- Email: [email protected]
P2-114 Cell-type specific processing of the APP protein family in drosophila
Poster Session P2: Animal and Cellular Models - Animal Models, Transgenic faster, were denser, and grew larger in AB-Arctic mice than in AB-Wt mice. Indeed, mice from a high expresser AB-Arctic line had massive cerebral amyloid deposits by two months of age. The results obtained so far demonstrate that the Arctic mutation elicits an aggressive cerebral amyloidosis in transgenic mice. Ongoing studies focus on a comparison of AB assembly and degradation in AB-Arctic mice and AB-Wt mice, as well as on neurodegenerative and behavioral alterations in the new models. This work was supported by the NIA and the N1NDS.
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CELL-TYPE SPECIFIC PROCESSING OF THE APP PROTEIN FAMILY IN DROSOPHILA
Alexander Loewer*, Peter Soba, Michaela V. Bilic, David Kuttenkeuler, Konrad Beyreuther, Renato Paro, Gunter Merdes. University of Heidelberg,
Heidelberg, Germany. Contact e-mail: [email protected] Background: In recent years, the secretases responsible for processing of APP and the release of the amyloidogenic A[3 peptide have been identified. However, it remains elusive, how these proteases are regulated and how they interact in the context of a living animal. Objectives: We choose Drosophila melanogaster as a model organism to study the processing of APP and related molecules in vivo, aiming to identify new regulatory mechanisms with the sophisticated genetic and molecular tools available for the fly. Methods: To analyze y-secretase dependent processing in-vivo, we established a GFP-based reporter system by expressing fusion constructs of the APP family with LexA and VP16 under the control of the tissue specific GAIA system. Upon processing of these transmembrane proteins, their intracelhilar domains are released and translocated to the nucleus where they activate a GFP reporter gene. Because of the similar cleavage mechanism, we also included fusion constructs of the Notch receptor in the reporter system to examine potential differences in the processing of these y-secretase substrates. Results: Using our reporter system in the developing Drosophila eye, we could demonstrate differences between the processing of APP and Notch. Most striking is the restriction of efficient APP processing to a subset of cells, which could be identified as the developing photoreceptor neurons. In contrast, the Notch receptor is only activated and processed in undetermined cells. By overexpressing ct- and 13-secretase and by using a pre-cleaved form of APP (C99), we could show that the efficiency of APP processing is regulated at the level of y-secretase cleavage. In accordance with these results, we find a similar cell-type dependent variation in the processing of APLP2 and the fly homolog APPL. Conclusions: Our results indicate that, in contrast to current models, the release of the intracellular domain of APE and thus of the amyloidogenic A[3 peptide, can be regulated in different cell types by modulated y-secretase activity. The isolation of factors responsible for this substrate and cell type specific processing by y-secretase should reveal new drug targets which can be used to lower A~3 production without affecting the processing of other y-secretase substrates.
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AXONAL DYSTROPHY IN CHOLINERGIC, GALANINERGIC AND DOPAMINERGIC PROJECTION SYSTEMS IN APPSWE/PSI?E9 TRANSGENIC MICE
Elliott J. Mufson*. Rush University Medical School, Chicago, IL, USA.
