- Email: [email protected]
S5-02-05 Involvement of aberrant regulation of PI3K and MAPK cascades in AD TAU phosphorylation
$90
Symposium $5-02: Molecular Pathology/Histopathology
hyperphosphorylated tau immunoreacfivity is almost indistinguishable from that seen in Alzheimer's disease. Moreover, the same amyloid associated proteins and inflammatory components seen in Alzheimer's lesions are also featured in FBD and FDD. All the above described findings support the notion that amyloid pepfides are of primary importance in the process of neurodegenerafion. Furthermore, they also indicate that amyloid peptides other than A~ can trigger similar pathological pathways resulting in neuronal loss and clinical dementia. The close spatial relationship between amyloid and pre-amyloid deposits and neurofibrillary pathology in FBD and FDD constitute an attractive paradigm for determining the molecular basis of neuronal damage and cell loss. We propose the chromosome 13 dementias FBD and FDD as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain. Supported by NIH grants NS38777 and AG05891 and by the Alzheimer Association
I $ 5 - 0 2 - 0 3 [ E N D O S O M A L PATHOLOGY IN EARLY STAGE A L Z H E I M E R ' S DISEASE Anne M. Cataldo* 1 Paul M. Mathews 2, Olivera M. Grbovic 3, Corrinne M. Peterhoff3, Ying Jiang 3, Ralph A. Nixon 2. tMcLean Hospital~Harvard
Medical School, Belmont, MA, USA; 2Nathan Kline Institute/NYU School of Medicine, Orangeburg, NY, USA; 3Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected]
Background: Internalized materials enter endosomes of the endocytic pathway and are selectively delivered to lysosomes or are recycled directly or by perinuclear recycling endosomes to the plasma membrane. Endosomes are also connected to the Golgi apparatus through nascent lysosomal enzymes and their receptors. We have previously shown that the earliest pathology in AD brain involves rab5-positive endosomes, which are enlarged and contain abnormally high levels of lysosomal hydrolases. Endocytic pathology in the neocortex precedes amyloid deposition and is exacerbated by APOE genotype and APP mutations but not influenced by presenilin mutations. In individuals with Down syndrome (DS), endocytic pathology develops decades before the onset of senile plaques and neuroflbrillary tangles. Objective: To investigate in model systems how the development of endosomal pathology relates to Abeta production. Methods: Brain tissue from mild AD and juvenile DS cases and from a genetic animal model of DS, the Ts65Dn mouse, which exhibits AD-like endocytic pathology, were analyzed using antibodies against markers of endocytic uptake and fusion (rab5, rabaptin5, early endosomal antigen 1) and recycling (tab4), MPR46, beta-cleaved APP fragments and Abeta peptides in combination with immunocytochemistry, immunoprecipitation, Western blotting, and ELISA. To reproduce the increased endocytic uptake or enhanced hydrolase delivery to endosomes seen in early stage AD, murine L cells were transfected with human tab5 or MPR46. Results: In cases with early stage AD and DS, the onset of endocytic pathology coincided with increased soluble Abeta levels in brain and with the presence of Abeta in abnormal endosomes of neurons in vulnerable brain regions. Cell models of either increased endocytic uptake and fusion or accentuated hydrolase delivery to endosomes reproduced aspects of the endocytic pathology seen in AD brain and also increased levels of Abeta production. Aging-related endocytic pathology in the Ts65Dn mouse, including intraendosomal Abeta accumulation, was dependent on triplication of the App gene. Conclusions: These results support an important contribution of endosomal pathology to beta-amyloidogenesis and indicate a critical influence of the App gene on endocytic function. AD-risk factors, including APE APOE, and cholesterol, which infuence amyloidogenesis, could exert their effects in part by perturbing trafficking within the endocytic pathway.
$5-02-04 ] SYMPOSIA SESSION Roger Nitsch*. University of Zurich, Zurich, Switzerland. Contact e-mail:
nitsch @bli.unizh, ch Abstract not received.
