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Novel regulation of interaction between APP and its binding proteins
Poster Presentation:
Molecular
DISREGULATION 18381MER’ S DISEASE
and Cellular
OF TUBERIN
Biology
III
AND Pz’Ras-GAP
Sl85
IN ALZHEI-
During the early stagea of Alzhenners disease (AD), dormant genes which are generally most active during neurogeneais and cell proliferation are reactivated. Current theories suggest that it is actually the reappearance of such mitogenic factors that lead to selected hallmarks of AD. Previous work using gene profiling techniques have suggested alterations in two such genes, tuberin and p*‘R”“-GAP Tubenn, the protein product of the TX2 gene, is involved in cell proliferation and act5 a&a tumor suppressor and is lost in patients suffering from the autosomal dominant disorder Tuberow Sclerosis. Ras molecules, such a.\ P”~“-GAP, play an important role in regulation of growth, differentiation. and proliferation of cells. Activation of p”“.“GAP is partially responsible for the post-translational modification of tau protein and amyloid precursor protein. Uring antibodies to tuberin and P”~“‘-GAP, results from immunohistological studies show alteration in protein expression in different region\ of the human brains of Alzheimer’s patients versus control patients. Cells are double stained with either PHF-I or TG3 antibodies to delineate disease progression. Cell counts across cerebellum, hippocampus, and cortex demonstrate whether tuberin or pL’R”“-GAP change m conjunction with the pathogen&s of Alzheimers disease. Western blot analyses provide quantitative information on the protein expression changes between Alzheimer’s samples and those from age matched controls. Alteratmn of these cell proliferation factors lend support to a growing hypothesis that the port-mltotic cells in patient\ suffering from Alzheimer’s disease are reentering or expressing signals generally associated with cell cycle activation.
AB DEPOSITION IN VASCULAR MYOCYTES CULTURED FROM APP TRANSGENIC MOUSE: THE ROLE OF CELL LIPID OXIDATION.
Amyloidosis in AD IS associated with aging not only in sporadic, but also familial cases, in which amyloidogenic mutations are compensated for decades of life. The major senescence-associated cell dysfunction is an increased cell vulnerability to oxidative stress. We tested the role of oxidative stress in AP deposition m APP-Swedish mutation transgenic mice (Hsiao mice)using vascular smooth muscle cells (SMC) cultured from the brain and periphery. In aged Hsiao mice, deposition of AP in meningeal blood vesels ia associated with SMC, similarly to that described m humanr with AD, DS and in aged dogs. Primary cultures of human and canine bran SMC from amyloid angiopathy-affected vessels were shown to accumulate AP during cell senewence. Primary culturer of SMC from tran%genic mice, isolated from the hram and peripheral vessels, contained 5 to 40% of cells that accumulated A!3 spontaneuously. The AD accumulating SMC overexpresxd APP. 4-5 times the APP levels in fibroblasts cultured from dura. Transformation of SMC with SV40 T large antigen retained APP over-expression and A@ accumulation. Further cloning yielded cultures with various levels of A@ accumulation and with distinct intracellular localization of AP deposits. SMC were tested for AP production and accumulation (by immunocytochemistry and ELISA) after oxidative stress induced by treatment with ImM ferric or ferrous ions for 72 hours. The treatment increased AP secretion and the proportion of cells with AD deposits (to 60 100%). Some deposits were immunoreactive with an antibody against tibrillar AD. In 5% of lines the induced accumulation of AP was very strong and led to cell damage. The treatment with lipid-soluble antioxidant u-tocopherol (vit. E) reduced AP deposition, both the spontaneou\ and iron-induced, but had little effect on Afi wxtion. The antiamyloidogenic effects of a water-soluble antioxidant melatonin were significantly less pronounced. The data indicate that oxidative modifications of cellular lipids influence the fate of the produced A@, its cellular localization and aggregation. Vlt. E may partially neutralize the effects of iron: it reduces the iron-induced AP aggregation, but not the iron-induced AP secretion.
