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Increased β-amyloid precursor protein mRNA in the rat cerebral cortex and hippocampus after chronic systemic atropine treatment
Neuroscience
Letters 2 10
(1996)13-16
Increased /3-amyloid precursor protein mRNA in the rat cerebral cortex and hippocampus after chronic systemic atropine treatment Thomas G. Beach%*, Douglas G. Walkerb, Max S. Cynaderc, Linda H. Hughesa “UniversiQ qf British Columbia, Vancouver Hospital and Health Sciences Centre, Department ofPathology, 855 West 12th Avenue, Vancouv(r, B.C. V5Z IM9, Canada bUniver.~ity#British Columbia, Vancouver Hospital and Health Sciences Centre, Department qf Psychiatry, Vancouver, B.C., Canada ‘University of British Columbia, Vancouver Hospital and Health Sciences Centre. Department of Ophthalmology. Vancouver, B.C., Canada Received
30 October
1995; revised version received 17 April 1996; accepted 17 April 1996
Abstract
Rats were treated with once-daily subcutaneous injections of atropine or normal saline for 10 days. Cryostat sections of fresh-frozen brain were subjected to quantitative muscarinic receptor ([3H]quinuc1idinylbenzilate (QNB)) binding autoradiography, and quantitative in-situ hybridization autoradiography for B-amyloid precursor protein @-APP) mRNA using an oligonucleotide probe recognizing all major isoforms. QNB binding in the atropine-treated group was increased 6-7% in the areas measured (dentate gyrus, CAl, and cerebral cortex), confirming that the treatment was effective in inducing muscarinic receptor upregulation. Hybridization signal for /3-APP mRNA was increased 15520% in the atropine-treated group in the same regions. As chronic atropine treatment models the muscarinic effects of cholinergic denervation, these results suggest that age-related cholinergic neuron loss may result in upregulation ofa-APP. Keywords:
Alzheimer’s
Deposition
disease; Aging brain; /3-Amyloid precursor protein; Cholinergic
of P-amyloid
(A/?) is a pathological
feature
(AD) arising from diverse etiologies. In Down’s syndrome, 48 deposition is thought to be secondary to overproduction of the /?amyloid precursor protein @I-APP) [2]. Mutations in or about the w-coding region of the /3-APP gene cosegregate with the disease in a number of families [7]. Although unproven as yet, there is evidence that other genotypes known to be associated with AD, including mutations on chromosome 1 and 14 [8,13] and the apolipoprotein E-s4 allele [ 161, also may act by affecting /3APP metabolism. The fact that A/3 deposition is almost universal in the aging human brain [5] and is a common feature of aging in other mammalian species [3], suggests, however, that an individual’s genotype merely influences the severity of an underlying process which is part of normal aging. The mechanism of this extremely common form of A@ deposition is unknown. We propose that @ is accumulated in normal aging as a result of age-related cholinergic terminal loss [12] causing common
to Alzheimer’s
* Corresponding
disease
author. Tel.: +I 604 8754381;
fax: +l 604 8754025.
