中枢神経系疾患の治療は正常細胞（ニューロン）の機能維持を目的とするが，脳血管障害のように機能障害の原因が細胞の死滅に基づくことは多い．一方，脳腫瘍の治療においては薬物療法や放射線療法といった腫瘍細胞の死滅を目標とするものが大きな位置を占める．いずれの場合にも，細胞死の機序を理解することは各種病態や治療法の理解のうえで重要である．現在のところ最も研究の進んでいる細胞死の型はアポトーシスである．そのなかで重要な位置を占めるミトコンドリアにおける反応および抗アポトーシス因子について概要を紹介する．

Neuroscience 細胞死：最近の知見

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the impairment of cognitive functions and by beta amyloid (Abeta) plaques in the cerebral cortex and the hippocampus. Our objective was to determine genes that are critical for cellular changes in AD progression, with particular emphasis on changes early in disease progression. We investigated an established amyloid precursor protein (APP) transgenic mouse model (the Tg2576 mouse model) for gene expression profiles at three stages of disease progression: long before (2 months of age), immediately before (5 months) and after (18 months) the appearance of Abeta plaques. Using cDNA microarray techniques, we measured mRNA levels in 11 283 cDNA clones from the cerebral cortex of Tg2576 mice and age-matched wild-type (WT) mice at each of the three time points. This gene expression analysis revealed that the genes related to mitochondrial energy metabolism and apoptosis were up-regulated in 2-month-old Tg2576 mice and that the same genes were up-regulated at 5 and 18 months of age. These microarray results were confirmed using northern blot analysis. Results from in situ hybridization of mitochondrial genes-ATPase-6, heat-shock protein 86 and programmed cell death gene 8-suggest that the granule cells of the hippocampal dentate gyrus and the pyramidal neurons in the hippocampus and the cerebral cortex are up-regulated in Tg2576 mice compared with WT mice. Results from double-labeling in situ hybridization suggest that in Tg2576 mice only selective, over-expressed neurons with the mitochondrial gene ATPase-6 undergo oxidative damage. These results, therefore, suggest that mitochondrial energy metabolism is impaired by the expression of mutant APP and/or Abeta, and that the up-regulation of mitochondrial genes is a compensatory response. These findings have important implications for understanding the mechanism of Abeta toxicity in AD and for developing therapeutic strategies for AD.

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Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease

Background and purpose:
The acetylcholinesterase inhibitor, donepezil, is also a high affinity σ1 receptor agonist. We examined the involvement of σ1 receptors in its anti-amnesic and neuroprotective properties against amyloid β25-35 peptide-induced toxicity in mice.

The anti-amnesic and neuroprotective effects of donepezil against amyloid β25-35 peptide-induced toxicity in mice involve an interaction with the σ1 receptor

A neural cell adhesion molecule-derived peptide reduces neuropathological signs and cognitive impairment induced by Abeta25-35.

We examined the effects of co-incubating nine different Abeta peptide fragments with full-length Abeta1-40 (Abeta40) on protein aggregation. Six fragments enhanced aggregation of Abeta40 (Abeta1-28, 12-28, 17-28, 10-20, 25-35 and 17-40), while three others did not (Abeta1-11, 1-16, and 20-29). All of the peptides that enhanced aggregation contained either residues 17-20 or 30-35, indicating the importance of these regions for promoting aggregation of full-length Abeta. Abeta25-35 in particular increased both the rate and extent of aggregation of Abeta40 considerably as indicated by fluorescence staining. Atomic force microscope imaging (AFM) indicates the increase in fluorescence staining with Abeta25-35 is primarily due to increased formation of oligomers and protofibrils rather than formation of large amyloid fibrils. AFM images of Abeta25-35 when incubated alone also indicate formation of aggregates and long thin filaments. The increase in formation of the small toxic oligomeric morphology of Abeta40, along with formation of Abeta25-35 oligomers and thin filaments, represent two different potential pathways for Abeta25-35 toxicity. The critical roles of residues 17-20 and 30-35 of Abeta provide further insight into mechanism that underlie the formation of toxic aggregates in Alzheimer Disease (AD) and suggest targets for the design of beta-sheet breakers to modulate the aggregation and inhibit toxicity of full-length Abeta.

