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Alzheimer cytoskeletal pathology: From microtubule associated protein tau to paired helical filaments
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BIOLOGICAL MARKERS/SYSTEMIC MARKERS The development of marker systems based on quantitative immunoassays or biological assays for the presence of EA4 or APP will be necessary. Preliminary steps in this area with radio-immunoassays, ELISAs, Western blotting based on second and third generation antisera have indicated the direction of further research. More sensitive and specific assays which dist'inguish the various species of APP are now being developed. Some innovative techniques are required to assay the insoluble forms of 5A4. At the most basic level, immunocytochemistry of BA4 has shown that deposition proceeds over a long period (probably exceeding 20 years in the lifetime of an "average" plaque) and that the age at onset of deposition may antedate the onset of clinical features by a similar interval. Thus, "pre-clinical" AD should be considered as a major component of the evolution of AD, and that this component needs to be taken into account when assessing peripheral (extra-neural) markers of 5A4 and APP.
322 ALZHEIMER CYTOSKELETAL PATHOLOGY: FROM MICROTUBULE ASSOCIATED PROTEIN TAU TO PAIRED HELICAL FILAMENTS. *lnge Grundke-lqbal and Khalid Iqbal, Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. Tau in Alzheimer disease (AD) brain is present in different modified forms. The earliest modification observed to date in neurons undergoing neurofibrillary changes is its abnormal phosphorylation. Based on their differential solubility in SDS, tangles have been classified as ANT I/PHF I and ANT II/PHF II for being made up of readily soluble and sparingly soluble PHF respectively. We have developed a method for the enrichment of ANT I and ANT II polypeptides from neuronal cell body fractions of Alzheimer brain. The neuronal cell bodies are isolated from cerebral cortex and extracted sequentially with 50raM Tris, 1% Triton X100, 8M urea/6OmM dithiothritol (D'I-F) and 1% SDS at room temperature, followed by boiling with 1% Uthium dodecylsulfate (LDS). Immunoblot analysis has revealed that most of the normal cytoskeletal proteins are extracted with urea/DTT. The SDS (ANT I) and LDS (ANT II) extracts from AD brain contain most of the abnormally phosphorylated tau (mAb Tau-1) and ubiquitin - reactive (mAb 5-25/3-39 to PHF) material whereas similar extracts from normal brain contained negligible levels of tau and ubiquitin immunoreactivity. The ANT I fraction seems to contain more of the fast moving species of the abnormally phosphorylated tau whereas the slowest moving 68kDa and 70kDa tau species are mostly present in the ANT II fractions. By densitometry of immunoblots the ubiquitin/tau ratio in the ANT II fractions is about 3-fold greater than in the ANT I fractions. In a separate study we have also isolated Alzheimer tau in a 27,000 to 100,000 xg pellet. This non-congophilic tau fraction although abnormally phosphorylated seems to be least modified by that it does not form aggregates on gel electrophoresis and does not contain ubiquitin. These studies suggest (1) that unpolymerized abnormal tau probably represent pre-tangles whereas ANT I and ANT II represent the mature tangle stages, containing tau with additional modifications and (2) that with increasing maturation the tangles become more difficult to depolymerize. (Supported in part by the Alzheimer's Disease Program of the American Health Assistance Foundation and NIH grants NS18105, AG05892 and AG04220). 323