receptor; Animal model
altered regulation of P-APP metabolism. Support for this hypothesis comes from in vitro experiments which have demonstrated that #?-APP production and processing can be regulated by cholinergic muscarinic receptor ligands [ 1 I] and from animal studies showing that lesions of the cholinergic basal forebrain result in upregulation of /?-APP in the cerebral cortex [ 181. In this experiment, we sought to determine whether chronic systemic administration of atropine, which would mimic the muscarinic effects of cholinergic denervation, would increase /3-APP mRNA content in the cerebral cortex and hippocampus. Atropine sulfate (or normal saline for controls, n = 6) was given by S.C. injection once daily for 10 days at a dose of 20 mg/kg (n = 7). The animals were sacrificed by thiopental overdose 24 h after the last injection and the brains dissected out and frozen on dry ice. To assess the efficacy of atropine treatment, [3H]quinuclidinylbenzilate (QNB) film autoradiography was performed as previously described [ 141; chronic atropine treatment is known to upregulate muscarinic receptors in the rat cerebral cortex [ 171. Cryostat sections (15 pm) were taken through the cerebral hemispheres to include parietal
0 1996 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/96/$12.00 PII: SO304-3940(96)12650-X
14
T.G. Beach et al. /Neuroscience
cortex and hippocampus at the level of the diencephalicmidbrain junction. Sections were incubated in the presence of 5 nM [3H]QNB for 60 min at 20°C; control sections were treated identically with the additional presence of cold ( 10T4 M) atropine. After brief washes in buffer and drying in a stream of cool air, dried sections were apposed to film sheets (Hyperfilm-3H, Amersham) for 3 weeks. Specific optical density was measured using image analysis of the developed film in two sections from each animal. Sections were coded so that the operator was blinded with respect to treatment category. Within each section, two sites were sampled in the dentate gyrus molecular layer (ML) and CA1 of the hippocampus; three sites were sampled in the parietal cortex (CX). The sampling sites were chosen randomly within the area to be sampled; each site was sampled within a rectangular box created on the computer monitor. The long axis of the box was coextensive with the depth of the region sampled (e.g. in the cortex the long axis of the sampling box extended from pia to white matter). Final optical density values for each animal represent the mean of four (ML, CAl) or six (CX) measurements from two sections, subtracted from the corresponding mean values obtained from control sections. These values were then related to radioactivity content using 3H brain paste standards (Amersham). /3-APP mRNA was quantified by in-situ hybridization film autoradiography, using the method of Dagerlind et al. [4]. A 33-base synthetic oligonucleotide probe was prepared corresponding to bases 168-201 of the rat P-APP sequence [15]. This sequence is N-terminal to the KPIspecifying region and would be expected to hybridize with all major p-APP mRNA isoforms. The probes were prepared by end-labelling with [35S]dATP using terminal transferase and purified by passing through Sephadex G- 10 gel filtration columns. Fresh-frozen cryostat sections were incubated overnight at 42°C with the labelled probes in hybridization buffer containing 200 nM D?T, washed with four changes of 1 X SSC at 50°C left to cool at room temperature for 30 min and then quickly dehydrated in alcohols and air-dried overnight. Specificity control sections were incubated under the same conditions using the sense probe for P-APP 168-201, or with a 30-base antisense sequence of the tyrosine hydroxylase (TH) gene (little or no TH mRNA is present in the cerebral cortex or hippocampus). Sections were apposed to film (Hyperfilm-Betamax, Amersham) for 12 days at 4°C. Specific optical density, defined as the difference of light transmission through film exposed to sections incubated with sense or antisense probes, was sampled, measured and quantified as detailed above for QNB. Analysis of the QNB autoradiograms showed that the atropine treatment was effective in producing the expected upregulation of muscarinic receptors. Muscarinic receptor binding was increased 6-7% in atropine-treated animals (Fig. 1). This difference was statistically significant (unpaired, two-tailed t-tests) in the molecular layer of the den-
Letrers 210 (1996) 13-16
QNB Receptor-Binding Autoradiography * *
n
CA1
ML Atropine
0
cx Saline
Fig. 1. QNB receptor binding autoradiograph (top) of rat cerebral hemisphere; the distribution of binding is as reported previously by others [14]. Data from quantification of binding by film optical density measurements are shown in graphic form (bottom). There was a significant (*) increase of binding (r-tests, P < 0.05) in the dentate gyms ML and cx.