Contact e-mail: emufson @rush.edu Background: The cortical and hippocampal cholinergic basal forebrain (CBF) and the nigro-striatal dopaminergic systems display axonal dystrophy in relation to amyloid beta (Ab) deposition in Alzheimer's (AD) and AD with Parkinson's disease (PD). Objective: To determine whether these projection systems exhibit axonal dystrophy in a transgenic model of amyloidosis. Methods: Axonal dystrophy in the cholinergic and dopaminergic projection systems were evaluated in double transgenic mice harboring the FAD-linked mutant APPswe/PSI?E9 genes at different ages using antibodies directed against choline acetyltransferase (CHAT), galanin (GAL; a neuropeptide associated with cholinergic systems) and tyrosine hydroxilase (a maker for dopaminergic profiles). Results and Conclusions: Amyloid plaques along with dystrophic CHAT, GAL immunoreactive (-Jr) neurites were first seen
$257
at 3 months of age in these mice. Interestingly, confocal microscopy of dual stained sections did not reveal an overlap between the ChAT and GAL neurites adjacent to Ab plaques. Moreover, beginning at 8 months there was a reduction in ChAT-ir fibers in the fornix, which connects the septal cholinergic neurons with the hippocampus. Ab plaques and dystrophic tyrosine hydroxylase-ir neurites within the striatum as well as swollen TH-ir axonal profiles in the nigro-striatal bundle were first found in 7-8 month old mice. Cortical and hippocampal tissue from 7-8 months old mice prepared for ultra-structural analysis revealed numerous dystrophic neurites in close apposition to Ab plaques as well as neurites far removed from these lesions. In 9-17 month-old mice, morphologic changes were also observed in the cholinergic neurons of the septal/nucleus basalis complex, striatal interneurons and, in dopaminergic neurons of the substantia nigra. These findings suggest that age-dependent axonal dystrophy occurs in the cholinergic and dopaminergic projection systems and is associated with the deposition of Ab plaques in the APPswe/PSI?E9 transgenic mouse. This transgenic mouse may provide a model to investigate whether axonal transport defects are related to the over expression of A[3.
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NEURONAL OVEREXPRESSION OF LRP SELECTIVELY INCREASES A DETERGENT-SOLUBLE POOL OF HIPPOCAMPAL ABETA IN PDAPP MICE
Celina V. Zerbinatti* 1, Kelly R. Bales 2, Steven M. Paul 2, Guojun Bu 1
1Washington University, Saint Louis, MO, USA," 2Lilly Research Laboratories, Indianapolis, IN, USA. Contact e-mail: zerbinatti c @kids.wustl, edu The low-density lipoprotein receptor-related protein (LRP) has been linked to Alzheimer's disease (AD) by both genetic and biochemical evidence. We generated a transgenic mouse that overexpress a functional minireceptor of LRP in the brain and bred it to PDAPP mice, an animal model that develops typical amyloid plaques. While A~ plaque burden was not altered by the overexpression of LRP, PDAPP/LRP + mice had increased carbonate-soluble brain A~ levels at old age. More important, A~ levels in carbonate-soluble extracts were highly correlated with deficits in spatial learning and memory in old mice. To further characterize the soluble pool of A~ altered by LRP, we performed a 3-step tissue extraction, which included: 1) a Tris-buffered saline (TBS) fraction (extracelhilar soluble A~); 2) a TBS/Triton X-100 fraction (membrane- and/or cell-associated soluble A~); and 3) a guanidine fraction (insoluble, plaque-associated A[~). Each extract was analyzed for A~40 and A~42 by ELISA. While TBS-sohible A[~ levels were similar between PDAPP/LRP + (n = 14) and PDAPP/LRP- (n = 15) (60.7 -4- 3.7 and 57.6 ± 2.8 pg/mg hippocampus, respectively; p = 0.51 by Student's t test), A~ levels in the TBS/Triton X-100-sohible fraction was significantly higher in PDAPP/LRP + than PDAPP/LRP- mice as young as 12 months of age (47.4 ± 3.8 and 37.5 ± 2.1 pg/mg hippocampus, respectively; p = 0.027 by Student's t test). Insoluble A~ levels did not differ significantly between the two groups (PDAPP/LRP + = 24,730 ± 2940 pg/mg hippocampus; PDAPP/LRP- = 21,170 ± 2186 pg/mg hippocampus; p = 0.33 by Student's t test). There were no significant correlations between TBS-soluble A~ levels or TBS/Triton X-100-soluble A[~ levels and insoluble A~ (R 2 = 0.03; p = 0.38 and R 2 = 0.03; p = 0.33, respectively), indicating that these soluble pools are independent of A~ plaques. By inmmnofluorescence of brain sections using A~-specific antibodies, we detected A~ staining in neuronal cell bodies and processes, suggesting that this cell-associated A~ is likely the pool represented in the detergent-soluble extract. In summary, LRP appears to preferentially increase a cell- or membrane-associated A~ pool, consistent with the hypothesis that LRP facilitates APP beta-cleavage in the endocytic pathway.
