1
THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE S61 (AB l-28, AB 12-28) and amphipathic (AI3 25-35) peptides. Among the fragments tested, AB 25-35 was found to mimic the effect of A6 l-40 on PKC activity. Al3 25-35 is homologous in sequence to the tachykinin neuropeptide family, and has been reported to act like Al3 l-40 in mediating neurotrophic and neurotoxic effects possibly by interacting with tachykinins (Yankner et al. Scince (199O),m, 279-282). In our experiments, the potent tachykinin antagonists-[[D-Pro’ , D-T~P’ .~] -Substance P and [D-At-g’ , D-Trp7s9* Leu”] -Substance P (spantide), were also found to influence PKC activity in a biphasic manner. Since PKC plays a key role in neuronal function and memory, it is proposed that (a) Al3at low concentrations may contribute to neurotrophic effects by stimulating PKC; and at higher concentrations it might lead to memory impairment and neuronal loss by inhibiting PKC activity and (b) dual action of A6 on PKC may be connected to its interaction with tachykinin neuropeptides system. 238 MULTIPLE ION CHANNEL BINDING SITES ARE DIFFERENTIALLY ALTERED IN ALZHEIMER’S DISEASE CORTEX. D. DEWAR, M. IKEDA, I. McCULLGCH. Wellcome Neuroscience Group, University of Glasgow, Glasgow, G61 IQH, U.K. Ion channels are important in normal neuronal function and may also mediate excitotoxic events leading to neurodegeneration. Three ion channel binding sites were examined using quantitative autoradiography in the temporal and occipital cmtices of 7 control subjects and 9 patients with neuropathologically-confirmed Alzheime,r;g disease (AD). The following ligands were used:- I-apamin to label calcium-sensitive potassium channels; 3H-glibenclamide to label ATP-sensitive potassium channels and 3H-PN200-110 to label L-type voltage - sensitive calcium channels. Each binding site exhibited a distinct laminar pattern of distribution in both areas of COrteX. In temporal cortex in AD, apamin binding sites were significantly increased in all cortical layers (e.g. superficial layers, control = 0.71 2 0.07; AD = 1.02 + 0.07 pmol/g; mean + SEM). In contrast glibenclamide binding was significantly reduced in all layers of the same patients (e.g. superficial layers, control = 25.3 + 1.7; AD = 17.9 & 1.4 pmol/g). PN200-110 binding was unchanged in AD in temporal cortex. In occipital cortex apamin binding was also increased in AD although less so (25%) than in temporal cortex. In this cortical area both glibenclamide and PN200-110 binding levels were no different from control values. In neither temporal or occipital cortex was there. a correlation between binding to any ion channel site and the density of neuritic plaques in the AD subjects. The results indicate the differential responses of these ion channel sites to the degenerative process of AD. This work was supported by the Wellcome Trust. 239 COUPLING OF HUMAN BRAIN CEREBRAL CORTICAL c+4DBENOCEPTORS AND %IYDROXYTRYPTAMINE, RECEPTORS TO G PROTEINS IN ALZHEIMER’S DISEASE. O’NeilI, C.,’ Cowburn, R.F.,’ Fowler, C.J.,S Wiehager, B.,’ Alafizoff, I.2 & Winblad, B. . 1 ‘Dept. of Geriatic Medicine & S>ept. of Pathology, Huddinge University Hospital, Sweden; ‘Preclinical Research, Astra Pain Control AB, Sadetilje, Sweden. Many of the neurotransmitter receptor systems that are altered in Alzheimer’s Disease (AD) activate appropriate signal transduction responses via GTP binding, G proteins. This study examined the interaction of h-adrenoceptora and 5-hydroxytryptamine, (5-HT,) receptors with G proteins in membranes prepared from frontal, temporal and parietal cortices of control and AD brain. The coupling of cq-adreno- ceptors to G proteins was investigated by characterising the effect of guanosine 5’-[DY-imidoldiphosphate (Gpp[NH]p) and MnCl, on ‘Nclonidine binding. 5-HT, receptor-G pro& ein interactions were examined by characterising the effect of Gpp[NH]p on 3[H]8-hydroxy-2-(di-n-propy1amino)-tetraIin (3[H18-OH-DPAT) binding parameters. Results show that Gpp[NH]p inhibited ?H]clonidine and ‘[H]B-OH-DPAT in a concentration dependent manner, the profiles of inhibition showing no significant difference between control and AD sampl&. Analysis of the effect of Gpp[NH]p on THlclonidine saturation curves showed no significant difference between control and AD cases in either frontal or temporal cortices. The number of 3[H]8-OH-DPAT binding sites was significantly decreased in the parietal cortex but remained unchanged in frontal cortex of AD samples compared to controls. However, Gpp[NH]p reduced the 3[H]8-OH-DPAT binding affinity and number of binding sites to the same degree in both regions in control and AD cases. 