Neuroscience Letters, 144 (1992) 75-78
75
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08924
Calcium/calmodulin dependent protein kinase II mRNA in the gerbil brain after cerebral ischemia David M. Hiestand a and M a r k S. Kindy b "Department of Biochemistry and bLaboratory of Cellular and Molecular Neurobiology, Chandler Medical Center, The University of Kentucky, Lexington, KY 40536-0084 (USA) (Received 13 March 1992; Revised version received 8 June 1992; Accepted 11 June 1992)
Key words." lschemia; Gerbil: Gene expression; CaM kinase II; Calcium The effect of transient cerebral ischemia on the expression of Ca2*/calmodulin dependent protein kinase II (CaM kinase II) mRNA in the gerbil brain was analyzed by Northern blots using cDNA clones for CaM kinase II. Ten minutes of bilateral carotid occlusion and 30 min of reperfusion resulted in reduced protein levels for ~ andfl subunits of the CaM kinase II, decreasing to 35% of control levels at 24 h. Recovery of immunoreactivity was detected in the cortex after 48 h. Eight to twelve hours after ischemia, the cortex showed a decrease in ~ and fl CaM kinase II mRNA levels. By 12 24 h of reperfusion the level of CaM kinase II mRNA was reduced to 26% of the control mRNA levels. CaM kinase II mRNA levels recovered by 48 h after ischemia, coinciding with the increase in CaM kinase II protein immunoreactivity. These results suggest that CaM kinase II is involved in neuronal survival through the reorganization of the neuroarchitecture and that the regulation of this role is controlled at the level of gene expression.
Cerebral ischemia and reperfusion injury results in selective vulnerability of the CA1 pyramidal cells in the hippocampus [8, 13]. During this time, a restructuring of the neuronal cytoskeleton and neural network takes place in response to calcium uptake [19, 20]. Calcium influx regulates the activation of a number of protein kinases which phosphorylate cytoskeletal components giving rise to a dynamic modulation of the neuronal membrane [17, 18]. Therefore, calcium may play a key role not only in the adaptive regulation of neurons in development and plasticity of the adult nervous system, but also in the neurodegenerative process of pathological conditions [25]. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is highly expressed in cortical and hippocampal neurons, and appears to be localized to the postsynaptic density [2, 9, 10]. Neuronal substrates of the CaM kinase II enzyme include tau, synapsin I, tyrosine hydroxylase and myelin basic protein suggesting a role in control of neuronal function [21-24]. Regulation of CaM kinase II activity in the brain following ischemia suggests that this enzyme may play a role in the neuronal alterations associated with ischemic injury [22]. Correspondence: M.S. Kindy, Department of Biochemistry, The University of Kentucky, Lexington, KY 40536, USA.
This study was designed to investigate the effects of forebrain ischemia on CaM kinase II mRNA levels. Recent studies have demonstrated the importance of gene regulation in the control of neuronal plasticity, and this may be an important factor in the mechanisms of CaM kinase II expression [11]. Ninety male gerbils (Tumblebrook Farm, West Brookfield, MA) were prepared as described previously [5]. Animals were anesthetized with pentobarbital (40 mg/kg) prior to surgery, and occluders were placed around the carotid arteries. After surgery, the animals were allowed to recover for 2 days to clear the pentobarbital. This allowed for occlusion of the arteries and production of ischemia in the absence of anesthetic. Transient forebrain ischemia was induced for 10 min in this preparation and then occluders were removed to allow for complete reperfusion. Animals were sacrificed by decapitation at specified times following ischemia (0-96 h), after which the brains were dissected and immediately frozen in liquid nitrogen and stored at -80°C. Ten animals were taken for histological assessment following cerebral ischemia and reperfusion. Following ischemia and reperfusion (14 days) animals were perfused and embedded in OCT freezing medium. Ten-/lm sections were analyzed for cellular viability using Cresyl violet staining of the hippocampal region. Body temperature was main-