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CELL-TYPE SPECIFIC PROCESSING OF THE APP PROTEIN FAMILY IN DROSOPHILA
Alexander Loewer*, Peter Soba, Michaela V. Bilic, David Kuttenkeuler, Konrad Beyreuther, Renato Paro, Gunter Merdes. University of Heidelberg,
Heidelberg, Germany. Contact e-mail: [email protected] Background: In recent years, the secretases responsible for processing of APP and the release of the amyloidogenic A[3 peptide have been identified. However, it remains elusive, how these proteases are regulated and how they interact in the context of a living animal. Objectives: We choose Drosophila melanogaster as a model organism to study the processing of APP and related molecules in vivo, aiming to identify new regulatory mechanisms with the sophisticated genetic and molecular tools available for the fly. Methods: To analyze y-secretase dependent processing in-vivo, we established a GFP-based reporter system by expressing fusion constructs of the APP family with LexA and VP16 under the control of the tissue specific GAIA system. Upon processing of these transmembrane proteins, their intracelhilar domains are released and translocated to the nucleus where they activate a GFP reporter gene. Because of the similar cleavage mechanism, we also included fusion constructs of the Notch receptor in the reporter system to examine potential differences in the processing of these y-secretase substrates. Results: Using our reporter system in the developing Drosophila eye, we could demonstrate differences between the processing of APP and Notch. Most striking is the restriction of efficient APP processing to a subset of cells, which could be identified as the developing photoreceptor neurons. In contrast, the Notch receptor is only activated and processed in undetermined cells. By overexpressing ct- and 13-secretase and by using a pre-cleaved form of APP (C99), we could show that the efficiency of APP processing is regulated at the level of y-secretase cleavage. In accordance with these results, we find a similar cell-type dependent variation in the processing of APLP2 and the fly homolog APPL. Conclusions: Our results indicate that, in contrast to current models, the release of the intracellular domain of APE and thus of the amyloidogenic A[3 peptide, can be regulated in different cell types by modulated y-secretase activity. The isolation of factors responsible for this substrate and cell type specific processing by y-secretase should reveal new drug targets which can be used to lower A~3 production without affecting the processing of other y-secretase substrates.
•
AXONAL DYSTROPHY IN CHOLINERGIC, GALANINERGIC AND DOPAMINERGIC PROJECTION SYSTEMS IN APPSWE/PSI?E9 TRANSGENIC MICE
Elliott J. Mufson*. Rush University Medical School, Chicago, IL, USA.
Contact e-mail: emufson @rush.edu Background: The cortical and hippocampal cholinergic basal forebrain (CBF) and the nigro-striatal dopaminergic systems display axonal dystrophy in relation to amyloid beta (Ab) deposition in Alzheimer's (AD) and AD with Parkinson's disease (PD). Objective: To determine whether these projection systems exhibit axonal dystrophy in a transgenic model of amyloidosis. Methods: Axonal dystrophy in the cholinergic and dopaminergic projection systems were evaluated in double transgenic mice harboring the FAD-linked mutant APPswe/PSI?E9 genes at different ages using antibodies directed against choline acetyltransferase (CHAT), galanin (GAL; a neuropeptide associated with cholinergic systems) and tyrosine hydroxilase (a maker for dopaminergic profiles). Results and Conclusions: Amyloid plaques along with dystrophic CHAT, GAL immunoreactive (-Jr) neurites were first seen
$257
at 3 months of age in these mice. Interestingly, confocal microscopy of dual stained sections did not reveal an overlap between the ChAT and GAL neurites adjacent to Ab plaques. Moreover, beginning at 8 months there was a reduction in ChAT-ir fibers in the fornix, which connects the septal cholinergic neurons with the hippocampus. Ab plaques and dystrophic tyrosine hydroxylase-ir neurites within the striatum as well as swollen TH-ir axonal profiles in the nigro-striatal bundle were first found in 7-8 month old mice. Cortical and hippocampal tissue from 7-8 months old mice prepared for ultra-structural analysis revealed numerous dystrophic neurites in close apposition to Ab plaques as well as neurites far removed from these lesions. In 9-17 month-old mice, morphologic changes were also observed in the cholinergic neurons of the septal/nucleus basalis complex, striatal interneurons and, in dopaminergic neurons of the substantia nigra. These findings suggest that age-dependent axonal dystrophy occurs in the cholinergic and dopaminergic projection systems and is associated with the deposition of Ab plaques in the APPswe/PSI?E9 transgenic mouse. This transgenic mouse may provide a model to investigate whether axonal transport defects are related to the over expression of A[3.
