$408 Poster Session P3: Histopathology

in cell-type specific accumulation of amino-terminally truncated amyloid beta. Strong intranenronal amyloid beta immunoreactivity over the entire lifespan was unrelated to neuronal death. The strongest intraneuronal amy- loid beta 17-42 immunoreactivity was observed in lateral geniculate body, dentate nucleus and nucleus olivaris inferior, known as structures with low susceptibility to fibrillar amyloid beta deposition or neurofibrillary changes. Conclusions: These observations indicate that a high level of intraneuronal amyloid beta immunoreactivity is not a predictor of cell susceptibility to neurofibrillary degeneration or involvement in fibrillar plaque formation.

• MICROVASCULAR DAMAGE IN ALZHEIMER'S DISEASE

Parag Butala* 1, Stephen Salloway I , Conrad E. Johanson 1 , Liliana Gonzalez 2, Rose Tavares 1, Virginia Hovanesian 1, John E. Donahue 1, Christine M. Hulette 3, Michael P. Vitek 3, Edward G. Stopa 1 . 1Brown University School of Medicine, Providence, RI, USA; 2 University of Rhode Island, Providence, RI, USA; SDuke University, Bryant Alzheimer Center, Durham, NC, USA. Contact e-mail: Parag_Butala@Brown.edu

Background: Alzheimer's Disease (AD) is a neurodegenerative disorder marked by characteristic pathology consisting of neurofibrillary tangles, beta-amyloid containing neocortical senile plaques, cerebral amyloid an- giopathy, and neuronal and synaptic loss. Clinically, the disease is defined by progressive dementia and memory loss and occurs in both familial and spo- radic forms. Between 10-15% of the population over the age of 65 develops AD, ranking it as the most prevalent neurodegenerative dementia and the fourth leading cause of death in the elderly. Objective(s): Microvasculature alterations and damage play an important role in AD progression, as cere- brovaseular plaque deposition accumulates along the basement membrane of cerebral vaseulature. Studies have shown that microvasculature damage, particularly through changes in smooth muscle cells, lead to progressively more severe forms of the disease. This study attempts to examine microvas- cular damage in AD by evaluating arteriolar smooth muscle. Methods: The present study analyzes smooth muscle in arterioles of AD patients through immunohistochemical staining for smooth muscle actin (SMA) and [~-amyloid. Arteriolar thickness is analyzed statistically. Results: By mea- suring the changes in the thickness of arterioles as well as the area of SMA and [~-amyloid staining across 86 AD cases and 20 age-matched controls, we have found statistically significant (p < 0.05) evidence of increased artefiolar thickness as a result of [~-amyloid plaque deposition in AD cases compared to age-matched controls. By taking the natural log of the thickness of the arterioles to equalize variance, we found a statistically significant (p < 0.02) difference between the mean thickness of the Braak III-IV cases and the Braak I-II cases, with the Braak I-II cases having evidence of increased amyloid deposition. We also found a nonsignificant trend towards increased arteriolar thickness with Braak V-VI cases compared to Braak III-IV cases. Conclusions: This study suggests evidence of a bimodal distribution of cerebrovascular plaque, with plaque clearance increasing in moderate forms of the disease and deposition increasing in the early and late portions of AD. These findings bear implications for amyloid-based immunization therapies to lower amyloid burden and treat AD.

~ T H E LOBAR DISTRIBUTION OF WHITE MATTER DISEASE IN ALZHEIMER'S DISEASE A NEUROPATHOLOGICAL IMAGE ANALYSIS STUDY

Mart~ Sjtbeck*, Mattias Haglund, Elisabet Enginnd. Jubileum Institute, Lund, Sweden. Contact e-mail: Martin.Sjobeck@pat. lu.se