76
tained constant throughout the experiments. Animals were monitored and those demonstrating seizure-like activity were not included in the study. Protein samples were prepared as described previously [6] and electrophoresed onto 10% polyacrylamide gels. The gels were transferred onto Imobilon membrane and Western blotted as described below. The membrane was blocked in phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA) and 100/aM CaCI> The blots were then incubated in blocking buffer containing 100/aM CaC12, 5/ag/ml calmodulin and 1/1,000 dilution of a monoclonal antibody directed against CaM kinase II [14]. The antibody detects both the phosphorylated and dephosphorylated proteins. The blots were washed and incubated with an t-'SI-labeled goat anti-mouse IgG antibody to detect the CaM kinase I1 ~ (60 kDa) and fl (50 kDa) subunit proteins. The blots were exposed to film following washing in phosphate buffer. All buffers contained 100/aM CaCI2. For RNA extractions, total cellular RNA was isolated from gerbil tissue as described by Auffray and Rougeon [1] after 10 min of bilateral carotid occlusion and various times of reperfusion (0-96 h). Briefly, tissue was homogenized in 3 M LiC1, 6 M urea with diethylpyrocarbonate (DEPC, 0.1%) for 1 min and precipitated overnight on ice. After centrifugation at 10,000 rpms for 20 min at 0°C, the RNA was resuspended in 10 mM Tris, pH 7.6 and 1% SDS plus DEPC and extracted twice with equal volumes of chloroform. RNA was precipitated with 0.3 M sodium acetate, pH 5.2 and 2.5 vols. of ethanol on dry ice for 2 h. The RNA was resuspended in 10 mM Tris, pH 7.6, 1 mM EDTA (TE) plus DEPC at a concentration of 3/ag//al. RNA (10 pg) was analyzed by electrophoresis through a 1.0% agarose-2.2 M formaldehyde gel
[12]. The RNA was transferred to nitrocellulose as previously described [I 6], and hybridized for specific gene expression. Blots were prepared as previously described [11]. Resulting blots were visualized by autoradiography. DNA probes for rat (:aM kinase Ii ~z 115] and fl [3] chains, and rat fl-actin [4] were prepared by isolation of excised fragments and random primer labeling using the method of Fienberg and Vogelstein [7]. All values presented are means _+ S.D. Results were analyzed by one-way analysis of variance. The CaM kinase II immunoreactivity is shown in Fig. 1. Our results are consistent with previous data demonstrating a decrease in CaM kinase I1 immunoreactivity in the brain following ischemia [19]. We detected a decrease in CaM kinase II immunoreactivity for both the 50 kDa ~ subunit) and 60 kDa (c~ subunit) proteins at 30 rain 2 h of reperfusion (Fig. IA, lane 3 and lane 4) and this decrease continued to 24 h with a 65% {0.35 _+ 0.05) reduction in protein levels (Fig. 1A, lane 6, B). By 48 h (Fig. IA, lane 7) the CaM kinase II levels increased to 74% (0.74 _+ 0.08) of control values (1.0 _+0.07). This suggests that the inactivation of CaM kinase 1I by ischemia may result in turnover of the CaM kinase I! enzyme, t\~llowed by new synthesis to return the CaM kinase 1I to control levels. To characterize the changes in CaM kinase II mRNA in the brain following ischemia, we analyzed the expression of CaM kinase II mRNA by Northern blot hybridization. Following 10 min of ischemia there was very little change in the amount of m R N A for both the 0t and fl subunit of CaM kinase II in the cortex at the early time points (Fig. 2A, top panel, lanes 2 4). By 12-24 h following ischemia, there was a 5-fold reduction in the ~ subunit m R N A of CaM kinase II and a small decrease in the II
r . = ~ ~ i n a s e II
A
B t--
- 60 kDa W
- 50 kDa
"O
Q
O
C
NR
30'
l h
2h
24h
48h
¢
NR
30'
lh
2h
24h
48h
Fig. I. C a M kinase II protein levels following ischemia and reperfusion. A: protein homogenates were electrophoresed onto 10% SDS-PAGE and Western blotted for C a M kinase II protein using monoclonal antibodies. Control (C), 5 rain ischemia and no reperfusion (NR), 30 rain, 1 h, 2 h, 24 h and 48 h reperfusion. B: graph of densitometric scan of Western blots for C a M kinase 1I protein. Data are presented in optical density units by scanning both bands. Data represent n : 4 for each time point, means + S.D.