•
NEURONAL OVEREXPRESSION OF LRP SELECTIVELY INCREASES A DETERGENT-SOLUBLE POOL OF HIPPOCAMPAL ABETA IN PDAPP MICE
Celina V. Zerbinatti* 1, Kelly R. Bales 2, Steven M. Paul 2, Guojun Bu 1
1Washington University, Saint Louis, MO, USA," 2Lilly Research Laboratories, Indianapolis, IN, USA. Contact e-mail: zerbinatti c @kids.wustl, edu The low-density lipoprotein receptor-related protein (LRP) has been linked to Alzheimer's disease (AD) by both genetic and biochemical evidence. We generated a transgenic mouse that overexpress a functional minireceptor of LRP in the brain and bred it to PDAPP mice, an animal model that develops typical amyloid plaques. While A~ plaque burden was not altered by the overexpression of LRP, PDAPP/LRP + mice had increased carbonate-soluble brain A~ levels at old age. More important, A~ levels in carbonate-soluble extracts were highly correlated with deficits in spatial learning and memory in old mice. To further characterize the soluble pool of A~ altered by LRP, we performed a 3-step tissue extraction, which included: 1) a Tris-buffered saline (TBS) fraction (extracelhilar soluble A~); 2) a TBS/Triton X-100 fraction (membrane- and/or cell-associated soluble A~); and 3) a guanidine fraction (insoluble, plaque-associated A[~). Each extract was analyzed for A~40 and A~42 by ELISA. While TBS-sohible A[~ levels were similar between PDAPP/LRP + (n = 14) and PDAPP/LRP- (n = 15) (60.7 -4- 3.7 and 57.6 ± 2.8 pg/mg hippocampus, respectively; p = 0.51 by Student's t test), A~ levels in the TBS/Triton X-100-sohible fraction was significantly higher in PDAPP/LRP + than PDAPP/LRP- mice as young as 12 months of age (47.4 ± 3.8 and 37.5 ± 2.1 pg/mg hippocampus, respectively; p = 0.027 by Student's t test). Insoluble A~ levels did not differ significantly between the two groups (PDAPP/LRP + = 24,730 ± 2940 pg/mg hippocampus; PDAPP/LRP- = 21,170 ± 2186 pg/mg hippocampus; p = 0.33 by Student's t test). There were no significant correlations between TBS-soluble A~ levels or TBS/Triton X-100-soluble A[~ levels and insoluble A~ (R 2 = 0.03; p = 0.38 and R 2 = 0.03; p = 0.33, respectively), indicating that these soluble pools are independent of A~ plaques. By inmmnofluorescence of brain sections using A~-specific antibodies, we detected A~ staining in neuronal cell bodies and processes, suggesting that this cell-associated A~ is likely the pool represented in the detergent-soluble extract. In summary, LRP appears to preferentially increase a cell- or membrane-associated A~ pool, consistent with the hypothesis that LRP facilitates APP beta-cleavage in the endocytic pathway.