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Stroke. 1995;26:1478-1489

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(Stroke. 1995;26:1478-1489.)
© 1995 American Heart Association, Inc.

Articles

Ischemic Delayed Neuronal Death

A Mitochondrial Hypothesis

K. Abe, MD; M. Aoki, MD; J. Kawagoe, PhD; T. Yoshida, MD; A. Hattori, MD; K. Kogure, MD Y. Itoyama, MD

From the Department of Neurology, Tohoku University School of Medicine, Sendai (K.A., M.A., J.K., Y.I.), and the Departments of Pathology at Mie University School of Medicine, Tsu (T.Y), Sapporo Medical College, Sapporo (A.H.), and the Institute of Neuropathology, Kumagaya (K.K.), Japan.

Correspondence to Koji Abe, MD, Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai 980-77, Japan.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowPrevious Hypotheses
 down arrowThe Mitochondrial Hypothesis
 down arrowFuture Directions
 down arrowReferences
 
Background A brief period of global brain ischemia causes cell death in hippocampal CA1 pyramidal neurons days after reperfusion in rodents and humans. Other neurons are much less vulnerable. This phenomenon is commonly referred to as delayed neuronal death, but the cause has not been fully understood although many mechanisms have been proposed.

Summary of Review Hippocampal CA1 neuronal death usually occurs 3 to 4 days after an initial ischemic insult. Such a delay is essential for the mechanism of this type of cell death. Previous hypotheses have not well explained the reason for the delay and the exact mechanism of the cell death, but a disturbance of mitochondrial gene expression could be a possibility. Reductions of mitochondrial RNA level and the activity of a mitochondrial protein, encoded partly by mitochondrial DNA, occurred exclusively in CA1 neurons at the early stage of reperfusion and were aggravated over time. In contrast, the activity of a nuclear DNA–encoded mitochondrial enzyme and the level of mitochondrial DNA remained intact in CA1 cells until death. Immunohistochemical staining for cytoplasmic dynein and kinesin, which are involved in the shuttle movement of mitochondria between cell body and the periphery, also showed early and progressive decreases after ischemia, and the decreases were found exclusively in the vulnerable CA1 subfield.

Conclusions A disturbance of mitochondrial DNA expression may be caused by dysfunction of the mitochondrial shuttle system and could cause progressive failure of energy production of CA1 neurons that eventually results in cell death. Thus, the mitochondrial hypothesis could provide a new and exciting potential for elucidating the mechanism of the delayed neuronal death of hippocampal CA1 neurons.


Key Words: cerebral ischemia • cytochrome c oxidase • mitochondria • neuronal death


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowPrevious Hypotheses
 down arrowThe Mitochondrial Hypothesis
 down arrowFuture Directions
 down arrowReferences
 
Brain cells, especially neurons, are very sensitive to various injuries, such as ischemia, hypoxia, hypoglycemia, infection, and trauma. These vulnerabilities of neurons often make it difficult to cure patients suffering from injuries to the central nervous system in clinical situations. The vulnerability is different even within neuronal populations. It is well known that a brief period of global brain ischemia causes cell death in hippocampal CA1 pyramidal neurons days after reperfusion (Fig 1Down). Other neurons, such as parietal cortical or hippocampal CA3, are much less vulnerable.1 2 This phenomenon is commonly referred to as delayed neuronal death.1 3 A similar phenomenon occurs after ischemic injury in humans.4 Prolonged ischemia extends the area of neuronal death to the caudate and thalamic nuclei.2 5 6 However, because hippocampal CA1 neurons are most vulnerable to ischemia, the cell death has been thought to represent a sensitivity of the neurons to injuries. Therefore, it is important to know the exact mechanism of hippocampal CA1 neuronal cell death after ischemia. The cause of such delayed neuronal damage in CA1 neurons has not been fully under-stood although many mechanisms have been proposed. Most of the previous hypotheses have some problems in explaining the reason why the eventual cell death occurs 3 to 4 days after the initial ischemic insult, the mechanism that should be essential to this type of neuronal death.



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  		Figure 1. Photomicrographs show delayed neuronal death of hippocampal CA1 neurons: cresyl violet staining of the gerbil hippocampal formation of sham control (A) and 7 days after reperfusion following a brief period of forebrain ischemia (B). C and D show high magnifications of the hippocampal CA1 pyramidal cell layer of A and B, respectively. Note almost complete loss of CA1 cells (B, large arrowheads, and D) in contrast to the survival of dentate granule cells (B, small arrowhead). Bar=50 µm in A and B, 20 µm in C and D.