Residues 17-20 and 30-35 of beta-amyloid play critical roles in aggregation.

Single intracerebroventricular administration of amyloid-beta (25-35) peptide induces impairment in short-term rather than long-term memory in rats.

Decreases in cognitive functions, particularly long-term (episodic) and working memory, are among the earliest prognostic signs of Alzheimer’s disease. The toxicity of β-amyloid peptide is regarded as a major cause of neurodegeneration and cognitive impairment in this disease. The present report describes studies of the effects of intracerebroventricular administration of β-amyloid peptide (25–35) (Aβ(25–35)) on the reproduction of a previously assimilated habit consisting of finding food in an eight-arm radial maze in rats. Aβ (25–35) was given bilaterally at doses of 15 and 30 nmol/animal seven days after preliminary training. Testing was performed 60 days after peptide administration. The results showed that Aβ(25–35) impaired working memory in rats without having any significant effect on the retention of responses. We were unable to demonstrate any relationship between memory impairment and the dose of peptide given. These data provide evidence of the ability of Aβ(25–35) to produce greater degradation of working memory function than long-term memory function.

Studies of the effects of fragment (25-35) of beta-amyloid peptide on the behavior of rats in a radial maze.

Studies of the mechanisms of development and methods of treatment of Alzheimer's disease (AD) are among the most relevant areas of contemporary biology and medicine. The major cognitive and neuropathological signs of AD include senile dementia, the formation of neurofibrillary tangles in neurons, the presence of deposits of/~-amyloid peptide in the form of amyloid \"plaques\" in the cerebral cortex and various subcortical structures (especially the hippocampus), and intensive neurodegenerative processes [6]. Neurodegenerative changes in AD primarily affect cholinergic neurons, especially in the region of the septohippocampal tract. The cholinergic deficit characteristic of AD is associated with disturbances in shortterm as well as in so-called \"working\" memory. Understanding of the mechanisms of development of AD is critically dependent on the production of an etiological model of the disease in animals, which would combine the cognitive and neuropathological signs. This is a complex task because of the multifactorial etiology of AD, including both genetic and nongenetic factors. Several different approaches to developing a model of AD in animals, especially rodents, have been addressed. First is the use of specific neurotoxins which predominantly kill cholinergic neurons [2]. This approach produces socalled analogous models, which model the behavioral and cholinergic deficit in animals, albeit without the characteristic pathomorphological picture of the course of AD. Second is a frequently used approach to modeling AD -creation of transgenic mice in which there is an additional copy of a gene (i.e., overexpression of the gene) encoding the protein precursor of amyloid peptide [4]. These animals usually show disturbances in spatial \"working\" memory, though the formation of plaques and neurofibrillary tangles has not been described in transgenic animals. These models can be used for studies of the mechanisms of development of AD and for searching for therapeutic substances for its treatmem. At the same time, transgenic animals cannot be used for developing prophylactic measures. The third approach consists of treating animals with fragments of amyloid peptide with the aim of triggering amyloidogenesis and cholinergic neurodegeneration. One study [3] showed that central administration of neurotoxic fragment (25-35) of B-amyloid peptide leads to Alzheimer's-like amnesia in mice, accompanied by neurodegeneration and cholinergic dysfunction. The aim of the present work was to study the behavior of rats given intracerebroventricular doses of fragment (25-35) of/3-amyloid peptide. Studies were carried out on adult male Wistar rats weighing 230-290 g, kept in an animal house in conditions of natural illumination in groups of five per cage, with free access to water and food. Rats were divided randomly into two groups: a sham-operated (SO, n = 13) group and an experimental group given amyloid peptide (AM, n = 14). Animals were anesthetized with ketamine HCI (150 mg/kg), placed in a stereotaxic apparatus, and the skin on the head was incised in the region of the sagittal suture. Rats were scalped and two openings were drilled in the skull bone at the following coordinates: 0.8 mm posterior to the bregma, 1.5 mm lateral to the sagittal suture, and 3.8 mm beneath the level of the skull bone. Animals of group AM were given 7.5 nmol of aqueous aggregated fragment (25-35) of/3-amyloid peptide (RBI, USA) at 1 #I/min into each ventricle. The syringe needle was left in situ for 3 min after injections. Peptide was aggregated as described in [3]. Animals of group SO received the same quantity of distilled water.