77 A
C
B
1
2
4
8 12 2 4 4 8
72
96
hr
hippo
cortex
C
e[ CaM
kinase
1 24 C
striat
1 24
C
1
mid
24 C
1
cereb
24C
1
24hr
II /
-actin
1
2
3
4
5
6
7
8
9
10
ct C a M k i r a s e
muwil411~UwUUuwuum-.o,io 1
2
3
4
5
6
7
8
9 1011
12 13 14 15
Fig. 2. RNA hybridization to CaM kinase II ~ and ,8 subunits in the brain. A: RNA was isolated from the cortex and analyzed for the CaM kinase II gene expression. Top panel: 10 pg of RNA from the cortex was examined by Northern blot analysis for expression of both ~ and ,8 CaM kinase II subunit mRNAs. Time points from 0 to 96 h of reperfusion are indicated in the figure. The time course was repeated 4 times with similar results for each experiment. Bottom panel: same blot probe with a,8-actin specific cDNA. B: RNA analysis from individual brain regions following ischemia and reperfusion. Top panel: for analysis at the regional level 10pg of RNA were isolated from cortex, hippocampus, striatum, midbrain and cerebellum from 4 animals and the tissue was pooled, then were subjected to gel electrophoresis and RNA analysis. Blots were hybridized with cDNA probes directed against the ~ and fl subunit of the CaM kinase II gene. Bottom panel: blot was hybridized with a ,8-actin cDNA. Autoradiograms were quantitated by scanning densitometer (LKB).
fl subunit m R N A suggesting inhibition at the level o f gene expression or increased m R N A turnover. Subsequent to the decrease in the ~ subunit m R N A there was an increase in both ~ and fl chains, approximately 5-fold for the ~ chain and 3-fold for the fl chain. This suggests that there are changes in the regulation o f the C a M kinase II genes following cerebral ischemia, These changes coincide with the increased enzyme activity and protein levels (Fig. 1, and ref. 6) in the brain following ischemia. Examination o f C a M kinase II m R N A f r o m the various brain regions d e m o n s t r a t e d similar findings shown in the cortex, with no change seen in the cerebellum, which was not ischemic (Fig. 2B, top panel, and ref. 11). Blots were stripped and rehybridized with a fl-actin specific c D N A probc and showed no change in actin m R N A (Fig. 2A, B, b o t t o m panel). The above results demonstrate differential regulation o f m R N A for the ~ and ,8 chains o f C a M kinase II and restoration o f near normal activity following a traumatic injury. The role C a M kinase II plays in the p h o s p h o r y l a t i o n o f cytoskeletal c o m p o n e n t s suggests that this enzyme has an i m p o r t a n t function in the maintenance o f neuronal function [19]. T u r n o v e r o f the C a M kinase II protein does not play a role in the rapid inactivation o f kinase function [6], however, this process m a y be involved in the availability o f C a M kinase II enzyme in neuronal function [19]. Fig. 1 showed that C a M kinase II protein levels do not change during the inactivation and reactivation process. L o n g - t e r m reperfusion studies demonstrate that the C a M kinase II protein decreased with time and correlated with the changes in m R N A levels (Figs. 1 and 2). These data suggest that ischemia and reperfusion injury results in decreased gene expression which alters the level
o f C a M kinase II protein available in the neuron. This causes a reduced level o f p h o s p h o r y l a t e d protein present in the post-ischemic brain. This decrease m a y be important for the lack o f function o f the neurons and result in neuronal injury following ischemia. The subsequent increase in m R N A and protein levels m a y be an attempt by the neuronal cells at recovery f r o m ischemic injury. Further studies using longer times o f ischemia and older animals should help to determine the potential role o f decreased kinase activity on neuronal function. The authors wish to thank Dr. J o h n M. Carney for his assistance, A r u n a Bhat for her expert technical work and M a r y K e n n e d y for providing the rat fl C a M kinase II and Michael Rosenfeld for the c~ C a M kinase II c D N A clones. This work was supported by a grant f r o m the Council for Tobacco Research (M.S.K.). 1 Auffray, C. and Rougeon, F., Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA, Eur. J. Biochem., 107 (1975) 303-314. 2 Bennett, M.K., Erondu, N.E. and Kenned3~, M.B., Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem., 258 (1983) 12735 12744. 3 Bennett, M.K. and Kennedy, M.B., Deduced primary structure of the ,8 subunit of brain type II Ca2*/calmodulin-dependent kinase determined by molecular cloning, Proc. Natl. Acad. Sci. USA, 84 (1987) 1794-1798. 4 Bond. J.F. and Farmer, S.R., Regulation of tubulin and actin mRNA production in rat brain: expression of a new ,8-tubulin mRNA with development, Mol. Cell. Biol., 3 (1983) 1333-1342. 5 Chandler, M.J., DeLeo, J. and Carney, J.M., An unanesthetized gerbil model of cerebral ischemia induced behavioral changes, J. Pharmacol. Methods, 14 (1985) 137 -146.
