis in the hippocampus persists despite normalization of energy metabolism and re-establishment of the membrane potential (reviewed in [9]). The mechanism by which the protein synthesis failure is maintained is unknown, but the effect appears to be exerted at the level of translation initiation [5,9,10]. The consequences of a persistent translation failure in neurons postischemia are unknown. Further, it is unclear whether the synthesis of all proteins is impaired. Because of the prolonged time course of the new synthesis inhibition, the localization of the persistent inhibi-

* Corresponding author. [email protected]

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tion to the most vulnerable cells, and the suggestive role of calcium in the mechanism of translation failure [10], protein synthesis failure has been implicated in the evolution of irreversible neuronal injury [1,9,31]. In contrast, several investigators have reported that blocking translation improves the outcome of ischemia in vivo [8,24], implicating the synthesis of a 'suicide' protein. Thus, the role of translation in response to ischemia is far from resolved. Elucidating the effects of ischemia on the synthesis and distribution of individual proteins throughout the recovery period, particularly within the first few hours after the insult, is thus likely to shed light on the role played by translation failure in the mechanism of neuronal injury. We have utilized the rat hippocampal slice as a model system to understand early events and effects of in vitro ischemia on protein synthesis. Previous work demonstrated that neurons within the hippocampal slice respond to in vitro ischemia (glucose and oxygen deprivation) with a pronounced and persistent inhibition of protein synthesis [25] that is very similar in degree and duration to that seen after ischemia in vivo. Synthesis failure is prevented by

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removing calcium and blocking NMDA receptors during the insult, suggesting that the inhibition is calcium-dependent. In addition, during the early postischemic period in the slice, the morphology of pyramidal neurons is compromised [3,25]. Further, deterioration of the neurons in the slice could be prevented by the same treatments that prevented the protein synthesis failure [25], suggesting that the morphological changes in the early postischemic period in the slice are mediated by calcium and NMDA receptor activation. To begin to investigate the role of cytoskeletal proteins in the morphological changes that occur after ischemia, we monitored levels of three neuronal cytoskeletal proteins, MAP-2, a-tubulin and a 68 kDa neurofilament protein. Specific changes in these proteins have been reported after ischemia in vivo [15,32], suggesting that the levels of these proteins might serve as markers of neuronal injury. Their responses to irreversible in vitro ischemia in the slice have not been studied. Finally, we studied the effects of this in vitro insult on the expression of HSP72/HSC73, thought to be markers of cellular injury. The 70 kDa family of stress proteins has been extensively studied following ischemia in vivo (reviewed in [23]), but relatively little work has been directed at the expression of these proteins following insults in the hippocampal slice. Our results demonstrate that the new synthesis of most proteins is substantially inhibited for several hours postischemia, with the notable exception of a 68/70 kDa protein whose new synthesis was transiently stimulated. In addition, the levels of cytoskeletal proteins MAP-2 and a-tubulin are depleted early in the postischemic period, while levels of a CA3 neuron neurofilament protein and most other proteins analyzed are not. Some of these results have appeared previously in abstract form [211.

2. Materials and methods

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for 45 min-1 h before beginning experimental procedures. The standard buffer contained (in raM): 124 NaCl, 26 NaHCO3, 1.2 KHEPO4, 3 KC1, 1.3 MgSO4, 1.2 CaCI2, 4 glucose, pH 7.4, 36°C. The modified buffer was the same except that it lacked CaC12 and contained 10 mM MgSO 4. 2.2. In vitro ischemia

Ten min prior to exposure to ischemic buffer (standard buffer lacking glucose and equilibrated with 95% N2-5% CO2), slices were transferred to standard oxygenated buffer lacking glucose, to deplete glucose from the extracellular space. The slices were rendered 'ischemic' by placing them in ischemic buffer for 5 rain. Slices were returned to standard buffer with glucose and allowed to recover for 30 min, 1 h or 3 h. Protein synthesis rates were determined by transferring slices for the last 30 min of their recovery time to buffer containing [35S]methionine (50 /xCi/ml; NENDuPont). Labeled slices were washed for 1-3 min in ice-cold buffer and total tissue protein extracted as described below. In some experiments, slices were not radiolabeled and were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 2 h at 4°C and prepared for immunocytochemistry (see below). 2.3. Protein extraction