3[H]-clonidine binding parameters, measured in the preeence of manganese were significantly different in the temporal cortex in AD compared to control samples. It is concluded that there are no gross disturbances in the qadrenoceptor, or 5-HT, receptor-G protein interaction mechanisms in AD cerebra1 cortex. 240 DEFECT COUPLING OF MUSCARINIC RECEPTORS To GTP PROTEINS IN ALZHEIMER BRAINS.. U.Warpmanl, I.Ala.fuzof@, A.Nordbergl. Lkpamnenlof Phannacologyl. University of Uppsala Box 591.S751 24 Uppsala and Department of Pa~hology~,Karolinska Instiute,Huddinge HospifaW 141 86 Huddinge, Sweden Alteration in the choline@ nervous system, such as in the release of acetvlcholine. acetvlcholine transferas activitv and number of nicotinic and muskinic keptok have in detail been investigated in Alzheimer’s disease/senile dementia of Alzheimer type (AD/SDAT) brains. The functional role of the cellular respons of muscarinic receptors via signal transduction mechanisms, their coupling to G proteins, has recieved very little attention. In the present study, regulation of the carbachol binding by the non- hydrolyzable GTP analogue Gpp(NH)p was investigated in AD/SDAT and age-matched control postmortem brains, in order to assess the efficacy of this aansduction step in the disease process. Membrane prepared of postmortem frontal, temporal cortices and thalamus tissue were obtained from 9 Alzheimer’s disease patients (mean 78X2 years; mean postmortem 227 h) and 6 control patients(mean age 75s years; mean postmortem 18fl h). The muscarinic receptorbinding study with [%jQNB and unlabelled carbachol in competing concentrations showed a Hill coefficient less than one and the computer assisted analysis revealed a best tit to a two-site model. A significant increase in high affinity agonist binding coefficient (Ki high) in the control thalamus tissue and an increase in the proportion of low agonist affinity binding sites (B,, tow) in the control temporal cortex were obtained in the presence of Gpp(NH)p, indicating a functional M2 muscarinic receptor-G protein coupling. No change in binding characteristics in the presence of the guanine nucleotide could be observed in the control kontal cortex tissue, probably due to the small amount of M2 muscarinic receptors(278). In the thalamus, temporal and frontal conices from Alzheimer’s disease tissue no change was observed in the binding properties after addition of Gpp(NH)p. The displacement curves in the thalamus of AD/SDAT showed a steepening in the slope in the absence and presence of the guanine nucleotide compared to thalamus tissue of control in the absence of Gpp(NH)p. Neither the total amount of muscarinic binding sites nor the proportion of M2 muscarinic receptors in the frontal corteqtempoml cortex and thalamus differed in Alzheimer’s disease compared to control groups. In conclusion, the muscarinic receptors coupling to G proteins can be studied in postmortem brain tissue. Furthermore&. M2 muscarinic receptors coupling to G proteins seemed to be altered in the brain of Alzheimer’s disease. These findings might be of importance in the development of selective choline@ drugs. 241 ALTERATIONS OF /3-ADRENERGIC-RECEPTOR--TED, G- PROTEIN-MEDIATED CAMP FORMATION IN CULTURED SKIN FIBROB- FROM ALZHEIMER DONORS, H.-M. Huang and G. E. Giison. Burke Medical Research Institute, Cornell University Medical College, White Plains, NY, USA. Signal transduction systems are critical to normal brain function, thus an alteration ia these processes in Alzheimer’s disease would likely be of patbophysiological importance. Since these dynamic transduction systems cannot be studied in autopsied brain, the current studies utilized cultured skin fibroblasts from Alzbeimer patients and age-matched controls. The accumulation of CAMP by the /3-adrenergic agonist isoproterenol was reduced approximately 65% in Alzheimer fibroblasts and no overlap occurred between multiple Alzbeimer and control lines. Both familial and