The purpose of this review is to summarize these previous hypotheses in terms of the mechanism of delayed neuronal death and propose a new hypothesis based on the disturbance of mitochondrial gene expression. Although previous hypotheses have some difficulties in explaining the exact mechanism of cell death, they are partly related to the new hypothesis.


*    Previous Hypotheses
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Previous Hypotheses
down arrowThe Mitochondrial Hypothesis
 down arrowFuture Directions
 down arrowReferences
 
Glutamate Excitotoxicity
In 1969 Olney7 first suggested glutamate as a powerful excitotoxin to neurons. A remarkable increase of extracellular excitatory neurotransmitter levels such as glutamate and aspartate was reported during brain ischemia and the early period of reperfusion in microdialysis studies in vivo.8 9 The glutamate release is thought to be mainly from presynaptic terminals although some are from postsynaptic terminals.10 11 Experiments with deafferentation of the presynaptic fibers partly protected against CA1 cell death.12 13 14 Dense excitatory and fewer inhibitory innervations in the CA1 neurons provide a background of hyperactivity after ischemia caused by an excitotoxic mechanism.15 16 17 18 The opening of both voltage-dependent and receptor-operated calcium channels results in unregulated excessive calcium influx into the cytoplasm. Calcium is released also from intracellular stores during ischemia.19 Such an unregulated increase of intracellular calcium concentration causes an activation of calcium ATPase, which results in further consumption of cellular ATP and of calcium-dependent proteases, phospholipases, and nucleases. All these processes cause catastrophic consequences in cells.20 21

However, even though the amount of glutamate release was very high,8 9 excessive glutamate release was only transient and returned to normal levels in 10 to 20 minutes of reperfusion. Although a very short period of nonlethal ischemia resulted in high-frequency discharge in the CA1 neurons,22 the phenomenon was not observed after longer periods of ischemia that were severe enough to damage CA1 neurons.23 24 25 Furthermore, recent reports suggest that the amount of N-methyl-D-aspartate receptor and mRNA for the receptor is not very different between the CA1 and CA3 regions.26 27 Thus, the excitotoxic mechanism does not completely explain the eventual cell death occurring so long after insult. However, it should be noted that excessive glutamate release causes a transient increase of intracellular calcium concentration. Although there still is a possibility that extracellular levels of glutamate measured by brain microdialysis may not represent levels in the synaptic cleft, it is important that glutamate returned to baseline levels in the early period of reperfusion, and the level is presumed to persist until cell death.

Prolonged Inhibition of Protein Synthesis
It is well known that the threshold for disturbance of protein synthesis is very sensitive28 and that the translational step is much more vulnerable to ischemia or other injuries than the transcriptional step.29 After a brief period of ischemia, protein synthesis is markedly inhibited in all neuronal cells but recovers during the course of reperfusion, except in vulnerable neurons such as CA1 cells.30 Polyribosomes, in which proteins are synthesized in the cell, become disaggregated into monosomes long after reperfusion. An increase in intracellular calcium results in an inactivation of eukaryotic initiation factor-2,20 21 29 and the problem could be in the activations of eukaryotic initiation factor-2 and guanine nucleotide exchange factor in the initiation of protein synthesis at the ribosomes.31 If ischemic cells cannot efficiently replace important proteins that are degraded by ischemic injury, the cells may eventually die. Thus, prolonged inhibition of protein synthesis has been recognized as an important aspect of delayed neuronal death.

However, this hypothesis did not specify particular proteins that play key roles in the mechanism of the death. Furthermore, this hypothesis does not explain why protein synthesis in the CA1 or other specific neuronal groups are selectively sensitive to ischemia. Although calcium overload was dominant in the CA1 area,5 the increase of calcium was also transient,32 making it difficult to explain the complete shutdown of protein synthesis for a long time. The process of protein synthesis requires extreme accuracy to avoid a mistake of amino acid sequence, which needs high energy consumption. Therefore, the prolonged inhibition of protein synthesis suggests that the neurons are in a condition in which they do not have enough energy to make most of the proteins or they have to save energy to use for another purpose.