Background: Concomitant white matter disease (WMD) of a non-focal character appears in more than 50% of Alzheimer's disease (AD) cases at neuropathological examination. White matter alterations are also frequently seen in-vivo with imaging methods in patients with AD, but the significance of these changes remains to be defined. There is increasing evidence that WMD alters the symptom profile in dementia by adding symptoms and signs of subcortical, not typically posterior, dysfunction. The lobar distribution of the WMD in the brain hypothetically affects the symptom

profile, but so far no quantitative study has addressed the differential regional engagement. Objective(s): To evaluate the distribution of WMD in AD on neuropathological specimens. Methods: Whole brain coronal stained sections from 16 cases with a neuropathological diagnosis of AD and concomitant WMD were scanned in a conventional flatbed desktop scanner. The WMD subtotal tissue loss was then measured with optical density (OD) values on the scanned images using a computer assisted image analysis software package. 9 AD cases void of WMD served as control cases. Results: The OD was significantly lower in the frontal, central, parietal mad temporal white matter of the AD-WMD cases compared with the pure AD cases.' OD difference between AD-WMD and AD was largest in the frontal lobes followed by parietal, central and temporal lobe white matter. No statistically significant difference was seen in occipital white matter between the groups. Conclusions: The WMD in AD is regionally variable and affects the lobes with different intensities, albeit preferentially in the frontal lobes. This finding could be helpful in further studies aimed at defining symptoms of WMD in AD.

• R E G I O N A L A L T E R A T I O N OF SYNAPSIN I IN THE H I P P O C A M P A L F O R M A T I O N OF ALZHEIMER PATIENTS

Song Qin*, Xiang-You Hu, Hao Xu, Jiang-Ning Zhou. School of Life Science, USTC, Hefei, China. Contact e-mail: qs@ustc.edu

Background: Synapsin I is a synaptic vesicle-associated protein, partici- pating synapse formation, regulating the synthesis of other synaptic vesicle proteins and promoting neurotransmitter release. The cognitive alterations in AD patients are considered to be associated with synaptic loss and neu- rofibtillary pathology in the limbic system and neocortex. The hippocampus, one of the most vulnerable regions in AD brain, plays an important role in the process of learning and memory. Objective(s): To detect whether regional alteration of synapsin I occurred in the hippocampal formation of Alzheimer's disease (AD) patients. Methods: Samples of hippocampus of 10 female AD patients and 10 non-demented female controls matched for age and postmortem delay were investigated immnnocytochemically. By the immunofluorescent labeling and laser confocal imaging technique, we examined the distribution of synapsin I in the CA1 and CA3 subfields of the hippocampus and the dentate gyrus (DG) in AD patients and controls. Results: There was a significant decrease of synapsin I immunoreactivity in the molecular layer of the dentate gyms and the stratum radiatum of the CA1 subfield in AD patients compared with controls (both P < 0.05). However, there was no difference in any other layers in the hippocampal formation of AD patients in comparison with the controls. In the present study, the puneta of synapsin I were widely distributed around the pretangles stained by Alz-50 and showed the same staining intensity as the pnncta surrounding the tangle-free neurons in AD hippocampus. Interestingly, some synapsin I immunoreactivity were colocalized with neuropile threads stained by Alz-50 especially in the stratum pyramidale of CA1 subfiedld of AD hippocampns. Conclusions: Our results are in agreement with previous study that the pathology of AD is not limited to a simple synaptic loss. The results imply that the regional decrease of synapsin I is associated with cytoskeletal changes as well as with ~-amyloid deposits in AD patients.

• CONCURRENT ALZHEIMER'S DISEASE-RELATED P A T H O L O G Y L O W E R S THE THRESHOLD F O R D E V E L O P I N G D E M E N T I A IN A R G Y R O P H I L I C GRAIN DISEASE

Dietmar R. Thal* i, Christian Schultz 2, Giovannina Botez 2, Mrak E. Robert 3 , Sue T. Griffin 3 , Otmar D. Wiesfler 1 , Heiko Braak 2, Esfifanos Ghehremedhin 2. 1 University of Bonn Medical Center, Bonn, Germany; 2Johann Wolfgang Goethe University, Frankfurt am Main, Germany; 3 University of Arkansas for Medical Sciences, Little Rock, AR, USA. Contact e-mail: Dietmar.Thal@uni-bonn.de

Background: Argyrophilic grain disease (AGD) constitutes a neurodegen- erative disorder that occurs in the brains of the elderly and affects 5% of

