- Email: [email protected]
P3-196
S432
Poster P3:: Tuesday Posters
“Italian woman”. Genealogical enquiry evidenced that she was born in Cairo (Egypt) in 1895 of Italian parents descending from the common ancestor of the PS1-Met146Leu family. Objective(s): To study tau pathology of PS1-Met146Leu Italian branch. Methods: Paraffin-embedded brain sections belonging to two PS1-Met146Leu Italian patients were immunohistochemically stained with anti-phospho tau antibody AT8, anti-3R (RD3) and anti-4Repeats tau (RD4) antibodies. Immunoblot of sarcosylinsoluble tau was also performed. Results: Immunohistochemical staining of the hippocampus and cortex revealed widespread tau pathology. Abundant neuritic plaques, neurofibrillary tangles and neuropil threads were observed in both areas by antibodies AT8 and RD3 while fewer deposits were stained by antibody RD4. Occasional Pick body-like inclusions were observed in the hippocampus; these structures were more numerous in the cortical area although their number was limited, compared to the other type of tau aggregates. Double staining with AT8 and RD4 showed that, differently from Pick bodies found in sporadic Pick disease, the observed inclusions could contain tau with 4 repeats. Immunoblot of sarcosylinsoluble tau extracted from parietal, frontal and temporal cortex showed the 4 bands of 60, 64, 68 and 72 kDa characteristic of Alzheimer’s disease cases. Although by immunohistochemistry RD3 antibody recognized more tau deposits than RD4, dephosphorylation of sarcosyl-insoluble tau with alkaline phosphatase showed the presence of all 6 tau isoforms in all investigated cortical areas. Conclusions: The Italian branch shows only occasional Pick body-like inclusions. Moreover, the presence of all tau isoforms account for a clear AD pathology.In view of the fact that Pick bodies have been indicated as an important feature in the Australian branch of the same family we can conclude that PSI-Met146Leu mutation can present variable neuropathological features suggesting that during centuries environmental and genetic factors can contribute to modify the phenotype. P3-194
OCCLUDIN EXPRESSION IN ALZHEIMER DISEASE AND VASCULAR DEMENTIA
Mihaela O. Romanitan1, Bengt Winblad1, Inga Volkmann1, Ovidiu Al. Bajenaru2, Nenad Bogdanovic1, 1Neurotec, Stockholm, Sweden; 2Emergency University Hospital, Bucharest, Romania. Contact e-mail: [email protected] Background: The blood brain barrier is essential for the maintenance and regulation of the neural microenvironment. The permeability of the BBB is determined by the inter-endothelial junctions. In mammalian cells, intercellular junctions are categorized into four types: adherens junctions (AJ), desmosomes (D), gap junctions (GJ) and tight junctions (TJ). TJ is a specialized membrane domain localized between endothelial cells of brain microvessels and epithelial cells of the choroid plexus, and is the main structure responsible for the barrier and fence functions of the BBB. Occludin, protein of 65 kDa, is the best-known integral membrane protein that contributes to the electrical barrier function of the TJs and likely to the formation of aqueous pores within TJ strands. Pathological conditions in Alzheimer disease, vascular dementia and other neurodegenerative disease can lead to increase of the permeability in the brain microvasculature and consecutively damage the BBB. Objectives: In this study we wanted to characterize the morphological expression of occludin in AD and vascular dementia comparing to matched control brains. Methods: Immunohistochemical analyses were applied to frontal and basal ganglia samples obtained from Huddinge Brain Bank. We performed immunostaining using anti-occludin antibodies. Results: In control brains occludin immunopositivity was found predominantly in oligodendrocytes ⬎ neurons ⬎ microvessels ⬎ astrocytes. Among the neuronal population the apical dendrites of pyramidal neurons were mainly positive. In AD occludin expression was especially increased in the white matter oligodendrocytes. In the pyramidal neurons increase of expression was obtained in somatic compartment ⬎ apical dendrite. In the microvessels occludin was localized exclusively in endothelium. In vascular dementia the occludin was apparently less expressed in oligodendrocyte in white matter while in the vessels the staining was found throughout entire wall. There was no difference in