PRESENILIN-CONTAINING AGGRESOME CHOLESTEROL MUTANT CELLS
FORMATION
IN
Imre Kovacs, Kristen M Lentini, Laura A MacKenzie Ingano. Luigi Pug/i& Rudolph E Tanzi, Dora M Kovncs, MA GermHasp and Harnrd Med Sch, Charlesrown, MA Intracellular inclusions are important neuropathological features ofneurodegenerative diseares. Neuronal inclusions in Alzheimer’r. disease are mainly represented by neurofibrillary tangles, but other inclusions are also present in specific brain regions (corpora amylacea, granulovacuolar bodies, Hiram bodies, etc.). A possible model for the formation of cytoplasmic inclusions is represented by a novel structure termed aggresome. Aggresomea are intracellular stable, multiubiquitmated protein aggregates
surrounded by a vimentin cage. These structures are formed in cells overproducing misfolded proteins m excess, like PSI or the cystic fibrosis CFTR, or in response to proteasomal inhibition. Given that both PS 1 and CFIX are membrane proteins, we hypothesized that cholesterol may have an effect on aggresome formation. Cholesterol metabolism is increasingly implicated in AD pathology and APP processing. Decreased levels of cholesterol in primary neurons and in APP-tranafected cell lines seem to reduce P-amyloid production. To investigate the role of cholesterol in aggresome formation, we used 25.RA and AC-29 Chinese hamster ovary (CHO) cells, both overproducing cholesterol. AC-29 cells, in addition to overproducing cholesterol, are unable to synthesize cholesterol esters. Both cell lines were stably transfected with wild type and FAD mutant Ml46L and L286V PSI. To detect aggresome formation, we treated cells with 20 pM ALLN for 12 hand double-stained them for vimentin and PSI. In untransfected AC-29 cells we found that vimentin collapsed at a ~uxtanuclear position, but PSI distribution did not change In untransfected 25.RA cells vimentin did not collapse in a juxtanuclear position. Elevated PSI expresrion m tranafected cells resulted in more aggresomes in AC-29 cells: 25% of cells contained aggresomes, as opposed to 50% in control transfected CHO cells. However, in PSI tranafected 25.RA cells we did not detect ‘classic’ aggresome formation, while PSI wa5 found characteristically in the ER. Our results \how that cholesterol metabolism affecta aggresome formatlon in stably tranafected CHO cells. We are currently studying the effect of PSI mutations on aggresome formation in thew cellr.
NOVEL REGULATION OF INTERACTION ITS BINDING PROTEINS
BETWEEN
APP AND
Amyloid precursor protein (APP) is an integral membrane protein with receptor-like structure. and cytoplasmic domam of APP contams several motifs for metabolism and function of APP. Many proteins are reported to bind its cytoplasmic domam but the regulation of binding of those proteins to APP is not analyzed sufficiently. We recently reported that APP Thr668 residue (numbering for APP695) of the cytoplasmic domain is phosphorylated in brain t&sues and neuronal-differentiated PC12 cells. We report here the mutation of Thr668 residue affects the binding affinity of APP to some of protein, which interact with APP. This finding suggest phosphoryolation of T668 may play an imponant role in biological function of APP.
pm/
STEM CELL BIOLOGY - THE SEARCH FOR A CELL REPLACEMENT PARADIGM.
Precursor stem cellr identified within particular anatomical regions of the mammalian brain have demonstrated an ability to amplify in vitro and to express specific phrnotyplc characteristic\ both in vitro and when transplanted in viva Such a discovery opens the door to the successful replacement of dying or non-functional cells acrow a spectrum of neurodegenerative diseases. As part of an international mvcsugation into the viability of such an approach we have exammed the ability of htppocampal precursor cells derived from rodent and non-human primates to amplify and to dlfferentmte without phenotypic ambiguity. This has involved mvesugating precursor cells taken at a range of neonatal ages (post natal days I- 7) amplifying them m culture and assessing the effects of different culture conditions upon survival. amplification, gene and protein expresrion and chromosomal stability. Within the common marmoset (Calhthrix jacchus), we have been able to demonstrate the ability of single hippocampal precursor stem cells to amplify from tissues at post natal days l-4. After thia age point, such cells survived and amplified for several months in vitro but failed to amplify as smgle isolated cells for the generation of clonal lines irrespective of culture conditions. Cells taken from marmosets at post natal days 1 and 2 amplified and continued to do so for over 18 months yielding an abundance of material for further analysis. Our results were consistent across all clonal lines and age ranges. While some lines expressed higher levels of glial markers throughout and other linea expressed higher levels of neuronal markers neither phenotypic marker wab exclnswely expressed. Independent of culture conditions and number of passages these cell$ remained phenotypically ambiguous. Details of analyses undertaken and repective rewlta wll be presented.