tate gyrus and in the cerebral cortex; the increase in area CA1 failed to reach the significance (P < 0.05) level. There was no apparent binding in the control sections. In-situ hybridization signal for p-APP was increased 1520% in the atropine-treated group relative to saline-treated animals (Fig. 2). The difference was significant (unpaired, two-tailed t-tests) in the granular cell layer (GCL) of the dentate gyrus and in the pyramidal cell layer of area CAl. The increase in cerebral cortex was not statistically signifi-
T.G. Beach et al. /Neuroscience
Letters 210 (1996) 13-16
15
effect on GAP-43 mRNA levels [9]. It should be noted that these results do not indicate whether the increase in j?-APP mRNA is due to increased transcription or decreased molecular turnover, or whether it is associated with a change in P-APP protein concentration. It is not possible to exclude that the observed effects of atropine on B-APP mRNA are mediated indirectly, as, for example, through effects of an-opine on other neurotransmitter systems. Atropine increases the spontaneous release of acetylcholine in cortical slices [lo]. Increased acetylcholine generated in this way may result in increased nicotinic receptor activation, as these receptors would not be blocked by atropine. Nicotinic receptor activation increases the release of aspartate and GABA in slice preparations [l]. Nicotine and acetylcholine may also increase norepinephrine release, possibly by a direct effect on locus coeruleus neurons [6]. The results of this experiment are relevant both for theories of AD pathogenesis and rationales for AD pharmacotherapy. Since chronic atropine treatment models the muscarinic effects of cholinergic denervation, declining cholinergic activity in the aging human cerebral cortex and hippocampus [ 121 may be accompanied by upregulation of B-APP. Although it is not known for certain whether simple upregulation of p-APP can lead to A/3 deposition, it is thought that this is the mechanism of A/? accumulation in Down’s syndrome [2]. If systemic administration of a muscarinic antagonist can upregulate #%APP, muscarinic agonists or other cholinergic enhancers might be used to downregulate /?-APP. This would constitute a new rationale for cholinergic pharmacotherapy in Alzheimer’s disease.
p-APP mRNA In-Situ Hybridization Autoradiography
The authors would like to express their thanks to Ms. Virginia Booth for technical assistance, Dr. W.-G. Hse for helpful advice, and Drs. P.L. and E.G. McGeer for the use of their laboratory facilities. This work was supported by the Alzheimer Society of British Columbia. CA1
GCL atropim
0
cx saline
Fig, 2. p-APP mRNA in-situ hybridization receptor binding autoradiograph (top) of rat cerebral hemisphere; the distribution is as previously reported [15]. Data from quantification of hybridization signal are shown (bottom). There was a significant (*) increase of signal (PC 0.05) in the dentate gyrus GCL and CA 1. The increase seen in CX did not reach the significance level.
cant. There was no significant hybridization signal over the control sections. These results suggest that a systemically-administered muscarinic receptor ligand can affect in vivo metabolism of p-APP. Specifically, blockade of muscarinic receptors elevates cortical P-APP mRNA levels in the intact animal. That this upregulation is not due simply to a general increase in metabolism is supported by a previous study showing that a similar atropine treatment regimen had no
111 Beani, L., Bianchi,
C., Ferraro, L., Nilsson, L., Nordberg, A., Romanelli, L., Spalluto, P., Sundwall, A. and Tanganelli, S., Effect of nicotine on the release of acetylcholine and amino acids in the brain. In A. Nordberg, K. Fuxe, B. Holmstedt and A. Sundwall (Eds.), Progress in Brain Research, Vol. 79, Elsevier, Amsterdam, 1989, pp. 149-155. I21 Beyreuther, K., Pollwein, P., Multhaup, G., Monning, U., Konig, G., Dyrks, T., Schubert, W. and Masters, CL., Regulation and expression of the Alzheimer’s /?/A4 amyloid protein precursor in health, disease and Down’s syndrome, Ann. N. Y. Acad. Sci., 695 (1993) 91-102. [31 Cummings, B.J., Su, J.H., Cotman, C.W., White, R. and Russell, M.J., /I-Amyloid accumulation in aged canine brain: a model of early plaque formation in Alzheimer’s disease, Neurobiol. Aging, 14 (1993) 547-560. 141 Dagerlind, A., Friberg, K., Bean, A.J. and Hokfelt, T., Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry, Histochemistry, 98 (1992) 39-49. r51 Davies, L., Wolska, B., Hilbich, C., Multhaup, G., Martins, R., Simms, G., Beyreuther, K. and Masters, CL., A4 amyloid protein
16
T.G. Beach et al. /Neuroscience
deposition and the diagnosis of Alzheimer’s disease: prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques, Neurology, 38 (1988) 1688-1693. @I Egan, T.M. and North, R.A., Actions of acetylcholine and nicotine on rat locus coeruleus neurons in vitro, Neuroscience, 19 (1986) 565-571. M.-C., Mullan, M., Brown, J., Craw171 Goate, A., Chattier-Harlin. ford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P.. Talbot, C., Pericak Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M. and Hardy, J., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease, Nature, 349 (1991) 704-706. PI Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D.M., Oshima, J., Pettingell, W.H., Yu, C-en, Jondro, P.D., Schmidt, SD., Wang, K., Crowley, A.C., Fu, Y.-H., Guenette, S.Y., Galas, D., Nemens, E, Wijsman, E.M., Bird, T.D., Schellenberg, G.D. and Tanzi, R.E., Candidate gene for the chromosome I familial Alzheimer’s disease locus, Science, 269 (1995) 973-977. 191 McKinney, M. and Robbins, M., Chronic atropine administration up-regulates rat cortical muscarinic ml receptor molecules: assessment with the RTIPCR, Mol. Brain Res., 12 (1992) 3945. [lOI Molenaar, P.C. and Polak, R.L., Stimulation by atropine of acetylcholine release and synthesis in cortical slices from rat brain, Br. J. Pharmacol., 40 (1970) 406-417. Ull Nitsch, R.M., Slack, B.E., Wurtman, R.J. and Growdon, J.H., Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors, Science, 258 (1992) 304-307.
