Nicastrin is an integral component of the high molecular weight presenilin complexes that control... more Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo--N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute ␥-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.
Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with famnilia... more Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with famnilial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS 1 as well as human wild-type (wtPSl) and the familial AD Metl46Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PSI antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PSI; (ii) significantly reduced levels of full-length human PS 1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organellespecific proteins revealed that PSI was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PSI showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PSI distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS 1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models.
Background: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease ... more Background: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease (FAD). Materials and Methods: We characterized the cellular localization and endoproteolysis of presenilin 2 (PS2) and presenilin 1 (PS1) in brains from 25 individuals with presenilin-mutations causing FAD, as well as neurologically normal individuals and individuals with sporadic Alzheimer's disease (AD). Results: Amino-terminal antibodies to both presenilins predominantly decorated large neurons. Regional differences between the broad distributions of the two presenilins were greatest in the cerebellum, where most Purkinje cells showed high levels of only PS2 immunoreactivity. PS2 endoproteolysis in brain yielded multiple amino-terminal fragments similar in size to the PS1 amino-terminal fragments detected in brain. In addition, two different PS2 amino-terminal antibodies also detected a prominent 42 kDa band that may represent a novel PS2 form in human brain. Similar to PS1 findings, neither amino-terminal nor antiloop PS2 antibodies revealed substantial full-length PS2 in brain. Immunocytochemical examination of brains from individuals with the N 141 I PS2 mutation or eight different PS1 mutations, spanning the molecule from the second transmembrane domain to the large cytoplasmic loop domain, revealed immunodecoration of no senile plaques and only neurofibrillary tangles in the M 139 I PS1 mutation stained with PS1 antibodies. Conclusions: Overall presenilin expression and the relative abundance of full-length and amino-terminal fragments in presenilin FAD cases were similar to control cases and sporadic AD cases. Thus, accumulation of full-length protein or other gross mismetabolism of neither PS2 nor PS1 is a consequence of the FAD mutations examined.
Nicastrin is an integral component of the high molecular weight presenilin complexes that control... more Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo--N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute ␥-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.
Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with famnilia... more Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with famnilial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS 1 as well as human wild-type (wtPSl) and the familial AD Metl46Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PSI antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PSI; (ii) significantly reduced levels of full-length human PS 1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organellespecific proteins revealed that PSI was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PSI showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PSI distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS 1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models.
Background: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease ... more Background: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease (FAD). Materials and Methods: We characterized the cellular localization and endoproteolysis of presenilin 2 (PS2) and presenilin 1 (PS1) in brains from 25 individuals with presenilin-mutations causing FAD, as well as neurologically normal individuals and individuals with sporadic Alzheimer's disease (AD). Results: Amino-terminal antibodies to both presenilins predominantly decorated large neurons. Regional differences between the broad distributions of the two presenilins were greatest in the cerebellum, where most Purkinje cells showed high levels of only PS2 immunoreactivity. PS2 endoproteolysis in brain yielded multiple amino-terminal fragments similar in size to the PS1 amino-terminal fragments detected in brain. In addition, two different PS2 amino-terminal antibodies also detected a prominent 42 kDa band that may represent a novel PS2 form in human brain. Similar to PS1 findings, neither amino-terminal nor antiloop PS2 antibodies revealed substantial full-length PS2 in brain. Immunocytochemical examination of brains from individuals with the N 141 I PS2 mutation or eight different PS1 mutations, spanning the molecule from the second transmembrane domain to the large cytoplasmic loop domain, revealed immunodecoration of no senile plaques and only neurofibrillary tangles in the M 139 I PS1 mutation stained with PS1 antibodies. Conclusions: Overall presenilin expression and the relative abundance of full-length and amino-terminal fragments in presenilin FAD cases were similar to control cases and sporadic AD cases. Thus, accumulation of full-length protein or other gross mismetabolism of neither PS2 nor PS1 is a consequence of the FAD mutations examined.
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