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1 : ) (Stress),,.,,,., stress, stress,,, 1-3). Stress,. stress,,,.,. Stressor. stress. ( ). 140 stress,,, stress., stress.. 4,5). stress,., 6). Stress (stressor),,,,., (palpita tion),

2 ,. stress,, 3..,,?. ( ). (mole cular mechanism).,, 7) ), ( ) (endorphin) (enkephalin) 8,9). (amyloid precursor protein, APP),..,.. 8,10).., ). [ (neurofibrillary. tangle), (neuritic plaque), (vascular amyloid)](fig. 1, 2). 12)...,, oli 14). gonucleotide cdna 6) ). pleated sheet amyloid, ). 4kd A4. 141

3 Fig. 1. Structure of amyloid precursor proteins(app695, APP751, APP770). Fig. 2. Pathology of alzheimer s disease. 142

4 Fig. 3. Formation of beta A4 protein. Overproduction of beta- APP could encourage the liberation of the amyloid beta- fragment. Ordinarily, only the enzymes that cut within the amyloid beta- region may react with beta- APP(left). When excess beta- APP appears, however, other enzymes that release the amyloid beta- fragment by cutting at its ends may also have the opportunity to act(right). This mechanism may occur in people with Down s syndrome, who carry an extra copy of the beta- APP gene. (APP695, APP714, APP751, APP770) (Fig. 2). APP protease APP A4 25,26), (Fig. 3). N stress (Swedish type) (Amyloid precursor protein ; APP) A4 (immobilization stress) 26).,. A4., 1) 30, 1, 2.. 2) (cerebral cortex), (cerebellum), (hippocampus),, (midbrain and thalamus), (corpus striatum). (brain stem) 143

5 mrna APP mrna. 3) APP mrna. ( ) 25-30g male C57BL/ cm tape 30 pooling, 5 group 15 1 cm acryl collection (20-25 ) g 10 ml denaturing solution [4M. stress 30, 1, 2,. cold plate Glowinski Iversen(1966) (cerebral cortex), (cerebellum), (hippocampus), (midbrain and thalamus), (corpus striatum) (brain stem) 6 3 group AGPC(acid guanidinium thio cyanate phenol chloroform) (Chomc zynski, 1987) total celluar RNA guanidinium thiocyanate, 25 mm Na-citrate(pH 7.0), 0.5% sarkosyl, 100 mm 2-mercaptoethanol] Potter-Elvjem homogenizer Fig. 4. Northern blot analysis showing the time course of APP mrna and 18s rrna expression in the cerebral cortex of the mouse brain after immobilization stress. The mice were treated with immobilization stress for the indicated times and total cellular RNA was isolated. Equal amoumts of RNA(20 g/lane) were analyzed on a Northern blot using P 32 labeled human APP770 cdna probes. 144

6 Fig. 5. Changes in APP mrna levels in the mouse cerebral cortex at different times after immobilization stress. the mice were treated with immobilization stress for the indicated times and total RNA were isolated. The levels of APP mrna and 18s rrna were determined by Northern analysis. Densitometric analysis of the autoradiograms exposed in the linear range of film density was made on a hoeffer densitometer. Results were expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats. * P 0.05 compared to non- immobilized control mice.. 1 ml 2M Na-acetate (ph 4.0) 0.1 ml, water saturated phenol 1 ml, chloroformisoamylalcohol (49 : 1) 0.2 ml, ,000 g 20 tube, isopropanol , 10,000 g 20 RNA pellet. RNA pellet 10, 50% formamide -20. Fig. 6. Changes in APP mrna levels in the mouse cerebellum at different times after immobilization stress. Conditions were the same as in figure legend 5. Total cellular RNA was isolated and equal amounts(20 g/lane) were analyzed for APP mrna and 18s rrna transcripts as described in figure legend 5. Results were expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats. RNA 30 50% formamide, 6.5% formal dehyde, denature. 6.6% formamide 1.2% agarose. 28s 18s ribosomal RNA capillary band EtBr, transfer nitrocellulose paper transfer 80, 2 baking. RNA filter prehybridization 2 incubation random primer labelling kit [a-32p] datp APP770 cdna probe