Slices were transferred to microfuge tubes and centrifuged at 2000 x g for 8 min at 4°C to gently compact the tissue. The buffer was removed and slices were homogenized in 5 volumes of homogenization buffer (10 mM Tris-HC1 (pH 7.6), 100 mM NaC1, 1 mM EDTA) containing phenylmethylsulfonyl fluoride (PMSF; 100/xM final concentration) to retard protease activity. The cell extract was dispensed into aliquots, frozen on dry ice, lyophilized to dryness and stored at -20°C until electrophoresis. Protein concentration was determined spectrophotometrically (OD at 595 nm) using the Bradford protein assay with y-globulin as a standard.

2.1. Slice preparation 2.4. SDS-PAGE

All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee. Adult (60 day) male SpragueDawley rats (Taconic Farms, New York) were decapitated without anesthesia; the cerebral hemispheres were removed within 20 s into ice-cold, oxygenated modified buffer (see below). Hippocampi were dissected out and kept chilled; 300-400 /xm slices were cut with a Gillette razor blade. Slices were placed on a nylon mesh stretched across a three-partitioned lucite platform submerged in modified buffer equilibrated with humidified, 95% 02-5% C O 2 gas and maintained for 45 min at 36°C. The slice platforms were then transferred to flasks containing standard buffer equilibrated with humidified 95% 02-5% CO 2

15 /zg total protein of each sample was solubilized in SDS sample buffer, heated at 80°C for 3 min, centrifuged briefly to pellet unsolubilized material and separated on 7.5% (29:1 acrylamide/bisacrylamide) resolving gel utilizing the Laemmli method [19]. Following electrophoresis, some gels were fixed and stained with silver [26] and gel images were digitized using a 300 dpi Hewlett-Packard flatbed scanner (Deskscan IIp). The gel was dried and exposed to fl-max Hyperfilm (Amersham) for 2 weeks to 1 month. Proteins from other gels were transferred to Immobilon (ISS, Inc.) membranes electrophoreticaUy, exposed to antibodies (see Results), dried, digitized as above and exposed to fl-max Hyperfilm for 1 month. All gels were run in duplicate and all lanes within a gel for immuno-

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blotting were run in duplicate. Films were developed with Kodak D-19 and fixer, washed, dried and digitized as above. All digitized images were analyzed densitometrically using Collage 2.0 software (Fotodyne, Inc.). We analyzed the pixel intensities of each band at a given apparent molecular weight (calibrated using Collage software) and subtracted the background surrounding each protein band. Data are presented as a percentage of withingel control bands, to account for gel to gel variations in background and staining intensity.

2.5. Immunoblotting After electrophoretic transfer of separated proteins to Immobilon membranes, membranes were equilibrated in TBSTM (50 mM Tris-HCl (pH 7.5), 0.15 M NaC1, 0.05% Tween-20, 5% nonfat dry milk) for 30 min to block nonspecific antibody binding. One-half of each membrane was exposed to the primary antibody (see below) in TBSTM for 2 h at room temperature; the other half of the membrane was exposed to nonspecific serum or 2% BSA for the same period of time and served as a negative control. Following several washes, membranes were exposed to goat anti-mouse IgGl conjugated to peroxidase (1:500; Sigma A3673) for 1 h at room temperature. Immunoreactive bands were visualized using a peroxidase substrate kit (Vectastain, Vector Laboratories) with DAB (diaminobenzidine) as a chromogenic substrate. Data were analyzed as described above.