Letters 210 (1996) 13-16 Perry, E.K., Court, J.A., Piggott, M.A., and Perry, R.H. Cholinergic component of dementia and aging. In F.A. Huppert, C. Brayne and D.W. O’Connor (Eds.), Dementia and Normal Aging, Cambridge University Press, Cambridge, 1994, pp. 437-469. Scheuner, D., Bird, T., Citron, M., Lannfelt, L., Schellenberg, G., Selkoe, D., Viitanen, M. and Younkin, S.G., Fibroblasts from carriers of familial AD linked to chromosome 14 show increased A#I production, Sot. Neurosci. Abstr., 21 (1995) 1500. Shaw, C., Needler, M.C. and Cynader, M., Ontogenesis of muscarinic acetylcholine binding sites in cat visual cortex: reversal of specific laminar distribution during the critical period, Dev. Brain Res., 14 (1984) 295-300. Shivers, B.D., Hilbich, C., Multhaup, G., Salbaum, M., Beyreuther, K. and Seeburg, P.H., Alzheimer’s disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact, EMBO J., 7 (1988) 1365-1370. Strittmatter, W.J., Saunders, A.M., Schmechel, D., Petit&-Vance, M., Enghild, J., Salvesen, G.S. and Roses, A.D., Apolipoprotein E: high avidity binding to #I-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. USA, 90 (1993) 1977-1981. Wall, S.J., Yasuda, R.P., Li, M., Ciesla, W. and Wolfe, B.B., Differential regulation of subtypes ml-m5 of muscarinic receptors in forebrain by chronic atropine administration, J. Pharmacol. Exp. Ther., 262 (1992) 584-588. Wallace, W., Ahlers, S.T., Gotlib, J., Bragin, V., Sugar, J., Gluck, R., Shea, P.A., Davis, K.L. and Haroutunian, V., Amyloid precursor protein in the cerebral cortex is rapidly and persistently induced by loss of subcortical innervation, Proc. Natl. Acad. Sci. USA, 90 (1993) 8712-8716.
