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506 Quantification of neurofibrillary damage in Alzheimer's disease, Down's syndrome and dementia pugilistica
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FIFTH INTERNATIONAL CONFERENCE ON A L Z H E I M E R ' S DISEASE
In our age matched population with no or only subjective cognitive complains (GDS=2) no subject (0% of 58 patients) had 414 isotype of APOE. In patients with probable AD, 15% of 117 subjects as well as 8% of 38 mildly impaired patients (GDS=3) had 4/4 APOE isotype. While patients with probable AD and 4/4 isotype had 8.30 _.+ 3.7 medical problems\conditions, individuals with only subjective cognitive decline had only 4.8 _.+ 2.8 medical conditions. In the AD group, females had significantly more medical conditions (9.3 + 3.4) than males (4.7 _.+2.5). In the group with only subjective cognitive complains, males and females had similar number of medical problems (4.8 _+ 2.8 and 4.2 + 3.2, respectively). Frequency of cardiovascular disorders, history of surgical procedures and that of electrolyte/endocrine disturbances were significantly different. In the AD population all patients (100%) had cardiovascular problems, in the GDS=2 group only 41% of subjects. 79% of AD patients have history of surgical procedures in the past, whereas 31% of subjects with only subjective cognitive complains have this history. 50% of patients with AD showed endocrine/electrolyte abnormalities in comparison to 28% of subjects with GDS=2. Elderly patients with dementia and ApoE 4/4 isotype have markedly more medical conditions and problems than individuals of the same age with only subjective cognitive decline.
Molecular Pathology H 506 Quantification of neurofibrillary damage in Alzheimer's disease, Down's syndrome and dementia pugilistica J.E. MeKenzie .1, G.W. Roberts2, M.C. Royston1 1. Departmentof Psychiatry,ChafingCross & WeslminsterMedical School, London,UK. 2. Depa_nmentof MolecularNeuropathologyResearch,SmlthKlineBeecham,Harlow,EssexUK. Cytoskeletal lesions are important features in disease classifications and investigations. The presence of neurofibrillary tangles is used in the diagnosis of Alzheimer's disease (AD). However these lesions are not pathognomonie and they have been reported in a range of other disorders such as Down's syndrome (DS) and dementia pugilistiea (DP). DS exhibits similar pathology to that seen in AD and adult DS patients develop the clinical and neuropathologieal features characteristic of AD. Although there are strong genetic links for the pathogenesis of AD these eases only account for ~ 20% of the total. The remaining sporadic eases are attributed to environmental factors (with/without genetic interactions), of which head injury is one of the most significant. With the discovery of plaques and neurofibriUary tangles in DP there has been some debate regarding the relationship between AD and DP (and head injury). One approach to determine whether AD, DS and DP share a common pathogenic mechanism is to undertake quantitative neurofibrillary studies. We have developed a novel technique, using colour image analysis, which can accurately quantify the total amount of neurofibrillary damage present. Furthermore we have developed a set of mathematically defined morphological criteria to allow objective discrimination between the three types of neurofibrillary damage seen in the cortex, namely neurofibrillary tangles, neurites and neuropil threads immunostained with Alz-50. Use of this novel technique provides a reliable, rational means for the classification of neurofibrillary lesions. We have quantified the extent of neurofibrillary damage in AD, DS, and DP. From our investigations it was found that neurofibrillary deposition was very similar in DS and AD highlighting the similarity in the underlying pathological mechanisms. However, more surprising, was the discovery of strong parallels in neurofibrillary deposition found between AD and DP. The conclusions from this investigation that both genetic (DS) and environmental (DP) risk factors for AD present with a similar pattern of neurofibrillary degeneration supports the theory that similar degenerative mechanisms are being triggered by both conditions.