Disturbed HSP Expression
HSPs are induced under stressful cell conditions when general protein syntheses are inhibited although some HSPs, such as HSC70, are normally expressed.33 34 35 36 37 HSPs are essentially involved in protein folding and therefore have been recognized as a "molecular chaperone."36 37 The cell-protective roles of HSPs have been shown in vitro by injection of antibody against HSP70 or transfection of a gene that constitutively expresses HSP70.38 39 Nowak40 first reported HSP70 synthesis after cerebral ischemia in gerbils in 1985. HSP70 is induced mainly in neurons but can also be induced in astroglial and capillary endothelial cells.41 42 43 44 Subsequent reports showed that CA1 neurons expressed a large amount of mRNA for HSP70 with less immunostaining of the protein.45 46 47 Although HSP70 gene expression genetically has priority over other general or housekeeping proteins under stressful conditions,33 48 HSP70 gene expression was disturbed at the translational level in CA1 neurons probably because of the severe injury.45 46 47 Recent reports have showed that pretreatment of animal brain with a very short period of ischemia that does not cause neuronal death partly saved the CA1 neuronal loss, with prominent expression of HSP70.49 50 51 52 53 54 These results suggest that the preexistence of HSP70 after pretreatment was related to the acceleration of subsequent robust and fast induction of HSP70 mRNA and to its effective translation into the protein.54

However, experiments with rats raised a question about the significance of the role of HSP70.52 55 Contrary to the case in gerbils, CA1 neurons of rats expressed a large amount of immunoreactive HSP70 with the mRNA,52 55 but such neurons eventually die like those in gerbils.2 52 55 Because immunoreactive staining does not provide a quantity of protein, it is difficult to know the significance of the protective effect of HSP70 and the difference of the protein induction between rat and gerbil. However, it can be said that a translational disturbance of HSP70 gene expression may not be a major cause of CA1 cell death in rats and perhaps not in gerbils. In addition, not only HSP70 gene expression is changed after pretreatment54 ; the setting level of many other gene expressions might also be changed.

Lipid Metabolism and Free Radicals
Free fatty acids are liberated from membrane phospholipids during ischemia.56 57 58 59 The level of free fatty acids decreases, and peroxidative derivatives of arachidonic acid increase during reperfusion.60 61 62 Activation of xanthine oxidase and dysfunction of mitochondria also generate free radicals. Because liberation of arachidonic acid during ischemia differs regionally63 and free arachidonic acid inhibits the reuptake of released gluamate without inhibition of that of {gamma}-aminobutyric acid,64 arachidonic acid may transneuronally enhance the differences in regional damage seen between CA1 and CA3 cells. Hillered and Chan65 reported that arachidonic acid was a potent uncoupler of and induced swelling in mitochondria. However, the difference in the amount of free arachidonic acid between CA1 and CA3 areas was only 10% to 30%,63 making it difficult to accept the essential differences in the prognosis for the neurons. Furthermore, free fatty acids returned to normal levels quickly, by 30 minutes to 1 hour.57

NO is a free radical produced in vascular endothelium and serves as an endothelium-derived relaxing factor that maintains and regulates vascular tone.66 67 68 69 However, it is also produced in neurons and glial cells.70 71 72 73 74 NO may mediate neuronal death caused by excitotoxic and hypoxic insults,75 76 77 and mice with knockouts of NO synthase were resistant to ischemia.78 However, inhibition of NO synthase showed dual effects on infarction size depending on the dose of the inhibitor.79 80 81 82 83 84 NO released from glial cells is long-lasting and could be cytotoxic.75 85 86 87 However, NO synthase is not rich in CA1 neurons88 89 and is inducible after a brief period of ischemia, with a peak at 1 day.89 Neurons that contain NO synthase are resistant to excitotoxic and ischemic injuries.90 91 Direct evidence of the relation between CA1 cell death and NO has not been reported.

Free radical scavengers including superoxide dismutase are induced during and after ischemia92 93 94 probably in response to superoxide generation.95 96 Administration of superoxide dismutase ameliorated the damage of CA1 neurons,96 reducing traumatic and ischemic damage.97 98 Transgenic mice with overexpression of human superoxide dismutase showed a reduction of infarcted size.99 100 The effects of the superoxide dismutase transgene on delayed neuronal death is under investigation by Chan et al. Recent data suggest that reaction of peroxynitrite (ONOO-) with superoxide dismutase may degenerate neuronal cells.101 102 103 However, protein tyrosine nitration by the reaction has not yet been reported in terms of delayed neuronal death.

