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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 is displaced from DRMs and cofractionates with APP in heavier membrane fractions of low membrane cholesterol AD hippocampi and hippocampal neurons in culture. (A) Immunoblots for APP, BACE 1, and flotilin1 in representative low membrane cholesterol AD hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 migration is shifted to the heavy APP-containing fraction 8. DRM modification is shown by a similar shift in the flotation characteristics of the DRM marker flotilin 1 (compare with Fig. 1 A). For quantification, the amount of BACE 1 in fractions 4 and 5 of sucrose gradients (DRM fraction) and fraction 8 (heavy APP-containing fraction) was measured by densitometry. The percentage of BACE 1 in DRMs is significantly reduced to 14% (graph, * indicates P < 0.005) compared with 24% in control samples (Fig. 1 A, graph). Conversely in the APP-containing fraction 8 BACE 1 content is increased to 15% compared with 10% in control samples (Fig. 1 A, graph). Data are means and SDs from 10 low cholesterol AD samples. (B) Moderate membrane cholesterol reduction in vitro displaces BACE 1 from DRMs. Hippocampal neurons grown for 5 d in culture were treated (low chol.) or not (control) with low concentrations of mevilonin and MCD for 5 d (see Materials and methods). This treatment induced <30% reduction in membrane cholesterol. Sucrose gradient fractionations after Lubrol WX extraction and Western blotting for APP and BACE 1 show that in control neurons BACE 1 peaks in fractions 4 and 5 (DRMs), whereas in low cholesterol neurons BACE 1 is spread along the gradient, with a relative enrichment in the APP-containing fraction 8. Disruption of DRMs is shown by the almost complete absence of flotilin 1 in fractions 4 and 5 and the relative enrichment in heavy fraction 8 (compare Flot-1 lines in control and low chol. samples).
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fig4: BACE 1 is displaced from DRMs and cofractionates with APP in heavier membrane fractions of low membrane cholesterol AD hippocampi and hippocampal neurons in culture. (A) Immunoblots for APP, BACE 1, and flotilin1 in representative low membrane cholesterol AD hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 migration is shifted to the heavy APP-containing fraction 8. DRM modification is shown by a similar shift in the flotation characteristics of the DRM marker flotilin 1 (compare with Fig. 1 A). For quantification, the amount of BACE 1 in fractions 4 and 5 of sucrose gradients (DRM fraction) and fraction 8 (heavy APP-containing fraction) was measured by densitometry. The percentage of BACE 1 in DRMs is significantly reduced to 14% (graph, * indicates P < 0.005) compared with 24% in control samples (Fig. 1 A, graph). Conversely in the APP-containing fraction 8 BACE 1 content is increased to 15% compared with 10% in control samples (Fig. 1 A, graph). Data are means and SDs from 10 low cholesterol AD samples. (B) Moderate membrane cholesterol reduction in vitro displaces BACE 1 from DRMs. Hippocampal neurons grown for 5 d in culture were treated (low chol.) or not (control) with low concentrations of mevilonin and MCD for 5 d (see Materials and methods). This treatment induced <30% reduction in membrane cholesterol. Sucrose gradient fractionations after Lubrol WX extraction and Western blotting for APP and BACE 1 show that in control neurons BACE 1 peaks in fractions 4 and 5 (DRMs), whereas in low cholesterol neurons BACE 1 is spread along the gradient, with a relative enrichment in the APP-containing fraction 8. Disruption of DRMs is shown by the almost complete absence of flotilin 1 in fractions 4 and 5 and the relative enrichment in heavy fraction 8 (compare Flot-1 lines in control and low chol. samples).