505 c~l-Antichymotrypsin genotypes modulate the risk of Alzheimer's disease associated with apolipoprotein E-~4 allele. A. Ueki*, M. Otsuka* and K. Ikeda *Departmet of Neurology, Jichi Medical School, Omiya Medical Center, 1-847 Amanuma, Omiya City, Saitama 330, Japan and Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, Setagaya, Tokyo 156, Japan Apolipoprotein E-E4 allele (APOE*4) is a major risk factor of Alzheimer's disease (AD). However, there are many cognitively normal elderly who carry APOE*4 implying the existence of other genetic factors which modify the effects of APOE*4 for developing AD. Recently, Kamboh et al. have shown that APOE*4 dosage effect associated with AD risk is significantly modulated by the o~l-antichymotrypsin (ACT) genotype (Nature Genet 10: 486,1995). To test their hypothesis we examined ACT genotypes in AD and age matched controls, and compared their effect s on APOE*4 carriers and non-carriers. A_POE and ACT were genotyped in 55 AD patients (mean age at onset,73.0; range, 60-84) and 90 healthy controls (mean age, 72.2; range, 60-91). ACT genotypes were determined by the methods according to Kamboh et al. Three common ACT genotypes were expressed as *AA, *AT, *TT. The distribution of ACT genotypes in total AD patients and controls did not differ significantly. The frequencies of *AA, *AT, *TT were 0.176, 0.456, 0.368, in total AD, and 0.111, 0.467, 0.422 in total contrlos, respectively. However, the distribution of ACT genotypes differed significantly when compared between subgroups of AD and controls earring APOE*4. The frequencies of *AA, *AT, *'IT in APOE*4 carriers of AD (n=36) were 0.194, 0.556, 0.250, and those in APOE*4 carriers of contrlos (n=15) were 0.000, 0.400, 0.600, respectively. Allele frequency of ACT*A in APOE*4 carriers of controls was signifcantly lower than that in APOE*4 carriers of AD (0.200 vs. 0.446, X2= 4.7 .p=0.03). The distribution patterns of ACT genotypes in APOE*4 non-carriers of AD and controls were almost identical. Our data show that ACT behaves as a modifier gene that alters the AD risk associated with the APOE*4 allele. Combination of the APOE and ACT genotypes might become a better susceptibility marker for AD than conventionally used test with APOE genotype alone.
507 Distribution of Phosphorylated Tau in Neonatal and Adult Rat Brains K. Tashiro*~, M. Hasegawa2, Y. lhara 2 and T. lwatsubo~ Department of Neuropathology and Neuroscience I, Faculty of Pharmaceutical Sciences, Department of Neuropathology z, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku Tokyo 113, Japan It has been widely accepted that normal tan in adult brains is poorly phosphorylated and localized predominantly in axons, whereas PHFs in Alzheimer's disease brains are composed of hyperphosphorylated isoforms of tan and deposit in the neuronal somata and dendrites, and tan in juvenile brains is more phosphorylated than adult tan. However, recently, the apparent poor phosphorylation of normal adult tan has been shown to result from postmortem dephosphorylation. Hence, we studied by immunocytochemistry the precise distribution of phosphorylated tan (ptau) in the cerebrum of neonatal and adult rat brains using following monoclonal antibodies that recognize distinct phosphorylated tau epitopes: AT8 (Ser202 and Ser 205, P+; amino acid numbers according to the longest human tau isoform indicate the phosphorylated residues recognized by each antibody; P+: phosphorylation state specific, P-: dephosphorylation state specific), tau 1 (Ser199 and Ser202, P-), M4 (Thr231, P+), C5 (Ser396, P+) and PHF-1 (Ser396 and Ser404, P+). Neonatal (1 day) and adult (8 weeks) rat brains were fixed transcardially by perfusion of 4% paraformaldehyde (PFA) and cut on a vibratome, or fixed by immersion in Bouin's solution and embedded in paraffin. To examine the effect of energy deprivation on the phosphorylation state of tan in situ, Ringer's solution was perfused for 1, 5, 10 and 30 min prior to fixation by perfusion of 4% PFA. In neonatal brains, ptau immunoreactivities were detected in the apical dendrites and somata of the pyramidal neurons as well as in the subcortical axons with all ptau antibodies regardless of the fixation methods. In adult brains, ptau immunoreactivities were faint or negative in specimens with prolonged preperfusion or with Bouin's fixation. However, briefly preperfused vibratome sections showed positive ptau immunoreactivities in apical dendrites and somata as well as in axons. In contrast, tau 1 immunoreactivity in dendrites and somata increased with prolonged preperfusion. Oligodendrocytes
