- Email: [email protected]
40 in chicken telencephalic cultures but specifically increases intracellular Abeta 1–38
Oral Session 04-04: Cellular and Animal Models 1
$80 I
04-03-08 I ROLE OF NICASTRIN IN P R O G R A M M E D C E L L 1
DEATH Raphaelle Pardossi-Piquard* 1, Gang Yu 2, Peter St George-Hyslop 3 , Christophe Bourdon 4, Cristine Alves da Costa 1, Frtdtric Checler 1 I lPMC
CNRS, Valbonne, France; 2University of Texas Southwestern Medical Center, Dallas, TX, USA; 3Center for Research in Neurodegenerative Diseases, Toronto, ON, Canada; 4Ninewells Hospital and Medical School, Dundee, United Kingdom. Contact e-mail: [email protected] Nicastrin is a component of a multiproteic complex containing presenilins, Aph-1 and Pen-2. This complex is involved in y-secretase cleavage of the I%amyloid precursor protein (~APP). The proteolytic processing of ~APP leads to the production of [3-amyloid peptides that are deposited in seniles plaques and that contribute to the neurodegeneration observed in brains of patients affected by Alzheimer disease. We and other previously showed that presehilins could also control programmed cell death. Therefore, we examined the putative role of nicastrin in the control of apoptotie response. We found that over-expression of nicastrin increases the viability of human embryonic kidney (HEK) 293 cells as shown by terminal deoxynucleotide transferase nick end labelling (TUNEL) and by propidium iodide incorporation FACS analyses. Furthermore, we observed by fluorimetric assay that nicastrin decreases staurosporine-induced caspase-3 activity in both HEK 293 cells and telencephaion specific routine neurons (TSM1). Interestingly, we also demonstrated that nicastrin lowers p53-transcriptional activity and p53-expression at both transcriptional and post-transcriptional levels. However, in ceils in which p53-gene had been knocked out, we still observed a significant nicastrin-mediated caspase inhibition. These findings suggest that this antiapoptotic response triggered by nicastrin is mediated through both p53-independent and -dependent pathways.
O r a l Session 0 4 - 0 4 : C e l l u l a r a n d A n i m a l M o d e l s I O4-04-01 l INDUCTION OF HEPARAN SULFATE PROTEOGLYCANS PRODUCTION BY AO Marcel M. Verbeek*, Irene Otte-Holler, Robert M. de Waal, Micha M. Wilhelmus, Jack van Horssen. University Medical Center Nijmegen,
Nijmegen, Netherlands. Contact e-mail: [email protected] Background: Heparan sulfate proteoglycans (HSPGs), in particular agrin and glypican, are associated with senile plaques, cerebral amyloid angiopathy and neurofibrillary tangles in Alzheimer's disease (AD) brains. It is yet unclear which cell types in the brain produce these HSPGs and what specific mechanisms are responsible for their excessive production and accumulation in AD brains. Objective(s): To study the ceil-specific production of various HSPG types (agrin, glypican-1, syndecanl-3, collagen XVIII) and to study the possibility that the amyloid f~ protein (A[~) induces the production of HSPGs. Methods: Primary human brain pericytes, neuroblastoma, astrocytoma and glioma cells were studied for their expression of the HSPGs agrin, glypican-1, syndecanl-3 and collagenXVIII by immunocytochemistry, westem blotting and electron microscopy. Western blotting was optimized for a reproducible detection of HSPGs in cell lysates. Human brain pericytes were treated with A[~40 carrying the Dutch mutation (D-A~40) to study its effects on HSPG expression by immunocytochemistry, western blotting and autoradiography after 35S-sulfate incorporation. Results: The four different cell types produced a cell-specific combination of HSPGs. Remarkably, western blot analysis demonstrated that the HSPGs predominantly consisted of the core protein only, and that only a minority of the immunoreactive bands contained glycosaminoglycan (GAG) sidechains, observed as high molecular weight immunoreactive smears. Incubation of human brain pericytes with D-A[340 resulted in a strong increase in both glypican-1 and agrin expression. Furthermore, significantly more 35S-sulfate was incorporated in a cetylpyridinium chloride-precipitable fraction of the cells. Finally, EM analysis demonstrated the close association of agrin with A~ fibrils accumulating at the cell surface of the pericytes. Conclusions: Cerebral cell types produce a cell-specific combination of HSPGs. The excessive production of HSPGs as observed in AD brains may be caused by the induction by AlL Since HSPG
GAGs interact with AI3 and may affect its fibrillogenesis, this indicates that enhanced HSPG production caused by A~ may contribute to the pathogenesis of AD.Supported by the Internationale Stichting Alzheimer Onderzoek, Zon-MW Innovational Research and the Hersenstichting Nederland.