the neuronal staining compared with the control brains. In basal ganglia occludin immunopositivity was found in AD merely in the astroglial-like cells. In contrast, in vascular dementia, occludin is expressed in vessels, oligoglial cells and neuronal fibers. Conclusions: Our finding suggests that occludin expression is cell-, region- and disease-specific indicating a selective TJ dysfunction and breakdown of BBB. P3-195
STEREOLOGICAL STUDY ON NEUROGLIAL CELLS IN THE INFERIOR OLIVARY NUCLEUS IN NORMAL AGING AND IN ALZHEIMER DISEASE
Helen Lasn, Bengt Winblad, Nenad Bogdanovic, Neurotec, Stockholm, Sweden. Contact e-mail: [email protected] Background: It is likely that neuronal loss occurs in certain brain regions in Alzheimer Disease (AD) without any neurofibrillary pathology. In the human, principal inferior olivary nucleus (PO) displays no NFTs in sporadic AD cases, but diffuse amyloid-plaques can be seen in affected individuals in relation to Braak staging. We have shown that neuronal loss is about 34% (Lasn, JAD, 2001), but the fate of neuroglial cells is unknown. Since the unique network of neurons and neuroglial cells and their cohabitation are essential for normal functioning in CNS, it is likely that an imbalance in one of these cell groups perturbs this morphofunctional network. Objective: The aim of this study is to estimate the total number of oligodendrocytes and astrocytes in human PO to obtain unbiased and comparable results. Methods: The study is based on 10 control and 11 post-mortem AD human brains from Huddinge Brain Bank. The sections were stained with cresyl-violet. An unbiased design-based fractionator method was applied. Results: We found significant oligodendroglial cell loss (46%) in AD as compared to control brains, while the total number of astrocytes remains unaffected. It is likely that the ratio of oligodendroglial cells to neurons is unchanged even in degenerative states, indicating that oligodendroglial cells parallel neuronal loss. Astroglial cells did not display astrocytosis, as often reported in other regions affected by neurofibrillary tangles and neuritic plaques. Conclusions: Using novel unbiased quantitative methods, we were able to estimate accurately neuronal and oligodendroglial loss in the PO. It is recognized that the proximity of oligodendroglial cells to neurons plays an important role in neuronal survival. It is still unclear which cell group degenerates first in AD. Some recent data suggest that oligodendrocytes are more vulnerable to amyloid toxicity. P3-196
CHARACTERIZATION OF UBIQUITINATED INTRANEURONAL INCLUSIONS IN A NOVEL BELGIAN FTD (FTLD-U) FAMILY
Daniel Pirici1, Julie van der Zee1, Rik Vandenberghe2, Rosa Rademakers1, Krist’l Vennekens1, Ivy Cuijt1, Ursula Lu¨bke3, Chantal Ceuterick3, Jean-Jacques Martin3, Christine Van Broeckhoven1, Samir Kumar-Singh1, 1Flanders Interuniversity Institute for Biotechnology, Institute Born Bunge, University of Antwerp, Antwerp, Belgium; 2University Hospital Gasthuisberg, Division of Neurology, Leuven, Belgium; 3Laboratory of Neuropathology, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium. Contact e-mail: [email protected] Background: The most common histological feature in patients with frontotemporal lobar degeneration (FTLD) is intracellular brain inclusions of yet uncharacterized protein that react with anti-ubiquitin (Ub) antibodies, but not with tau or synuclein (FTLD-U). There is a growing consensus that FTLD-U entity might be much more common than originally thought and described. Objective: To characterize clinically, genetically, and pathologically a 4-generation Belgian FTLD family. Methods: Clinical diagnosis was based on neurological examination, medical history and informant interview. Mutation analysis on genomic DNA was performed for the MAPT, PSEN1, PSEN2, APP and PRNP genes. One patient was available for neuropathological analysis and a detailed histological and immunohistochemical examination was performed here utilizing more than
