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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 membrane cholesterol reduction in vitro enhances APP-β-cleavage and Aβ production. (A) 10 d in vitro hippocampal neurons were not treated (C) or treated as indicated in Materials and methods to lower the membrane cholesterol up to 30% (Chol−). Cholesterol was added back to some of the treated cells for 15 min (Chol+) as cholesterol–MCD inclusion complexes. The amount of total APP and APP-β-CTF fragment in the different cell extracts was determined by Western blot. Densitometry of the β-CTF fragment normalized to the amount of total APP revealed a significant 39% increase in low cholesterol neurons with respect to untreated neurons. This effect was reverted by cholesterol replenishment. Thus, a 15-min treatment with the cholesterol inclusion complexes results in the production of similar amount of β-CTF fragment than control neurons (91% of control). Data shown in the graph are means and SDs from three different experiments. (B) Crude membrane pellets and conditioned media of control or low membrane cholesterol CHO-7w cells (stably expressing human APP) were submitted to PAGE-SDS in 10% Bis-Tris NuPage gels. Western blot detection was performed with an anti-APP COOH-terminal antibody to visualize holo-APP and with anti-APP mAb (WO2) to detect the amyloid peptide. Aβ peptide production was increased in cases of moderate low cholesterol an average of 47% over the control (lanes 1 and 3 in the blot and <25% bar in the graph, n = 6). Confirming previous data from other groups, extensive cholesterol loss over 35% leads to a strong decrease in Aβ production (lane 2 in the blot and >35% bar in the graph, n = 2).
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fig6: Moderate membrane cholesterol reduction in vitro enhances APP-β-cleavage and Aβ production. (A) 10 d in vitro hippocampal neurons were not treated (C) or treated as indicated in Materials and methods to lower the membrane cholesterol up to 30% (Chol−). Cholesterol was added back to some of the treated cells for 15 min (Chol+) as cholesterol–MCD inclusion complexes. The amount of total APP and APP-β-CTF fragment in the different cell extracts was determined by Western blot. Densitometry of the β-CTF fragment normalized to the amount of total APP revealed a significant 39% increase in low cholesterol neurons with respect to untreated neurons. This effect was reverted by cholesterol replenishment. Thus, a 15-min treatment with the cholesterol inclusion complexes results in the production of similar amount of β-CTF fragment than control neurons (91% of control). Data shown in the graph are means and SDs from three different experiments. (B) Crude membrane pellets and conditioned media of control or low membrane cholesterol CHO-7w cells (stably expressing human APP) were submitted to PAGE-SDS in 10% Bis-Tris NuPage gels. Western blot detection was performed with an anti-APP COOH-terminal antibody to visualize holo-APP and with anti-APP mAb (WO2) to detect the amyloid peptide. Aβ peptide production was increased in cases of moderate low cholesterol an average of 47% over the control (lanes 1 and 3 in the blot and <25% bar in the graph, n = 6). Confirming previous data from other groups, extensive cholesterol loss over 35% leads to a strong decrease in Aβ production (lane 2 in the blot and >35% bar in the graph, n = 2).

Mentions: To determine whether the enhanced biochemical and spatial association between BACE 1 and APP resulting from moderate cholesterol reduction is functionally relevant, the production of the β-COOH-terminal fragment (β-CTF) of APP and of Aβ was measured in APP constitutively expressing hippocampal neurons and in APP stably transfected fibroblast-like cells (CHO) in culture, with normal or a moderate to high membrane cholesterol reduction. The level of β-CTF in hippocampal neurons with a 25–30% reduction of membrane cholesterol is 39% higher than in cells with normal membrane cholesterol content (P < 0.005; Fig. 6 A). To test if the increased production is the direct consequence of the loss of cholesterol different from a secondary, unspecific effect of the added drugs, cholesterol was replenished to the treated neurons in the form of cyclodextrin–cholesterol inclusion complexes (Simons et al., 1998). Such treatment for 15 min restores membrane cholesterol levels (110% of control). Notably, the level of APP β-cleavage in this condition is reestablished to the nontreated situation (91% of control; Fig. 6 A).