Multiple ion channel binding sites are differentially altered in Alzheimer's disease cortex

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THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE S61

(AB l-28, AB 12-28) and amphipathic (AI3 25-35) peptides. Among the fragments tested, AB 25-35 was found to mimic the effect of A6 l-40 on PKC activity. Al3 25-35 is homologous in sequence to the tachykinin neuropeptide family, and has been reported to act like Al3 l-40 in mediating neurotrophic and neurotoxic effects possibly by interacting with tachykinins (Yankner et al. Scince (199O),m, 279-282). In our experiments, the potent tachykinin antagonists-[[D-Pro’, D-T~P’.~] -Substance P and [D-At-g’, D-Trp7s9* Leu”] -Substance P (spantide), were also found to influence PKC activity in a biphasic manner. Since PKC plays a key role in neuronal function and memory, it is proposed that (a) Al3 at low concentrations may contribute to neurotrophic effects by stimulating PKC; and at higher concentrations it might lead to memory impairment and neuronal loss by inhibiting PKC activity and (b) dual action of A6 on PKC may be connected to its interaction with tachykinin neuropeptides system.

238 MULTIPLE ION CHANNEL BINDING SITES ARE DIFFERENTIALLY ALTERED IN ALZHEIMER’S DISEASE CORTEX. D. DEWAR, M. IKEDA, I. McCULLGCH. Wellcome Neuroscience Group, University of Glasgow, Glasgow, G61 IQH, U.K.

Ion channels are important in normal neuronal function and may also mediate excitotoxic events leading to neurodegeneration. Three ion channel binding sites were examined using quantitative autoradiography in the temporal and occipital cmtices of 7 control subjects and 9 patients with neuropathologically-confirmed Alzheime,r;g disease (AD). The following ligands were used:- I-apamin to label calcium-sensitive potassium channels; 3H-glibenclamide to label ATP-sensitive potassium channels and 3H-PN200-110 to label L-type voltage - sensitive calcium channels. Each binding site exhibited a distinct laminar pattern of distribution in both areas of COrteX.

In temporal cortex in AD, apamin binding sites were significantly increased in all cortical layers (e.g. superficial layers, control = 0.71 2 0.07; AD = 1.02 + 0.07 pmol/g; mean + SEM). In contrast glibenclamide binding was significantly reduced in all layers of the same patients (e.g. superficial layers, control = 25.3 + 1.7; AD = 17.9 & 1.4 pmol/g). PN200-110 binding was unchanged in AD in temporal cortex. In occipital cortex apamin binding was also increased in AD although less so (25%) than in temporal cortex. In this cortical area both glibenclamide and PN200-110 binding levels were no different from control values. In neither temporal or occipital cortex was there. a correlation between binding to any ion channel site and the density of neuritic plaques in the AD subjects. The results indicate the differential responses of these ion channel sites to the degenerative process of AD.

This work was supported by the Wellcome Trust.

239 COUPLING OF HUMAN BRAIN CEREBRAL CORTICAL c+4DBENOCEPTORS AND %IYDROXYTRYPTAMINE, RECEPTORS TO G PROTEINS IN ALZHEIMER’S DISEASE. O’NeilI, C.,’ Cowburn, R.F.,’ Fowler, C.J.,S Wiehager, B.,’ Alafizoff, I.2 & Winblad, B. . 1 ‘Dept. of Geriatic Medicine & S>ept. of Pathology, Huddinge University Hospital, Sweden; ‘Preclinical Research, Astra Pain Control AB, Sadetilje, Sweden. Many of the neurotransmitter receptor systems that are altered in Alzheimer’s Disease (AD) activate appropriate signal transduction responses via GTP binding, G proteins. This study examined the interaction of h-adrenoceptora and 5-hydroxytryptamine, (5-HT,) receptors with G proteins in membranes prepared from frontal, temporal and parietal cortices of control and AD brain. The coupling of cq-adreno- ceptors to G proteins was investigated by characterising the effect of guanosine 5’-[DY-imidoldiphosphate (Gpp[NH]p) and MnCl, on ‘Nclonidine binding. 5-HT, receptor-G pro& ein interactions were examined by characterising the effect

