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Continuous remodelling of synapses in Alzheimer disease and enhanced neurotrophic activity of Alzheimer brain tissue extract in culture
THIRD
INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
DISEASE
relationship of depletion of parent phospholipids and accumulation of catabolic breakdown product suggests accelerated breakdown of brain cell membranes in AD. The product-precursor ratio for PtdCho and PtdEtn was significantly increased two - three-fold in AD in all examined brain areas, which is also consistent with the idea of accelerated membrane phospholipid breakdown in AD brain. In addition, the initial precursors of these phospholipids, choline and ethanolamine, were decreased by 50% in all examined brain areas, suggesting that the synthesis of both phospholipids and acetylcholine may by slowed AD, since the reactions involved in these pathways are precursor dependent. We did not find similar alterations in brains from individuals with other neurodegenerative diseases including Down’s syndrome, Huntington’s disease, and Parkinson’s disease, suggesting that this biochemical membrane lesion may be specific to the pathophysiology of neurodegeneration in AD. The exact causes of this abnormality of membrane metabolism in AD brain are unknown but may include alterations in the regulation of phospholipase activities, toxic events which damage membranes, and impaired utilization of the membrane precursors, choline and ethanolamine. PM. Nitsch et al. PNAS 69, 1671-l 675, 1992.
175 GLUTAMATERGIC NEUROTRANSMISSION IN RAT BRAIN SYNAPTOSOMES: EFFECT OF AGING AND INHIBITION OF ENERGY METABOLISM, F. Dagani, R. Ferrari, and L Canevari. Istituto di Farmacologia, Facolta di Scienze, Universih di Pavia, Piazza Botta 11, 27100 Pavia, Italy. An impairment in energy metabolism of presynaptic terminals in brain leads to an unbalance of intra- and extra-cellular concentration of excitatory amino acid (EAA) neurotransmitters like glutamate and aspartate. This phenomenon causes the excitotoxic damage due to a prolonged activation of EAA channel-receptors complexes. Whereas this mechanism was described for brain ischemia/hypoxia, it is still hypothetical for aging and related disorders. In this investigation we studied the possibility that glutamate release and presynaptic energy machinery governing it can be modified by aging and inhibition of energy metabolism in synaptosomes isolated from rat brains at different ages. Synaptosomes, obtained by differential and density-gradient centrifugations, were incubated in isoosmotic saline (Krebs-Henseleit HEPES); oxygen consumption rate, intracellular free calcium levels membrane potential, ATP/ADP conentrations and glutamate release were evaluated both at rest and following veratridine depolarization. Glutamate release was also evaluated in conditions of inhibition of mitochondrial respiration operated by addition, of NaCN or NaN3+iodoacetic acid (IA4 to block glycolysis) (“chemical ischemia”). The results show that basal and veratridine-stimulated oxygen consumption rate were not significantly affected by aging. ATP levels decreased with aging but neither veratridine nor “chemical ischemia” effects were affected by aging. Glutamate release stimulated by veratridine and “chemical ischemia” was decreased by both conditions during aging. The “in vitro” effect of some drugs like GM1 and adenosine analogs have been also studied. GM1 did not affect the veratridineinduced release of glutamate. Adenosine analogs showed heterogeneous effects according to the receptor selectivity and dose. These observations provide new information concerning the potential utilization of these drugs in affecting age-related and chemical ischemia related modifications which may be of interest in the understanding of age related neurological disorders.