II
78 6 Churn, S.B., Taft, W.C., Billingsley, M.S., Blair, R.E. and DeLorenzo, R.J., Temperature modulation of ischemic neuronal death and inhibition of calcium/calmodulin-dependent protein kinase II in gerbils, Stroke, 21 (1988) 17t5 1721. 7 Fienberg, A. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132 (1983) 6-13. 8 Kahn, K., The natural course of experimental cerebral infarction in the gerbil, Neurology, 22 (1972) 510 515. 9 Kelly, P.T., McGuinness, T.L. and Greengard, P., Evidence that the major post synaptic density protein is a component of calcium/ calmodulin protein kinase, Proc. Natl. Acad. Sci. USA. 81 (1984) 945 949. 10 Kennedy, M.B., Bennett, M.K. and Erondu, N.E., Biochemical and immunochemical evidence that the 'major postsynaptic density protein' is a subunit of a calmodulin-dependent protein kinase, Proc. Natl. Acad. Sci. USA, 80 (1983) 5604-5608. 1l Kindy, M.S., Carney, J.E, Dempsey, R.J. and Carney, J.M., Ischemic induction of protooncogene expression in gerbil brain, J. Mol. Neurosci., 2 (1991) 217 228. 12 Kindy, M.S. and Verma, I.M., Developmental expression of the Xenopus laevis fos protooncogene, Cell. Diff. Dev., 1 (1990) 27 38. 13 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57 69. 14 LeVine, H,, Su, J.-L. and Sahyoun, N.E., Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin, Biochemistry, 27 (1988) 6612-6617. 15 Lin, C.R., Kapiloff, M.S., Durgerian, S., Tatemoto, K., Russo, A.F., Hanson, E, Schulman, H. and Rosenfeld, M.G.. Molecular cloning of a brain specific calcium/calmodulin-dependent protein kinase, Proc. Natl. Acad. Sci. USA. 84 (1987) 59626966.
16 Maniatis, T., Fritsch, E.F. and Sambrook, J., In: Molecular Clon+ ing: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 382 386. 17 Mattson, M.E, Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca +++influx in cultured hippocampal neurons, Neuron, 4 (1990) 105 117. 18 Mattson, M.R, Guthrie, t~B., Hayes, B.C. and Kater, S.B.. Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture, J. Neurosci., 9 (1989) 3728 3740. 19 Onodera, H., Hara, H., Kogure, K., Fukunaga, K., Ohta, Y. and Miyamoto, E., Ca>/calmodulin-dependent protein kinase II immunoreaclivity in the rat hippocampus after forebrain ischemia, Neurosci. Lett., 113 (1990) 134 138. 20 Raichle, M.E., The pathobiology of brain ischemia, Ann. Neurol., 13(1989) 2 10. 21 Schulman, H., Phosphorylation of microtubule associated proteins by Ca2÷/calmodulin-dependent protein kinase, J. Cell. Biol.. 99 (1984) 11 19. 22 Schulman, H., Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cAME Mol. Cell. Biol., 4 (1984) 1175 1178. 23 Schulman, H., The multifunctional Ca2"/calmodulin-dependent protein kinase, Adv. Cyclic Nucleotide Protein Phosphorylation Res., 22 (1988) 3% 112. 24 Schwartz, J.H. and Schulman, H., Molecular mechanisms tbr memory: second messenger induced modifications of protein kinases in nerve cells, Annu. Rev. Neurosci., 10 (1987) 459476. 25 Siesjo, B.K., Mechanisms of ischemic brain damage, Critical Care Med., 16 (1988) 954-963.