7 Fig. 7. Changes in APP mrna levels in the mouse hippocampus at different times after immobilization stress. Conditions were the same as in figure legend 5. Total cellular RNA was isolated and equal amounts(20 g/lane) were analyzed for APP mrna and 18s rrna transcripts as described in figure legend t. Results were expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats * P 0.05 compared to control. Fig. 8. Changes in APP mrna levels in the mouse midbrain and thalamus at different times after stress. Conditions were the same as in figure legend 5. Total cellular RNA was isolated and equal amounts(20 g/lane) were analyzed for APP mrna and 18s rrna transcripts as described in figure legend 5. Results were expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats. *P 0.05 compared to control. hybridization. hybridization filter 2, X-ray film(kodak XR) autoradiography. APP mrna band 18s rrna band. random primer labelling kit [a-32p] datp APP770 cdna probe 12 hybridization. hybridization filter 2, X-ray film(kodak XR) autoradiography. RNA 20 4% formaldehyde, 6 SSC, denature. 20 SSC, filtration manifold NC paper. 80, 2 baking. RNA filter prehybridization 2 incubation APP mrna band 18s rrna band Analysis of Variancre(ANOVA) Bonferroni t-. 146

8 Fig. 9. Changes in APP mrna levels in the mouse corpus striatum at different times after stress. Conditions were the same as in figure legend 5. Total cellular RNA was isolated and equal amounts(20 g/lane) were analyzed for APP mrna and 18s rrna transcripts as described in figure legend 5. Results weree expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats. Fig. 10. Changes in APP mrna levels in the mouse brain stem at different times after stress. Conditions were the same as in figure legend 5. Total cellular RNA was isolated and equal amounts(20 g/lane) were analyzed for APP mrna and 18s rrna transcripts as described in figure legend 5. Results were expressed as ratio of OD X mm(% of control). The values represented mean SD for 5 groups of 3 rats. *P 0.05 compared to control. ( ) C57BL/6 immobilization stress 30, 1, 2 (cerebral cortex), (cerebellum), (hippocampus), (midbrain & thalamus), (corpus striatum), (brain stem) RNA mrna 18s rrna 18s rrna APP mrna Northern blot (Fig. 4). ( ) APP mrna Nor thrn blot Image Analyzer (Fig. 5). 147

9 , stress,, ( ) APP mrna,, ). 1 Stress,, 2, (Fig. 6)., stress glucocorticoid, ( ) APP mrna 30 1, 2 catecholamine, energy stress 7-11) (Fig. 7). glucocorticoid stress hormone cytokine ( ) ( ) 30, 1, 2, cytokine,, stress stress APP mrna 12-15). (Fig. 9)., stress, processing. ( ) APP mrna 30 stress., stress, 1, 2 15). Stress (Fig. 9).,. ( ) APP mrna 30, 1 2 (Fig. 10). (immediate early gene), stress 16-20). stress Stress 1-3), Ca++, stress Stress. 148

10 Ca++, Ca++ (Ca++ load) 28-30)., stress glucocorticoid Ca ). glutamate NMDA Ca++, glutamate 28,29).,, 35-37). stress,. stress,,., (endogenous neurotoxin)., MPTP-, (, Alzheimer s disease : AD) A4 25,38-40). A4 amyloid plaque, neurofibrillary tangle, cerebrovascular amyloid deposition, A4 110kd-140kd (APP) 41,21-24). AD APP, AD APP A4 25,38)., APP., interleukin-1(il-1) heat shock APP 42-45), AD IL-1 cytokine, heat shock., heat shock, heat shock stress, stress protein 19,20,46)., stress APP, stress APP, stress, stress. APP APP,., AD stress, ( ) stress, APP,. APP, A4, A4. mrna. 149

11 APP mrna. (structural protein) (stress protein).. 1,, APP mrna. 2 APP mrna. APP mrna. 2 APP mrna (adaptation).,..,..,. APP 26,38). APP. (protease) APP A4 (Fig. 3). Down... APP mrna. APP mrna