$5-02-05 ] INVOLVEMENT O F A B E R R A N T REGULATION OF PI3K AND MAPK CASCADES IN AD TAU PHOSPHORYLATION Jin Jing Pei* 1, Wen-Lin An 1, Richard F. Cowburn 1, Inge Gnandke-Iqbal 2, Khalid Iqbal 2, Bengt Winblad 1.1Karolinska Institutet, Huddinge, Sweden;
2NYS Institute for Basic Research, Staten Island, Ny USA. Contact e-mail: Jin-,[email protected]
Background: Deposition of abnormally hyperphosphorylated tau in the form of paired helical filaments (PHF-tau) in neuroflbrillary tangles (NFT) is one of the major lesions in Alzheimer's disease (AD) brain. Glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinases (MAPK) are considered to be among the prime tau candidate kinases involved in NFT formation. To test this hypothesis, activities of tan ldnases must be associated with PI-IF-tan in neurons. Phospho-specific antibodies to tau and to the active forms of tan kinases have provided opportunities to visualize these biochemical changes in sire prior to NPT formation in AD. Objective(s): To investigate the mechanism of tan hyperphosphorylation in AD. Methods: Regional distributions of active forms of GSK-3 and MAPK, and other components in PI3 and MAPK cascades such as protein kinase B (PKB), MAPK kinase (MEK), and p70 $6 kinase were investigated by immnnohistochemistry using human brains staged for neurofibrillary degeneration according to Braak criteria. Intraneuronal changes of these kinases and tau were distinguished by double immtmofluorescent confocal microscopy. Levels of these kinases and tan were analyzed with ELISA and/or Western blots. Systems such as metabolically active rat brain slice culture, and zinc-treated SH-SY5Y cells were used to investigate the mechanisms behind the changes. Results: We found that 1) aberrant accumulation of the active forms of PKB, GSK-3, MEKll2, MAPK (ERK1/2, JNK, and p38), and p70 $6 kinase coincided with the occurrence of neumfibrillary degeneration in a sequential order in brains staged according to Braak neurofibrillary criteria; 2) using immunofluorescent confocal microscopy, four patterns of neurons from pretangle to classic tangles were distinguished by antibodies to PHF-tau (AT8) and active forms of these enzymes; 3) in SH-SY5Y neuroblastoma cells, zinc induced activation of MAPK (ERK, JNK, p38), PKB, and p70 $6 kinase following by increased levels and phosphorylation of tan; 4) in brain slices, activation of MEK1/2, MAPK, and p70 $6 kinase correlated with tau phosphorylation at several sites seen in AD when PP-2A was selectively inhibited. Conclusions: These data suggest that tan hyperphosphorylation might involve aberrant regulation of both PI3K and MAPK cascades in AD brain.
$5-02-06 1 UNDERSTANDING THE ROLE OF APP F R A G M E N T S IN N E U R O D E G E N E R A T I O N IN TRANSGENIC M O D E L S O F A L Z H E I M E R ' S DISEASE Eliezar Masliah*. University of California, San Diego, La JoUa, CA, USA.