75
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08924
Calcium/calmodulin dependent protein kinase II mRNA in the gerbil brain after cerebral ischemia David M. Hiestand a and M a r k S. Kindy b "Department of Biochemistry and bLaboratory of Cellular and Molecular Neurobiology, Chandler Medical Center, The University of Kentucky, Lexington, KY 40536-0084 (USA) (Received 13 March 1992; Revised version received 8 June 1992; Accepted 11 June 1992)
Key words." lschemia; Gerbil: Gene expression; CaM kinase II; Calcium The effect of transient cerebral ischemia on the expression of Ca2*/calmodulin dependent protein kinase II (CaM kinase II) mRNA in the gerbil brain was analyzed by Northern blots using cDNA clones for CaM kinase II. Ten minutes of bilateral carotid occlusion and 30 min of reperfusion resulted in reduced protein levels for ~ andfl subunits of the CaM kinase II, decreasing to 35% of control levels at 24 h. Recovery of immunoreactivity was detected in the cortex after 48 h. Eight to twelve hours after ischemia, the cortex showed a decrease in ~ and fl CaM kinase II mRNA levels. By 12 24 h of reperfusion the level of CaM kinase II mRNA was reduced to 26% of the control mRNA levels. CaM kinase II mRNA levels recovered by 48 h after ischemia, coinciding with the increase in CaM kinase II protein immunoreactivity. These results suggest that CaM kinase II is involved in neuronal survival through the reorganization of the neuroarchitecture and that the regulation of this role is controlled at the level of gene expression.
Cerebral ischemia and reperfusion injury results in selective vulnerability of the CA1 pyramidal cells in the hippocampus [8, 13]. During this time, a restructuring of the neuronal cytoskeleton and neural network takes place in response to calcium uptake [19, 20]. Calcium influx regulates the activation of a number of protein kinases which phosphorylate cytoskeletal components giving rise to a dynamic modulation of the neuronal membrane [17, 18]. Therefore, calcium may play a key role not only in the adaptive regulation of neurons in development and plasticity of the adult nervous system, but also in the neurodegenerative process of pathological conditions [25]. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is highly expressed in cortical and hippocampal neurons, and appears to be localized to the postsynaptic density [2, 9, 10]. Neuronal substrates of the CaM kinase II enzyme include tau, synapsin I, tyrosine hydroxylase and myelin basic protein suggesting a role in control of neuronal function [21-24]. Regulation of CaM kinase II activity in the brain following ischemia suggests that this enzyme may play a role in the neuronal alterations associated with ischemic injury [22]. Correspondence: M.S. Kindy, Department of Biochemistry, The University of Kentucky, Lexington, KY 40536, USA.
This study was designed to investigate the effects of forebrain ischemia on CaM kinase II mRNA levels. Recent studies have demonstrated the importance of gene regulation in the control of neuronal plasticity, and this may be an important factor in the mechanisms of CaM kinase II expression [11]. Ninety male gerbils (Tumblebrook Farm, West Brookfield, MA) were prepared as described previously [5]. Animals were anesthetized with pentobarbital (40 mg/kg) prior to surgery, and occluders were placed around the carotid arteries. After surgery, the animals were allowed to recover for 2 days to clear the pentobarbital. This allowed for occlusion of the arteries and production of ischemia in the absence of anesthetic. Transient forebrain ischemia was induced for 10 min in this preparation and then occluders were removed to allow for complete reperfusion. Animals were sacrificed by decapitation at specified times following ischemia (0-96 h), after which the brains were dissected and immediately frozen in liquid nitrogen and stored at -80°C. Ten animals were taken for histological assessment following cerebral ischemia and reperfusion. Following ischemia and reperfusion (14 days) animals were perfused and embedded in OCT freezing medium. Ten-/lm sections were analyzed for cellular viability using Cresyl violet staining of the hippocampal region. Body temperature was main-