12 30, 1 2.,,,,,, RNA Nothern blotting., mrna Kap Sung Oh, M.D. 4, Woong Choi, M.D. 1 Seong Hun Kim, M.D., Ph.D. 1, Sung Jin Jeong, M.S. 1 Young Hae Chong, Ph.D. 1, Young Jae Lee, Ph.D 1.. Sang Hyung Lee, M.D., Ph.D. 1, Hye Sun Kim, Ph.D. 1, Sung Su Kim, Ph.D. 1, Cheol Hyoung Park, M.S. 1., Chin Whan Kim, M.D. 3 and Yoo-Hun Suh, M.D. 1.,. 151 = Abstract = Effect of Immobilization Stress on the Expression of Amyloid Precursor Protein in the Mouse Brain Department of Pharmacol, College of Medicine, Seoul National University 1, Department of Neurosugery, College of Medicine, Seoul National University 2, Department of Plasticsugery, College of Medicine, Seoul National University 3, Department of Plasticsugery, College of Medicine, Inju University Background : The Central nervous system(cns) plays a essential role in mediating stress responses. However, the exact mechanism of the CNS in mediating stress responses has not been clarified sufficiently as yet. Stress may cause brain dysfunction including cognitive dysfunction which was most commonly found in Alzheimer s dementia. Amyloid precursor protein(app) is a large, ubiquitously distributed and evolutionarily conserved molecule whose funtion remains unknown. Although the precise function of APP following injury to the CNS such as stab and kainic acid lesion. However, there have not been reports on the effects of stress on the expression of amyloid precursor protein in the brain. This study was under taken to elucidate the effects of stress on the expres sion of APP in the mouse brain. Methods : The several brain region was isolated from the mouse that was in the immoblization stress for 30 min, 1 hour, and 2 hours. The mouse brain was divided into 5 regions, cerebrsl cortex, cerebel-

13 lum hippocampus, midbrain and thalamus, corpus striatum and brain stem. The change of mrna was examined in the several brain regions using Northern blot hybridization. Results : The amounts of APP mrna in the cerebral cortex, hippocampus and brain stem were found to be significantly increased after stress for 30 minutes and to reach a maximum after stress for 1 hour and to normal range at stress for 2 hours. On the contray, the contents of APP mrna in midbrain and thalamus were decreased after stress for 30 minutes and sustained after stress for 2 hours. Conclusion : These findings suggest that APP may not be static but functional protein reactive to stress and stress may increase the levels of APP mrna especially in Alzheimer disease associated sites such as cerebral cortex and hippocampus, which may contriute to the pathogenesis of Alzheimer disease. Key Words : Alzheimer amyloid precursor protein, immobilization stress, mouse brain regions. REFERENCES 1) Burchfield SR: The stress response; A new pers pective Psychosom. Med 41: , ) Henry JP: Present concept of stress theory. In Usdin E, Kvetnansky R and Axelrod J(eds) "Catecholamines and stress: Resent Advances". New York: Pergamon , ) Levine S: Definition of stress? In: Noberg GP (eds). "Animal stress". Bethesda Maryland: American Physiological Society 51-69, ) Elliott GR, Eisdorfer C: Stress and Human health. New York: Springer 1-20, ) Gatcherl RJ, Baum A: An inrtoduction to health psychology. Reading. Massachusetts: Addison- Wiley 10-20, ) Vogel WH: Coping, stress, stressors and health consequence. Neuropsychobiology 13: , ) McCarty R, Horvatt K, Konarska M: Chronic stress and sympathetid-adrenal medullary responsiveness. Soc. Sci. Med 26: , ) Van Loon GR, Kvetnansky R, McCarty R Axelrod J, Editors: "Stress: Neurochemical and humoral mechanism". New York: Gordon and Breach , ) Henry JP: Biological basis of the stress response. Integr. Physiol. Behav. Sci 27:66-83, ) Usdin E, Kvetnansky R, Axelrod J, Editors "Stress: The role of catecholamines and other neurotransmitters". New York: Gordon and Breach , ) Kavelaars A, Bellieux RE, Heijnen CJ: In vitro beta-adrenergic stimulation of lymphoytes indu ces the release of immunoreactive beta-endor phin. Endocrinol. 126: , ) Okuse, Anzai T: Psychosomatic aspects of stress. Rinsho-Byori. 40: , ) Yokoyama MM: Neuro-immunomodulation. Nippon-Rinsho 50: , ) Dohms JE, Metz A: Stress, Mechanisms o immunosupression. Vet. Immunol. Immunopathol 30:89-109, ) Irwin MR, Hauger RL: Adaptation to chronic stress. Temporal pattern of immune and neuro endocrine coeelates, neuropsychopharmacol 1: , ) Smith MA, Banerjee S, Gold PW, Glowa J Induction of c-fos mrnas in rat brain by condi rioned and unconditioned stressors. Brein Res 578: , ) Imaki T, Shibasaki T, Motta M, Demura H Early induction of c-fos precedes increased ex pression of corticotropin-releasing factor mrnas in paraventricular nucleus after immobilization stress. Endocrinol 131: , ) Schreiber SS, Tocco G, Shors TJ, Thompson FR: Activation of immediated early genes after acute stress. Neuroreport 2:17-20, ) Nowak TS Jr(1990): Protein synthesis and the heat shock/stress response after ischemia. Cere brovasc. Bruin. Metab. Rev 2: , ) Dragnow M, Currie RW, Robertson HA, Faul RL: Heat shock induces c-fos protein-like immunoreactivity in glial cells in adult rat brain. Exp. Neurol 106: ,