Mentions: To determine if the cleavage of APP can be affected by changes in membrane cholesterol content we analyzed the flotation density of BACE 1 and APP in hippocampal membranes from the brains of a group of AD patients that present a moderate, still significant, reduction of brain membrane cholesterol (30% less than controls; Ledesma et al., 2003). Fig. 4 A shows that in these membranes APP remains concentrated in detergent-soluble fractions, like in the control situation. Quite differently from controls, BACE 1 is significantly reduced from the light detergent-resistant fractions and increased in the soluble fractions (Fig. 4 A, compare the partitioning profile of both proteins with Fig. 1 A). Densitometric quantification reveals that in the low cholesterol membranes there is a 50% increase (P < 0.005) in the amount of BACE 1 present in the soluble fractions where APP also concentrates (compare Fig. 1 A with Fig. 4 A). The fact that the total amount of BACE 1, measuring both insoluble and soluble fractions, is similar in low cholesterol AD samples and controls, indicates that the loss of BACE 1 from DRMs is not the consequence of changes in its rates of synthesis or degradation but results from a change in DRM organization. In agreement, a significant pool of flotilin 1 is displaced to the soluble fractions in the low cholesterol human membranes (Fig. 4 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)

BACE 1 is displaced from DRMs and cofractionates with APP in heavier membrane fractions of low membrane cholesterol AD hippocampi and hippocampal neurons in culture. (A) Immunoblots for APP, BACE 1, and flotilin1 in representative low membrane cholesterol AD hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 migration is shifted to the heavy APP-containing fraction 8. DRM modification is shown by a similar shift in the flotation characteristics of the DRM marker flotilin 1 (compare with Fig. 1 A). For quantification, the amount of BACE 1 in fractions 4 and 5 of sucrose gradients (DRM fraction) and fraction 8 (heavy APP-containing fraction) was measured by densitometry. The percentage of BACE 1 in DRMs is significantly reduced to 14% (graph, * indicates P < 0.005) compared with 24% in control samples (Fig. 1 A, graph). Conversely in the APP-containing fraction 8 BACE 1 content is increased to 15% compared with 10% in control samples (Fig. 1 A, graph). Data are means and SDs from 10 low cholesterol AD samples. (B) Moderate membrane cholesterol reduction in vitro displaces BACE 1 from DRMs. Hippocampal neurons grown for 5 d in culture were treated (low chol.) or not (control) with low concentrations of mevilonin and MCD for 5 d (see Materials and methods). This treatment induced <30% reduction in membrane cholesterol. Sucrose gradient fractionations after Lubrol WX extraction and Western blotting for APP and BACE 1 show that in control neurons BACE 1 peaks in fractions 4 and 5 (DRMs), whereas in low cholesterol neurons BACE 1 is spread along the gradient, with a relative enrichment in the APP-containing fraction 8. Disruption of DRMs is shown by the almost complete absence of flotilin 1 in fractions 4 and 5 and the relative enrichment in heavy fraction 8 (compare Flot-1 lines in control and low chol. samples).
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fig4: BACE 1 is displaced from DRMs and cofractionates with APP in heavier membrane fractions of low membrane cholesterol AD hippocampi and hippocampal neurons in culture. (A) Immunoblots for APP, BACE 1, and flotilin1 in representative low membrane cholesterol AD hippocampal sample after Lubrol WX extraction and sucrose gradient centrifugation. Note that BACE 1 migration is shifted to the heavy APP-containing fraction 8. DRM modification is shown by a similar shift in the flotation characteristics of the DRM marker flotilin 1 (compare with Fig. 1 A). For quantification, the amount of BACE 1 in fractions 4 and 5 of sucrose gradients (DRM fraction) and fraction 8 (heavy APP-containing fraction) was measured by densitometry. The percentage of BACE 1 in DRMs is significantly reduced to 14% (graph, * indicates P < 0.005) compared with 24% in control samples (Fig. 1 A, graph). Conversely in the APP-containing fraction 8 BACE 1 content is increased to 15% compared with 10% in control samples (Fig. 1 A, graph). Data are means and SDs from 10 low cholesterol AD samples. (B) Moderate membrane cholesterol reduction in vitro displaces BACE 1 from DRMs. Hippocampal neurons grown for 5 d in culture were treated (low chol.) or not (control) with low concentrations of mevilonin and MCD for 5 d (see Materials and methods). This treatment induced <30% reduction in membrane cholesterol. Sucrose gradient fractionations after Lubrol WX extraction and Western blotting for APP and BACE 1 show that in control neurons BACE 1 peaks in fractions 4 and 5 (DRMs), whereas in low cholesterol neurons BACE 1 is spread along the gradient, with a relative enrichment in the APP-containing fraction 8. Disruption of DRMs is shown by the almost complete absence of flotilin 1 in fractions 4 and 5 and the relative enrichment in heavy fraction 8 (compare Flot-1 lines in control and low chol. samples).
Mentions: To determine if the cleavage of APP can be affected by changes in membrane cholesterol content we analyzed the flotation density of BACE 1 and APP in hippocampal membranes from the brains of a group of AD patients that present a moderate, still significant, reduction of brain membrane cholesterol (30% less than controls; Ledesma et al., 2003). Fig. 4 A shows that in these membranes APP remains concentrated in detergent-soluble fractions, like in the control situation. Quite differently from controls, BACE 1 is significantly reduced from the light detergent-resistant fractions and increased in the soluble fractions (Fig. 4 A, compare the partitioning profile of both proteins with Fig. 1 A). Densitometric quantification reveals that in the low cholesterol membranes there is a 50% increase (P < 0.005) in the amount of BACE 1 present in the soluble fractions where APP also concentrates (compare Fig. 1 A with Fig. 4 A). The fact that the total amount of BACE 1, measuring both insoluble and soluble fractions, is similar in low cholesterol AD samples and controls, indicates that the loss of BACE 1 from DRMs is not the consequence of changes in its rates of synthesis or degradation but results from a change in DRM organization. In agreement, a significant pool of flotilin 1 is displaced to the soluble fractions in the low cholesterol human membranes (Fig. 4 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