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LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery.

Brachet A, Norwood S, Brouwers JF, Palomer E, Helms JB, Dotti CG, Esteban JA - J. Cell Biol. (2015)

Bottom Line: A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation.This process is accompanied by an increase of NMDAR function and an enhancement of LTP.These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.

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Affiliation: Departamento de Neurobiología, Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.

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Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.
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fig1: Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.

Mentions: LTP was induced on hippocampal slice cultures using a well-characterized pharmacological approach that mimics biochemical and electrophysiological properties of electrically induced LTP (chemical LTP [cLTP]; Otmakhov et al., 2004; Kopec et al., 2006; Arendt et al., 2014). This pharmacological approach was used to maximize the number of synapses undergoing plasticity. For biochemical quantification of endogenous cholesterol, slices were homogenized just after cLTP induction or after a recovery period of 20 min and compared with control (untreated) slices. Liquid chromatography (LC)/mass spectrometry (MS) analysis was used to separately measure cholesterol levels in the plasma membrane and microsomal fractions (enriched in intracellular membranes; see Materials and methods). As shown in Fig. 1 A (left-most graph), no significant change was observed in the plasma membrane fraction. In contrast, a rapid and sustained decrease in cholesterol content was observed in intracellular membranes (Fig. 1 A, microsomal fraction, left graph). This decrease required NMDAR activation, as it was blocked when cLTP was induced in the presence of the NMDAR antagonist AP5 (2-amino-5-phosphonopentanoate; Fig. 1 A, +AP5 graphs).


LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery.

Brachet A, Norwood S, Brouwers JF, Palomer E, Helms JB, Dotti CG, Esteban JA - J. Cell Biol. (2015)

Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.
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fig1: Cholesterol reduction from intracellular membranes after cLTP induction. (A) Quantification of cholesterol content by LC/MS from hippocampal slices immediately after cLTP induction (cLTP), 20 min recovery after cLTP induction (Rec), or from untreated slices (baseline [Bsln]). Cholesterol (Chol) content was normalized to major phospholipids (phosphocholine [PC] plus sphingomyelin [SM]) and plotted relative to baseline values. Experiments were performed with or without the NMDAR antagonist AP5, as indicated. Symbols connected with lines represent time course experiments performed in parallel. Different lines represent independent experiments. Quantifications were performed from plasma membrane fraction (left) or from microsomal fractions (right). Statistical significance was evaluated with the Kruskal–Wallis test. (B) Quantification of Bodipy-cholesterol fluorescence from hippocampal slices undergoing cLTP induction (blue-shaded area), with or without AP5, as indicated. Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Representative images of Bodipy-cholesterol–labeled neurons are shown on the left. (C) Mean values of Bodipy-cholesterol fluorescence immediately at the end of the cLTP induction period (cLTP) and after 10-min recovery, from the time course shown in B. The p-value was determined with the Mann–Whitney test. n represents the number of cells. (D) Diffuse and clustered mCherry-D4 fluorescence was quantified (see Materials and methods) from hippocampal slices undergoing cLTP induction (blue-shaded area). Fluorescence values are normalized to the mean fluorescence before cLTP induction. n represents the number of cells. Left shows representative images of a CA1 neuron expressing mCherry-D4 (top) and EGFP (bottom), before (Baseline) and after 15-min cLTP induction. (E) Mean values of mCherry-D4 diffuse or clustered fluorescence immediately before (Baseline), at the end of the cLTP induction period (cLTP), or after 10 min of recovery, from the time course shown in D. The p-value was determined with the Mann–Whitney test. n represent the number of cells. Error bars show means ± SEM. Bars, 10 µm.
Mentions: LTP was induced on hippocampal slice cultures using a well-characterized pharmacological approach that mimics biochemical and electrophysiological properties of electrically induced LTP (chemical LTP [cLTP]; Otmakhov et al., 2004; Kopec et al., 2006; Arendt et al., 2014). This pharmacological approach was used to maximize the number of synapses undergoing plasticity. For biochemical quantification of endogenous cholesterol, slices were homogenized just after cLTP induction or after a recovery period of 20 min and compared with control (untreated) slices. Liquid chromatography (LC)/mass spectrometry (MS) analysis was used to separately measure cholesterol levels in the plasma membrane and microsomal fractions (enriched in intracellular membranes; see Materials and methods). As shown in Fig. 1 A (left-most graph), no significant change was observed in the plasma membrane fraction. In contrast, a rapid and sustained decrease in cholesterol content was observed in intracellular membranes (Fig. 1 A, microsomal fraction, left graph). This decrease required NMDAR activation, as it was blocked when cLTP was induced in the presence of the NMDAR antagonist AP5 (2-amino-5-phosphonopentanoate; Fig. 1 A, +AP5 graphs).

Bottom Line: A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation.This process is accompanied by an increase of NMDAR function and an enhancement of LTP.These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Neurobiología, Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.

Show MeSH
Related in: MedlinePlus