Poster P3:: Tuesday Posters 40 antibodies directed against, for instance, transcription factors, cellspecific antigens (p62, HLA-DR, GFAP, NeuN), heat-shock proteins (HSP), and cytoskeletal components. Stereologic point-counting techniques and Western blotting were used to quantify neuronal loss and soluble tau protein, respectively. Results: Clinically, 8 patients had FTLD. Behavioral problems and aphasia were an important finding and at least three patients suffered from parkinsonian features. No mutations were identified in MAPT, APP, PS1, PS2, and PRNP. We showed frontotemporal atrophy with filamentous Ub-positive intracellular inclusions in absence of tau pathology or any alterations in the levels of soluble tau. We characterized their cellular and subcellular localization and morphology. Ub-positive inclusions predominantly occurred within neurons (⬎97%), but were also observed within oligodendroglia (approx. 2%), microglia (⬍1%), but not within astroglia. Regarding the subcellular localization, the intranuclear inclusions (INI) were up to approx. 4 fold more frequent than the cytoplasmic inclusions, although the latter were more specific to neurons. The INIs frequently appeared spindle-shaped and 3-D confocal reconstructions identified flattened, leaf-like structures. Ultrastructurally, straight 10-18 nm diameter filaments constituted the spindle-shaped inclusions that occurred in close proximity to the nuclear membrane. Staining for HSP40, p62, and valosin/p97 was observed in only a minority of the inclusions. Conclusion: While the precise nature of the protein remains
S433
elusive, characterization of such familial FTLD-U patients would be helpful in identifying a common denominator in the pathogenesis of familial and the more prevalent sporadic FTLD-U. P3-197
CYSTIC BORRELIA IN ALZHEIMER’S DISEASE AND IN NON-DEMENTIA NEUROBORRELIOSIS
Alan B. MacDonald1,2, 1Columbia University, Smithtown, NY, USA; 2St Catherine of Siena Medical Center, Smithtown, NY, USA. Contact email: [email protected] Background: A cystic form for Borrelia burgdoferi (Bb) was initially reported in 1988 in an autopsy study of Alzheimer’s disease tissue obtained from Dr. George Glenner’s brain bank. Cystic profiles were documented with silver stains, and with Murine Monoclonal antibodies to a Flagellin Epitope specific for Bb and B. hermseii [H9724 from Dr Alan Barbour]. Objective(s): Fresh frozen hippocampus tissues from Alzheimer’s disease cases provided by the Harvard University McLean Hospital brain bank were cultured in BSK M medium to attempt to grow spirochetes in vitro. Methods: Triturated fresh hippocampus was cultured in vitro in sterile BSK M liquid media at 24 degrees C for one year. Darkfield microscopy examination and Acridine orange staining with epifluorescence microscopy was completed. Detection of specific Flagellin DNA sequences from ORF BBO147 using a Molecular Beacon DNA probe was used to measure incremental increases in Borrelia specific Flagellin DNA in cultures, as compared with the original fresh tissue retained uncultured DNA Extracts. Results: Cystic borrelia structures were recovered from in vitro cultures of fresh Alzheimer disease hippocampus tissue. Incremental increases in Borrelia burgdorferi flagellin B DNA were documented in cultured tissues. Conclusions: A subset of Alzheimer’s disease is related to chronic Neuroborreliosis in the human host.