Energy Metabolism and Cerebral Circulation
Immediately after the findings of delayed neuronal death in gerbil and rat, many groups examined changes of energy metabolism and cerebral blood flow. Many reports suggested that energy metabolism recovered quickly after reperfusion.104 105 106 107 108 Several reports indicated that vascular supply to the hippocampus is inferior to that of other regions of the brain109 and that perfused capillaries and the volume of circulating blood are 20% to 30% lower in the CA1 than CA3 region.110 However, the data of microcirculatory capacity were obtained only at 3 minutes and 7 days after reperfusion,110 which does not explain why cell death takes 3 to 4 days. In addition, cerebral blood flow was not significantly different between vulnerable and more resistant areas during and after ischemia.111 112 Thus, it is now commonly thought that disturbances of energy metabolism and cerebral circulation are not mainly involved in the mechanism of delayed neuronal death. However, it should be noted that Munekata and Hossmann107 have reported a relative delay of recovery from acidosis in the CA1 area compared with the CA3 and cortical areas. This suggests an enhanced glycolysis, with possible impairment of mitochondrial functions.

Apoptosis and Neurotrophic Factors
Apoptosis requires active synthesis of mRNA and protein and has been observed typically in the processes of lymphocyte maturation in thymus, of cultured cells, and of developing central nervous system.113 114 115 On the other hand, necrosis involves destruction of tissue, including neurons, glial cells, and capillary blood vessels. Deprivation of neurotrophic factors activates a putative "killer protein" that causes cell death with an apoptotic mechanism.116 117 118 Fas antigen mediates apoptosis,119 and a proto-oncogene product, bcl-2, inhibits apoptotic cell death in vitro.120 121 The apoptotic cell death takes a few days without the formation of edema, and a 180-bp DNA ladder produced by Ca2+-dependent endonuclease usually precedes cell death.114 115 Several lines of evidence have suggested an apoptotic mechanism of CA1 cell death, including a protective effect of administration of protein synthesis inhibitor122 or nerve growth factor,123 124 an observation of a DNA ladder,125 decreases of neurotrophic factors,126 127 128 129 and bcl-2 inductions.130 Thus, hippocampal CA1 neuronal death has some aspects that are similar to apoptotic cell death.

However, ultrastructural analyses suggest that CA1 cell death was not accompanied by typical pathological changes seen in apoptosis, such as early extensive chromatin condensation and nuclear displacement.131 132 133 Inhibitors of protein and RNA syntheses did not reproducibly prevent neuronal damage.133 134 An infusion of nerve growth factor did not rescue CA1 neurons in rats.135 DNA ladders can also be found in other types of neuronal cell death, such as cold injury and necrosis.136 137 The profile of bcl-2 expression was not specific to this protein and was similar to other proto-oncogenes, such as c-fos, in the CA1 region.130 138 Glutamate-related neuronal death was not a programmed cell death in cerebellar culture.139 Apoptotic cells may not express a stress response such as HSP70 for survival. In fact, cultured neurons died days after withdrawal of serum, with an apoptotic mechanism expressing no HSP70 inductions (F.R. Sharp, personal communication, 1994). Furthermore, there is no compensatory advantage, which is required of cells dying with the apoptotic mechanism, to the brain or the individual animals for losing important CA1 neurons. These data provide evidence against the apoptotic hypothesis. Thus, the exact mechanism should be essentially different from that of apoptotic cell death as well as from typical necrosis.


*    The Mitochondrial Hypothesis
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowPrevious Hypotheses
 *The Mitochondrial Hypothesis
down arrowFuture Directions
 down arrowReferences
 