Molecular
DISREGULATION 18381MER’ S DISEASE
and Cellular
OF TUBERIN
Biology
III
AND Pz’Ras-GAP
Sl85
IN ALZHEI-
During the early stagea of Alzhenners disease (AD), dormant genes which are generally most active during neurogeneais and cell proliferation are reactivated. Current theories suggest that it is actually the reappearance of such mitogenic factors that lead to selected hallmarks of AD. Previous work using gene profiling techniques have suggested alterations in two such genes, tuberin and p*‘R”“-GAP Tubenn, the protein product of the TX2 gene, is involved in cell proliferation and act5 a&a tumor suppressor and is lost in patients suffering from the autosomal dominant disorder Tuberow Sclerosis. Ras molecules, such a.\ P”~“-GAP, play an important role in regulation of growth, differentiation. and proliferation of cells. Activation of p”“.“GAP is partially responsible for the post-translational modification of tau protein and amyloid precursor protein. Uring antibodies to tuberin and P”~“‘-GAP, results from immunohistological studies show alteration in protein expression in different region\ of the human brains of Alzheimer’s patients versus control patients. Cells are double stained with either PHF-I or TG3 antibodies to delineate disease progression. Cell counts across cerebellum, hippocampus, and cortex demonstrate whether tuberin or pL’R”“-GAP change m conjunction with the pathogen&s of Alzheimers disease. Western blot analyses provide quantitative information on the protein expression changes between Alzheimer’s samples and those from age matched controls. Alteratmn of these cell proliferation factors lend support to a growing hypothesis that the port-mltotic cells in patient\ suffering from Alzheimer’s disease are reentering or expressing signals generally associated with cell cycle activation.
AB DEPOSITION IN VASCULAR MYOCYTES CULTURED FROM APP TRANSGENIC MOUSE: THE ROLE OF CELL LIPID OXIDATION.
Amyloidosis in AD IS associated with aging not only in sporadic, but also familial cases, in which amyloidogenic mutations are compensated for decades of life. The major senescence-associated cell dysfunction is an increased cell vulnerability to oxidative stress. We tested the role of oxidative stress in AP deposition m APP-Swedish mutation transgenic mice (Hsiao mice)using vascular smooth muscle cells (SMC) cultured from the brain and periphery. In aged Hsiao mice, deposition of AP in meningeal blood vesels ia associated with SMC, similarly to that described m humanr with AD, DS and in aged dogs. Primary cultures of human and canine bran SMC from amyloid angiopathy-affected vessels were shown to accumulate AP during cell senewence. Primary culturer of SMC from tran%genic mice, isolated from the hram and peripheral vessels, contained 5 to 40% of cells that accumulated A!3 spontaneuously. The AD accumulating SMC overexpresxd APP. 4-5 times the APP levels in fibroblasts cultured from dura. Transformation of SMC with SV40 T large antigen retained APP over-expression and A@ accumulation. Further cloning yielded cultures with various levels of A@ accumulation and with distinct intracellular localization of AP deposits. SMC were tested for AP production and accumulation (by immunocytochemistry and ELISA) after oxidative stress induced by treatment with ImM ferric or ferrous ions for 72 hours. The treatment increased AP secretion and the proportion of cells with AD deposits (to 60 100%). Some deposits were immunoreactive with an antibody against tibrillar AD. In 5% of lines the induced accumulation of AP was very strong and led to cell damage. The treatment with lipid-soluble antioxidant u-tocopherol (vit. E) reduced AP deposition, both the spontaneou\ and iron-induced, but had little effect on Afi wxtion. The antiamyloidogenic effects of a water-soluble antioxidant melatonin were significantly less pronounced. The data indicate that oxidative modifications of cellular lipids influence the fate of the produced A@, its cellular localization and aggregation. Vlt. E may partially neutralize the effects of iron: it reduces the iron-induced AP aggregation, but not the iron-induced AP secretion.