E PROMOTES THE IO4-04-021 APOLIPOPROTEIN DEGRADATION OF DEPOSITED AMYLOID-~ PEPTIDES BY ADULT MOUSE ASTROCYTES Milla Koistinaho* 1,2, Suizhen Lin 1, Xin Wu 1, Michail Esterman 1, Jeffrey Hanson 1, Kelly R. Bales 1, Steven M. Paul 1 1Eli Lilly and
Company, Indianapolis, 1N, USA; ecurrent employer: Cerebricon Ltd, Kuopio, Finland. Contact e-mail: [email protected] Background: Deposition of amyloid-~ (A~) peptides and the presence of large numbers of activated astrocytes in close proximity to A~ deposits are characteristic neuropathological features found in Alzheimer's disease (AD) brain, however the relationship between A[~ deposition and astrocytosis is not completely understood. Apolipoprotein E (apoE) is a lipid carrier protein that is synthesized and secreted mainly by astrocytes within the central nervous system. The ~4 allele of apoE represents a major genetic risk factor for the age of onset as well as relative risk to develop AD. Additionally, brain A~ burden is dramatically increased in AD patients carrying the e4 allele. Previous studies have shown that astrocytic expression of human apoE suppresses A~ deposition in a PDAPP mouse model of AD without having any effect on brain A~ synthesis, suggesting that apoE affects the clearance of A[~ from brain parenchyma. Objectives: Given the recently discovered capacity of adult astrocytes to degrade A~, the role of apoE in astrocyte-mediated clearance of deposited human A~ peptides was investigated. Methods: To study the association and possible degradation of human A[~ by primary astrocytes prepared from adult wild type or apoE knockout mice, an in vitro assay in which exogenous astrocytes are cultured on top of cryostat sections from old PDAPP transgenic mice, was used. Phase-contrast and confocal microscopy were used to assess morphological changes in astrocytes following incubation with deposited human A~ peptides at various time points. Quantitative immunohistochemical and as well as ELISA methods were used to quantify the ability of astrocytes to remove AI3 from brain sections. Results: Astrocytes lacking apoE were unable to associate with, respond to, internalize and degrade human A~ present in A~ plaque-bearing brain sections. Antibodies to both A~ and apoE, as well as RAP, an LDL receptor family antagonist, blocked the clearance of A~ by adult astrocytes. Conclusions: ApoE is critical for adult astrocytes to find and clear A~ in brain parenchyma. Furthermore, our data suggests that deficits in apoE-mediated clearance of A~ by astrocytes may contribute to the pathogenesis of AD.
O4-04-03 I LITHIUM DECREASES SECRETION OF ABETA 1-42 AND C-TRUNCATED SPECIES ABETA 1-37138139140 IN C H I C K E N TELENCEPHALIC CULTURES BUT S P E C I F I C A L L Y INCREASES INTRACELLULAR ABETA 1-38 Hermann Esselmann .1 , Nikolans Kunz 1, Juan M. Maler 2, Piotr Lewczuk 2, Markns Otto I , Eckart Ruether 1, Johannes Komhuber 2, Jens Wiltfang a. 1University of Goettingen, Goettingen, Germany;
2University of Erlangen-Nuremberg, Erlangen, Germany. Contact e-mail: hesselm @gwdg.de Background: We studied endogenous amyloid precursor protein (APP) processing and amyloid beta (A[3) peptide formation in primary chicken telencephalic neurons, because their A[3 peptide sequence is identical to humans. Objective and Methods: As detected by quantitative A[~-SDSPAGE/immunoblot, AI3 peptides 1-40/42 and three additional C-truncated species, namely A~ 1-37/38/39 were regularly released into the supematant. The highly conserved A~ quintett strongly resembles the pattern of A~ peptides found in human cerebrospinal fluid (CSF). Furthermore, the Cterminally shorter AI3 peptides 1-33/34/35 could be readily detected. These findings were confirmed by surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry. Recent evidence indicates