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 membrane cholesterol reduction in vitro enhances APP-β-cleavage and Aβ production. (A) 10 d in vitro hippocampal neurons were not treated (C) or treated as indicated in Materials and methods to lower the membrane cholesterol up to 30% (Chol−). Cholesterol was added back to some of the treated cells for 15 min (Chol+) as cholesterol–MCD inclusion complexes. The amount of total APP and APP-β-CTF fragment in the different cell extracts was determined by Western blot. Densitometry of the β-CTF fragment normalized to the amount of total APP revealed a significant 39% increase in low cholesterol neurons with respect to untreated neurons. This effect was reverted by cholesterol replenishment. Thus, a 15-min treatment with the cholesterol inclusion complexes results in the production of similar amount of β-CTF fragment than control neurons (91% of control). Data shown in the graph are means and SDs from three different experiments. (B) Crude membrane pellets and conditioned media of control or low membrane cholesterol CHO-7w cells (stably expressing human APP) were submitted to PAGE-SDS in 10% Bis-Tris NuPage gels. Western blot detection was performed with an anti-APP COOH-terminal antibody to visualize holo-APP and with anti-APP mAb (WO2) to detect the amyloid peptide. Aβ peptide production was increased in cases of moderate low cholesterol an average of 47% over the control (lanes 1 and 3 in the blot and <25% bar in the graph, n = 6). Confirming previous data from other groups, extensive cholesterol loss over 35% leads to a strong decrease in Aβ production (lane 2 in the blot and >35% bar in the graph, n = 2).
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fig6: Moderate membrane cholesterol reduction in vitro enhances APP-β-cleavage and Aβ production. (A) 10 d in vitro hippocampal neurons were not treated (C) or treated as indicated in Materials and methods to lower the membrane cholesterol up to 30% (Chol−). Cholesterol was added back to some of the treated cells for 15 min (Chol+) as cholesterol–MCD inclusion complexes. The amount of total APP and APP-β-CTF fragment in the different cell extracts was determined by Western blot. Densitometry of the β-CTF fragment normalized to the amount of total APP revealed a significant 39% increase in low cholesterol neurons with respect to untreated neurons. This effect was reverted by cholesterol replenishment. Thus, a 15-min treatment with the cholesterol inclusion complexes results in the production of similar amount of β-CTF fragment than control neurons (91% of control). Data shown in the graph are means and SDs from three different experiments. (B) Crude membrane pellets and conditioned media of control or low membrane cholesterol CHO-7w cells (stably expressing human APP) were submitted to PAGE-SDS in 10% Bis-Tris NuPage gels. Western blot detection was performed with an anti-APP COOH-terminal antibody to visualize holo-APP and with anti-APP mAb (WO2) to detect the amyloid peptide. Aβ peptide production was increased in cases of moderate low cholesterol an average of 47% over the control (lanes 1 and 3 in the blot and <25% bar in the graph, n = 6). Confirming previous data from other groups, extensive cholesterol loss over 35% leads to a strong decrease in Aβ production (lane 2 in the blot and >35% bar in the graph, n = 2).
Mentions: To determine whether the enhanced biochemical and spatial association between BACE 1 and APP resulting from moderate cholesterol reduction is functionally relevant, the production of the β-COOH-terminal fragment (β-CTF) of APP and of Aβ was measured in APP constitutively expressing hippocampal neurons and in APP stably transfected fibroblast-like cells (CHO) in culture, with normal or a moderate to high membrane cholesterol reduction. The level of β-CTF in hippocampal neurons with a 25–30% reduction of membrane cholesterol is 39% higher than in cells with normal membrane cholesterol content (P < 0.005; Fig. 6 A). To test if the increased production is the direct consequence of the loss of cholesterol different from a secondary, unspecific effect of the added drugs, cholesterol was replenished to the treated neurons in the form of cyclodextrin–cholesterol inclusion complexes (Simons et al., 1998). Such treatment for 15 min restores membrane cholesterol levels (110% of control). Notably, the level of APP β-cleavage in this condition is reestablished to the nontreated situation (91% of control; Fig. 6 A).

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