IMPAIRMENTS IN CELLULAR FIBROUS PROTEINS: BIOLOGIC MARKER FOR ALZHEIMER DISEASE? *S.S. Matsuyama, West Los Angeles VA Medical Center, Brentwood Division, Los Angeles, CA 9007:5 and UCLA NPI, Los Angeles, CA 90024 USA. The search for a biologic marker For Alzheimer disease (AD) is being actively pursued. Although considered a neurodegenerative disease of the brain, there is mounting evidence oF abnormalities in peripheral tissues including red blood cells, ]ymphocytes, granulocytes, platelets and fibroblasts. Unlike brain tissue, which is obtained at autopsy or rarely biopsy, these tissues are readily available and Facilitate research investigations. In our laboratory, we are evaluating the possibility of a diagnostic test For AD based
331
on immunocytochemical examination oF cellular fibrous proteins in skin Fibr0blasts Following disruption with specific pharmacological agents, W e previously reported that Following colchicine treatment the reappearance of the microtubule network was significantly delayed in AD fibroblasts compared to normal ceils (AGE 12:127-132, 1989)suggesting an impairment in the microtubule system, Recently, we have Found evidence that the microFilamen[ system may also be involved (BondareFF and Matsuyama, unpublished observations), and are continuing investigations of microtubules and microFilaments in Fibroblasts From AD, non-AD demented patients and normal controls, 324 BRAIN NICOTINIC RECEPTOR DEFICITS IN ALZHEIMER PATIENTS AS STUDIED BY POSITRON EMISSION TOMOGRAPHY TECHNIQUE *A. Nordberg ~'e, P. Hartvlg 5, A. Litja 3, M. Viitanen 8, H. Lundqvlst4, J. Ulln 2, Y. Andersson =, B. Wlnbled', B.Langstrom 2, Depts of Pharmacology', Organic Chemistry~, Diagnostic Radiology~, The Svedberg Laboratory4, Uppsala University, Hospital Pharmacy~, University Hospital, Uppsala and Dept of Geriatric Medicine', Karolinska Institute, Huddinge Hospital, Huddinge, Sweden. Positron emission tomography (PET) is a technique suitable for In vlvo studies of central neuronal activity in brain. Until now the technique has been used In Alzhetmer patients in studies concerning cerebral blood flow and metabolism. Attempts have recently been made to measure cholinerglc nicotinic receptors using "C-nicotine in monkey and human brain by PET technique. We have eediar found a reduced number of high affinity nicotinic receptors In postmortem cortical brain tissues from Alzheimer patients. In order to visualize the nicotinic receptor defects in viv0 the (+)(R) and (-)(L) Isomers of '~C-nicotine have been given Intravenously in tracer doses (< 1/10 of the content of a cigarette) to Alzheimer patients and age matched healthy volunteers. The clinical diagnosis of Alzheimer's disease was made according to Diagnosis and Statistical Manual of Mental Disorders, DSM 111 criteria. Clinical examinations include blood and CSF laboratory, EEG, CT and MRI investigations. All patients and controls had undergone careful neuropsychometric testings. Seven Alzheimer patients (mean age 69+--2 ys; range 63-75, MMSE 17+-2) and six age-matched controls (mean age 69+-2 range 61-72) received an Intravenous bonus dose of "C-nicotine. The "C(-)-nicotine which peaks in brain within 2-5 mins and then decline during the following 15-20 mins shows a high content in brain areas as the thalamus, caudate nucleus, putamen, frontal and temporal cortex, intermediate in the occipital cortex, cerebellum and low in white matter. The uptake of "C-i-) nicotine Is lower and the time course is less steep in temporal cortex of Alzheimer patients compared to controls. In addition, a significant lower uptake of "C-i+) nicotine is found In the Alzheimer brains but not in the controls. "C-butanol was used as a marker for cerebral blood flow and the time course was different from that of both (+) and (-) "C-nicotine. The regional uptake of "C-butanol in control brains was rather homogenous while in Alzheimer brains a lower uptake of "C-butanol was found in temporal cortex, frontal cortex compared to thalamus.
325 NEL~OIRAN~'I'I'~ ~ IN THE C~RE~q~SPINAL FLUID OF PATI~I~TS ~ HISIDLOGICALL¥ VERIFIED ~ ' S DISEASE. * K.J. Reinikainen, H. Soininen, L. Pal j&rvi §, P.J. Riekkinen. Departments of Neurology and Pathology §, University of K~opio, 70211 Kuopio, Finland.