Oral Session 04-04: Cellular and Animal Models 1 that lithium specifically inhibits secretion of the amyloidogenic A[3 142 peptide in cultured permanent cells transfected with human APP. We therefore investigated the effect of lithium on A[3 peptide secretion as well as intracellular A[3 peptides in our untransfected primary cell culture system. Results: Our data shows that lithium leads to a dose dependent reduction of A[3 1-37/38/39/40/42 secretion. Surprisingly, intracellular analysis revealed that lithium specifically increases a band co-migrating with synthetic A[3 1-38 while A[~ 1-40 and AI3 1-42 remained almost unaffected. These results demonstrate for the first time that lithium treatment decreases Af3 peptides secretion in primary chicken neuronal cells but specifically elevates intracellular A[3 1-38. Conclusions: Therefore, we conclude two independent mechanisms of lithium on intra- and extracellular A[3 peptide production. This work was supported by DIADEM
04-04-041
$81
littermate controls. Genotypes were determined by PCR analyses specific for hAPPswe and hX1 lalpha. Results: Immunofluorescence microscopy of primary cultures of cortical neurons and immunohistochemistry of brain sections revealed that hXllalpha was expressed only in neurons and that hXllalpha, hAPPswe, and mufine APP colocalized in perinuctear and vesicular compartments. ELISA of human Abeta40 and Abeta42 in conditioned media from double transgenic versus hAPPswe transgenic neuronal cultures revealed a significantly lower concentration of Abeta42, and a lower Abeta42/Abeta40 ratio, in media from double transgenic cultures. Conclusions: These data suggest a modulatory role of the Xllalpha:APP interaction in neurons, particularly on the specific gamma-cleavage site of APP and its C-terminal fragments.
04-04-06 ] fI-AMYLOID P R E C U R S O R P R O T E I N ACUTE PHASE REACTANTS RECAPITULATE HEME OXYGENASE-1 SUPPRESSOR (HOS) ACTIVITY IN ALZHEIMER PLASMA
Olivier C. Maes*, Howard Chertkow, Howard Bergman, Hyman M. Schipper. Lady Davis Institute for Medical Research, Sir Mortimer B.
Davis Jewish General Hospital McGill University, Montreal PQ, Canada. Contact e-mail: [email protected]
Background: Up-regulation of heme oxygenase-1 (HO-1), induced by amyloid-t3 or pro-inflammatory cytokines, may promote pathological iron deposition and oxidative mitochondrial injury in Alzheimer (AD)-affected neural tissues (Exp Gerontol 35: 821-830, 2000). Conversely, blood HO-1 mRNA and protein levels are lower in AD than in normal elderly control (NEC) subjects (Neurology 54: 1297-1304, 2000). Three acute phase reactants, transferrin (Tf), hemopexin (Hpx) and alphal-antitrypsin (AT), were found to be over-expressed in AD plasma fractions with proven HO-1 suppressor (HOS) activity by 2D gel electrophoresis and mass spectrometry (Proteomics 3: 2240-2248, 2003). Objective(s): To determine whether and the extent to which purified Tf, Hpx and AT proteins recapitulate the HOS activity of AD plasma. Methods: The effects of various doses and exposure times of Tf, Hpx and AT on the HO-1 mRNA response to 880 IxM cysteamine challenge in cultured rat astroglia (HOS bioassay) was quantified by densitometry and the data expressed as percentage HOS activity. Results: Physiologically-relevant concentrations of AT and Tf alone yielded robust HOS activity, whereas Hpx and human albumin alone were devoid of any such activity. In contrast, Hpx and albumin administered in combination yielded intense HOS activity similar to that of native AD plasma, and albumin further augmented HOS activity mediated by AT. Conclusions: Our data ascribe a novel functional link between systemic HO-1 expression and the acute phase response in AD subjects. We conjecture that, in AD, acute phase proteins may attenuate the CNS HO-I response and downstream mitochondrial damage accruing from exposure to amyloid-13 or pro-inflammatory cytokines.
04-04-05 [ XllALPHA/MINT-1 MODULATES GAMMA-CLEAVAGE O F A M Y L O I D P R E C U R S O R PROTEIN Raymond S. Turner* 1, Inderjeet Saluja 2, Ye Lu 2, Thomas L. Saunders 2.