Letters 2 10
(1996)13-16
Increased /3-amyloid precursor protein mRNA in the rat cerebral cortex and hippocampus after chronic systemic atropine treatment Thomas G. Beach%*, Douglas G. Walkerb, Max S. Cynaderc, Linda H. Hughesa “UniversiQ qf British Columbia, Vancouver Hospital and Health Sciences Centre, Department ofPathology, 855 West 12th Avenue, Vancouv(r, B.C. V5Z IM9, Canada bUniver.~ity#British Columbia, Vancouver Hospital and Health Sciences Centre, Department qf Psychiatry, Vancouver, B.C., Canada ‘University of British Columbia, Vancouver Hospital and Health Sciences Centre. Department of Ophthalmology. Vancouver, B.C., Canada Received
30 October
1995; revised version received 17 April 1996; accepted 17 April 1996
Abstract
Rats were treated with once-daily subcutaneous injections of atropine or normal saline for 10 days. Cryostat sections of fresh-frozen brain were subjected to quantitative muscarinic receptor ([3H]quinuc1idinylbenzilate (QNB)) binding autoradiography, and quantitative in-situ hybridization autoradiography for B-amyloid precursor protein @-APP) mRNA using an oligonucleotide probe recognizing all major isoforms. QNB binding in the atropine-treated group was increased 6-7% in the areas measured (dentate gyrus, CAl, and cerebral cortex), confirming that the treatment was effective in inducing muscarinic receptor upregulation. Hybridization signal for /3-APP mRNA was increased 15520% in the atropine-treated group in the same regions. As chronic atropine treatment models the muscarinic effects of cholinergic denervation, these results suggest that age-related cholinergic neuron loss may result in upregulation ofa-APP. Keywords:
Alzheimer’s
Deposition
disease; Aging brain; /3-Amyloid precursor protein; Cholinergic
of P-amyloid
(A/?) is a pathological
feature
(AD) arising from diverse etiologies. In Down’s syndrome, 48 deposition is thought to be secondary to overproduction of the /?amyloid precursor protein @I-APP) [2]. Mutations in or about the w-coding region of the /3-APP gene cosegregate with the disease in a number of families [7]. Although unproven as yet, there is evidence that other genotypes known to be associated with AD, including mutations on chromosome 1 and 14 [8,13] and the apolipoprotein E-s4 allele [ 161, also may act by affecting /3APP metabolism. The fact that A/3 deposition is almost universal in the aging human brain [5] and is a common feature of aging in other mammalian species [3], suggests, however, that an individual’s genotype merely influences the severity of an underlying process which is part of normal aging. The mechanism of this extremely common form of A@ deposition is unknown. We propose that @ is accumulated in normal aging as a result of age-related cholinergic terminal loss [12] causing common
to Alzheimer’s
* Corresponding
disease
author. Tel.: +I 604 8754381;
fax: +l 604 8754025.
receptor; Animal model
altered regulation of P-APP metabolism. Support for this hypothesis comes from in vitro experiments which have demonstrated that #?-APP production and processing can be regulated by cholinergic muscarinic receptor ligands [ 1 I] and from animal studies showing that lesions of the cholinergic basal forebrain result in upregulation of /?-APP in the cerebral cortex [ 181. In this experiment, we sought to determine whether chronic systemic administration of atropine, which would mimic the muscarinic effects of cholinergic denervation, would increase /3-APP mRNA content in the cerebral cortex and hippocampus. Atropine sulfate (or normal saline for controls, n = 6) was given by S.C. injection once daily for 10 days at a dose of 20 mg/kg (n = 7). The animals were sacrificed by thiopental overdose 24 h after the last injection and the brains dissected out and frozen on dry ice. To assess the efficacy of atropine treatment, [3H]quinuclidinylbenzilate (QNB) film autoradiography was performed as previously described [ 141; chronic atropine treatment is known to upregulate muscarinic receptors in the rat cerebral cortex [ 171. Cryostat sections (15 pm) were taken through the cerebral hemispheres to include parietal
0 1996 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/96/$12.00 PII: SO304-3940(96)12650-X
14
T.G. Beach et al. /Neuroscience
cortex and hippocampus at the level of the diencephalicmidbrain junction. Sections were incubated in the presence of 5 nM [3H]QNB for 60 min at 20°C; control sections were treated identically with the additional presence of cold ( 10T4 M) atropine. After brief washes in buffer and drying in a stream of cool air, dried sections were apposed to film sheets (Hyperfilm-3H, Amersham) for 3 weeks. Specific optical density was measured using image analysis of the developed film in two sections from each animal. Sections were coded so that the operator was blinded with respect to treatment category. Within each section, two sites were sampled in the dentate gyrus molecular layer (ML) and CA1 of the hippocampus; three sites were sampled in the parietal cortex (CX). The sampling sites