14 21) Tanzi R, McClatchey AI, Lamperti ED, Villa- Komaroff L, Gusella JF, Neve RL: Protease inhibitor domain encoded by an amyloid protein precursor mrna associated with Alzheimer s disease. Nature 331: , ) Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Milthaup G Beyreuther K, Muller-Hill B: The precursor o Alzhermer s disease amyloid A4 protein resem bles cell surface receptor. Nature. 325: ) Robakis NK, Ramakrishna N, Wolfe G, Wisnie wski HM: Molecular cloning and characteri zation of a cdna encoding the cerebrovascular and the neurite plaque amyloid peptides. Pro Natl. Acad. Sci. USA 84: , ) Goldgaber D, Lerman MI, McBride W, Saffiott U, Gajdusek DC: Characterization and chromo somal localization of a cdna encoding brain amyloid Alzheimer s disease. Science 235: , ) Selkoe DJ: Deciphering Alzheimer s disease: the amyloid precursor protein yields new clues. Science 248: , ) Selkoe DJ: Amyloid protein and Alzheimer s disease. Scientific American. November 68-72, ) Nakajima T, Davel JL, Gleiter CH, Deckert J Post RM, Marangos PJ: C-fos mrna expression following electrical-induced seizure and acute nociceptive stress in mouse brain. Epilepsy Res 4: , ) Choi D W: The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Ann. Rev. Neurosci 13: , ) Nemoto EM: Brain ischemia, Lajtha A(ed), Alterations of metabolites in the nervous system (Handbook of Neurochemistry, vol 9) New York Plenum , ) Rothman SM, Olney JW: Glutemate and the pathphysiology of hypoxic-ischemic brain da mage. Ann. Neurol 19: , ) De KLoet ER, Reul JM, Sutanto W: Corticos teroid and brain. J. Steroid Biochem. Mol. Biol 34: , ) Reul JM, Sutanto W, van Elkelen JA, Rothuizen J, de Kloet ER: Central action of adrenal steroids during stress and adaptation. Adv. Exp. Med. Biol 274: , ) Tombaugh GC, Yang SH, Swanson RA, Sapoi sky RM: Glucocoticoid exacerbate hypoxic and hypoglycemic hippocampal injury in vitro: Bio chemical correlates and a role for astrocytes. J Neurochem 59: , ) Elloit EM, Sapolsky RM: Cortocosterone en hances kainic acid-induced calcium elevation in cultured hippocampal neurons. Neurochem 59: , ) Flamm ES, Harry B, Demoplulous HB, et al Free radicals in cerebral ischemia Stroke 7: , ) Hall ED, Braughler JM: Central nervous system trauma and stroke. Physiological and phar macological evidence for involvement of oxygen redicals and lipid peroxidation. Free Radic Biol. Med 6: , ) Guliaeva NV: Inhibition of free-radical lipid oxidation in the mechanism of immediate and long-term adaptation to stress. Biol. Nauki 4:5-14, ) Selkoe DJ: Aging, amyloid and Alzheimer s disease. N. Engl. J. Med. 1989, 320: : the amyloid precursor protein yields new clues Science 248: , ) Yankner BA, Dawes LR, Fischer S, Villa- Komaroff L, Oster-Granite ML, Neve RL: Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer s disease. Science 245: , ) Kowall NW, Beal MF, Busciglio J, Duff LK Yankner BA: An in vivo model for the neurode generative effects of beta amyloid akd protection by substance P. Proc. Natl. Acad. Sci. USA , ) Glenner GG, Wong CW: Initial report of the purification and characterization of a novel cerebrovascular amyloid protion. Biochem. Biopphys. Res. Commun 120: , ) Altstiel LD, Sperber K: Cytokine and Alzhei mer s disease Prog. Neuro. Psycholpharmacol. Biol. Psychiat 15: , ) Dickson DW, Lee SC, Mattiace LA, Yen SH 153

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