1VA and Univ. of Michigan, Ann Arbor, MI, USA; 2 Univ. of Michigan, Ann Arbor, MI, USA. Contact e-mail: [email protected]
Background: Amyloid precursor protein processing to Abeta plays a pivotal role in Alzheimer's disease pathogenesis. In transfected non-neuronal cells, the PTB domain of the neuronal adaptor protein X1 lalpha/mint-1 interacts with the cytoplasmic tail of APP to prolong its half-life and inhibit Abeta40 and Abeta42 secretion by impairing alpha- and gamma- but not beta-cleavage of APR Objective(s): To test the hypothesis that X1 lalpha modulates APP metabolism in neurons, we generated novel laX i l alpha transgenic mice and crossed them with Tg2576 mice (expressing the Swedish mutation of hAPP, hAPPswe, or K651N/M652L in the 751 isoform). Methods: Embryonic (El4-16) neuronal cultures were generated from hX1 lalpha/hAPPswe double transgenic, hX1 lalpha transgenic, hAPPswe transgenic, and wild type
I N T R A C E L L U L A R DOMAIN (AICD) STRONGLY
ENHANCES RESTING FREE CYTOSOLIC CALCIUM LEVELS Runa Hamid 1, Michael WiUem 2, Dieter Edbaner 2, Harald Steiner 2, Ulrike Mtiller 3, Gerda Wiinsch 1, Hans Kretzschmar 1, Christian Haass 2, Jochen Herms .4. 1Department of Neuropathology, University Munich,
81377Munich, Germany; 2Department of Biochemistry, Adolf-Butenandt-Institute, University Munich, 81377 Munich, Germany; 3Department of Neurochemistry, MPI for Brain Resarch, 60528 Frankfurt, Germany; 4Ludwig Maximilians Universitiit, 81377 Munich, Germany. Contact e-mail: [email protected]
Background: Loss of APP has been shown to affect calcium release from intracellular stores most likely due to a reduction of the intracellular fragment of APP lAPP intracellular domain (AICD)] (Leissring et al. PNAS 99:4697ff; 2002). Objectives: Here we aim to analyse this mechanism in more detail by analysing the intracelhilar free Ca 2+ concentration in HEK 293 cells expressing both APP with the Swedish mutation (APPsw) as well as the insulin-degrading enzyme (IDE) which is known to degrade AICDs (Edbauer et al. JBC 277:13389ff; 2002). Results: In comparison to nontransfected HEK 293 cells as well as cells expressing APP with the Swedish mutation (APPsw) alone the cytoplasmic free calcium concentration was dramatically increased in two cell lines expressing both APPsw and IDE. Moreover the application of cyclopiazonic acid, known to block the Ca 2+ATPase that pumps Ca 2+ from the cytosol into the endoplasmatic reticulum (ER) did not induce any Ca 2+ transient in these cells. Consequently the capacitative calcium entry from the extracelhilar space, which is activated when the ER-calcium pool levels are low, was found to be dramatically enhanced in APPsw/IDE cells. Similar observations were made in HEK 293 cells expressing a "dominant negative " PS1 mutant (PSl[D385N]) were the generation of AICD is inhibited as well as in dermal fibroblasts from APP deficient mice. These results strongly indicate that lack of AICD blocks the uptake of cytosolic calcium into the ER and leads to a prominent increase of the resting free cytosolic calcium concentration. Conclusions: An enhancement of the resting cytosolic calcium concentration and a decrease of calcium within the ER in neurons have been well described to occur during aging and they are central for the "Ca 2+ hypothesis" of neuronal aging. Thus it is tempting to speculate that neuronal aging might be at least partly caused by an enhanced AICD degradation.
04-04-07 ] CORTICAL DYSPLASIA RESEMBLING HUMAN TYPE 2 LISSENCEPHALY IN M I C E L A C K I N G A L L T H R E E APP-FAMILY M E M B E R S Ulrike Miiller* 1, Brigitte Anliker I , Sabine Heber I , Martin Fuhrmann 2, Hans Kretzschmar 2, Sangram Sisodia 3, Jochen Herms 2.