Studies of neurotransmitter markers in the Q~T~xu~Knal fluid (CSF) of patients with Alzheimex's disease (AD) have given inoonsisterfc f ~ . SJ/ice most stLKiies have bees1 performsd in clinically diagnosed patierfcs, inaccuracies in the diagnostics or coincidental presence of some other disease pathology may have confoLuK]ed the findings. 11%erefore, to study this point further we have reanalyzed the neurotrarmmdtter findirmjs of autopsied dementia cases with the histological verification of AD. In the group of 33 definite AD patients (25 F, 8 M) the data of CSF ~ t t e ~ markers w~re avail~]e ~ 5 ~o 18 ~ and these values were o o ~ a r e d with the f i r ~ of c o ~ control series. ~%e activity of cholinesterase, as marker of cholinergic neurons, was sig~ificarrtly lower (-27%) in ~D p ~ (mean + SO, 26.1 + 6.7 rn~ol/ml/min, n=12) as O ~ @ 8 L ~ to conL~uls (35.6 + 8.9, n=29, [3<0.01). T~e level of main dopsmine mst~dhnllte, homovanillic acid (HVA), was reduosd (-46%) significantly in the total group of AD patients (42.0 + 20.5 ng/ml, n=18, vs. 78.0 _+ 25.2, n=13, p<0.001). In the histopathologicsl ~ five oN& of these 18 AD cases shoved coincident pathology of Parkinson's disease (PD) and HVA in the CSF in these patients (18.3 + 3.1) was significantly lower c c ~ both to AD cases without PD pathology (-64%, 51.0 + 14.0, n=13, [3<0.01) and to the ~ s ~ u l s (-77%,p<0.001). The concentration of serotonin metabolite, 5hydroxyindoleacetic acid, was also slightly lower (-22%) in AD
322 ALZHEIMER CYTOSKELETAL PATHOLOGY: FROM MICROTUBULE ASSOCIATED PROTEIN TAU TO PAIRED HELICAL FILAMENTS. *lnge Grundke-lqbal and Khalid Iqbal, Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. Tau in Alzheimer disease (AD) brain is present in different modified forms. The earliest modification observed to date in neurons undergoing neurofibrillary changes is its abnormal phosphorylation. Based on their differential solubility in SDS, tangles have been classified as ANT I/PHF I and ANT II/PHF II for being made up of readily soluble and sparingly soluble PHF respectively. We have developed a method for the enrichment of ANT I and ANT II polypeptides from neuronal cell body fractions of Alzheimer brain. The neuronal cell bodies are isolated from cerebral cortex and extracted sequentially with 50raM Tris, 1% Triton X100, 8M urea/6OmM dithiothritol (D'I-F) and 1% SDS at room temperature, followed by boiling with 1% Uthium dodecylsulfate (LDS). Immunoblot analysis has revealed that most of the normal cytoskeletal proteins are extracted with urea/DTT. The SDS (ANT I) and LDS (ANT II) extracts from AD brain contain most of the abnormally phosphorylated tau (mAb Tau-1) and ubiquitin - reactive (mAb 5-25/3-39 to PHF) material whereas similar extracts from normal brain contained negligible levels of tau and ubiquitin immunoreactivity. The ANT I fraction seems to contain more of the fast moving species of the abnormally phosphorylated tau whereas the slowest moving 68kDa and 70kDa tau species are mostly present in the ANT II fractions. By densitometry of immunoblots the ubiquitin/tau ratio in the ANT II fractions is about 3-fold greater than in the ANT I fractions. In a separate study we have also isolated Alzheimer tau in a 27,000 to 100,000 xg pellet. This non-congophilic tau fraction although abnormally phosphorylated seems to be least modified by that it does not form aggregates on gel electrophoresis and does not contain ubiquitin. These studies suggest (1) that unpolymerized abnormal tau probably represent pre-tangles whereas ANT I and ANT II represent the mature tangle stages, containing tau with additional modifications and (2) that with increasing maturation the tangles become more difficult to depolymerize. (Supported in part by the Alzheimer's Disease Program of the American Health Assistance Foundation and NIH grants NS18105, AG05892 and AG04220). 323
IMPAIRMENTS IN CELLULAR FIBROUS PROTEINS: BIOLOGIC MARKER FOR ALZHEIMER DISEASE? *S.S. Matsuyama, West Los Angeles VA Medical Center, Brentwood Division, Los Angeles, CA 9007:5 and UCLA NPI, Los Angeles, CA 90024 USA. The search for a biologic marker For Alzheimer disease (AD) is being actively pursued. Although considered a neurodegenerative disease of the brain, there is mounting evidence oF abnormalities in peripheral tissues including red blood cells, ]ymphocytes, granulocytes, platelets and fibroblasts. Unlike brain tissue, which is obtained at autopsy or rarely biopsy, these tissues are readily available and Facilitate research investigations. In our laboratory, we are evaluating the possibility of a diagnostic test For AD based
331