P3-198
A CASE OF EARLY ONSET ALZHEIMER’S DISEASE WITH COTTON WOOL PLAQUES BUT WITHOUT SPASTIC PARAPARESIS
Beata Sikorska1, Pawel P. Liberski1, Herbert Budka2, 1Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland; 2Institute of Neurology, Medical University of Vienna, Vienna, Austria. Contact e-mail: [email protected] Background: We report a case of 32-year-old man with myoclonus, rapidly progressive dementia, apraxia, ataxic gait and subtle right hemiparesis. The clinical and first pathological diagnosis was Creutzfeldt Jakob disease. Methods: A brain biopsy was obtained from the left temporoparietal area. Autopsy was performed one hour after death. Formalin-fixed, paraffin embedded tissues were used for routine and immunohistochemical microscopic stainings. Part of the autopsy material was fixed in 4% glutaraldehyde in cacodylate buffer for electron microscopy. Results: On
Poster P3:: Tuesday Posters
“Italian woman”. Genealogical enquiry evidenced that she was born in Cairo (Egypt) in 1895 of Italian parents descending from the common ancestor of the PS1-Met146Leu family. Objective(s): To study tau pathology of PS1-Met146Leu Italian branch. Methods: Paraffin-embedded brain sections belonging to two PS1-Met146Leu Italian patients were immunohistochemically stained with anti-phospho tau antibody AT8, anti-3R (RD3) and anti-4Repeats tau (RD4) antibodies. Immunoblot of sarcosylinsoluble tau was also performed. Results: Immunohistochemical staining of the hippocampus and cortex revealed widespread tau pathology. Abundant neuritic plaques, neurofibrillary tangles and neuropil threads were observed in both areas by antibodies AT8 and RD3 while fewer deposits were stained by antibody RD4. Occasional Pick body-like inclusions were observed in the hippocampus; these structures were more numerous in the cortical area although their number was limited, compared to the other type of tau aggregates. Double staining with AT8 and RD4 showed that, differently from Pick bodies found in sporadic Pick disease, the observed inclusions could contain tau with 4 repeats. Immunoblot of sarcosylinsoluble tau extracted from parietal, frontal and temporal cortex showed the 4 bands of 60, 64, 68 and 72 kDa characteristic of Alzheimer’s disease cases. Although by immunohistochemistry RD3 antibody recognized more tau deposits than RD4, dephosphorylation of sarcosyl-insoluble tau with alkaline phosphatase showed the presence of all 6 tau isoforms in all investigated cortical areas. Conclusions: The Italian branch shows only occasional Pick body-like inclusions. Moreover, the presence of all tau isoforms account for a clear AD pathology.In view of the fact that Pick bodies have been indicated as an important feature in the Australian branch of the same family we can conclude that PSI-Met146Leu mutation can present variable neuropathological features suggesting that during centuries environmental and genetic factors can contribute to modify the phenotype. P3-194
OCCLUDIN EXPRESSION IN ALZHEIMER DISEASE AND VASCULAR DEMENTIA
Mihaela O. Romanitan1, Bengt Winblad1, Inga Volkmann1, Ovidiu Al. Bajenaru2, Nenad Bogdanovic1, 1Neurotec, Stockholm, Sweden; 2Emergency University Hospital, Bucharest, Romania. Contact e-mail: [email protected] Background: The blood brain barrier is essential for the maintenance and regulation of the neural microenvironment. The permeability of the BBB is determined by the inter-endothelial junctions. In mammalian cells, intercellular junctions are categorized into four types: adherens junctions (AJ), desmosomes (D), gap junctions (GJ) and tight junctions (TJ). TJ is a specialized membrane domain localized between endothelial cells of brain microvessels and epithelial cells of the choroid plexus, and is the main structure responsible for the barrier and fence functions of the BBB. Occludin, protein of 65 kDa, is the best-known integral membrane protein that contributes to the electrical barrier function of the TJs and likely to the formation of aqueous pores within TJ strands. Pathological conditions in Alzheimer disease, vascular dementia and other neurodegenerative disease can lead to increase of the permeability in the brain microvasculature and consecutively damage the BBB. Objectives: In this study we wanted to characterize the morphological expression of occludin in AD and vascular dementia comparing to matched control brains. Methods: Immunohistochemical analyses were applied to frontal and basal ganglia samples obtained from Huddinge Brain Bank. We