Poster Session P3: Molecular Mechanisms of Neurodegeneration - Apoptosis $409

all patients with dementia, tan-protein-containing lesions known as argy- rophilic grains in neuronal processes and coiled bodies in oligodendrocytes characterize this disease. Dementia is encountered in only a subset of cases that display the morphological pattern of AGD at autopsy. Objective: To de- termine the role of concurrent Alzheimer's disease (AD)-related pathology for the development of dementia in AGD patients. Methods: Autopsy brains from 30 demented and 49 non-demented AGD patients, from 46 AD patients and from 86 non-demented controls without AGD were staged for the dis- tribution of AD-related neurofibrillary changes and amyloid 13-protein (AI3) deposits. To identify differences in the distribution of AD-related pathology between demented and non-demented AGD cases, and to differentiate the pattern of AD-related changes in AGD cases from that seen in AD and non-demented controls, we compared the distribution of AI3-deposits and neurofibrillary changes among these groups of cases. Differences in the stages of the distribution of neurofibrillary changes and A[3-deposits among these groups were analyzed statistically. Conclusions: Demented AGD patients exhibited significantly higher stages of AD-related AI3-deposition and neurnfibrillary tangle pathology as compared to non-demented AGD cases, but significantly lower stages of both lesions compared to AD without AGD. In conclusion, AGD is a clinically relevant neurodegenerative entity that significantly lowers the threshold for developing dementia when it is associated with AD-related pathology.

• REDUCED SENSITIVITY TO APOPTOS1S IN AGING CORTICAL NEURONS

Seon Eui Hong*, Hyo Jung Kang, Byoung Jot Gwag. Ajou university, Suwon, Republic of Korea. Contact e-mail: ladykate@navercom

Background: While apoptosis as well as necrosis has been proposed to participate in neuronal death in neurodegenerative diseases including Alzheimer's disease, several lines of evidence suggests that aged cells appear to be resistant to apoptosis. Objective(s): The present study was aimed to delineate age-dependent response of neuronal cells to apoptosis-inducing insults. Methods: Cortical cells culture, Western blotting, Immunostaining, Calcium imaging Results: Cortical cells cultured for 10-12 days in vitro (DIV 10 - 12) underwent neuronal cell apoptosis over next 24 hr after exposure to stanrosporine, cyclosporin A, and beta amyloid. However, aged cortical cell cultures (DIV 30-32) did not show neuronal death following exposure to the pro-apoptotic agents. Activation of caspase-3 and release of cytochrome C were observed in young cortical neurons after exposure to stanrosporine but not in aged cortical neurons, suggesting that upstream events of mitochondria-dependent apoptosis signaling pathways may underlie reduced sensitivity to apoptosis with aging. The resistance to apoptosis in aging cortical neurons was associated with increased levels of [Ca2+]i. Conclusions: We propose that altered [Ca2+]i in aging plays a role in reduced sensitivity to apoptosis.

Poster Session P3: Molecular Mechani sms of Neurodegenerat ion - Apoptosis

• MUTAGENESIS-TOXICITY STUDIES OF THE AMYLOID BETA PEPTIDE OF A L Z H E I M E R ' S DISEASE

Giuseppe D. Ciccotosto* 1, Cyril C. Curtain 1,2, Deborah Tew 1 , Darryl Carrington 1, Colin L. Masters 1 , Robert A. Cberny l, Ashley I. Bush 1, Roberto Cappai 1, Kevin J. Barnham 1.1Department of Pathology, The University of Melbourne, and The Mental Health Research Institute of Victoria, Parkville, Australia; 2School of Physics and Materials Engineering, Monash University, Clayton, Australia. Contact e-mail: jcicco@unimelb.edu.au