CMax-Planck-Institute for Brain Research, Frankfurt, Germany; 2Centre for NeuropathoIogy, University of Munich, Munich, Germany; 3Dept. of NeurobioIogy, University of Chicago, Chicago, IL, USA. Contact e-mail: umueller @mpih-frankfurt.mpg.de
Background: The amyloid precursor protein APP plays a central rote in the pathogenesis of Alzheimer's diseae. By contrast, its physiological function
$80 I
04-03-08 I ROLE OF NICASTRIN IN P R O G R A M M E D C E L L 1
DEATH Raphaelle Pardossi-Piquard* 1, Gang Yu 2, Peter St George-Hyslop 3 , Christophe Bourdon 4, Cristine Alves da Costa 1, Frtdtric Checler 1 I lPMC
CNRS, Valbonne, France; 2University of Texas Southwestern Medical Center, Dallas, TX, USA; 3Center for Research in Neurodegenerative Diseases, Toronto, ON, Canada; 4Ninewells Hospital and Medical School, Dundee, United Kingdom. Contact e-mail: [email protected] Nicastrin is a component of a multiproteic complex containing presenilins, Aph-1 and Pen-2. This complex is involved in y-secretase cleavage of the I%amyloid precursor protein (~APP). The proteolytic processing of ~APP leads to the production of [3-amyloid peptides that are deposited in seniles plaques and that contribute to the neurodegeneration observed in brains of patients affected by Alzheimer disease. We and other previously showed that presehilins could also control programmed cell death. Therefore, we examined the putative role of nicastrin in the control of apoptotie response. We found that over-expression of nicastrin increases the viability of human embryonic kidney (HEK) 293 cells as shown by terminal deoxynucleotide transferase nick end labelling (TUNEL) and by propidium iodide incorporation FACS analyses. Furthermore, we observed by fluorimetric assay that nicastrin decreases staurosporine-induced caspase-3 activity in both HEK 293 cells and telencephaion specific routine neurons (TSM1). Interestingly, we also demonstrated that nicastrin lowers p53-transcriptional activity and p53-expression at both transcriptional and post-transcriptional levels. However, in ceils in which p53-gene had been knocked out, we still observed a significant nicastrin-mediated caspase inhibition. These findings suggest that this antiapoptotic response triggered by nicastrin is mediated through both p53-independent and -dependent pathways.
O r a l Session 0 4 - 0 4 : C e l l u l a r a n d A n i m a l M o d e l s I O4-04-01 l INDUCTION OF HEPARAN SULFATE PROTEOGLYCANS PRODUCTION BY AO Marcel M. Verbeek*, Irene Otte-Holler, Robert M. de Waal, Micha M. Wilhelmus, Jack van Horssen. University Medical Center Nijmegen,
Nijmegen, Netherlands. Contact e-mail: [email protected] Background: Heparan sulfate proteoglycans (HSPGs), in particular agrin and glypican, are associated with senile plaques, cerebral amyloid angiopathy and neurofibrillary tangles in Alzheimer's disease (AD) brains. It is yet unclear which cell types in the brain produce these HSPGs and what specific mechanisms are responsible for their excessive production and accumulation in AD brains. Objective(s): To study the ceil-specific production of various HSPG types (agrin, glypican-1, syndecanl-3, collagen XVIII) and to study the possibility that the amyloid f~ protein (A[~) induces the production of HSPGs. Methods: Primary human brain pericytes, neuroblastoma, astrocytoma and glioma cells were studied for their expression of the HSPGs agrin, glypican-1, syndecanl-3 and collagenXVIII by immunocytochemistry, westem blotting and electron microscopy. Western blotting was optimized for a reproducible detection of HSPGs in cell lysates. Human brain pericytes were treated with A[~40 carrying the Dutch mutation (D-A~40) to study its effects on HSPG expression by immunocytochemistry, western blotting and autoradiography after 35S-sulfate incorporation. Results: The four different cell types produced a cell-specific combination of HSPGs. Remarkably, western blot analysis demonstrated that the HSPGs predominantly consisted of the core protein only, and that only a minority of the immunoreactive bands contained glycosaminoglycan (GAG) sidechains, observed as high molecular weight immunoreactive smears. Incubation of human brain pericytes with D-A[340 resulted in a strong increase in both glypican-1 and agrin expression. Furthermore, significantly more 35S-sulfate was incorporated in a cetylpyridinium chloride-precipitable fraction of the cells. Finally, EM analysis demonstrated the close association of agrin with A~ fibrils accumulating at the cell surface of the pericytes. Conclusions: Cerebral cell types produce a cell-specific combination of HSPGs. The excessive production of HSPGs as observed in AD brains may be caused by the induction by AlL Since HSPG
GAGs interact with AI3 and may affect its fibrillogenesis, this indicates that enhanced HSPG production caused by A~ may contribute to the pathogenesis of AD.Supported by the Internationale Stichting Alzheimer Onderzoek, Zon-MW Innovational Research and the Hersenstichting Nederland.