on immunocytochemical examination oF cellular fibrous proteins in skin Fibr0blasts Following disruption with specific pharmacological agents, W e previously reported that Following colchicine treatment the reappearance of the microtubule network was significantly delayed in AD fibroblasts compared to normal ceils (AGE 12:127-132, 1989)suggesting an impairment in the microtubule system, Recently, we have Found evidence that the microFilamen[ system may also be involved (BondareFF and Matsuyama, unpublished observations), and are continuing investigations of microtubules and microFilaments in Fibroblasts From AD, non-AD demented patients and normal controls, 324 BRAIN NICOTINIC RECEPTOR DEFICITS IN ALZHEIMER PATIENTS AS STUDIED BY POSITRON EMISSION TOMOGRAPHY TECHNIQUE *A. Nordberg ~'e, P. Hartvlg 5, A. Litja 3, M. Viitanen 8, H. Lundqvlst4, J. Ulln 2, Y. Andersson =, B. Wlnbled', B.Langstrom 2, Depts of Pharmacology', Organic Chemistry~, Diagnostic Radiology~, The Svedberg Laboratory4, Uppsala University, Hospital Pharmacy~, University Hospital, Uppsala and Dept of Geriatric Medicine', Karolinska Institute, Huddinge Hospital, Huddinge, Sweden. Positron emission tomography (PET) is a technique suitable for In vlvo studies of central neuronal activity in brain. Until now the technique has been used In Alzhetmer patients in studies concerning cerebral blood flow and metabolism. Attempts have recently been made to measure cholinerglc nicotinic receptors using "C-nicotine in monkey and human brain by PET technique. We have eediar found a reduced number of high affinity nicotinic receptors In postmortem cortical brain tissues from Alzheimer patients. In order to visualize the nicotinic receptor defects in viv0 the (+)(R) and (-)(L) Isomers of '~C-nicotine have been given Intravenously in tracer doses (< 1/10 of the content of a cigarette) to Alzheimer patients and age matched healthy volunteers. The clinical diagnosis of Alzheimer's disease was made according to Diagnosis and Statistical Manual of Mental Disorders, DSM 111 criteria. Clinical examinations include blood and CSF laboratory, EEG, CT and MRI investigations. All patients and controls had undergone careful neuropsychometric testings. Seven Alzheimer patients (mean age 69+--2 ys; range 63-75, MMSE 17+-2) and six age-matched controls (mean age 69+-2 range 61-72) received an Intravenous bonus dose of "C-nicotine. The "C(-)-nicotine which peaks in brain within 2-5 mins and then decline during the following 15-20 mins shows a high content in brain areas as the thalamus, caudate nucleus, putamen, frontal and temporal cortex, intermediate in the occipital cortex, cerebellum and low in white matter. The uptake of "C-i-) nicotine Is lower and the time course is less steep in temporal cortex of Alzheimer patients compared to controls. In addition, a significant lower uptake of "C-i+) nicotine is found In the Alzheimer brains but not in the controls. "C-butanol was used as a marker for cerebral blood flow and the time course was different from that of both (+) and (-) "C-nicotine. The regional uptake of "C-butanol in control brains was rather homogenous while in Alzheimer brains a lower uptake of "C-butanol was found in temporal cortex, frontal cortex compared to thalamus.
325 NEL~OIRAN~'I'I'~ ~ IN THE C~RE~q~SPINAL FLUID OF PATI~I~TS ~ HISIDLOGICALL¥ VERIFIED ~ ' S DISEASE. * K.J. Reinikainen, H. Soininen, L. Pal j&rvi §, P.J. Riekkinen. Departments of Neurology and Pathology §, University of K~opio, 70211 Kuopio, Finland.
Studies of neurotransmitter markers in the Q~T~xu~Knal fluid (CSF) of patients with Alzheimex's disease (AD) have given inoonsisterfc f ~ . SJ/ice most stLKiies have bees1 performsd in clinically diagnosed patierfcs, inaccuracies in the diagnostics or coincidental presence of some other disease pathology may have confoLuK]ed the findings. 11%erefore, to study this point further we have reanalyzed the neurotrarmmdtter findirmjs of autopsied dementia cases with the histological verification of AD. In the group of 33 definite AD patients (25 F, 8 M) the data of CSF ~ t t e ~ markers w~re avail~]e ~ 5 ~o 18 ~ and these values were o o ~ a r e d with the f i r ~ of c o ~ control series. ~%e activity of cholinesterase, as marker of cholinergic neurons, was sig~ificarrtly lower (-27%) in ~D p ~ (mean + SO, 26.1 + 6.7 rn~ol/ml/min, n=12) as O ~ @ 8 L ~ to conL~uls (35.6 + 8.9, n=29, [3<0.01). T~e level of main dopsmine mst~dhnllte, homovanillic acid (HVA), was reduosd (-46%) significantly in the total group of AD patients (42.0 + 20.5 ng/ml, n=18, vs. 78.0 _+ 25.2, n=13, p<0.001). In the histopathologicsl ~ five oN& of these 18 AD cases shoved coincident pathology of Parkinson's disease (PD) and HVA in the CSF in these patients (18.3 + 3.1) was significantly lower c c ~ both to AD cases without PD pathology (-64%, 51.0 + 14.0, n=13, [3<0.01) and to the ~ s ~ u l s (-77%,p<0.001). The concentration of serotonin metabolite, 5hydroxyindoleacetic acid, was also slightly lower (-22%) in AD