Contact e-mail: emasliah@ ucsd.edu
Background: Amyloid-b peptides (Ab) are widely presumed to play a causal role in Alzheimer's disease (AD). Release of Ab and C-terminal fragments from the arnyloid precursor protein (APP) requires proteolysis by the b-site APP-cleaving enzyme (BACE1). While increased BACE1 activity has been identified in AD brains, it is unclear to what extent and by what mechanism(s) this molecular alteration might contribute to the pathogenesis of AD and how regulation of this process might be used develop new treatment targets for AD. Objectives: To investigate the differential toxic effects of Ab and C-terminal products in the neurodegenerative process of AD in transgenic animal models and to develop new potential treatment approaches. Results: hBACE1 and hBACE1/hAPP mice showed progressive neurological deficits, degeneration of neurons in the neocortex and hippocampus, degradation of myelin, and atrophy of nerves and muscles, Notably, increased BACE1 activity did not significantly alter Ab levels. However, it markedly increased the levels of APP C-terminal fragments and decreased the levels of fulllength APP in the brains of singly and doubly TG mice. Treatment with a BACE inhibitor reduced the levels of C-terminal APP fragments and ameliorated the neurological deficits. Similarly genetic manipulations
Symposium $5-02: Molecular Pathology/Histopathology
hyperphosphorylated tau immunoreacfivity is almost indistinguishable from that seen in Alzheimer's disease. Moreover, the same amyloid associated proteins and inflammatory components seen in Alzheimer's lesions are also featured in FBD and FDD. All the above described findings support the notion that amyloid pepfides are of primary importance in the process of neurodegenerafion. Furthermore, they also indicate that amyloid peptides other than A~ can trigger similar pathological pathways resulting in neuronal loss and clinical dementia. The close spatial relationship between amyloid and pre-amyloid deposits and neurofibrillary pathology in FBD and FDD constitute an attractive paradigm for determining the molecular basis of neuronal damage and cell loss. We propose the chromosome 13 dementias FBD and FDD as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain. Supported by NIH grants NS38777 and AG05891 and by the Alzheimer Association
I $ 5 - 0 2 - 0 3 [ E N D O S O M A L PATHOLOGY IN EARLY STAGE A L Z H E I M E R ' S DISEASE Anne M. Cataldo* 1 Paul M. Mathews 2, Olivera M. Grbovic 3, Corrinne M. Peterhoff3, Ying Jiang 3, Ralph A. Nixon 2. tMcLean Hospital~Harvard
Medical School, Belmont, MA, USA; 2Nathan Kline Institute/NYU School of Medicine, Orangeburg, NY, USA; 3Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected]
Background: Internalized materials enter endosomes of the endocytic pathway and are selectively delivered to lysosomes or are recycled directly or by perinuclear recycling endosomes to the plasma membrane. Endosomes are also connected to the Golgi apparatus through nascent lysosomal enzymes and their receptors. We have previously shown that the earliest pathology in AD brain involves rab5-positive endosomes, which are enlarged and contain abnormally high levels of lysosomal hydrolases. Endocytic pathology in the neocortex precedes amyloid deposition and is exacerbated by APOE genotype and APP mutations but not influenced by presenilin mutations. In individuals with Down syndrome (DS), endocytic pathology develops decades before the onset of senile plaques and neuroflbrillary tangles. Objective: To investigate in model systems how the development of endosomal pathology relates to Abeta production. Methods: Brain tissue from mild AD and juvenile DS cases and from a genetic animal model of DS, the Ts65Dn mouse, which exhibits AD-like endocytic pathology, were analyzed using antibodies against markers of endocytic uptake and fusion (rab5, rabaptin5, early endosomal antigen 1) and recycling (tab4), MPR46, beta-cleaved APP fragments and Abeta peptides in combination with immunocytochemistry, immunoprecipitation, Western blotting, and ELISA. To reproduce the increased endocytic uptake or enhanced hydrolase delivery to endosomes seen in early stage AD, murine L cells were transfected with human tab5 or MPR46. Results: In cases with early stage AD and DS, the onset of endocytic pathology coincided with increased soluble Abeta levels in brain and with the presence of Abeta in abnormal endosomes of neurons in vulnerable brain regions. Cell models of either increased endocytic uptake and fusion or accentuated hydrolase delivery to endosomes reproduced aspects of the endocytic pathology seen in AD brain and also increased levels of Abeta production. Aging-related endocytic pathology in the Ts65Dn mouse, including intraendosomal Abeta accumulation, was dependent on triplication of the App gene. Conclusions: These results support an important contribution of endosomal pathology to beta-amyloidogenesis and indicate a critical influence of the App gene on endocytic function. AD-risk factors, including APE APOE, and cholesterol, which infuence amyloidogenesis, could exert their effects in part by perturbing trafficking within the endocytic pathway.
$5-02-04 ] SYMPOSIA SESSION Roger Nitsch*. University of Zurich, Zurich, Switzerland. Contact e-mail:
nitsch @bli.unizh, ch Abstract not received.