2.6. Immunocytochemistry Following fixation in 4% paraformaldehyde for 2 h at 4°C, slices were cryoprotected and 36 /xm sections made with a freezing stage sliding microtome. Sections were mounted on gelatin-coated microscope slides, air-dried for 1 h and frozen with desiccant overnight at - 20°C. Thawed sections were permeabilized and blocked with PBS containing 2% BSA and 0.5% Triton X-100, washed and exposed to primary antibodies (see below) for 3 h at room temperature. Following three washes, slides were exposed to rat-adsorbed biotinylated-anti-mouse IgG~ (Vector Laboratories) for 1 h at room temperature. Following extensive washes, slides were developed using the ABC procedure (Vector Laboratories) with DAB as a substrate for the peroxidase reaction. Stained sections were dehydrated in graded alcohols, cleared in xylene and coverslipped with Permount. Immunoreactivity was analyzed qualitatively.

2. 7. Antibodies All primary antibodies were raised in mouse and are available commercially. The following antibodies were used: monoclonal anti-68 kDa neurofilament (Amersham RPN 1105) used at 1:200 for immunoblotting and at 1:60 for i m m u n o c y t o c h e m i s t r y ; m o n o c l o n a l anti-

HSP72/HSC73 (Sigma H5147) used at 1:2500 for immunoblotting; monoclonal anti-HSP72 (Amersham RPN 1197) used at 1:60 for immunoblotting; monoclonal antiMAP-2 (Boehringer-Mannheim AP20) used at 1:500 for immunoblotting and at 1:400 for immunocytochemistry; monoclonal anti-o~-tubulin (Amersham N356) used at 1:500 for immunoblotting.

2.8. Statistics All densitometric data were analyzed using ANOVA followed by the Fisher PLSD post-hoc test, using Statview 4.0 (Abacus Software, Inc.). A P-value of 0.05 or smaller was considered statistically significant. At least 3 - 4 slices per treatment were used for each experiment and at least 3-5 separate experiments (i.e., n = 3-5 rats) were performed to ensure statistical validity.

3. Results

3.1. Densitometric analysis of silver-stained proteins Hippocampal slices subjected to 5 min in vitro ischemia were allowed to recover for various times and proteins were extracted and analyzed by SDS-PAGE. Measurement of the total amount of protein extracted indicated that the ischemic insult did not alter the total amount of protein per tissue dry weight. The effects of in vitro ischemia on levels of individual proteins separated by SDS-PAGE were analyzed densitometrically and compared with control levels (at the same molecular weight) from slices taken from the same rat and run on the same gel. The densitometric analysis measured the grayscale pixel intensity of each band and subtracted the background surrounding each individual band, thus taking into account differing levels of background silver staining. As a test of the analysis procedure, we loaded differing amounts of total protein from the same control tissue sample. The analysis procedure reliably detected differences in protein levels of 5-10% (data not shown), which are not visible by eye. Measurement of varying amounts of molecular weight standards (where the identity and precise amount of each protein was known) yielded similar results. Thus, by several different criteria, our analysis procedure reliably and without bias detected small differences in staining intensity. As a final control for variations between rats and between experiments, all data are presented relative to the control slice tissue run concurrently on the same gel. Densitometric analysis of silver stained protein levels following an irreversible in vitro ischemia insult indicated that the total amount of most proteins was not altered by in vitro ischemia. The average level of all proteins analyzed was 112% + 2.6% (S.E.M.) of corresponding control levels. There were, however, several notable exceptions. A

K.M. Raley-Susman, J. Murata / Brain Research 694 (1995) 94-102

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B Fig. 1. Autoradiographic analysis of new protein synthesis of proteins separated by SDS-PAGE. Slices were prepared and incubated as described in section 2. Experimental slices were then exposed to 5 min in vitro ischemia and allowed to recover in standard buffer for 30 rain, 1 h or 3 h. New protein synthesis was determined by exposing slices to 50 /~Ci/ml [35S]methionine for the last 30 rain of the recovery period. Identical amounts (15 /,~g) of extracted proteins were separated by SDS-PAGE, on a 7.5% resolving gel. Gels were silver-stained, as described in section 2, dried and exposed to fl-max film for 1 month at room temperature. The figure is representative of 4 separate experiments. Lane 1 = 30 min recovery from 5 rain in vitro ischemia. Lane 2 = 1 h recovery. Lane 3 = control tissue protein. Lane 4 = 3 h recovery. Arrow: increased synthesis of 6 8 / 7 0 kDa protein.