Characteristics of the mtDNA System
Most mitochondrial proteins are encoded by nuclear DNA, synthesized on the cytoplasmic ribosomes, and then imported into mitochondria (Fig 2Down). However, unlike other organelles in the cell, mitochondria have a DNA system that is different from the nuclear DNA system. Some proteins encoded by mtDNA are synthesized on the mitochondrial ribosomes. Although mitochondria have their own genome, the gene expression is completely controlled by the nuclear DNA system. The protein traffic seems to be unidirectional from cytoplasm to mitochondria. However, the nuclear and mitochondrial genetic systems communicate to coordinate their contributions to form the energy-converting enzyme complexes.140 Transcription of mRNA from mtDNA needs mitochondrial transcription factor and mitochondrial RNA polymerase, which are entirely encoded by nuclear DNA. mtDNA is different from nuclear DNA in several ways: (1) mtDNA is circular and nuclear DNA is linear; the size is approximately 16.5 kb in mammals, which is 105 times smaller than that of nuclear DNA; (2) each mitochondrion has multiple copies of mtDNA, ranging from 100 to 1000 in a cell, which makes up to 1% of the total cellular DNA; (3) mtDNA is packaged without histones and is usually distributed in several clusters in the matrix of the mitochondria, where they are thought to be attached to the inner membrane; and (4) because of the low fidelity of replication and lack of repair of mtDNA, the amount of normal mtDNA may decrease and deleted mtDNA increases during cell aging, which may cause a problem in nondividing cells such as neurons and cardiac cells.141 142 143



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  		Figure 2. Schematic shows nuclear and mitochondrial genetic systems in the cell. Some proteins derived from mitochondrial and nuclear genomes form a complex to work as a holoenzyme, and some work as each enzyme in mitochondria.

The mitochondrial respiratory complex is essential for the production of ATP by oxidative phosphorylation and consists of complex proteins that are encoded by both mtDNA and nuclear DNA. The mtDNA encodes for 13 essential oxidative phosphorylation polypeptides as well as containing 2 rRNA and 22 tRNA genes.144 COX is a mitochondrial enzyme forming complex IV for the electron transfer system and is composed of 13 subunits; three of them (COX-I, -II, and -III) are encoded by mtDNA. Therefore, COX-I mRNA is transcribed from mtDNA and translated into the protein within mitochondria. The activity of COX protein is an overall activity of these 13 subunits. COX activity is high in the oriens layer, stratum radiatum, and lacunosum moleculare layer of the CA1 subfield as well as in the molecular layer of dentate gyrus in gerbils (Fig 3aDown). With in situ hybridization, the amount of COX-I mRNA and DNA can be measured.145 COX-I mRNA is abundantly present in hippocampal neuronal cell layers in control brain (Fig 3bDown); COX-I DNA is scarcely present in hippocampal pyramidal cell layers in control brain (Fig 3cDown). It is rich in the oriens layer and stratum radiatum of the CA1 subfield and especially dense in the lacunosum moleculare layer of the CA1 subfield and molecular layer of dentate gyrus, similar to the case of COX activity. COX activity, COX-I mRNA, and COX-I DNA should be in the same mitochondrial capsules. Therefore, these different regional distributions between COX activity and COX-I DNA and COX-I mRNA (Fig 3Down) support the previous hypothesis of the presence of a mitochondrial shuttle system.146 Mitochondria are periodically shuttled to and from the cell body to obtain newly synthesized nuclear-encoded mitochondrial proteins, such as other COX subunits (COX-IV through -XIII), succinate dehydrogenase, and mitochondrial transcription factor.



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  		Figure 3. Photomicrographs show hippocampal areas with cytochrome c oxidase (COX) activity (a), COX-I mRNA (b), and COX-I DNA (c) in sham control gerbil. Panel d shows a section hybridized with the COX-I probe after treatment with RNase without subsequent denaturation of cellular DNA. Or indicates oriens layer; Py, pyramidal cell layer; Rad, stratum radiatum; and LMol, lacunosum moleculare layer of the CA1 subfield.

Disturbance of mtDNA Expression
Many biochemical analyses of the activities of mitochondrial respiratory function have been reported in brains with experimental ischemia.147 148 149 However, regional changes of mitochondrial enzyme activity, mRNA, and DNA after ischemia have not been extensively examined in relation to the delayed neuronal death of CA1 neurons. Recent reports suggest a selective and progressive decrease of mtDNA expression in CA1 neurons after 3.5 minutes of forebrain ischemia.150 151 152 Compared with other areas of hippocampal neurons such as CA3 and dentate granule cells, COX-I mRNA level began to decrease at an early stage of reperfusion in the CA1 subfield (Fig 4Down, left, arrowheads), progressively deteriorated, and was completely gone at 7 days (arrowheads), whereas other hippocampal neurons showed almost no change during the reperfusion period. A previous report with a biotinylated probe suggested no change of COX-I mRNA levels in hippocampal CA1 neurons up to 48 hours after reperfusion.153 However, nonspecific signals after RNase treatment seem to be too high to detect small changes of COX-I mRNA.153