E PROMOTES THE IO4-04-021 APOLIPOPROTEIN DEGRADATION OF DEPOSITED AMYLOID-~ PEPTIDES BY ADULT MOUSE ASTROCYTES Milla Koistinaho* 1,2, Suizhen Lin 1, Xin Wu 1, Michail Esterman 1, Jeffrey Hanson 1, Kelly R. Bales 1, Steven M. Paul 1 1Eli Lilly and
Company, Indianapolis, 1N, USA; ecurrent employer: Cerebricon Ltd, Kuopio, Finland. Contact e-mail: [email protected] Background: Deposition of amyloid-~ (A~) peptides and the presence of large numbers of activated astrocytes in close proximity to A~ deposits are characteristic neuropathological features found in Alzheimer's disease (AD) brain, however the relationship between A[~ deposition and astrocytosis is not completely understood. Apolipoprotein E (apoE) is a lipid carrier protein that is synthesized and secreted mainly by astrocytes within the central nervous system. The ~4 allele of apoE represents a major genetic risk factor for the age of onset as well as relative risk to develop AD. Additionally, brain A~ burden is dramatically increased in AD patients carrying the e4 allele. Previous studies have shown that astrocytic expression of human apoE suppresses A~ deposition in a PDAPP mouse model of AD without having any effect on brain A~ synthesis, suggesting that apoE affects the clearance of A[~ from brain parenchyma. Objectives: Given the recently discovered capacity of adult astrocytes to degrade A~, the role of apoE in astrocyte-mediated clearance of deposited human A~ peptides was investigated. Methods: To study the association and possible degradation of human A[~ by primary astrocytes prepared from adult wild type or apoE knockout mice, an in vitro assay in which exogenous astrocytes are cultured on top of cryostat sections from old PDAPP transgenic mice, was used. Phase-contrast and confocal microscopy were used to assess morphological changes in astrocytes following incubation with deposited human A~ peptides at various time points. Quantitative immunohistochemical and as well as ELISA methods were used to quantify the ability of astrocytes to remove AI3 from brain sections. Results: Astrocytes lacking apoE were unable to associate with, respond to, internalize and degrade human A~ present in A~ plaque-bearing brain sections. Antibodies to both A~ and apoE, as well as RAP, an LDL receptor family antagonist, blocked the clearance of A~ by adult astrocytes. Conclusions: ApoE is critical for adult astrocytes to find and clear A~ in brain parenchyma. Furthermore, our data suggests that deficits in apoE-mediated clearance of A~ by astrocytes may contribute to the pathogenesis of AD.
O4-04-03 I LITHIUM DECREASES SECRETION OF ABETA 1-42 AND C-TRUNCATED SPECIES ABETA 1-37138139140 IN C H I C K E N TELENCEPHALIC CULTURES BUT S P E C I F I C A L L Y INCREASES INTRACELLULAR ABETA 1-38 Hermann Esselmann .1 , Nikolans Kunz 1, Juan M. Maler 2, Piotr Lewczuk 2, Markns Otto I , Eckart Ruether 1, Johannes Komhuber 2, Jens Wiltfang a. 1University of Goettingen, Goettingen, Germany;
2University of Erlangen-Nuremberg, Erlangen, Germany. Contact e-mail: hesselm @gwdg.de Background: We studied endogenous amyloid precursor protein (APP) processing and amyloid beta (A[3) peptide formation in primary chicken telencephalic neurons, because their A[3 peptide sequence is identical to humans. Objective and Methods: As detected by quantitative A[~-SDSPAGE/immunoblot, AI3 peptides 1-40/42 and three additional C-truncated species, namely A~ 1-37/38/39 were regularly released into the supematant. The highly conserved A~ quintett strongly resembles the pattern of A~ peptides found in human cerebrospinal fluid (CSF). Furthermore, the Cterminally shorter AI3 peptides 1-33/34/35 could be readily detected. These findings were confirmed by surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry. Recent evidence indicates