c-

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132 kDa protein was significantly but reversibly depleted at 1 h postischemia. In addition, 30 min after ischemia, the amount of protein migrating to 50 kDa was significantly increased in 3 out of 5 experiments.

3.2. New protein synthesis following in vitro ischemia Although overall levels of most proteins are stable postischemia, de novo synthesis of most proteins was dramatically and significantly reduced to 40-60% of con-

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~ Fig. 2. Densitometric analysis of new protein synthesis. Data are summaries of autoradiographs depicted in Fig. 1 from 4 similar experiments. Data are presented as percentages of densities of corresponding bands from control tissue samples (mean density + S.E.M.). A: 30 min after 5 min in vitro ischemia. B: 1 h after 5 min ischemia. C: 3 h after 5 min ischemia. In all cases, autoradiographs were digitized and analyzed densitometrically, as described in section 2. * Indicates P < 0.05 when compared with control tissue using A N O V A followed by post-hoc analysis. n = 4 separate experiments (4 slices per group). Note that the y-axis scales are different.

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hsc73

cx-tubulin

MAP-2 9

Fig. 3. Western blot analysis of protein responses to 5 min in vitro ischemia. Slices were exposed to in vitro ischemia as described in section 2 and Fig. 1. Following SDS-PAGE, proteins were transferred to Immobilon and treated with antibodies as described in section 2. Immunoblots were dried, digitized and analyzed densitometrically. Blots are representative of 3-5 similar experiments. Lanes 1 = 30 min recovery from 5 min in vitro ischemia. Lanes 2 = 1 h recovery. Lanes 3 = control slice tissue protein. Lanes 4 = 3 h recovery.

Fig. 4. Immunocytochemical analysis of response of MAP-2 to 5 min in vitro ischemia. Slices were treated as described in section 2. Sections are representative of 4 similar experiments. In the figure, ' p ' refers to the CA1 stratum pyramidale and 'r' refers to the stratum radiatum. A: MAP-2 immunoreactivity in CA1 pyramidal neurons from control slices, B: MAP-2 immunoreactivity in CA1 pyramidal neurons from ischemic slices after 3 h recovery. Scale bar = 34 /zm. Note the depletion of staining in somata, with retention of dendritic immunoreactivity.

trol rates after 5 min in vitro ischemia (Fig. l, Fig. 2A). A notable exception was a protein of apparent molecular weight 68/70 kDa, which exhibited a significant increase in new synthesis at 30 min postischemia. The stimulation persisted for at least 1 h postischemia (Fig. 2B), but new synthesis of this protein was depressed to 60% of control levels by 3 h postischemia (Fig. 2C). A number of other proteins exhibited an early suppression of new synthesis, with a subsequent return toward control levels by 3 h. Thus, the synthesis rates of individual proteins in response to in vitro ischemia varied.

3.3. Stress protein responses to in vitro ischemia

Because stress proteins are implicated in the response to many types of insult, we examined the responses of two of these proteins to in vitro ischemia, using a monoclonal antibody recognizing both HSP72, the stress-inducible heat shock protein, and HSC73, a constitutively expressed heat shock cognate protein. This antibody recognized only one protein band in our slices (Fig. 3). We detected no signal

Table 1 Responses of HSC73, MAP-2, and a-tubulin to 5 min in vitro ischemia Density (% control) of immunoreactive protein at different recovery times after 5 min in vitro ischemia (mean + S.E.M.)