176 CONTINUOUS BEMODELLIh’G OF SYNAPSES IN ALZHEIMER DISEASE AND ENHANCED h’EUBOTBOPHIC ACTIVITY OF ALZHEIMEB BRAIN TISSUE EXTRACT IN CULTURE, O.S. Jorgensen’ and R. EWzs*. lInstitute of Nemopsychiatry, University of Copenhagen, KigshospitaJet, DK-2100 Copenhagen, Denmark; ‘Netherlands Institute for Brain Research, 1105 AZ Amsterdam ZO, The Netherlands. Evidence has accumulated that in Alzheimer disease (AD) the neuronal and synaptic degeneration in the neighbourhood of the dendddc plagues is accompanied by neudtic sprouting and synaptic remodelling. It has previously been demonstrated that the ratio between two membrane proteins: NCAM (neural cell adhesion molecule; marker for new neuronal membranes) and D3protein (marker for mature neuronal structures) can be used to monitor the extent
s45
of synaptic remodelling in brain tissue. In AD frontal cortex the ratio of NCAM to D3-protein was significantly increased by 8.5% (Jorgensen, Brooksbank and B&&s, J Nemo1 Sci, 1990,98:63-79). In a new group of brain samples obtained from the Netherlands Brain Bank this study was replicated and extended to other brain areas. Furthermore, high molecular weight extracts were produced from the samples and tested for growth-promoting (trophic) and toxic activity towards mouse cerebellar granule cells in culture using acute 2-deoxy-D-glucose accumulation as a sensitive test parameter. The assay system demonstrated enhanced glucose accumulation in the presence of the trophic substancebFGF (maximally 20% for 12DIV cultures grown with more than 50 nglml) but was not responsive to NGF, EGF, IGF-1, beta-amyloid protein (l-40) or to the Kunitz inhibitor sequence of the amyloid precursor protein. Soluble proteins from AD frontal cortex stimulated the glucose accumulation by up to 20% (peak maximum at 25 ug protein/ml). The cells were significantly more stimulated by AD extracts than by extracts of control frontal cortex analyzed in parallel (8% average difference over the concentration range l-100 “g/ml; P=O.Ol; 8 AD and 4 control patients). We interpret these results as a further demonstration of the increased capacity for generalized synaptic remodelling in AD. However, in the light of recent findings (Yankner et al., PNAS, 1990, 879020~9023) it remains to be proven whether such remodelling can be considered an advantage to the AD brain or alternatively beta-amyloid protein and trophic factors interact and aggravate the disease. (Supported by grants from the Danish Medical Research Council and from NW0 and the Van den Houten Foundation).
177 DYSTROPHIC NEURITE- AND AMYLOID-CONTAINING PLAQUES IN THE PULVINAR NUCLEUS FROM PATIENTS WITH ALZHEIMER'S DISEASE. Rodrigo 0. Kuljis. Department of Neurology, The University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242-1053, USA. Patients with Alzheimer's disease (AD) suffer a deterioration of complex cognitive operations, including those mediated by the occipital, temporal and parietal association cortices. Such deficits often correlate with a high density of senile plaques and neurofibrillary tangles in those regions, justifying their interpretation as cortical dysfunction due mainly to intracortical lesions. This interpretation rests on the notion that the thalamus is "spared" in AD, which has been challenged by the finding of Ah&positive terminals in the reticular nucleus (Tourtellotte et al., Brain Res. 515:227) and neurofibrillary tangles and neuritic plaques in the medioventral and paraventricular nuclei (May et al., Sot. Neurosci. Abstr. 17:690). The pulvinar (Pu), lateral geniculate nucleus (LGN), and visual cortices of ten patients with histologically-proven AD and ten age-matched controls (with and without dementia) were examined using antibodies for A68, and the thioflavine, Bielschowsky, Campbell-Switzer, and Gallyas methods. Patients with AD exhibit abundant neuritic and amyloid plaques throughout the various subdivisions of the Pu complex, especially the lateral and inferior nuclei. A linear correlation was observed between the density of these lesions in the thalamus and in the visual cortices. Neurofibrillary tangles, neuropil threads, and A6Blike immunoreactivity are absent from the Pu in all cases. Virtually no lesions were observed in controls, in patients with non-AD dementia, and in the LGN of AD patients, These observations indicate a distinct involvement of the pulvinar that is relatively selective to AD, and which is in contrast to the "sparing" of most other thalamic regions reported previously. The lesions in the pulvinar coexist with lesions in a vast array of association cortices that normally maintain reciprocal synaptic relations with this nucleus. Therefore, lesions in the Pu appear to be in a strategic position to aggravate the deficits mediated by occipito-par&to-temporal association cortices heavily laden with lesions themselves. These tandem lesions may play a" important role in the pathophysiology of the clinical deficits that characterize AD. Support: American Federation for Aging Research and NIH NS29856.