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  		Figure 4. Photomicrographs show in situ hybridizations for cytochrome c oxidase subunit I (COX-I) mRNA, COX-I DNA, and 60-kD heat-shock protein (HSP60) mRNA in gerbil hippocampus after 3.5 minutes of forebrain ischemia. Note the early onset and progressive decrease of the COX-I mRNA level in the CA1 area, whereas COX-I DNA remained normal until 1 day in the same cells. S indicates sham control. HSP60 mRNA was induced mainly in CA1 (arrowheads at 1 day) and dentate granule (arrowheads at 3 and 8 hours) cells.

In contrast, the amount of COX-I DNA began to decrease in the oriens layer of the CA1 subfield at 2 days and significantly decreased in the entire CA1 subfield at 7 days (Fig 4Up, middle, arrowheads). Unlike COX-I mRNA, COX-I DNA did not completely disappear even at 7 days. The amount of COX-I DNA remained normal in other areas of hippocampus and cerebral cortex during the reperfusion period. Southern blot analysis confirmed the change in the CA1 region.151 These data may be consistent with data from brain mtDNA, which was not damaged after prolonged cardiac arrest and reperfusion.154

HSP60, also known as cpn60, is an E coli GroEL homologue and works in conjunction with the 10-kD chaperonin (cpn10) in the mitochondrial matrix.36 37 HSP60 is a mitochondrial protein but is encoded by nuclear DNA; therefore, the protein is synthesized on cytoplasmic ribosomes and is imported in mitochondria. HSP60 mRNA is also induced in CA1 neurons with a peak at 1 day (Fig 4Up, right), and the increase may be related to the interaction with denatured or unfolded protein in mitochondria by analogy with the roles of HSP70 and HSC70.36 37 152 In fact, the increase of HSP60 mRNA is associated with the inductions of HSP70 and HSC70 mRNAs and the decrease of COX-I mRNA,152 which may represent the mitochondrial damage.

Changes in Mitochondrial Enzyme Activities
COX activities also show an early, selective, and progressive decrease in the hippocampal CA1 subfield until 7 days after ischemia (Fig 5Down, left, arrowheads). Developmental changes of COX activity in brain regions and marked decreases of the activity in hyperglycemic brain after anoxia have been reported in cats.149 155 However, such an early decrease in the CA1 region has not been reported. The early decrease of mitochondrial COX activity in the stratum radiatum may be related to the high glucose uptake in the same region at the early period of reperfusion,156 suggesting an enhanced glycolysis in the region with a disturbed mitochondrial function. The delayed recovery from acidosis in the CA1 region107 might be related to this possible upregulation of glycolysis.



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  		Figure 5. Photomicrographs of histochemistry for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) show changes in activities after forebrain ischemia in the gerbil. Note the early onset and progressive decrease of COX activity in the CA1 area, whereas SDH activity began to decrease from 2 days in the CA1 subfield and markedly decreased at 7 days especially in the lacunosum moleculare layer (arrowheads).

Distribution of succinate dehydrogenase activity (Fig 5Up, right) in the sham control hippocampus is almost the same as that of the COX activity (Fig 5Up, left). In contrast to the change of COX activity, succinate dehydrogenase activity begins to decrease in the oriens layer at 2 days and markedly decreases at 7 days in the entire CA1 subfield, especially in the lacunosum moleculare layer (arrowheads). An activity of pyruvate dehydrogenase complex that is also encoded by nuclear DNA was increased during the early period of reperfusion.157 158 Histological examination shows that most of the CA1 cells are still present at 2 days although the staining began to decrease compared with staining in sham controls.150 151 152 Only the CA1 cells were completely gone at 7 days (Fig 1Up) in the present study after 3.5 minutes of ischemia.150 151 152

Motor Proteins
So-called motor proteins, such as CD and kinesin, are linear motors that convert the energy of ATP hydrolysis into mechanical work and move cellular organelles such as mitochondria along microtubules (Fig 6Down). CD, also referred to as MAP1C,159 translocates organelles to the "minus" (proximal) end of microtubules.159 160 161 162 163 Kinesin is thought to mediate an axonal transport to the "plus" (peripheral) end of microtubules. The heavy chain contains all of the mechanochemical elements necessary for generating microtubule-based movements and has microtubule-stimulated ATP activity.164 165 166 167 168 169



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  		Figure 6. Schematic of organelle movement along the microtubule. Kinesin and cytoplasmic dynein take part in the antegrade and retrograde transport, respectively, of organelles such as mitochondria. Both movements require ATP. d indicates cytoplasmic dynein.