of Gpp[NH]p on 3[H]8-hydroxy-2-(di-n-propy1amino)-tetraIin (3[H18-OH-DPAT) binding parameters. Results show that Gpp[NH]p inhibited ?H]clonidine and ‘[H]B-OH-DPAT in a concentration dependent manner, the profiles of inhibition showing no significant difference between control and AD sampl&. Analysis of the effect of Gpp[NH]p on THlclonidine saturation curves showed no significant difference between control and AD cases in either frontal or temporal cortices. The number of 3[H]8-OH-DPAT binding sites was significantly decreased in the parietal cortex but remained unchanged in frontal cortex of AD samples compared to controls. However, Gpp[NH]p reduced the 3[H]8-OH-DPAT binding affinity and number of binding sites to the same degree in both regions in control and AD cases. 3[H]-clonidine binding parameters, measured in the preeence of manganese were significantly different in the temporal cortex in AD compared to control samples. It is concluded that there are no gross disturbances in the qadrenoceptor, or 5-HT, receptor-G protein interaction mechanisms in AD cerebra1 cortex.

240 DEFECT COUPLING OF MUSCARINIC RECEPTORS To GTP PROTEINS IN ALZHEIMER BRAINS.. U.Warpmanl, I.Ala.fuzof@, A.Nordbergl. Lkpamnenl of Phannacologyl. University of Uppsala Box 591.S751 24 Uppsala and Department of Pa~hology~,Karolinska Instiute,Huddinge HospifaW 141 86 Huddinge, Sweden

Alteration in the choline@ nervous system, such as in the release of acetvlcholine. acetvlcholine transferas activitv and number of nicotinic and muskinic keptok have in detail been investigated in Alzheimer’s disease/senile dementia of Alzheimer type (AD/SDAT) brains. The functional role of the cellular respons of muscarinic receptors via signal transduction mechanisms, their coupling to G proteins, has recieved very little attention.

In the present study, regulation of the carbachol binding by the non- hydrolyzable GTP analogue Gpp(NH)p was investigated in AD/SDAT and age-matched control postmortem brains, in order to assess the efficacy of this aansduction step in the disease process. Membrane prepared of postmortem frontal, temporal cortices and thalamus tissue were obtained from 9 Alzheimer’s disease patients (mean 78X2 years; mean postmortem 227 h) and 6 control patients(mean age 75s years; mean postmortem 18fl h). The muscarinic receptorbinding study with [%jQNB and unlabelled carbachol in competing concentrations showed a Hill coefficient less than one and the computer assisted analysis revealed a best tit to a two-site model. A significant increase in high affinity agonist binding coefficient (Ki high) in the control thalamus tissue and an increase in the proportion of low agonist affinity binding sites (B,, tow) in the control temporal cortex were obtained in the presence of Gpp(NH)p, indicating a functional M2 muscarinic receptor-G protein coupling. No change in binding characteristics in the presence of the guanine nucleotide could be observed in the control kontal cortex tissue, probably due to the small amount of M2 muscarinic receptors(278). In the thalamus, temporal and frontal conices from Alzheimer’s disease tissue no change was observed in the binding properties after addition of Gpp(NH)p. The displacement curves in the thalamus of AD/SDAT showed a steepening in the slope in the absence and presence of the guanine nucleotide compared to thalamus tissue of control in the absence of Gpp(NH)p. Neither the total amount of muscarinic binding sites nor the proportion of M2 muscarinic receptors in the frontal corteqtempoml cortex and thalamus differed in Alzheimer’s disease compared to control groups.

In conclusion, the muscarinic receptors coupling to G proteins can be studied in postmortem brain tissue. Furthermore&. M2 muscarinic receptors coupling to G proteins seemed to be altered in the brain of Alzheimer’s disease. These findings might be of importance in the development of selective choline@ drugs.

241 ALTERATIONS OF /3-ADRENERGIC-RECEPTOR--TED, G- PROTEIN-MEDIATED CAMP FORMATION IN CULTURED SKIN FIBROB- FROM ALZHEIMER DONORS, H.-M. Huang and G. E. Giison. Burke Medical Research Institute, Cornell University Medical College, White Plains, NY, USA. Signal transduction systems are critical to normal brain function, thus an alteration ia these processes in Alzheimer’s disease would likely be of patbophysiological importance. Since these dynamic transduction systems cannot be studied in autopsied brain, the current studies utilized cultured skin fibroblasts from Alzbeimer patients and age-matched controls. The accumulation of CAMP by the /3-adrenergic agonist isoproterenol was reduced approximately 65% in Alzheimer fibroblasts and no overlap occurred between multiple Alzbeimer and control lines. Both familial and