HSC 73 MAP-2 a-tubulin

30 rain

1h

3h

76 + 9.4 * 86 + 3.5 * 76 + 18.9

67_+ 6.3 * 78.8 + 5.1 * 78.7 ± 23.7

65 + 2.5 * 78.2 5:3.5 * 55.4 + 11.6 *

Slices were treated as described in Fig. 3. Immunoblots were digitized and analyzed densitometrically as described in section 2. Data are presented as percent of within-blot control immunoreactivity + S.E.M. n = 3-5 separate experiments. * Indicates P = 0.05 when compared with control densities using ANOVA followed by Fisher PLSD test.

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with an antibody specific for HSP72 (Amersham RPN 1197) which has been used in immunocytochemical analyses (reviewed in [7]), consistent with previous reports [3]. Thus, it is likely that the antibody we used recognized only the HSC73 protein. The total amount of protein at this apparent molecular weight, as measured by densitometry of immunoblots, was reduced to 76% of control levels within 30 min postischemia, with a further reduction to 67% and 65% of control levels by 1 h and 3 h recovery, respectively (Table 1). Further, the new synthesis of the protein migrating to this molecular weight was dramatically reduced, to ,-, 40-50% of control rates throughout the recovery period. 3.4. Neuronal cytoskeletal protein responses to in vitro ischemia The monoclonal antibody against MAP-2 recognized several bands (Fig. 3) and specifically labeled dendritic processes of hippocampal neurons (Fig. 4A). These reactions were specific as judged by the use of non-specific antibodies or by the presence of nonimmune serum in the

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absence of primary antibody (data not shown). Densitometric analysis of the major immunoreactive band indicated a reduction in the amount of protein to 86% of control levels at 30 min recovery, and a further reduction to ~ 78% of control levels at 1 h and 3 h recovery. While numerous dendritic processes stained with the MAP-2 antibody, the somata and basal dendrites of the CA1 pyramidal neurons were depleted of MAP-2 (Fig. 4B) by 3 h postischemia, perhaps accounting for the reduction measured in slice homogenates. Levels of a 68 kDa neurofilament protein (which appeared distinct from the protein ( 6 8 / 7 0 kDa) exhibiting a stimulated synthesis postischemia) were not altered by in vitro ischemia (data not shown). The 68 kDa neurofilament antibody stained dendritic processes and the somata (except for the nucleus) of CA3 pyramidal neurons in the slice (Fig. 5A). The pattern of staining closely resembled the reported in vivo distribution [22]. In vitro ischemia had no discernible effect on the degree or pattern of staining (Fig. 5B). In contrast, immunoblotting analysis of c~-tubulin showed a reduction to 76% and 78% of control levels at 30 min and 1 h, respectively, followed by a significant depletion to 55% of control levels by 3 h recovery (Fig. 3; Table 1). Thus, a-tubulin is more dramatically affected by 5 min in vitro ischemia than is MAP-2, which, in turn, is more sensitive to in vitro ischemia than the 68 kDa neurofilament protein.

4. Discussion

Fig. 5. Immunocytochemical analysis of 68 kDa neurofilament after 5 min in vitro ischemia. Slices treated as described in section 2. Sections are representative of 4 similar experiments. A: control CA3 neurons. B: CA3 neurons from ischemic slices after 1 h recovery from 5 rain in vitro ischemia. Scale bar = 34 btm.