Immunohistochemical analyses for CD and kinesin are shown in Fig 7Down (left and middle). Immunoreactivity for CD is observed in all hippocampal pyramidal cells, whereas it is scarcely expressed in dentate granule cells.170 The immunoreactivity begins to decrease at 1 hour in CA1 cells; the decrease becomes more evident at 3 hours after ischemia, whereas in other neurons immunoreactivity remains at normal levels (Fig 7Down left). The immunoreactivity progressively decreased and was almost gone as early as 1 day. Immunoreactive kinesin is present in all hippocampal cells, especially in cell bodies, of control brain. Immunoreactivity for kinesin shows an apparent change in hippocampal CA1 cells after 8 hours of reperfusion and continuously decreases (Fig 7Down, middle). However, other hippocampal neurons show almost no change during the reperfusion period. Thus, CD shows a progressive decrease from 1 to 3 hours, and kinesin shows a decrease from 8 hours of reperfusion; these changes in CD and kinesin are found only in the vulnerable CA1 sector. Two minutes of ischemia did not significantly change the immunoreactivities of CD and kinesin in CA1 cells.170 MAP2, which is abundant in neuronal dendrites, is thought to be one of the microtubule cross-linking proteins.171 Previous studies revealed that immunoreactivity of MAP2 was more susceptible to ischemia than that of {alpha}-tubulin and should serve as a marker of cell injury after ischemia.172 173 174 175 176 However, staining of MAP2 was maintained at control levels in CA1 cells until 2 days but was completely gone at 7 days.



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  		Figure 7. Left, Photomicrographs show immunohistochemical change of cytoplasmic dynein in the hippocampal CA1 and CA3 subfields after 3.5 minutes of forebrain ischemia in the gerbil. Arrows represent CA1 pyramidal cell layer; arrowhead represents CA3 cells. Note early decrease of cytoplasmic dynein immunoreactivity in CA1 neurons, whereas that in CA3 was not affected. Middle, Photomicrographs show immunoreactive distribution of kinesin in gerbil hippocampus and the change in CA1 pyramidal cells (arrows). Note significant and progressive decrease from 8 hours of reperfusion. DG indicates dentate granule cells. Right, Photomicrographs show immunohistochemical change of microtubule-associated protein 2 in gerbil hippocampal CA1 layer (arrowheads) and dentate gyrus (arrowhead). Note normal staining until 2 days and complete disappearance of staining at 7 days in contrast to preservation in the dentate gyrus. In all panels, C indicates control.

Thus, the motor proteins CD and kinesin are much more susceptible to ischemia compared with nonmotor or constitutive proteins such as MAP2 and tubulin. CD and kinesin are thought to be normally attached to the lipid membrane of cell organelles. The early reduction of immunoreactivity suggests that CD and kinesin become translocated from the lipid membrane or that these motor proteins are sensitive to a transient increase of calcium during and after ischemia. Recent works identified a new kinesin superfamily (KIF1-5), and some of the members are involved in mitochondrial movement (KIF1b).177 178 The present data provide evidence for the early disturbance of the mitochondrial shuttle system in CA1 neurons.

A Speculative Synthesis
The previous150 151 152 and present data show an early onset and progressive reduction of COX-I mRNA levels and COX activity in contrast to succinate dehydrogenase activity and COX-I DNA level. Because the mitochondrial shuttle system is essential in mtDNA expressions and the renewal of proteins, disturbances of motor proteins must result in the progressive failure of energy production in CA1 neurons that eventually causes a final catastrophe and cell death. Although the reason why the immunoreactivities of motor proteins are susceptible to ischemia remains to be resolved, it is possible that biological activities of motor proteins are disturbed. Because CA1 neurons may need more ATP to repair the disturbed intracellular environment and denatured protein and lipids, ATP may be difficult to be sufficiently distributed to motor proteins (Fig 6Up). Impairment of kinesin activity caused by mutations decreased action potential propagation and neurotransmitter release in Drosophila.179 Kirino et al180 suggested that a slight reduction of membrane resting potential (25% of control) persisted in CA1 neurons after ischemia and preceded cell death. Recent data suggest that ambient levels of glutamate become toxic to cells under mild energy failure.181 182 183 184 After an initial increase, normal glutamate levels were maintained long after reperfusion, probably until cell death at 3 to 4 days.8 9 Hippocampal CA1 neurons have uniquely longer dendrites and axonal projects than other hippocampal neurons, making them more vulnerable to the disturbance of motor proteins and mild energy failure.