Effects of doses of fragment (25-35) of beta-amyloid peptide on behavior in rats.

In experiments with animals, a group that is injected with the vehicle in which a drug of interest is dissolved is often used as a control. However, even a single injection of a vehicle is a stressor, i.e., “treatment stress,” which may significantly affect some stress-sensitive indices. In the present study, we report some data on the effects of a single intraperitoneal injection of isotonic saline solution on the contents of corticosterone, nitric oxide metabolites, and oxidative capacity, as well as on the expression of proteins and mRNAs of proinflammatory cytokines in the blood and brain regions of rats within one day after the injection as compared to intact animals. At the early time points after the injection, corticosterone contents were substantially elevated in the blood and ventral hippocampus. The content of nitric oxide metabolites decreased in the blood and remained stably low within 2–24 h after the injection. The injection did not affect the contents of proinflammatory cytokines in the blood; however, early after the injection the expression of IL-1s mRNA decreased in the ventral hippocampus and frontal cortex, whereas 24 h after this treatment, the expression of TNF-a mRNA increased by a factor of 4 in the frontal cortex. Thus, a single injection of isotonic saline solution had a clear stress-producing effect, which was observed at the systemic level and in stress-sensitive brain regions. The strength of this stressful event was sufficient to activate the hypothalamus–pituitary–adrenal axis but not sufficient to induce a significant inflammatory response. The frontal cortex was most sensitive to this treatment; the alterations in the ventral hippocampus were less expressed, whereas the dorsal hippocampus was most stress resistant. Our data show that it is important to consider and thoroughly analyze the effects of “treatment stress” in experiments using injections of biologically active substances.

The stress effects of a single injection of isotonic saline solution: systemic (blood) and central (frontal cortex and dorsal and ventral hippocampus)

Most ischemic strokes are caused by the occlusion of the middle cerebral artery (MCAO), which results in focal brain lesions in different areas of the neocortex. Secondary damage develops in brain regions located out of the infarct area, including the hippocampus. Hippocampal lesion may lead to cognitive impairments and post-stroke depression. Here, we studied the time course of changes in the levels of corticosterone and proinflammatory cytokine interleukine-1β (IL-1β) in the blood and hippocampus of rats after transient focal brain ischemia. Activation of the hypothalamo–pituitary–adrenal axis, which causes a release of corticosterone into blood, was observed at the early stage after MCAO and was accompanied by the presence of the stress hormone in the hippocampi of both the ischemic and contralateral hemispheres. We show for the first time that this effect was observed only in the ventral hippocampus (VH) but not in the dorsal hippocampus (DH). MCAO induced accumulation of the proinflammatory cytokine IL-1β, which coexisted with the elevated level of corticosterone at the early and delayed stages after reperfusion and was also observed in the VH of both hemispheres. Our data show that the VH is more vulnerable to remote damage induced by MCAO compared to the DH and corticosteroid response and neuroinflammation may be detected in the VH of both ischemic and contralateral hemispheres.

Accumulation of corticosterone and interleukin-1β in the hippocampus after focal ischemic damage of the neocortex: Selective vulnerability of the ventral hippocampus

Yu. V. Moiseeva

Papers

Single intracerebroventricular administration of amyloid-beta (25-35) peptide induces impairment in short-term rather than long-term memory in rats.

Studies of the effects of fragment (25-35) of beta-amyloid peptide on the behavior of rats in a radial maze.

Effects of doses of fragment (25-35) of beta-amyloid peptide on behavior in rats.

The stress effects of a single injection of isotonic saline solution: systemic (blood) and central (frontal cortex and dorsal and ventral hippocampus)

Accumulation of corticosterone and interleukin-1β in the hippocampus after focal ischemic damage of the neocortex: Selective vulnerability of the ventral hippocampus