Oral Session 04-04: Cellular and Animal Models 1 that lithium specifically inhibits secretion of the amyloidogenic A[3 142 peptide in cultured permanent cells transfected with human APP. We therefore investigated the effect of lithium on A[3 peptide secretion as well as intracellular A[3 peptides in our untransfected primary cell culture system. Results: Our data shows that lithium leads to a dose dependent reduction of A[3 1-37/38/39/40/42 secretion. Surprisingly, intracellular analysis revealed that lithium specifically increases a band co-migrating with synthetic A[3 1-38 while A[~ 1-40 and AI3 1-42 remained almost unaffected. These results demonstrate for the first time that lithium treatment decreases Af3 peptides secretion in primary chicken neuronal cells but specifically elevates intracellular A[3 1-38. Conclusions: Therefore, we conclude two independent mechanisms of lithium on intra- and extracellular A[3 peptide production. This work was supported by DIADEM
04-04-041
$81
littermate controls. Genotypes were determined by PCR analyses specific for hAPPswe and hX1 lalpha. Results: Immunofluorescence microscopy of primary cultures of cortical neurons and immunohistochemistry of brain sections revealed that hXllalpha was expressed only in neurons and that hXllalpha, hAPPswe, and mufine APP colocalized in perinuctear and vesicular compartments. ELISA of human Abeta40 and Abeta42 in conditioned media from double transgenic versus hAPPswe transgenic neuronal cultures revealed a significantly lower concentration of Abeta42, and a lower Abeta42/Abeta40 ratio, in media from double transgenic cultures. Conclusions: These data suggest a modulatory role of the Xllalpha:APP interaction in neurons, particularly on the specific gamma-cleavage site of APP and its C-terminal fragments.
04-04-06 ] fI-AMYLOID P R E C U R S O R P R O T E I N ACUTE PHASE REACTANTS RECAPITULATE HEME OXYGENASE-1 SUPPRESSOR (HOS) ACTIVITY IN ALZHEIMER PLASMA
Olivier C. Maes*, Howard Chertkow, Howard Bergman, Hyman M. Schipper. Lady Davis Institute for Medical Research, Sir Mortimer B.
Davis Jewish General Hospital McGill University, Montreal PQ, Canada. Contact e-mail: [email protected]
Background: Up-regulation of heme oxygenase-1 (HO-1), induced by amyloid-t3 or pro-inflammatory cytokines, may promote pathological iron deposition and oxidative mitochondrial injury in Alzheimer (AD)-affected neural tissues (Exp Gerontol 35: 821-830, 2000). Conversely, blood HO-1 mRNA and protein levels are lower in AD than in normal elderly control (NEC) subjects (Neurology 54: 1297-1304, 2000). Three acute phase reactants, transferrin (Tf), hemopexin (Hpx) and alphal-antitrypsin (AT), were found to be over-expressed in AD plasma fractions with proven HO-1 suppressor (HOS) activity by 2D gel electrophoresis and mass spectrometry (Proteomics 3: 2240-2248, 2003). Objective(s): To determine whether and the extent to which purified Tf, Hpx and AT proteins recapitulate the HOS activity of AD plasma. Methods: The effects of various doses and exposure times of Tf, Hpx and AT on the HO-1 mRNA response to 880 IxM cysteamine challenge in cultured rat astroglia (HOS bioassay) was quantified by densitometry and the data expressed as percentage HOS activity. Results: Physiologically-relevant concentrations of AT and Tf alone yielded robust HOS activity, whereas Hpx and human albumin alone were devoid of any such activity. In contrast, Hpx and albumin administered in combination yielded intense HOS activity similar to that of native AD plasma, and albumin further augmented HOS activity mediated by AT. Conclusions: Our data ascribe a novel functional link between systemic HO-1 expression and the acute phase response in AD subjects. We conjecture that, in AD, acute phase proteins may attenuate the CNS HO-I response and downstream mitochondrial damage accruing from exposure to amyloid-13 or pro-inflammatory cytokines.
04-04-05 [ XllALPHA/MINT-1 MODULATES GAMMA-CLEAVAGE O F A M Y L O I D P R E C U R S O R PROTEIN Raymond S. Turner* 1, Inderjeet Saluja 2, Ye Lu 2, Thomas L. Saunders 2.