PRESENILIN-CONTAINING AGGRESOME CHOLESTEROL MUTANT CELLS
FORMATION
IN
Imre Kovacs, Kristen M Lentini, Laura A MacKenzie Ingano. Luigi Pug/i& Rudolph E Tanzi, Dora M Kovncs, MA GermHasp and Harnrd Med Sch, Charlesrown, MA Intracellular inclusions are important neuropathological features ofneurodegenerative diseares. Neuronal inclusions in Alzheimer’r. disease are mainly represented by neurofibrillary tangles, but other inclusions are also present in specific brain regions (corpora amylacea, granulovacuolar bodies, Hiram bodies, etc.). A possible model for the formation of cytoplasmic inclusions is represented by a novel structure termed aggresome. Aggresomea are intracellular stable, multiubiquitmated protein aggregates
surrounded by a vimentin cage. These structures are formed in cells overproducing misfolded proteins m excess, like PSI or the cystic fibrosis CFTR, or in response to proteasomal inhibition. Given that both PS 1 and CFIX are membrane proteins, we hypothesized that cholesterol may have an effect on aggresome formation. Cholesterol metabolism is increasingly implicated in AD pathology and APP processing. Decreased levels of cholesterol in primary neurons and in APP-tranafected cell lines seem to reduce P-amyloid production. To investigate the role of cholesterol in aggresome formation, we used 25.RA and AC-29 Chinese hamster ovary (CHO) cells, both overproducing cholesterol. AC-29 cells, in addition to overproducing cholesterol, are unable to synthesize cholesterol esters. Both cell lines were stably transfected with wild type and FAD mutant Ml46L and L286V PSI. To detect aggresome formation, we treated cells with 20 pM ALLN for 12 hand double-stained them for vimentin and PSI. In untransfected AC-29 cells we found that vimentin collapsed at a ~uxtanuclear position, but PSI distribution did not change In untransfected 25.RA cells vimentin did not collapse in a juxtanuclear position. Elevated PSI expresrion m tranafected cells resulted in more aggresomes in AC-29 cells: 25% of cells contained aggresomes, as opposed to 50% in control transfected CHO cells. However, in PSI tranafected 25.RA cells we did not detect ‘classic’ aggresome formation, while PSI wa5 found characteristically in the ER. Our results \how that cholesterol metabolism affecta aggresome formatlon in stably tranafected CHO cells. We are currently studying the effect of PSI mutations on aggresome formation in thew cellr.
NOVEL REGULATION OF INTERACTION ITS BINDING PROTEINS
BETWEEN
APP AND
Amyloid precursor protein (APP) is an integral membrane protein with receptor-like structure. and cytoplasmic domam of APP contams several motifs for metabolism and function of APP. Many proteins are reported to bind its cytoplasmic domam but the regulation of binding of those proteins to APP is not analyzed sufficiently. We recently reported that APP Thr668 residue (numbering for APP695) of the cytoplasmic domain is phosphorylated in brain t&sues and neuronal-differentiated PC12 cells. We report here the mutation of Thr668 residue affects the binding affinity of APP to some of protein, which interact with APP. This finding suggest phosphoryolation of T668 may play an imponant role in biological function of APP.
pm/
STEM CELL BIOLOGY - THE SEARCH FOR A CELL REPLACEMENT PARADIGM.
Precursor stem cellr identified within particular anatomical regions of the mammalian brain have demonstrated an ability to amplify in vitro and to express specific phrnotyplc characteristic\ both in vitro and when transplanted in viva Such a discovery opens the door to the successful replacement of dying or non-functional cells acrow a spectrum of neurodegenerative diseases. As part of an international mvcsugation into the viability of such an approach we have exammed the ability of htppocampal precursor cells derived from rodent and non-human primates to amplify and to dlfferentmte without phenotypic ambiguity. This has involved mvesugating precursor cells taken at a range of neonatal ages (post natal days I- 7) amplifying them m culture and assessing the effects of different culture conditions upon survival. amplification, gene and protein expresrion and chromosomal stability. Within the common marmoset (Calhthrix jacchus), we have been able to demonstrate the ability of single hippocampal precursor stem cells to amplify from tissues at post natal days l-4. After thia age point, such cells survived and amplified for several months in vitro but failed to amplify as smgle isolated cells for the generation of clonal lines irrespective of culture conditions. Cells taken from marmosets at post natal days 1 and 2 amplified and continued to do so for over 18 months yielding an abundance of material for further analysis. Our results were consistent across all clonal lines and age ranges. While some lines expressed higher levels of glial markers throughout and other linea expressed higher levels of neuronal markers neither phenotypic marker wab exclnswely expressed. Independent of culture conditions and number of passages these cell$ remained phenotypically ambiguous. Details of analyses undertaken and repective rewlta wll be presented.