76
tained constant throughout the experiments. Animals were monitored and those demonstrating seizure-like activity were not included in the study. Protein samples were prepared as described previously [6] and electrophoresed onto 10% polyacrylamide gels. The gels were transferred onto Imobilon membrane and Western blotted as described below. The membrane was blocked in phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA) and 100/aM CaCI> The blots were then incubated in blocking buffer containing 100/aM CaC12, 5/ag/ml calmodulin and 1/1,000 dilution of a monoclonal antibody directed against CaM kinase II [14]. The antibody detects both the phosphorylated and dephosphorylated proteins. The blots were washed and incubated with an t-'SI-labeled goat anti-mouse IgG antibody to detect the CaM kinase I1 ~ (60 kDa) and fl (50 kDa) subunit proteins. The blots were exposed to film following washing in phosphate buffer. All buffers contained 100/aM CaCI2. For RNA extractions, total cellular RNA was isolated from gerbil tissue as described by Auffray and Rougeon [1] after 10 min of bilateral carotid occlusion and various times of reperfusion (0-96 h). Briefly, tissue was homogenized in 3 M LiC1, 6 M urea with diethylpyrocarbonate (DEPC, 0.1%) for 1 min and precipitated overnight on ice. After centrifugation at 10,000 rpms for 20 min at 0°C, the RNA was resuspended in 10 mM Tris, pH 7.6 and 1% SDS plus DEPC and extracted twice with equal volumes of chloroform. RNA was precipitated with 0.3 M sodium acetate, pH 5.2 and 2.5 vols. of ethanol on dry ice for 2 h. The RNA was resuspended in 10 mM Tris, pH 7.6, 1 mM EDTA (TE) plus DEPC at a concentration of 3/ag//al. RNA (10 pg) was analyzed by electrophoresis through a 1.0% agarose-2.2 M formaldehyde gel
[12]. The RNA was transferred to nitrocellulose as previously described [I 6], and hybridized for specific gene expression. Blots were prepared as previously described [11]. Resulting blots were visualized by autoradiography. DNA probes for rat (:aM kinase Ii ~z 115] and fl [3] chains, and rat fl-actin [4] were prepared by isolation of excised fragments and random primer labeling using the method of Fienberg and Vogelstein [7]. All values presented are means _+ S.D. Results were analyzed by one-way analysis of variance. The CaM kinase II immunoreactivity is shown in Fig. 1. Our results are consistent with previous data demonstrating a decrease in CaM kinase I1 immunoreactivity in the brain following ischemia [19]. We detected a decrease in CaM kinase II immunoreactivity for both the 50 kDa ~ subunit) and 60 kDa (c~ subunit) proteins at 30 rain 2 h of reperfusion (Fig. IA, lane 3 and lane 4) and this decrease continued to 24 h with a 65% {0.35 _+ 0.05) reduction in protein levels (Fig. 1A, lane 6, B). By 48 h (Fig. IA, lane 7) the CaM kinase II levels increased to 74% (0.74 _+ 0.08) of control values (1.0 _+0.07). This suggests that the inactivation of CaM kinase 1I by ischemia may result in turnover of the CaM kinase I! enzyme, t\~llowed by new synthesis to return the CaM kinase 1I to control levels. To characterize the changes in CaM kinase II mRNA in the brain following ischemia, we analyzed the expression of CaM kinase II mRNA by Northern blot hybridization. Following 10 min of ischemia there was very little change in the amount of m R N A for both the 0t and fl subunit of CaM kinase II in the cortex at the early time points (Fig. 2A, top panel, lanes 2 4). By 12-24 h following ischemia, there was a 5-fold reduction in the ~ subunit m R N A of CaM kinase II and a small decrease in the II
r . = ~ ~ i n a s e II
A
B t--
- 60 kDa W
- 50 kDa
"O
Q
O
C
NR
30'
l h
2h
24h
48h
¢
NR
30'
lh
2h
24h
48h
Fig. I. C a M kinase II protein levels following ischemia and reperfusion. A: protein homogenates were electrophoresed onto 10% SDS-PAGE and Western blotted for C a M kinase II protein using monoclonal antibodies. Control (C), 5 rain ischemia and no reperfusion (NR), 30 rain, 1 h, 2 h, 24 h and 48 h reperfusion. B: graph of densitometric scan of Western blots for C a M kinase 1I protein. Data are presented in optical density units by scanning both bands. Data represent n : 4 for each time point, means + S.D.