1VA and Univ. of Michigan, Ann Arbor, MI, USA; 2 Univ. of Michigan, Ann Arbor, MI, USA. Contact e-mail: [email protected]
Background: Amyloid precursor protein processing to Abeta plays a pivotal role in Alzheimer's disease pathogenesis. In transfected non-neuronal cells, the PTB domain of the neuronal adaptor protein X1 lalpha/mint-1 interacts with the cytoplasmic tail of APP to prolong its half-life and inhibit Abeta40 and Abeta42 secretion by impairing alpha- and gamma- but not beta-cleavage of APR Objective(s): To test the hypothesis that X1 lalpha modulates APP metabolism in neurons, we generated novel laX i l alpha transgenic mice and crossed them with Tg2576 mice (expressing the Swedish mutation of hAPP, hAPPswe, or K651N/M652L in the 751 isoform). Methods: Embryonic (El4-16) neuronal cultures were generated from hX1 lalpha/hAPPswe double transgenic, hX1 lalpha transgenic, hAPPswe transgenic, and wild type
I N T R A C E L L U L A R DOMAIN (AICD) STRONGLY
ENHANCES RESTING FREE CYTOSOLIC CALCIUM LEVELS Runa Hamid 1, Michael WiUem 2, Dieter Edbaner 2, Harald Steiner 2, Ulrike Mtiller 3, Gerda Wiinsch 1, Hans Kretzschmar 1, Christian Haass 2, Jochen Herms .4. 1Department of Neuropathology, University Munich,
81377Munich, Germany; 2Department of Biochemistry, Adolf-Butenandt-Institute, University Munich, 81377 Munich, Germany; 3Department of Neurochemistry, MPI for Brain Resarch, 60528 Frankfurt, Germany; 4Ludwig Maximilians Universitiit, 81377 Munich, Germany. Contact e-mail: [email protected]
Background: Loss of APP has been shown to affect calcium release from intracellular stores most likely due to a reduction of the intracellular fragment of APP lAPP intracellular domain (AICD)] (Leissring et al. PNAS 99:4697ff; 2002). Objectives: Here we aim to analyse this mechanism in more detail by analysing the intracelhilar free Ca 2+ concentration in HEK 293 cells expressing both APP with the Swedish mutation (APPsw) as well as the insulin-degrading enzyme (IDE) which is known to degrade AICDs (Edbauer et al. JBC 277:13389ff; 2002). Results: In comparison to nontransfected HEK 293 cells as well as cells expressing APP with the Swedish mutation (APPsw) alone the cytoplasmic free calcium concentration was dramatically increased in two cell lines expressing both APPsw and IDE. Moreover the application of cyclopiazonic acid, known to block the Ca 2+ATPase that pumps Ca 2+ from the cytosol into the endoplasmatic reticulum (ER) did not induce any Ca 2+ transient in these cells. Consequently the capacitative calcium entry from the extracelhilar space, which is activated when the ER-calcium pool levels are low, was found to be dramatically enhanced in APPsw/IDE cells. Similar observations were made in HEK 293 cells expressing a "dominant negative " PS1 mutant (PSl[D385N]) were the generation of AICD is inhibited as well as in dermal fibroblasts from APP deficient mice. These results strongly indicate that lack of AICD blocks the uptake of cytosolic calcium into the ER and leads to a prominent increase of the resting free cytosolic calcium concentration. Conclusions: An enhancement of the resting cytosolic calcium concentration and a decrease of calcium within the ER in neurons have been well described to occur during aging and they are central for the "Ca 2+ hypothesis" of neuronal aging. Thus it is tempting to speculate that neuronal aging might be at least partly caused by an enhanced AICD degradation.
04-04-07 ] CORTICAL DYSPLASIA RESEMBLING HUMAN TYPE 2 LISSENCEPHALY IN M I C E L A C K I N G A L L T H R E E APP-FAMILY M E M B E R S Ulrike Miiller* 1, Brigitte Anliker I , Sabine Heber I , Martin Fuhrmann 2, Hans Kretzschmar 2, Sangram Sisodia 3, Jochen Herms 2.
CMax-Planck-Institute for Brain Research, Frankfurt, Germany; 2Centre for NeuropathoIogy, University of Munich, Munich, Germany; 3Dept. of NeurobioIogy, University of Chicago, Chicago, IL, USA. Contact e-mail: umueller @mpih-frankfurt.mpg.de
Background: The amyloid precursor protein APP plays a central rote in the pathogenesis of Alzheimer's diseae. By contrast, its physiological function