Background: The amyloid beta peptide (AI3) is toxic to neuronal cells and it is strongly believed that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease (AD). What remains unknown is the nature of the toxic A[3 species and its precise mechanism of action. The importance of the methionine residue at position 35 (M35) and its reported role in the toxicity of AI3 is contentious. We have previously found that the oxidation of Met35 did not diminish its toxicity on neuronal cell cultures and it had a similar profile to the native peptide. Objective(s): To examine the effect of mutating the Met to Val at position 35 in AI342 (AI3M35V). Methods/Results: A[3M35V displayed a dose and time related increase in neuronal cell toxicity compared to the native All42. The tox- icity of A[3M35V was similarly rescued by the H202 inhibitor, catalase. AI342M35V produced similar amounts of H202 as the native AI3 peptide in an in vitro test tube assay. Cell binding studies also showed that AI3M35V had a faster rate of peptide uptake and binding from conditioned media. EPR spectra, which was used to examined the interaction between AI3 and Cu in a lipid environment, revealed that the valine substitution resulted in the peptide becoming less rigid in the bilayer region. The Cu 2+ EPR spectra showed that the copper coordination site of At3 was not affected. A reduction of Cu 2+ to :the EPR silent Cu + is much slower with the AI3M35V peptide, confirming earlier observations that the M35 residue in AI3 plays an important part in the redox behavior of the peptide in solution. Circular dichroism spectroscopy showed that both the native and mutant peptides displayed a mixture of a-helical and 13-sbeet conformations yet the native peptide was predomi- nantly a- helical while the AI~M35V was predominantly 13 sheet smacture. Conclusion: Our findings for AI3M35V support our previous observations seen for Met(O)AI3 and this has important implications for the mechanism of AI3 toxicity. These results suggest that the toxicity that we have observed for both A~, AI3Met(O) and A[~M35V occur via similar mechanisms.

~ T H E EFFECTS OF THE CHLAMYDIA PNEUMONIAE INFECTIOUS PROCESS ON NEURONAL CELLS: IMPLICATIONS FOR A L Z H E I M E R ' S DISEASE

Denah M. Appelt*, Mafia Roupas, Christine J. Hammond, Christopher S. Little, Brian J. Balin. Philadelphia College of Osteopathic Medicine, Philadelphia, PA, USA. Contact e-mail: DenahA @pcom.edu

Background: Chlamydia pneumoniae has been implicated in the patho- genesis of Alzheimer's disease (AD). While controversial, the focus of this study is the relationship between infection and neurodegeneration. Typically, obligate intracellular organisms, such as C. pneumoniae, if not cleared by the immune system will evoke an apoptotic process of the infected cell. Intriguingly, it has been demonstrated that infection with C. pneumoniae, of monocytes and epithelial cells, inhibits the apoptotic process. Objective(s): The importance of this work may be the correlation of infection with the apoptotic process observed in AD. The apoptotic process in AD has been shown to be initiated although there is debate whether it goes to completion. Our current study with infection in neuronal cells may provide insight into this process. Methods: In this current study, SK-N-MC neuroblastoma cells were infected with C. pneumoniae followed by the addition of stanrosporine to induce apoptosis. These cells were monitored for apoptotic changes by immunocytochemistry, immunoblotting, and electron microscopy. Re- suits: Our earlier studies found that human monocytes, endothelia, and glia infected with C. pneumoniae demonstrated increased production and processing of amyloid (A[3) 1-42; and yet, both proinflammatory molecules and At3 1-42 have been shown to induce apoptosis in neuronal cells in vitro. Hence, an infection with C pneumoniae may inhibit the apoptotic process while promoting a buildup of A[3 within neuronal cells. In our novel mufine model in which we infected non-transgenic mice with C. pneumoniae, we found deposits of A[3 in areas of the brain most affected in AD. The neurons in these areas showed intracellular A[3 1-42 immunoreactivity and these neu- rons did not appear to be apoptotic. Our results showed that both neuronal cells directly infected with C. pneumoniae and uninfected neighboring cells, in close contact, were less vulnerable to apoptosis. A persistent infection was maintained, in this paradigm, for up to three months. Conclusions: Inhibition of apoptosis may be one mechanism by which C. pneumoniae can sustain an infection in the host to maintain an optimal intracellular environment. This cumulative data will further our knowledge into the relationship between infection and apoptosis in the pathogenesis of AD.