Our previous work established the rat hippocampal slice as a useful model system for exploring the effects of in vitro ischemia on neuronal protein synthesis [25]; others have also utilized this model system [2,3]. We chose a 5 min insult because it elicits irreversible changes in both protein metabolism and electrophysiological parameters. A 5 rain insult causes a persistent inhibition of overall protein synthesis in neurons of the slice, but a 3-fold increase in synthesis rates in glia and capillary endothelial cells [25]. In the present study, our goal was to begin to elucidate the time course of these changes and the responses of individual proteins to this insult. We focused on MAP-2, a-tubulin and a 68 kDa neurofilament protein as cytoskeletal markers for neurons and on HSP72/HSC73 as a marker of tissue injury. This is the first demonstration of changes in the synthesis and expression of particular proteins for a number of hours following in vitro ischemia in rat hippocampal slices. In this report, we have established that this model system can be used to examine the effects of this irreversible insult on the levels, synthesis and distribution of individual proteins in the hippocampus. In addition, we observed that changes in these proteins occurred at different times during the recovery, indicating differential threshold effects of in vitro ischemia.

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4.1. Protein synthesis after 5 min in vitro ischemia

The new synthesis of most proteins remained suppressed for at least 3 h postischemia. The impact of a 50% inhibition of new synthesis of most proteins was not observable at 3 h recovery as there was no significant change in the overall levels of most proteins analyzed. The lack of change in the amount of silver-stained protein was not because of limitations in the densitometry measurement technique. Our control studies indicated that the analysis technique can reliably detect as little as a 10% change in the amount of silver-stained material. Changes as little as 5% were also detectable, but were close to the reliable limit of detectability. An important caveat of the measurement technique used in this study is that it is quite likely that more than one protein migrates to the same apparent molecular weight using one-dimensional electrophoresis. Indeed, for at least one protein, migrating to 6 8 / 7 0 kDa, this appears to be true. The individual protein measurements reflect a summation of all proteins migrating to the same molecular weight. It is important, therefore, to combine the electrophoretic analysis with a more specific measure of a given protein, using immunoblotting or immunocytochemical procedures.

been reported to be induced by these insults [13,14]. Finally, a recent study demonstrated a lower threshold for changes in HSC73 mRNA, compared with induction of HSP72 mRNA [12]. The changes in HSC73 mRNA develop sooner than changes in HSP72 mRNA. The new synthesis of the HSC73 immunoreactive band was reduced, not stimulated, by in vitro ischemia in this study. HSC73 has been reported to be a clathrin uncoating ATPase [17], and may be involved in constitutive protein folding and processing and the response to cell injury [11]. Consistent with a possible role in membrane-based vesicle trafficking, two studies have demonstrated a reduction in the amount of unassembled clathrin postischemia [17] and a corresponding increase in membrane associated clathrin [33], implicating a disruption in the clathrin vesicle cycling process. Thus, if HSC73 is necessary for this process in the hippocampus, then a reduction in HSC73 might lead to disruptions in this process after ischemia. In contrast to the reduced synthesis of HSC73, the synthesis of a 6 8 / 7 0 kDa protein was dramatically increased after ischemia. The identity of this protein is unknown. One candidate molecule is a 68 kDa member of the 70 kDa stress protein family [7]. While the precise identity of this protein is currently under investigation, the protein appears distinct from the HSP72/HSC73 immunoreactive band.

4.2. Stress protein responses to in vitro ischemia 4.3. Neuronal cytoskeletal protein responses

Numerous investigators have shown that transcription of HSP70, HSP72 and HSP68 are induced in the hippocampus following ischemia in vivo [7]. Others, using a commercially available antibody that specifically recognizes HSP72 demonstrated that, while mRNA expression of this species increases in all neuronal subfields of the hippocampus, the protein is synthesized only in the more resistant neurons [30], suggesting that the ability to synthesize HSP72 is associated with cell survival (reviewed in [23]). In our study, HSP72 was not detected following slice preparation or following in vitro ischemia, in keeping with these reports. However, variation in expression patterns is observed depending on the insult, the recovery time, the technique used to measure expression changes and the species of animal studied [7]. Thus, a number of investigators have demonstrated the appearance of HSP72 postischemia, even in those neurons destined to die [4,27,29]. Further, methodological considerations and antibody choice have a dramatic influence on the results obtained. For example, a recent review indicated that the commercially available antibody used in many of these studies (Amersham RPN 1197) reacts differently in different cell types [7]. Thus, the role and response of HSP72 following ischemia remains to be elucidated. Based on our findings, the most likely candidate for the single immunoreactive band at 72/73 kDa is HSC73. This protein was present in both control slices and slices exposed to in vitro ischemia. Further, HSC73 mRNA has