performed immunostaining using anti-occludin antibodies. Results: In control brains occludin immunopositivity was found predominantly in oligodendrocytes ⬎ neurons ⬎ microvessels ⬎ astrocytes. Among the neuronal population the apical dendrites of pyramidal neurons were mainly positive. In AD occludin expression was especially increased in the white matter oligodendrocytes. In the pyramidal neurons increase of expression was obtained in somatic compartment ⬎ apical dendrite. In the microvessels occludin was localized exclusively in endothelium. In vascular dementia the occludin was apparently less expressed in oligodendrocyte in white matter while in the vessels the staining was found throughout entire wall. There was no difference in
the neuronal staining compared with the control brains. In basal ganglia occludin immunopositivity was found in AD merely in the astroglial-like cells. In contrast, in vascular dementia, occludin is expressed in vessels, oligoglial cells and neuronal fibers. Conclusions: Our finding suggests that occludin expression is cell-, region- and disease-specific indicating a selective TJ dysfunction and breakdown of BBB. P3-195
STEREOLOGICAL STUDY ON NEUROGLIAL CELLS IN THE INFERIOR OLIVARY NUCLEUS IN NORMAL AGING AND IN ALZHEIMER DISEASE
Helen Lasn, Bengt Winblad, Nenad Bogdanovic, Neurotec, Stockholm, Sweden. Contact e-mail: [email protected] Background: It is likely that neuronal loss occurs in certain brain regions in Alzheimer Disease (AD) without any neurofibrillary pathology. In the human, principal inferior olivary nucleus (PO) displays no NFTs in sporadic AD cases, but diffuse amyloid-plaques can be seen in affected individuals in relation to Braak staging. We have shown that neuronal loss is about 34% (Lasn, JAD, 2001), but the fate of neuroglial cells is unknown. Since the unique network of neurons and neuroglial cells and their cohabitation are essential for normal functioning in CNS, it is likely that an imbalance in one of these cell groups perturbs this morphofunctional network. Objective: The aim of this study is to estimate the total number of oligodendrocytes and astrocytes in human PO to obtain unbiased and comparable results. Methods: The study is based on 10 control and 11 post-mortem AD human brains from Huddinge Brain Bank. The sections were stained with cresyl-violet. An unbiased design-based fractionator method was applied. Results: We found significant oligodendroglial cell loss (46%) in AD as compared to control brains, while the total number of astrocytes remains unaffected. It is likely that the ratio of oligodendroglial cells to neurons is unchanged even in degenerative states, indicating that oligodendroglial cells parallel neuronal loss. Astroglial cells did not display astrocytosis, as often reported in other regions affected by neurofibrillary tangles and neuritic plaques. Conclusions: Using novel unbiased quantitative methods, we were able to estimate accurately neuronal and oligodendroglial loss in the PO. It is recognized that the proximity of oligodendroglial cells to neurons plays an important role in neuronal survival. It is still unclear which cell group degenerates first in AD. Some recent data suggest that oligodendrocytes are more vulnerable to amyloid toxicity. P3-196
CHARACTERIZATION OF UBIQUITINATED INTRANEURONAL INCLUSIONS IN A NOVEL BELGIAN FTD (FTLD-U) FAMILY
Daniel Pirici1, Julie van der Zee1, Rik Vandenberghe2, Rosa Rademakers1, Krist’l Vennekens1, Ivy Cuijt1, Ursula Lu¨bke3, Chantal Ceuterick3, Jean-Jacques Martin3, Christine Van Broeckhoven1, Samir Kumar-Singh1, 1Flanders Interuniversity Institute for Biotechnology, Institute Born Bunge, University of Antwerp, Antwerp, Belgium; 2University Hospital Gasthuisberg, Division of Neurology, Leuven, Belgium; 3Laboratory of Neuropathology, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium. Contact e-mail: [email protected] Background: The most common histological feature in patients with frontotemporal lobar degeneration (FTLD) is intracellular brain inclusions of yet uncharacterized protein that react with anti-ubiquitin (Ub) antibodies, but not with tau or synuclein (FTLD-U). There is a growing consensus that FTLD-U entity might be much more common than originally thought and described. Objective: To characterize clinically, genetically, and pathologically a 4-generation Belgian FTLD family. Methods: Clinical diagnosis was based on neurological examination, medical history and informant interview. Mutation analysis on genomic DNA was performed for the MAPT, PSEN1, PSEN2, APP and PRNP genes. One patient was available for neuropathological analysis and a detailed histological and immunohistochemical examination was performed here utilizing more than
