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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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Moderate cholesterol reduction induces coclustering of BACE 1 and APP on the plasma membrane of cultured hippocampal neurons. BACE 1–APP colocalization was studied in 10 d in vitro hippocampal neurons using the copatching technique (see Materials and methods). (A) In control neurons BACE 1 (red clusters) and APP (green clusters) are extensively segregated to different membrane domains. (B) In contrast, low cholesterol neurons (treated as indicated in Materials and methods to lower the membrane cholesterol up to 30%) show a clear enhancement of BACE 1–APP colocalization. (C) For quantification, the degree of intersection among APP and BACE 1 clusters was considered as “copatching” (intersection >80%), “partial copatching” (intersection between 30% and 50%),“contact” (intersection between 0% and 30%), or random (no intersection; not depicted). For control cells 7% of APP clusters copatch and 12% partially copatch with BACE 1 (gray bars). These values are significantly increased to 19% and 21%, respectively (* indicates P < 0.005) in low cholesterol neurons (black bars). Data are means and SDs of three independent experiments.
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fig5: Moderate cholesterol reduction induces coclustering of BACE 1 and APP on the plasma membrane of cultured hippocampal neurons. BACE 1–APP colocalization was studied in 10 d in vitro hippocampal neurons using the copatching technique (see Materials and methods). (A) In control neurons BACE 1 (red clusters) and APP (green clusters) are extensively segregated to different membrane domains. (B) In contrast, low cholesterol neurons (treated as indicated in Materials and methods to lower the membrane cholesterol up to 30%) show a clear enhancement of BACE 1–APP colocalization. (C) For quantification, the degree of intersection among APP and BACE 1 clusters was considered as “copatching” (intersection >80%), “partial copatching” (intersection between 30% and 50%),“contact” (intersection between 0% and 30%), or random (no intersection; not depicted). For control cells 7% of APP clusters copatch and 12% partially copatch with BACE 1 (gray bars). These values are significantly increased to 19% and 21%, respectively (* indicates P < 0.005) in low cholesterol neurons (black bars). Data are means and SDs of three independent experiments.

Mentions: Because differences in detergent partitioning cannot be used as the sole parameter to conclude that a given protein is present or excluded from a particular domain of the membrane (Zurzolo et al., 2003), we investigated next the distribution of endogenous APP and BACE 1 on the plasma membrane of living neurons in culture. Thus, antibody “copatching” (Harder et al., 1998; Ehehalt et al., 2003) for these molecules was performed in live rodent hippocampal neurons in culture (see Materials and methods). This work revealed that only 7% of APP positive dots exactly colocalize with BACE 1 clusters (Fig. 3 B; Fig. 5 for details and quantitation; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1). Because APP is not detected in DRMs by biochemistry (Fig. 1), it is quite likely that the few coclusters revealed in the microscopy assay reflect non-DRM coexistence. Further confirming the biochemical differences, a large proportion of BACE 1 on the neuronal surface is in DRMs, as judged from the extensive copatching with Thy-1 (Fig. 3 A). Together, with the previously demonstrated paucity of APP in the DRMs of Golgi and endosomal membranes (Fig. 1 C), this first series of results is consistent with the view that non-DRM domains of the plasma or internal membranes are the preferred sites for BACE 1–APP interaction.


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)

Moderate cholesterol reduction induces coclustering of BACE 1 and APP on the plasma membrane of cultured hippocampal neurons. BACE 1–APP colocalization was studied in 10 d in vitro hippocampal neurons using the copatching technique (see Materials and methods). (A) In control neurons BACE 1 (red clusters) and APP (green clusters) are extensively segregated to different membrane domains. (B) In contrast, low cholesterol neurons (treated as indicated in Materials and methods to lower the membrane cholesterol up to 30%) show a clear enhancement of BACE 1–APP colocalization. (C) For quantification, the degree of intersection among APP and BACE 1 clusters was considered as “copatching” (intersection >80%), “partial copatching” (intersection between 30% and 50%),“contact” (intersection between 0% and 30%), or random (no intersection; not depicted). For control cells 7% of APP clusters copatch and 12% partially copatch with BACE 1 (gray bars). These values are significantly increased to 19% and 21%, respectively (* indicates P < 0.005) in low cholesterol neurons (black bars). Data are means and SDs of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172459&req=5

fig5: Moderate cholesterol reduction induces coclustering of BACE 1 and APP on the plasma membrane of cultured hippocampal neurons. BACE 1–APP colocalization was studied in 10 d in vitro hippocampal neurons using the copatching technique (see Materials and methods). (A) In control neurons BACE 1 (red clusters) and APP (green clusters) are extensively segregated to different membrane domains. (B) In contrast, low cholesterol neurons (treated as indicated in Materials and methods to lower the membrane cholesterol up to 30%) show a clear enhancement of BACE 1–APP colocalization. (C) For quantification, the degree of intersection among APP and BACE 1 clusters was considered as “copatching” (intersection >80%), “partial copatching” (intersection between 30% and 50%),“contact” (intersection between 0% and 30%), or random (no intersection; not depicted). For control cells 7% of APP clusters copatch and 12% partially copatch with BACE 1 (gray bars). These values are significantly increased to 19% and 21%, respectively (* indicates P < 0.005) in low cholesterol neurons (black bars). Data are means and SDs of three independent experiments.
Mentions: Because differences in detergent partitioning cannot be used as the sole parameter to conclude that a given protein is present or excluded from a particular domain of the membrane (Zurzolo et al., 2003), we investigated next the distribution of endogenous APP and BACE 1 on the plasma membrane of living neurons in culture. Thus, antibody “copatching” (Harder et al., 1998; Ehehalt et al., 2003) for these molecules was performed in live rodent hippocampal neurons in culture (see Materials and methods). This work revealed that only 7% of APP positive dots exactly colocalize with BACE 1 clusters (Fig. 3 B; Fig. 5 for details and quantitation; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404149/DC1). Because APP is not detected in DRMs by biochemistry (Fig. 1), it is quite likely that the few coclusters revealed in the microscopy assay reflect non-DRM coexistence. Further confirming the biochemical differences, a large proportion of BACE 1 on the neuronal surface is in DRMs, as judged from the extensive copatching with Thy-1 (Fig. 3 A). Together, with the previously demonstrated paucity of APP in the DRMs of Golgi and endosomal membranes (Fig. 1 C), this first series of results is consistent with the view that non-DRM domains of the plasma or internal membranes are the preferred sites for BACE 1–APP interaction.

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