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Neuronal membrane cholesterol loss enhances amyloid peptide generation.

Abad-Rodriguez J, Ledesma MD, Craessaerts K, Perga S, Medina M, Delacourte A, Dingwall C, De Strooper B, Dotti CG - J. Cell Biol. (2004)

Bottom Line: Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol.Their increased colocalization is associated with elevated production of amyloid peptide.These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cavalieri Ottolenghi Scientific Institute, Universita degli Studi di Torino, Orbassano, Italy.

ABSTRACT
Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

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BACE 1 floats in the DRM fraction from control human and mice brain membranes, whereas APP remains in non-DRM heavy fractions. (A) Immunoblots for APP, BACE 1, and flotilin1 in a representative control human hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin 1 (Flot-1). APP, on the contrary, is only detected in heavy fraction 8. The percentages of total BACE 1 in DRMs and fraction 8 are shown in the graph as means and SDs from 10 control human samples. (B) Immunoblots for APP, BACE 1, and flotilin 1 of hippocampal extracts from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. As for the human brain, BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin1 (Flot-1), whereas APP is only detected in heavy fraction 8. (C) Immunoblots for APP, BACE 1, and flotilin 1 of Golgi-endosomal–enriched brain membranes from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. Although BACE 1 floats to DRM light fractions similar to flotilin 1, APP remains in the heavy fractions of the gradient.
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fig1: BACE 1 floats in the DRM fraction from control human and mice brain membranes, whereas APP remains in non-DRM heavy fractions. (A) Immunoblots for APP, BACE 1, and flotilin1 in a representative control human hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin 1 (Flot-1). APP, on the contrary, is only detected in heavy fraction 8. The percentages of total BACE 1 in DRMs and fraction 8 are shown in the graph as means and SDs from 10 control human samples. (B) Immunoblots for APP, BACE 1, and flotilin 1 of hippocampal extracts from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. As for the human brain, BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin1 (Flot-1), whereas APP is only detected in heavy fraction 8. (C) Immunoblots for APP, BACE 1, and flotilin 1 of Golgi-endosomal–enriched brain membranes from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. Although BACE 1 floats to DRM light fractions similar to flotilin 1, APP remains in the heavy fractions of the gradient.

Mentions: To determine the possible involvement of membrane cholesterol in APP processing in physiological conditions, we started by analyzing the buoyant flotation density of this protein and that of its β-secretase BACE 1 in human brain hippocampal membranes. Sucrose gradient centrifugation of membranes extracted with the detergents Lubrol, CHAPS, or Triton X-100, followed by SDS-PAGE and Western blotting with NH2 terminus antibody (see Materials and methods), revealed that most APP is in heavy, detergent-soluble membrane fractions (Fig. 1 A; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1). By contrast, a significant amount of BACE 1 is also present in light, detergent-resistant fractions, similarly to the DRM marker flotilin 1 (Fig. 1 A). The existence of a pool of BACE 1 in DRMs and another in non-DRMs, similarly to other canonical DRM markers (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1), confirms that the protein is in a dynamic balance on the membrane. On the other hand, the almost complete absence of APP in DRMs suggests that this protein is largely restricted to the most fluid environment of non-DRMs.


Neuronal membrane cholesterol loss enhances amyloid peptide generation.

Abad-Rodriguez J, Ledesma MD, Craessaerts K, Perga S, Medina M, Delacourte A, Dingwall C, De Strooper B, Dotti CG - J. Cell Biol. (2004)

BACE 1 floats in the DRM fraction from control human and mice brain membranes, whereas APP remains in non-DRM heavy fractions. (A) Immunoblots for APP, BACE 1, and flotilin1 in a representative control human hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin 1 (Flot-1). APP, on the contrary, is only detected in heavy fraction 8. The percentages of total BACE 1 in DRMs and fraction 8 are shown in the graph as means and SDs from 10 control human samples. (B) Immunoblots for APP, BACE 1, and flotilin 1 of hippocampal extracts from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. As for the human brain, BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin1 (Flot-1), whereas APP is only detected in heavy fraction 8. (C) Immunoblots for APP, BACE 1, and flotilin 1 of Golgi-endosomal–enriched brain membranes from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. Although BACE 1 floats to DRM light fractions similar to flotilin 1, APP remains in the heavy fractions of the gradient.
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Related In: Results  -  Collection

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fig1: BACE 1 floats in the DRM fraction from control human and mice brain membranes, whereas APP remains in non-DRM heavy fractions. (A) Immunoblots for APP, BACE 1, and flotilin1 in a representative control human hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin 1 (Flot-1). APP, on the contrary, is only detected in heavy fraction 8. The percentages of total BACE 1 in DRMs and fraction 8 are shown in the graph as means and SDs from 10 control human samples. (B) Immunoblots for APP, BACE 1, and flotilin 1 of hippocampal extracts from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. As for the human brain, BACE 1 is enriched in fractions 4 and 5 corresponding to DRMs as indicated by the presence of the DRM marker flotilin1 (Flot-1), whereas APP is only detected in heavy fraction 8. (C) Immunoblots for APP, BACE 1, and flotilin 1 of Golgi-endosomal–enriched brain membranes from mice expressing the human APP after Lubrol WX extraction and sucrose gradient centrifugation. Although BACE 1 floats to DRM light fractions similar to flotilin 1, APP remains in the heavy fractions of the gradient.
Mentions: To determine the possible involvement of membrane cholesterol in APP processing in physiological conditions, we started by analyzing the buoyant flotation density of this protein and that of its β-secretase BACE 1 in human brain hippocampal membranes. Sucrose gradient centrifugation of membranes extracted with the detergents Lubrol, CHAPS, or Triton X-100, followed by SDS-PAGE and Western blotting with NH2 terminus antibody (see Materials and methods), revealed that most APP is in heavy, detergent-soluble membrane fractions (Fig. 1 A; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1). By contrast, a significant amount of BACE 1 is also present in light, detergent-resistant fractions, similarly to the DRM marker flotilin 1 (Fig. 1 A). The existence of a pool of BACE 1 in DRMs and another in non-DRMs, similarly to other canonical DRM markers (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1), confirms that the protein is in a dynamic balance on the membrane. On the other hand, the almost complete absence of APP in DRMs suggests that this protein is largely restricted to the most fluid environment of non-DRMs.

Bottom Line: Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol.Their increased colocalization is associated with elevated production of amyloid peptide.These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cavalieri Ottolenghi Scientific Institute, Universita degli Studi di Torino, Orbassano, Italy.

ABSTRACT
Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Show MeSH
Related in: MedlinePlus