$5-02-05 ] INVOLVEMENT O F A B E R R A N T REGULATION OF PI3K AND MAPK CASCADES IN AD TAU PHOSPHORYLATION Jin Jing Pei* 1, Wen-Lin An 1, Richard F. Cowburn 1, Inge Gnandke-Iqbal 2, Khalid Iqbal 2, Bengt Winblad 1.1Karolinska Institutet, Huddinge, Sweden;
2NYS Institute for Basic Research, Staten Island, Ny USA. Contact e-mail: Jin-,[email protected]
Background: Deposition of abnormally hyperphosphorylated tau in the form of paired helical filaments (PHF-tau) in neuroflbrillary tangles (NFT) is one of the major lesions in Alzheimer's disease (AD) brain. Glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinases (MAPK) are considered to be among the prime tau candidate kinases involved in NFT formation. To test this hypothesis, activities of tan ldnases must be associated with PI-IF-tan in neurons. Phospho-specific antibodies to tau and to the active forms of tan kinases have provided opportunities to visualize these biochemical changes in sire prior to NPT formation in AD. Objective(s): To investigate the mechanism of tan hyperphosphorylation in AD. Methods: Regional distributions of active forms of GSK-3 and MAPK, and other components in PI3 and MAPK cascades such as protein kinase B (PKB), MAPK kinase (MEK), and p70 $6 kinase were investigated by immnnohistochemistry using human brains staged for neurofibrillary degeneration according to Braak criteria. Intraneuronal changes of these kinases and tau were distinguished by double immtmofluorescent confocal microscopy. Levels of these kinases and tan were analyzed with ELISA and/or Western blots. Systems such as metabolically active rat brain slice culture, and zinc-treated SH-SY5Y cells were used to investigate the mechanisms behind the changes. Results: We found that 1) aberrant accumulation of the active forms of PKB, GSK-3, MEKll2, MAPK (ERK1/2, JNK, and p38), and p70 $6 kinase coincided with the occurrence of neumfibrillary degeneration in a sequential order in brains staged according to Braak neurofibrillary criteria; 2) using immunofluorescent confocal microscopy, four patterns of neurons from pretangle to classic tangles were distinguished by antibodies to PHF-tau (AT8) and active forms of these enzymes; 3) in SH-SY5Y neuroblastoma cells, zinc induced activation of MAPK (ERK, JNK, p38), PKB, and p70 $6 kinase following by increased levels and phosphorylation of tan; 4) in brain slices, activation of MEK1/2, MAPK, and p70 $6 kinase correlated with tau phosphorylation at several sites seen in AD when PP-2A was selectively inhibited. Conclusions: These data suggest that tan hyperphosphorylation might involve aberrant regulation of both PI3K and MAPK cascades in AD brain.
$5-02-06 1 UNDERSTANDING THE ROLE OF APP F R A G M E N T S IN N E U R O D E G E N E R A T I O N IN TRANSGENIC M O D E L S O F A L Z H E I M E R ' S DISEASE Eliezar Masliah*. University of California, San Diego, La JoUa, CA, USA.
Contact e-mail: emasliah@ ucsd.edu
Background: Amyloid-b peptides (Ab) are widely presumed to play a causal role in Alzheimer's disease (AD). Release of Ab and C-terminal fragments from the arnyloid precursor protein (APP) requires proteolysis by the b-site APP-cleaving enzyme (BACE1). While increased BACE1 activity has been identified in AD brains, it is unclear to what extent and by what mechanism(s) this molecular alteration might contribute to the pathogenesis of AD and how regulation of this process might be used develop new treatment targets for AD. Objectives: To investigate the differential toxic effects of Ab and C-terminal products in the neurodegenerative process of AD in transgenic animal models and to develop new potential treatment approaches. Results: hBACE1 and hBACE1/hAPP mice showed progressive neurological deficits, degeneration of neurons in the neocortex and hippocampus, degradation of myelin, and atrophy of nerves and muscles, Notably, increased BACE1 activity did not significantly alter Ab levels. However, it markedly increased the levels of APP C-terminal fragments and decreased the levels of fulllength APP in the brains of singly and doubly TG mice. Treatment with a BACE inhibitor reduced the levels of C-terminal APP fragments and ameliorated the neurological deficits. Similarly genetic manipulations