FIFTH INTERNATIONAL CONFERENCE ON A L Z H E I M E R ' S DISEASE
In our age matched population with no or only subjective cognitive complains (GDS=2) no subject (0% of 58 patients) had 414 isotype of APOE. In patients with probable AD, 15% of 117 subjects as well as 8% of 38 mildly impaired patients (GDS=3) had 4/4 APOE isotype. While patients with probable AD and 4/4 isotype had 8.30 _.+ 3.7 medical problems\conditions, individuals with only subjective cognitive decline had only 4.8 _.+ 2.8 medical conditions. In the AD group, females had significantly more medical conditions (9.3 + 3.4) than males (4.7 _.+2.5). In the group with only subjective cognitive complains, males and females had similar number of medical problems (4.8 _+ 2.8 and 4.2 + 3.2, respectively). Frequency of cardiovascular disorders, history of surgical procedures and that of electrolyte/endocrine disturbances were significantly different. In the AD population all patients (100%) had cardiovascular problems, in the GDS=2 group only 41% of subjects. 79% of AD patients have history of surgical procedures in the past, whereas 31% of subjects with only subjective cognitive complains have this history. 50% of patients with AD showed endocrine/electrolyte abnormalities in comparison to 28% of subjects with GDS=2. Elderly patients with dementia and ApoE 4/4 isotype have markedly more medical conditions and problems than individuals of the same age with only subjective cognitive decline.
Molecular Pathology H 506 Quantification of neurofibrillary damage in Alzheimer's disease, Down's syndrome and dementia pugilistica J.E. MeKenzie .1, G.W. Roberts2, M.C. Royston1 1. Departmentof Psychiatry,ChafingCross & WeslminsterMedical School, London,UK. 2. Depa_nmentof MolecularNeuropathologyResearch,SmlthKlineBeecham,Harlow,EssexUK. Cytoskeletal lesions are important features in disease classifications and investigations. The presence of neurofibrillary tangles is used in the diagnosis of Alzheimer's disease (AD). However these lesions are not pathognomonie and they have been reported in a range of other disorders such as Down's syndrome (DS) and dementia pugilistiea (DP). DS exhibits similar pathology to that seen in AD and adult DS patients develop the clinical and neuropathologieal features characteristic of AD. Although there are strong genetic links for the pathogenesis of AD these eases only account for ~ 20% of the total. The remaining sporadic eases are attributed to environmental factors (with/without genetic interactions), of which head injury is one of the most significant. With the discovery of plaques and neurofibriUary tangles in DP there has been some debate regarding the relationship between AD and DP (and head injury). One approach to determine whether AD, DS and DP share a common pathogenic mechanism is to undertake quantitative neurofibrillary studies. We have developed a novel technique, using colour image analysis, which can accurately quantify the total amount of neurofibrillary damage present. Furthermore we have developed a set of mathematically defined morphological criteria to allow objective discrimination between the three types of neurofibrillary damage seen in the cortex, namely neurofibrillary tangles, neurites and neuropil threads immunostained with Alz-50. Use of this novel technique provides a reliable, rational means for the classification of neurofibrillary lesions. We have quantified the extent of neurofibrillary damage in AD, DS, and DP. From our investigations it was found that neurofibrillary deposition was very similar in DS and AD highlighting the similarity in the underlying pathological mechanisms. However, more surprising, was the discovery of strong parallels in neurofibrillary deposition found between AD and DP. The conclusions from this investigation that both genetic (DS) and environmental (DP) risk factors for AD present with a similar pattern of neurofibrillary degeneration supports the theory that similar degenerative mechanisms are being triggered by both conditions.