Poster P3:: Tuesday Posters 40 antibodies directed against, for instance, transcription factors, cellspecific antigens (p62, HLA-DR, GFAP, NeuN), heat-shock proteins (HSP), and cytoskeletal components. Stereologic point-counting techniques and Western blotting were used to quantify neuronal loss and soluble tau protein, respectively. Results: Clinically, 8 patients had FTLD. Behavioral problems and aphasia were an important finding and at least three patients suffered from parkinsonian features. No mutations were identified in MAPT, APP, PS1, PS2, and PRNP. We showed frontotemporal atrophy with filamentous Ub-positive intracellular inclusions in absence of tau pathology or any alterations in the levels of soluble tau. We characterized their cellular and subcellular localization and morphology. Ub-positive inclusions predominantly occurred within neurons (⬎97%), but were also observed within oligodendroglia (approx. 2%), microglia (⬍1%), but not within astroglia. Regarding the subcellular localization, the intranuclear inclusions (INI) were up to approx. 4 fold more frequent than the cytoplasmic inclusions, although the latter were more specific to neurons. The INIs frequently appeared spindle-shaped and 3-D confocal reconstructions identified flattened, leaf-like structures. Ultrastructurally, straight 10-18 nm diameter filaments constituted the spindle-shaped inclusions that occurred in close proximity to the nuclear membrane. Staining for HSP40, p62, and valosin/p97 was observed in only a minority of the inclusions. Conclusion: While the precise nature of the protein remains
S433
elusive, characterization of such familial FTLD-U patients would be helpful in identifying a common denominator in the pathogenesis of familial and the more prevalent sporadic FTLD-U. P3-197
CYSTIC BORRELIA IN ALZHEIMER’S DISEASE AND IN NON-DEMENTIA NEUROBORRELIOSIS
Alan B. MacDonald1,2, 1Columbia University, Smithtown, NY, USA; 2St Catherine of Siena Medical Center, Smithtown, NY, USA. Contact email: [email protected] Background: A cystic form for Borrelia burgdoferi (Bb) was initially reported in 1988 in an autopsy study of Alzheimer’s disease tissue obtained from Dr. George Glenner’s brain bank. Cystic profiles were documented with silver stains, and with Murine Monoclonal antibodies to a Flagellin Epitope specific for Bb and B. hermseii [H9724 from Dr Alan Barbour]. Objective(s): Fresh frozen hippocampus tissues from Alzheimer’s disease cases provided by the Harvard University McLean Hospital brain bank were cultured in BSK M medium to attempt to grow spirochetes in vitro. Methods: Triturated fresh hippocampus was cultured in vitro in sterile BSK M liquid media at 24 degrees C for one year. Darkfield microscopy examination and Acridine orange staining with epifluorescence microscopy was completed. Detection of specific Flagellin DNA sequences from ORF BBO147 using a Molecular Beacon DNA probe was used to measure incremental increases in Borrelia specific Flagellin DNA in cultures, as compared with the original fresh tissue retained uncultured DNA Extracts. Results: Cystic borrelia structures were recovered from in vitro cultures of fresh Alzheimer disease hippocampus tissue. Incremental increases in Borrelia burgdorferi flagellin B DNA were documented in cultured tissues. Conclusions: A subset of Alzheimer’s disease is related to chronic Neuroborreliosis in the human host.
P3-198
A CASE OF EARLY ONSET ALZHEIMER’S DISEASE WITH COTTON WOOL PLAQUES BUT WITHOUT SPASTIC PARAPARESIS
Beata Sikorska1, Pawel P. Liberski1, Herbert Budka2, 1Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland; 2Institute of Neurology, Medical University of Vienna, Vienna, Austria. Contact e-mail: [email protected] Background: We report a case of 32-year-old man with myoclonus, rapidly progressive dementia, apraxia, ataxic gait and subtle right hemiparesis. The clinical and first pathological diagnosis was Creutzfeldt Jakob disease. Methods: A brain biopsy was obtained from the left temporoparietal area. Autopsy was performed one hour after death. Formalin-fixed, paraffin embedded tissues were used for routine and immunohistochemical microscopic stainings. Part of the autopsy material was fixed in 4% glutaraldehyde in cacodylate buffer for electron microscopy. Results: On