were chosen randomly within the area to be sampled; each site was sampled within a rectangular box created on the computer monitor. The long axis of the box was coextensive with the depth of the region sampled (e.g. in the cortex the long axis of the sampling box extended from pia to white matter). Final optical density values for each animal represent the mean of four (ML, CAl) or six (CX) measurements from two sections, subtracted from the corresponding mean values obtained from control sections. These values were then related to radioactivity content using 3H brain paste standards (Amersham). /3-APP mRNA was quantified by in-situ hybridization film autoradiography, using the method of Dagerlind et al. [4]. A 33-base synthetic oligonucleotide probe was prepared corresponding to bases 168-201 of the rat P-APP sequence [15]. This sequence is N-terminal to the KPIspecifying region and would be expected to hybridize with all major p-APP mRNA isoforms. The probes were prepared by end-labelling with [35S]dATP using terminal transferase and purified by passing through Sephadex G- 10 gel filtration columns. Fresh-frozen cryostat sections were incubated overnight at 42°C with the labelled probes in hybridization buffer containing 200 nM D?T, washed with four changes of 1 X SSC at 50°C left to cool at room temperature for 30 min and then quickly dehydrated in alcohols and air-dried overnight. Specificity control sections were incubated under the same conditions using the sense probe for P-APP 168-201, or with a 30-base antisense sequence of the tyrosine hydroxylase (TH) gene (little or no TH mRNA is present in the cerebral cortex or hippocampus). Sections were apposed to film (Hyperfilm-Betamax, Amersham) for 12 days at 4°C. Specific optical density, defined as the difference of light transmission through film exposed to sections incubated with sense or antisense probes, was sampled, measured and quantified as detailed above for QNB. Analysis of the QNB autoradiograms showed that the atropine treatment was effective in producing the expected upregulation of muscarinic receptors. Muscarinic receptor binding was increased 6-7% in atropine-treated animals (Fig. 1). This difference was statistically significant (unpaired, two-tailed t-tests) in the molecular layer of the den-
Letrers 210 (1996) 13-16
QNB Receptor-Binding Autoradiography * *
n
CA1
ML Atropine
0
cx Saline
Fig. 1. QNB receptor binding autoradiograph (top) of rat cerebral hemisphere; the distribution of binding is as reported previously by others [14]. Data from quantification of binding by film optical density measurements are shown in graphic form (bottom). There was a significant (*) increase of binding (r-tests, P < 0.05) in the dentate gyms ML and cx.
tate gyrus and in the cerebral cortex; the increase in area CA1 failed to reach the significance (P < 0.05) level. There was no apparent binding in the control sections. In-situ hybridization signal for p-APP was increased 1520% in the atropine-treated group relative to saline-treated animals (Fig. 2). The difference was significant (unpaired, two-tailed t-tests) in the granular cell layer (GCL) of the dentate gyrus and in the pyramidal cell layer of area CAl. The increase in cerebral cortex was not statistically signifi-
T.G. Beach et al. /Neuroscience
Letters 210 (1996) 13-16
15
effect on GAP-43 mRNA levels [9]. It should be noted that these results do not indicate whether the increase in j?-APP mRNA is due to increased transcription or decreased molecular turnover, or whether it is associated with a change in P-APP protein concentration. It is not possible to exclude that the observed effects of atropine on B-APP mRNA are mediated indirectly, as, for example, through effects of an-opine on other neurotransmitter systems. Atropine increases the spontaneous release of acetylcholine in cortical slices [lo]. Increased acetylcholine generated in this way may result in increased nicotinic receptor activation, as these receptors would not be blocked by atropine. Nicotinic receptor activation increases the release of aspartate and GABA in slice preparations [l]. Nicotine and acetylcholine may also increase norepinephrine release, possibly by a direct effect on locus coeruleus neurons [6]. The results of this experiment are relevant both for theories of AD pathogenesis and rationales for AD pharmacotherapy. Since chronic atropine treatment models the muscarinic effects of cholinergic denervation, declining cholinergic activity in the aging human cerebral cortex and hippocampus [ 121 may be accompanied by upregulation of B-APP. Although it is not known for certain whether simple upregulation of p-APP can lead to A/3 deposition, it is thought that this is the mechanism of A/? accumulation in Down’s syndrome [2]. If systemic administration of a muscarinic antagonist can upregulate #%APP, muscarinic agonists or other cholinergic enhancers might be used to downregulate /?-APP. This would constitute a new rationale for cholinergic pharmacotherapy in Alzheimer’s disease.