505 c~l-Antichymotrypsin genotypes modulate the risk of Alzheimer's disease associated with apolipoprotein E-~4 allele. A. Ueki*, M. Otsuka* and K. Ikeda *Departmet of Neurology, Jichi Medical School, Omiya Medical Center, 1-847 Amanuma, Omiya City, Saitama 330, Japan and Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, Setagaya, Tokyo 156, Japan Apolipoprotein E-E4 allele (APOE*4) is a major risk factor of Alzheimer's disease (AD). However, there are many cognitively normal elderly who carry APOE*4 implying the existence of other genetic factors which modify the effects of APOE*4 for developing AD. Recently, Kamboh et al. have shown that APOE*4 dosage effect associated with AD risk is significantly modulated by the o~l-antichymotrypsin (ACT) genotype (Nature Genet 10: 486,1995). To test their hypothesis we examined ACT genotypes in AD and age matched controls, and compared their effect s on APOE*4 carriers and non-carriers. A_POE and ACT were genotyped in 55 AD patients (mean age at onset,73.0; range, 60-84) and 90 healthy controls (mean age, 72.2; range, 60-91). ACT genotypes were determined by the methods according to Kamboh et al. Three common ACT genotypes were expressed as *AA, *AT, *TT. The distribution of ACT genotypes in total AD patients and controls did not differ significantly. The frequencies of *AA, *AT, *TT were 0.176, 0.456, 0.368, in total AD, and 0.111, 0.467, 0.422 in total contrlos, respectively. However, the distribution of ACT genotypes differed significantly when compared between subgroups of AD and controls earring APOE*4. The frequencies of *AA, *AT, *'IT in APOE*4 carriers of AD (n=36) were 0.194, 0.556, 0.250, and those in APOE*4 carriers of contrlos (n=15) were 0.000, 0.400, 0.600, respectively. Allele frequency of ACT*A in APOE*4 carriers of controls was signifcantly lower than that in APOE*4 carriers of AD (0.200 vs. 0.446, X2= 4.7 .p=0.03). The distribution patterns of ACT genotypes in APOE*4 non-carriers of AD and controls were almost identical. Our data show that ACT behaves as a modifier gene that alters the AD risk associated with the APOE*4 allele. Combination of the APOE and ACT genotypes might become a better susceptibility marker for AD than conventionally used test with APOE genotype alone.
507 Distribution of Phosphorylated Tau in Neonatal and Adult Rat Brains K. Tashiro*~, M. Hasegawa2, Y. lhara 2 and T. lwatsubo~ Department of Neuropathology and Neuroscience I, Faculty of Pharmaceutical Sciences, Department of Neuropathology z, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku Tokyo 113, Japan It has been widely accepted that normal tan in adult brains is poorly phosphorylated and localized predominantly in axons, whereas PHFs in Alzheimer's disease brains are composed of hyperphosphorylated isoforms of tan and deposit in the neuronal somata and dendrites, and tan in juvenile brains is more phosphorylated than adult tan. However, recently, the apparent poor phosphorylation of normal adult tan has been shown to result from postmortem dephosphorylation. Hence, we studied by immunocytochemistry the precise distribution of phosphorylated tan (ptau) in the cerebrum of neonatal and adult rat brains using following monoclonal antibodies that recognize distinct phosphorylated tau epitopes: AT8 (Ser202 and Ser 205, P+; amino acid numbers according to the longest human tau isoform indicate the phosphorylated residues recognized by each antibody; P+: phosphorylation state specific, P-: dephosphorylation state specific), tau 1 (Ser199 and Ser202, P-), M4 (Thr231, P+), C5 (Ser396, P+) and PHF-1 (Ser396 and Ser404, P+). Neonatal (1 day) and adult (8 weeks) rat brains were fixed transcardially by perfusion of 4% paraformaldehyde (PFA) and cut on a vibratome, or fixed by immersion in Bouin's solution and embedded in paraffin. To examine the effect of energy deprivation on the phosphorylation state of tan in situ, Ringer's solution was perfused for 1, 5, 10 and 30 min prior to fixation by perfusion of 4% PFA. In neonatal brains, ptau immunoreactivities were detected in the apical dendrites and somata of the pyramidal neurons as well as in the subcortical axons with all ptau antibodies regardless of the fixation methods. In adult brains, ptau immunoreactivities were faint or negative in specimens with prolonged preperfusion or with Bouin's fixation. However, briefly preperfused vibratome sections showed positive ptau immunoreactivities in apical dendrites and somata as well as in axons. In contrast, tau 1 immunoreactivity in dendrites and somata increased with prolonged preperfusion. Oligodendrocytes