178 Henaria-Binding Growth Factors Exhibit BiBeeentiaJ AfBnities for B/A4 AmyJOid-Containing Struduees in Alzhiemer’s Disease. *E.G. Stopa, A. Baird, A.M. Canxalea, V. Kuo-LeBlanc, RX. Corona,I. Kovesdi, and P. Boblen. Departments of WtholeBy, SUNY-HBC,Syraeuse and Albany Msdieal College, Alby, NY, Dept. of Molecular and Cellular Growth Biology, The Whittier Institute, L&Jolla, CA, Brain Tiiue Besource Ctr., Me&an Hospital, Belmont, MA mid Me&at Besea& Biviskm, American Cyanamid Company, Pearl River, NY,USA. Heparin-binding is a common biochemical pmpeay of several rsently
INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
DISEASE
relationship of depletion of parent phospholipids and accumulation of catabolic breakdown product suggests accelerated breakdown of brain cell membranes in AD. The product-precursor ratio for PtdCho and PtdEtn was significantly increased two - three-fold in AD in all examined brain areas, which is also consistent with the idea of accelerated membrane phospholipid breakdown in AD brain. In addition, the initial precursors of these phospholipids, choline and ethanolamine, were decreased by 50% in all examined brain areas, suggesting that the synthesis of both phospholipids and acetylcholine may by slowed AD, since the reactions involved in these pathways are precursor dependent. We did not find similar alterations in brains from individuals with other neurodegenerative diseases including Down’s syndrome, Huntington’s disease, and Parkinson’s disease, suggesting that this biochemical membrane lesion may be specific to the pathophysiology of neurodegeneration in AD. The exact causes of this abnormality of membrane metabolism in AD brain are unknown but may include alterations in the regulation of phospholipase activities, toxic events which damage membranes, and impaired utilization of the membrane precursors, choline and ethanolamine. PM. Nitsch et al. PNAS 69, 1671-l 675, 1992.
175 GLUTAMATERGIC NEUROTRANSMISSION IN RAT BRAIN SYNAPTOSOMES: EFFECT OF AGING AND INHIBITION OF ENERGY METABOLISM, F. Dagani, R. Ferrari, and L Canevari. Istituto di Farmacologia, Facolta di Scienze, Universih di Pavia, Piazza Botta 11, 27100 Pavia, Italy. An impairment in energy metabolism of presynaptic terminals in brain leads to an unbalance of intra- and extra-cellular concentration of excitatory amino acid (EAA) neurotransmitters like glutamate and aspartate. This phenomenon causes the excitotoxic damage due to a prolonged activation of EAA channel-receptors complexes. Whereas this mechanism was described for brain ischemia/hypoxia, it is still hypothetical for aging and related disorders. In this investigation we studied the possibility that glutamate release and presynaptic energy machinery governing it can be modified by aging and inhibition of energy metabolism in synaptosomes isolated from rat brains at different ages. Synaptosomes, obtained by differential and density-gradient centrifugations, were incubated in isoosmotic saline (Krebs-Henseleit HEPES); oxygen consumption rate, intracellular free calcium levels membrane potential, ATP/ADP conentrations and glutamate release were evaluated both at rest and following veratridine depolarization. Glutamate release was also evaluated in conditions of inhibition of mitochondrial respiration operated by addition, of NaCN or NaN3+iodoacetic acid (IA4 to block glycolysis) (“chemical ischemia”). The results show that basal and veratridine-stimulated oxygen consumption rate were not significantly affected by aging. ATP levels decreased with aging but neither veratridine nor “chemical ischemia” effects were affected by aging. Glutamate release stimulated by veratridine and “chemical ischemia” was decreased by both conditions during aging. The “in vitro” effect of some drugs like GM1 and adenosine analogs have been also studied. GM1 did not affect the veratridineinduced release of glutamate. Adenosine analogs showed heterogeneous effects according to the receptor selectivity and dose. These observations provide new information concerning the potential utilization of these drugs in affecting age-related and chemical ischemia related modifications which may be of interest in the understanding of age related neurological disorders.