We analyzed the effects of in vitro ischemia on a 68 kDa putative neurofilament protein, using both immunoblotting and immunocytochemical analysis. Immunoblots indicated little change in the amount of this protein, and this protein is apparently distinct from the 6 8 / 7 0 kDa protein whose synthesis was transiently stimulated postischemia. Immunocytochemical analysis of this 68 kDa protein showed that this putative neurofilament protein is localized to processes of the less vulnerable CA3 and hilar neurons, with very little immunoreactivity in CA1 pyramidal neurons. This distribution agrees with that seen in vivo [20,22], although the intensity and degree of staining is lower in the slice. The reduced signal could reflect differences in staining procedure or a depletion of this protein upon slicing. The latter is unlikely because MAP-2, a protein that has been reported to be vulnerable to rapid degradation following a host of insults [18], is not depleted noticeably after slice preparation in our studies. Our immunocytochemical analysis of MAP-2 distribution in the rat hippocampal slice revealed a pattern and intensity of staining very similar to that seen in vivo [33]. This pattern persisted in control slices after many hours of incubation. Thus, the slice makes an excellent system for studying early effects of insults on MAP-2 levels and distribution. The immunoblot densitometric analysis revealed a detectable and progressive depletion of MAP-2, beginning as early as 30 min postischemia. In addition, the

K.M. Raley-Susman, J. Murata / Brain Research 694 (1995) 94-102

distribution of MAP-2 staining (determined immunocytochemically) was altered by 3 h postischemia. While staining persisted in dendrites of the CA1 pyramidal neurons, the staining in somata was reduced considerably. A recent study demonstrated a similar loss of somatic staining for MAP-2 following in vivo ischemia in the gerbil [20]. Thus, even in the early hours after in vitro ischemia, detectable changes in MAP-2 have occurred in the slice, indicating that depletion of MAP-2 serves as a sensitive index of neuronal injury in slices. Immunoblot analysis of a-tubulin levels demonstrated a pronounced depletion of tubulin by 3 h postischemia. The degradation of tubulin agrees well with studies of in vivo ischemia in gerbils [32] and thus underscores the usefulness of the slice to monitor changes in cytoskeletal proteins following ischemic insults. These results, taken together with those for MAP-2, indicate that there are early changes in cytoskeletal protein synthesis, levels and distribution within the first hours after an energy deprivation insult. These changes occur at a time when neuronal morphology is severely altered in postischemic slices [25], implying that changes in cytoskeletal protein synthesis and distribution are translated into morphological changes. In summary, this study has demonstrated that many proteins in the hippocampal slice are not depleted within the first 3 h following in vitro ischemia. However, levels of key neuronal cytoskeletal proteins, MAP-2 and a-tubulin, begin to be depleted within the first three hours of recovery from in vitro ischemia in the hippocampal slice. Further, new synthesis of most proteins is drastically reduced. Intriguingly the synthesis of one protein, 68/70 kDa, is dramatically increased postischemia. The identity and localization of this protein are currently under investigation. Overall, the slice preparation responds to in vitro ischemia with many of the changes documented in vivo and is a useful system for studying the influence of energy depletion on protein expression.

Acknowledgements Support was provided by AHA Grant-in-Aid 92-014GB and NIH Grant 1R55NS30790-01 to K.M.R.-S. and a Clare Boothe Luce Undergraduate Fellowship (J.M.). We thank Dr. Robert M. Sapolsky of the Department of Biological Sciences at Stanford University and Dr. Nancy J. Pokrywka for their helpful pre-review of the manuscript. We also thank Mr. Jerry Calvin at Vassar College for expert assistance in the preparation of the figures.

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