The present hypothesis is not contrary to previous hypotheses. An initial increase of intracellular calcium concentration caused by excitatory neurotransmission first damages motor proteins; then the mitochondrial shuttle system is disturbed, followed by a gradual decrease of energy production in mitochondria that eventually causes an energy crisis of the cells, which are increasingly demanding ATP for recovery. These processes finally result in cell death. Thus, this hypothesis cooperates with the previous hypotheses and explains the reason why cell death takes 3 to 4 days after the initial ischemic insult. The delayed neuronal death of hippocampal CA1 neurons seems to be a particular type of necrosis.


*    Future Directions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowPrevious Hypotheses
 up arrowThe Mitochondrial Hypothesis
 *Future Directions
down arrowReferences
 
The present data suggest a progressive change of energy metabolism within a specific region of the CA1 area. Therefore, further analyses on regional energy metabolism and quantitative topographic measurement of ATP that are more detailed than previous reports104 105 106 107 108 185 and possible measurement of the intracellular regional metabolic rate of ATP will be required to understand the mechanism more clearly. Recent data suggest that an mtDNA deletion and the reduction of mitochondrial enzyme activity are also associated with ischemic, hypoxic, and aged tissue.186 187 188 189 190 191 192 Although COX-I DNA is not located in the hot spot of such mtDNA deletions,141 142 143 193 194 deleted mtDNA might be increasing in CA1 neurons after ischemia if it is examined with probes that detect hot spots. Problems in mitochondrial transcription factor and mitochondrial RNA polymerase could also take part in the disturbances of mtDNA expression.195 196 197 Delayed neuronal death is found in other neuronal populations, such as small neurons in lateral caudate, neurons in cortical 4-6 layers, and Purkinje cells.1 2 3 5 Further studies are required to determine whether the present hypothesis could also be applied to those cells with a longer duration of ischemia.


*    Selected Abbreviations and Acronyms
 
CD = cytoplasmic dynein
COX = cytochrome c oxidase
HSC = heat-shock cognate protein
HSP = heat-shock protein
MAP = microtubule-associated protein
mtDNA = mitochondrial DNA
NO = nitric oxide



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  		Figure 8. Schematic of the mitochondrial shuttle and formation of mitochondrial enzyme complexes. Mitochondria are transported from the cell periphery to cell body where they receive nuclear-encoded mitochondrial transcription factor (mtTF) and mitochondrial RNA (mtRNA) polymerase that then transcribe RNA in mitochondria for protein synthesis. Some nuclear DNA–encoded proteins are synthesized, partly maturated in cytoplasm with the chaperoning activity of the 70-kD heat-shock cognate protein (HSC70), and transported into mitochondria with the assistance of the 60-kD heat-shock protein (HSP60) and mitochondrial HSP70 (mtHSP70). COX indicates cytochrome c oxidase; mt, mitochondrial; ETS, electron transfer system; and SDH, succinate dehydrogenase.


*    Acknowledgments
 
This work was partly supported by Grant-in-Aid for Scientific Research on Priority Areas (I. Kanazawa) 06272204; Grant-in-Aid for Scientific Research (C) 06807055 from the Ministry of Education, Science and Culture of Japan; and a grant (S. Hirai) from the Ministry of Health and Welfare of Japan. The authors would like to thank Profs T. Kirino, T. Yoshimoto, A. Tamura, and T. Yanagihara and Drs T. Tominaga, H. Kinouchi, and H. Kato for helpful discussions and support. The authors also thank Profs B.K. Siesjö, K.A. Hossmann, W.A. Pulsinelli, M.D. Ginsberg, D. Choi, P.H. Chan, F.R. Sharp, T. Wieloch, M.A. Moskowitz, N.G. Bazan, and T.S. Nowak, Jr, for their suggestions to the present hypothesis.

Received November 9, 1994; revision received January 11, 1995; accepted March 30, 1995.


*    References
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 *References
 

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