176 CONTINUOUS BEMODELLIh’G OF SYNAPSES IN ALZHEIMER DISEASE AND ENHANCED h’EUBOTBOPHIC ACTIVITY OF ALZHEIMEB BRAIN TISSUE EXTRACT IN CULTURE, O.S. Jorgensen’ and R. EWzs*. lInstitute of Nemopsychiatry, University of Copenhagen, KigshospitaJet, DK-2100 Copenhagen, Denmark; ‘Netherlands Institute for Brain Research, 1105 AZ Amsterdam ZO, The Netherlands. Evidence has accumulated that in Alzheimer disease (AD) the neuronal and synaptic degeneration in the neighbourhood of the dendddc plagues is accompanied by neudtic sprouting and synaptic remodelling. It has previously been demonstrated that the ratio between two membrane proteins: NCAM (neural cell adhesion molecule; marker for new neuronal membranes) and D3protein (marker for mature neuronal structures) can be used to monitor the extent
s45
of synaptic remodelling in brain tissue. In AD frontal cortex the ratio of NCAM to D3-protein was significantly increased by 8.5% (Jorgensen, Brooksbank and B&&s, J Nemo1 Sci, 1990,98:63-79). In a new group of brain samples obtained from the Netherlands Brain Bank this study was replicated and extended to other brain areas. Furthermore, high molecular weight extracts were produced from the samples and tested for growth-promoting (trophic) and toxic activity towards mouse cerebellar granule cells in culture using acute 2-deoxy-D-glucose accumulation as a sensitive test parameter. The assay system demonstrated enhanced glucose accumulation in the presence of the trophic substancebFGF (maximally 20% for 12DIV cultures grown with more than 50 nglml) but was not responsive to NGF, EGF, IGF-1, beta-amyloid protein (l-40) or to the Kunitz inhibitor sequence of the amyloid precursor protein. Soluble proteins from AD frontal cortex stimulated the glucose accumulation by up to 20% (peak maximum at 25 ug protein/ml). The cells were significantly more stimulated by AD extracts than by extracts of control frontal cortex analyzed in parallel (8% average difference over the concentration range l-100 “g/ml; P=O.Ol; 8 AD and 4 control patients). We interpret these results as a further demonstration of the increased capacity for generalized synaptic remodelling in AD. However, in the light of recent findings (Yankner et al., PNAS, 1990, 879020~9023) it remains to be proven whether such remodelling can be considered an advantage to the AD brain or alternatively beta-amyloid protein and trophic factors interact and aggravate the disease. (Supported by grants from the Danish Medical Research Council and from NW0 and the Van den Houten Foundation).
177 DYSTROPHIC NEURITE- AND AMYLOID-CONTAINING PLAQUES IN THE PULVINAR NUCLEUS FROM PATIENTS WITH ALZHEIMER'S DISEASE. Rodrigo 0. Kuljis. Department of Neurology, The University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242-1053, USA. Patients with Alzheimer's disease (AD) suffer a deterioration of complex cognitive operations, including those mediated by the occipital, temporal and parietal association cortices. Such deficits often correlate with a high density of senile plaques and neurofibrillary tangles in those regions, justifying their interpretation as cortical dysfunction due mainly to intracortical lesions. This interpretation rests on the notion that the thalamus is "spared" in AD, which has been challenged by the finding of Ah&positive terminals in the reticular nucleus (Tourtellotte et al., Brain Res. 515:227) and neurofibrillary tangles and neuritic plaques in the medioventral and paraventricular nuclei (May et al., Sot. Neurosci. Abstr. 17:690). The pulvinar (Pu), lateral geniculate nucleus (LGN), and visual cortices of ten patients with histologically-proven AD and ten age-matched controls (with and without dementia) were examined using antibodies for A68, and the thioflavine, Bielschowsky, Campbell-Switzer, and Gallyas methods. Patients with AD exhibit abundant neuritic and amyloid plaques throughout the various subdivisions of the Pu complex, especially the lateral and inferior nuclei. A linear correlation was observed between the density of these lesions in the thalamus and in the visual cortices. Neurofibrillary tangles, neuropil threads, and A6Blike immunoreactivity are absent from the Pu in all cases. Virtually no lesions were observed in controls, in patients with non-AD dementia, and in the LGN of AD patients, These observations indicate a distinct involvement of the pulvinar that is relatively selective to AD, and which is in contrast to the "sparing" of most other thalamic regions reported previously. The lesions in the pulvinar coexist with lesions in a vast array of association cortices that normally maintain reciprocal synaptic relations with this nucleus. Therefore, lesions in the Pu appear to be in a strategic position to aggravate the deficits mediated by occipito-par&to-temporal association cortices heavily laden with lesions themselves. These tandem lesions may play a" important role in the pathophysiology of the clinical deficits that characterize AD. Support: American Federation for Aging Research and NIH NS29856.
178 Henaria-Binding Growth Factors Exhibit BiBeeentiaJ AfBnities for B/A4 AmyJOid-Containing Struduees in Alzhiemer’s Disease. *E.G. Stopa, A. Baird, A.M. Canxalea, V. Kuo-LeBlanc, RX. Corona,I. Kovesdi, and P. Boblen. Departments of WtholeBy, SUNY-HBC,Syraeuse and Albany Msdieal College, Alby, NY, Dept. of Molecular and Cellular Growth Biology, The Whittier Institute, L&Jolla, CA, Brain Tiiue Besource Ctr., Me&an Hospital, Belmont, MA mid Me&at Besea& Biviskm, American Cyanamid Company, Pearl River, NY,USA. Heparin-binding is a common biochemical pmpeay of several rsently