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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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Overexpression of human APP leads to the incorporation of the protein in DRMs from rodent neuroblastoma N2A cells but not in human neuroblastoma SH-SY5Y cells or rat hippocampal neurons in primary culture. Nondifferentiated N2A cells (A), nondifferentiated SH-SY5Y cells (B) and mature primary rat hippocampal neurons (C) were infected with SFV-APP for 8 h. Cell extracts were detergent extracted at 4°C and centrifuged in sucrose gradients. DRMs were obtained in fractions 4 and 5 as indicated by the enrichment of the DRM marker flotilin1. Although a small amount of human APP (<5%) appears in DRMs from overexpressing N2A cells, no significant amount of the protein was found in the DRM fractions from SH-SY5Y or primary neurons even when the levels of APP overexpression are very high (see fractions 7 and 8 of the gradients). The absence of overexpressed APP in detergent insoluble membranes of primary hippocampal neurons was further confirmed along the biosynthetic pathway with a pulse-chase experiment after metabolic labeling (D). Mature neurons were infected either with Fowl plague virus to express the DRM marker HA (D, a) or with SFV-APP (D, b) and extracted with 20 mM CHAPS at 4°C. CHAPS-soluble material (S) and insoluble (P) was resolved in SDS-PAGE (6%) and the images obtained by autoradiography. Although HA CHAPS insolubility increases during the biosynthetic pathway (D, a) and is evident after 90-min chase, when the protein has already reached the plasma membrane, APP remains CHAPS soluble along the biosynthetic pathway and transport to the membrane (D, b).
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fig2: Overexpression of human APP leads to the incorporation of the protein in DRMs from rodent neuroblastoma N2A cells but not in human neuroblastoma SH-SY5Y cells or rat hippocampal neurons in primary culture. Nondifferentiated N2A cells (A), nondifferentiated SH-SY5Y cells (B) and mature primary rat hippocampal neurons (C) were infected with SFV-APP for 8 h. Cell extracts were detergent extracted at 4°C and centrifuged in sucrose gradients. DRMs were obtained in fractions 4 and 5 as indicated by the enrichment of the DRM marker flotilin1. Although a small amount of human APP (<5%) appears in DRMs from overexpressing N2A cells, no significant amount of the protein was found in the DRM fractions from SH-SY5Y or primary neurons even when the levels of APP overexpression are very high (see fractions 7 and 8 of the gradients). The absence of overexpressed APP in detergent insoluble membranes of primary hippocampal neurons was further confirmed along the biosynthetic pathway with a pulse-chase experiment after metabolic labeling (D). Mature neurons were infected either with Fowl plague virus to express the DRM marker HA (D, a) or with SFV-APP (D, b) and extracted with 20 mM CHAPS at 4°C. CHAPS-soluble material (S) and insoluble (P) was resolved in SDS-PAGE (6%) and the images obtained by autoradiography. Although HA CHAPS insolubility increases during the biosynthetic pathway (D, a) and is evident after 90-min chase, when the protein has already reached the plasma membrane, APP remains CHAPS soluble along the biosynthetic pathway and transport to the membrane (D, b).

Mentions: Considering that a pool of APP has been reported to partition in DRMs in a number of cell types under overexpression conditions (Ehehalt et al., 2003), we analyzed if increasing APP levels in neurons would suffice for its incorporation into these domains. Thus, human APP was expressed transiently via the Semliki Forest virus vector (De Strooper et al., 1995) in pseudo-neuronal and primary neurons in culture. Fig. 2 A shows that only a small percentage of the protein is in DRMs (<5%) in overexpressing undifferentiated N2A rodent neuroblastoma cells. On the other hand, APP remains undetectable in DRMs of both overexpressing SH-SY5Y human neuroblastoma cells and rat hippocampal neurons in primary culture (Fig. 2, B–D).


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)

Overexpression of human APP leads to the incorporation of the protein in DRMs from rodent neuroblastoma N2A cells but not in human neuroblastoma SH-SY5Y cells or rat hippocampal neurons in primary culture. Nondifferentiated N2A cells (A), nondifferentiated SH-SY5Y cells (B) and mature primary rat hippocampal neurons (C) were infected with SFV-APP for 8 h. Cell extracts were detergent extracted at 4°C and centrifuged in sucrose gradients. DRMs were obtained in fractions 4 and 5 as indicated by the enrichment of the DRM marker flotilin1. Although a small amount of human APP (<5%) appears in DRMs from overexpressing N2A cells, no significant amount of the protein was found in the DRM fractions from SH-SY5Y or primary neurons even when the levels of APP overexpression are very high (see fractions 7 and 8 of the gradients). The absence of overexpressed APP in detergent insoluble membranes of primary hippocampal neurons was further confirmed along the biosynthetic pathway with a pulse-chase experiment after metabolic labeling (D). Mature neurons were infected either with Fowl plague virus to express the DRM marker HA (D, a) or with SFV-APP (D, b) and extracted with 20 mM CHAPS at 4°C. CHAPS-soluble material (S) and insoluble (P) was resolved in SDS-PAGE (6%) and the images obtained by autoradiography. Although HA CHAPS insolubility increases during the biosynthetic pathway (D, a) and is evident after 90-min chase, when the protein has already reached the plasma membrane, APP remains CHAPS soluble along the biosynthetic pathway and transport to the membrane (D, b).
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fig2: Overexpression of human APP leads to the incorporation of the protein in DRMs from rodent neuroblastoma N2A cells but not in human neuroblastoma SH-SY5Y cells or rat hippocampal neurons in primary culture. Nondifferentiated N2A cells (A), nondifferentiated SH-SY5Y cells (B) and mature primary rat hippocampal neurons (C) were infected with SFV-APP for 8 h. Cell extracts were detergent extracted at 4°C and centrifuged in sucrose gradients. DRMs were obtained in fractions 4 and 5 as indicated by the enrichment of the DRM marker flotilin1. Although a small amount of human APP (<5%) appears in DRMs from overexpressing N2A cells, no significant amount of the protein was found in the DRM fractions from SH-SY5Y or primary neurons even when the levels of APP overexpression are very high (see fractions 7 and 8 of the gradients). The absence of overexpressed APP in detergent insoluble membranes of primary hippocampal neurons was further confirmed along the biosynthetic pathway with a pulse-chase experiment after metabolic labeling (D). Mature neurons were infected either with Fowl plague virus to express the DRM marker HA (D, a) or with SFV-APP (D, b) and extracted with 20 mM CHAPS at 4°C. CHAPS-soluble material (S) and insoluble (P) was resolved in SDS-PAGE (6%) and the images obtained by autoradiography. Although HA CHAPS insolubility increases during the biosynthetic pathway (D, a) and is evident after 90-min chase, when the protein has already reached the plasma membrane, APP remains CHAPS soluble along the biosynthetic pathway and transport to the membrane (D, b).
Mentions: Considering that a pool of APP has been reported to partition in DRMs in a number of cell types under overexpression conditions (Ehehalt et al., 2003), we analyzed if increasing APP levels in neurons would suffice for its incorporation into these domains. Thus, human APP was expressed transiently via the Semliki Forest virus vector (De Strooper et al., 1995) in pseudo-neuronal and primary neurons in culture. Fig. 2 A shows that only a small percentage of the protein is in DRMs (<5%) in overexpressing undifferentiated N2A rodent neuroblastoma cells. On the other hand, APP remains undetectable in DRMs of both overexpressing SH-SY5Y human neuroblastoma cells and rat hippocampal neurons in primary culture (Fig. 2, B–D).

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