77 A
C
B
1
2
4
8 12 2 4 4 8
72
96
hr
hippo
cortex
C
e[ CaM
kinase
1 24 C
striat
1 24
C
1
mid
24 C
1
cereb
24C
1
24hr
II /
-actin
1
2
3
4
5
6
7
8
9
10
ct C a M k i r a s e
muwil411~UwUUuwuum-.o,io 1
2
3
4
5
6
7
8
9 1011
12 13 14 15
Fig. 2. RNA hybridization to CaM kinase II ~ and ,8 subunits in the brain. A: RNA was isolated from the cortex and analyzed for the CaM kinase II gene expression. Top panel: 10 pg of RNA from the cortex was examined by Northern blot analysis for expression of both ~ and ,8 CaM kinase II subunit mRNAs. Time points from 0 to 96 h of reperfusion are indicated in the figure. The time course was repeated 4 times with similar results for each experiment. Bottom panel: same blot probe with a,8-actin specific cDNA. B: RNA analysis from individual brain regions following ischemia and reperfusion. Top panel: for analysis at the regional level 10pg of RNA were isolated from cortex, hippocampus, striatum, midbrain and cerebellum from 4 animals and the tissue was pooled, then were subjected to gel electrophoresis and RNA analysis. Blots were hybridized with cDNA probes directed against the ~ and fl subunit of the CaM kinase II gene. Bottom panel: blot was hybridized with a ,8-actin cDNA. Autoradiograms were quantitated by scanning densitometer (LKB).
fl subunit m R N A suggesting inhibition at the level o f gene expression or increased m R N A turnover. Subsequent to the decrease in the ~ subunit m R N A there was an increase in both ~ and fl chains, approximately 5-fold for the ~ chain and 3-fold for the fl chain. This suggests that there are changes in the regulation o f the C a M kinase II genes following cerebral ischemia, These changes coincide with the increased enzyme activity and protein levels (Fig. 1, and ref. 6) in the brain following ischemia. Examination o f C a M kinase II m R N A f r o m the various brain regions d e m o n s t r a t e d similar findings shown in the cortex, with no change seen in the cerebellum, which was not ischemic (Fig. 2B, top panel, and ref. 11). Blots were stripped and rehybridized with a fl-actin specific c D N A probc and showed no change in actin m R N A (Fig. 2A, B, b o t t o m panel). The above results demonstrate differential regulation o f m R N A for the ~ and ,8 chains o f C a M kinase II and restoration o f near normal activity following a traumatic injury. The role C a M kinase II plays in the p h o s p h o r y l a t i o n o f cytoskeletal c o m p o n e n t s suggests that this enzyme has an i m p o r t a n t function in the maintenance o f neuronal function [19]. T u r n o v e r o f the C a M kinase II protein does not play a role in the rapid inactivation o f kinase function [6], however, this process m a y be involved in the availability o f C a M kinase II enzyme in neuronal function [19]. Fig. 1 showed that C a M kinase II protein levels do not change during the inactivation and reactivation process. L o n g - t e r m reperfusion studies demonstrate that the C a M kinase II protein decreased with time and correlated with the changes in m R N A levels (Figs. 1 and 2). These data suggest that ischemia and reperfusion injury results in decreased gene expression which alters the level
o f C a M kinase II protein available in the neuron. This causes a reduced level o f p h o s p h o r y l a t e d protein present in the post-ischemic brain. This decrease m a y be important for the lack o f function o f the neurons and result in neuronal injury following ischemia. The subsequent increase in m R N A and protein levels m a y be an attempt by the neuronal cells at recovery f r o m ischemic injury. Further studies using longer times o f ischemia and older animals should help to determine the potential role o f decreased kinase activity on neuronal function. The authors wish to thank Dr. J o h n M. Carney for his assistance, A r u n a Bhat for her expert technical work and M a r y K e n n e d y for providing the rat fl C a M kinase II and Michael Rosenfeld for the c~ C a M kinase II c D N A clones. This work was supported by a grant f r o m the Council for Tobacco Research (M.S.K.). 1 Auffray, C. and Rougeon, F., Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA, Eur. J. Biochem., 107 (1975) 303-314. 2 Bennett, M.K., Erondu, N.E. and Kenned3~, M.B., Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem., 258 (1983) 12735 12744. 3 Bennett, M.K. and Kennedy, M.B., Deduced primary structure of the ,8 subunit of brain type II Ca2*/calmodulin-dependent kinase determined by molecular cloning, Proc. Natl. Acad. Sci. USA, 84 (1987) 1794-1798. 4 Bond. J.F. and Farmer, S.R., Regulation of tubulin and actin mRNA production in rat brain: expression of a new ,8-tubulin mRNA with development, Mol. Cell. Biol., 3 (1983) 1333-1342. 5 Chandler, M.J., DeLeo, J. and Carney, J.M., An unanesthetized gerbil model of cerebral ischemia induced behavioral changes, J. Pharmacol. Methods, 14 (1985) 137 -146.
II
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