p-APP mRNA In-Situ Hybridization Autoradiography
The authors would like to express their thanks to Ms. Virginia Booth for technical assistance, Dr. W.-G. Hse for helpful advice, and Drs. P.L. and E.G. McGeer for the use of their laboratory facilities. This work was supported by the Alzheimer Society of British Columbia. CA1
GCL atropim
0
cx saline
Fig, 2. p-APP mRNA in-situ hybridization receptor binding autoradiograph (top) of rat cerebral hemisphere; the distribution is as previously reported [15]. Data from quantification of hybridization signal are shown (bottom). There was a significant (*) increase of signal (PC 0.05) in the dentate gyrus GCL and CA 1. The increase seen in CX did not reach the significance level.
cant. There was no significant hybridization signal over the control sections. These results suggest that a systemically-administered muscarinic receptor ligand can affect in vivo metabolism of p-APP. Specifically, blockade of muscarinic receptors elevates cortical P-APP mRNA levels in the intact animal. That this upregulation is not due simply to a general increase in metabolism is supported by a previous study showing that a similar atropine treatment regimen had no
111 Beani, L., Bianchi,
C., Ferraro, L., Nilsson, L., Nordberg, A., Romanelli, L., Spalluto, P., Sundwall, A. and Tanganelli, S., Effect of nicotine on the release of acetylcholine and amino acids in the brain. In A. Nordberg, K. Fuxe, B. Holmstedt and A. Sundwall (Eds.), Progress in Brain Research, Vol. 79, Elsevier, Amsterdam, 1989, pp. 149-155. I21 Beyreuther, K., Pollwein, P., Multhaup, G., Monning, U., Konig, G., Dyrks, T., Schubert, W. and Masters, CL., Regulation and expression of the Alzheimer’s /?/A4 amyloid protein precursor in health, disease and Down’s syndrome, Ann. N. Y. Acad. Sci., 695 (1993) 91-102. [31 Cummings, B.J., Su, J.H., Cotman, C.W., White, R. and Russell, M.J., /I-Amyloid accumulation in aged canine brain: a model of early plaque formation in Alzheimer’s disease, Neurobiol. Aging, 14 (1993) 547-560. 141 Dagerlind, A., Friberg, K., Bean, A.J. and Hokfelt, T., Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry, Histochemistry, 98 (1992) 39-49. r51 Davies, L., Wolska, B., Hilbich, C., Multhaup, G., Martins, R., Simms, G., Beyreuther, K. and Masters, CL., A4 amyloid protein
16
T.G. Beach et al. /Neuroscience
deposition and the diagnosis of Alzheimer’s disease: prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques, Neurology, 38 (1988) 1688-1693. @I Egan, T.M. and North, R.A., Actions of acetylcholine and nicotine on rat locus coeruleus neurons in vitro, Neuroscience, 19 (1986) 565-571. M.-C., Mullan, M., Brown, J., Craw171 Goate, A., Chattier-Harlin. ford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P.. Talbot, C., Pericak Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M. and Hardy, J., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease, Nature, 349 (1991) 704-706. PI Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D.M., Oshima, J., Pettingell, W.H., Yu, C-en, Jondro, P.D., Schmidt, SD., Wang, K., Crowley, A.C., Fu, Y.-H., Guenette, S.Y., Galas, D., Nemens, E, Wijsman, E.M., Bird, T.D., Schellenberg, G.D. and Tanzi, R.E., Candidate gene for the chromosome I familial Alzheimer’s disease locus, Science, 269 (1995) 973-977. 191 McKinney, M. and Robbins, M., Chronic atropine administration up-regulates rat cortical muscarinic ml receptor molecules: assessment with the RTIPCR, Mol. Brain Res., 12 (1992) 3945. [lOI Molenaar, P.C. and Polak, R.L., Stimulation by atropine of acetylcholine release and synthesis in cortical slices from rat brain, Br. J. Pharmacol., 40 (1970) 406-417. Ull Nitsch, R.M., Slack, B.E., Wurtman, R.J. and Growdon, J.H., Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors, Science, 258 (1992) 304-307.
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