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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: However, little is known about the contribution of lipid metabolism during these processes.We found that N-methyl-d-aspartate-type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron.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.

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Effect of cholesterol removal on synaptic function. (A and B) Quantification of cholesterol content of hippocampal slices treated with cholesterol oxidase (Chol Ox) or methyl-β-cyclodextrin (MBCD) from plasma membrane fraction (A) or from microsomal fraction (B). Values are normalized to untreated (control) slices. n represents number of independent experiments. Statistical significance is calculated according to the Wilcoxon test for slices treated in parallel. (C and D) Input resistance (C) and whole-cell capacitance (D) from hippocampal slices treated as indicated. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (E) Paired-pulse facilitation (PPF) ratios from whole-cell synaptic responses evoked by stimulation with different interstimulus intervals (50, 100, 200, and 400 ms). Hippocampal slices were treated as indicated. Representative traces at 50-ms interstimulus interval are shown above the graph. Bars: (vertical) 50 pA; (horizontal) 50 ms. (F) Mean ratio of synaptic responses mediated by AMPARs and GABAARs (AMPA/GABA ratio). Representative traces are shown above the histogram. Bars: (vertical) 50 pA; (horizontal) 10 ms. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (G) Cumulative distribution of mEPSC amplitude from dissociated neuronal cultures treated with Chol Ox or untreated controls, as indicated. Mean mEPSC amplitude from the same data is plotted as a histogram in the inset. n represents number of miniature responses recorded from four cells for each condition. Representative traces are shown above the graphs. Bars: (horizontal) 1 s; (vertical) 10 pA. (H) Similar to G for mIPSC amplitudes. Bars: (horizontal) 1 s; (vertical) 50 pA. Error bars show means ± SEM.
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fig2: Effect of cholesterol removal on synaptic function. (A and B) Quantification of cholesterol content of hippocampal slices treated with cholesterol oxidase (Chol Ox) or methyl-β-cyclodextrin (MBCD) from plasma membrane fraction (A) or from microsomal fraction (B). Values are normalized to untreated (control) slices. n represents number of independent experiments. Statistical significance is calculated according to the Wilcoxon test for slices treated in parallel. (C and D) Input resistance (C) and whole-cell capacitance (D) from hippocampal slices treated as indicated. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (E) Paired-pulse facilitation (PPF) ratios from whole-cell synaptic responses evoked by stimulation with different interstimulus intervals (50, 100, 200, and 400 ms). Hippocampal slices were treated as indicated. Representative traces at 50-ms interstimulus interval are shown above the graph. Bars: (vertical) 50 pA; (horizontal) 50 ms. (F) Mean ratio of synaptic responses mediated by AMPARs and GABAARs (AMPA/GABA ratio). Representative traces are shown above the histogram. Bars: (vertical) 50 pA; (horizontal) 10 ms. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (G) Cumulative distribution of mEPSC amplitude from dissociated neuronal cultures treated with Chol Ox or untreated controls, as indicated. Mean mEPSC amplitude from the same data is plotted as a histogram in the inset. n represents number of miniature responses recorded from four cells for each condition. Representative traces are shown above the graphs. Bars: (horizontal) 1 s; (vertical) 10 pA. (H) Similar to G for mIPSC amplitudes. Bars: (horizontal) 1 s; (vertical) 50 pA. Error bars show means ± SEM.

Mentions: After observing that LTP induction leads to a rapid decrease in cholesterol content in CA1 neurons, we decided to test whether an acute cholesterol loss may in turn affect synaptic function. To this end, we used pharmacological approaches, rather than slower genetic interventions or inhibitors of enzymatic activities, to bypass compensatory effects and/or accumulation of intermediary products (Kotti et al., 2006). Endogenous cholesterol was reduced by short time applications (30 min) of MBCD (10 mM) or cholesterol oxidase (Chol Ox) enzyme (10 U/ml). These two manipulations offer complementary information to control for potential nonspecific effects because MBCD is only partially specific for cholesterol (Ohtani et al., 1989) but does not generate additional metabolites, whereas Chol Ox is very specific for cholesterol but generates H2O2 and a ketone derivative of cholesterol as byproducts. As shown in Fig. 2 A, both protocols produced a moderate but significant decrease of cholesterol levels in hippocampal slices, as probed by a fluorescence enzymatic assay. This decrease was stable 1 h after the end of the treatment (Fig. S2 A). Of note, these manipulations initially target plasma membrane cholesterol, but they produced a similar reduction of cholesterol from intracellular membranes (Fig. 2 B), probably because of the extensive exchange between these two pools of cholesterol (Zidovetzki and Levitan, 2007). Importantly, this pharmacological reduction in cholesterol from intracellular membranes was quantitatively similar to the one observed upon cLTP induction (compare Fig. 1 A and Fig. 2 B).


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)

Effect of cholesterol removal on synaptic function. (A and B) Quantification of cholesterol content of hippocampal slices treated with cholesterol oxidase (Chol Ox) or methyl-β-cyclodextrin (MBCD) from plasma membrane fraction (A) or from microsomal fraction (B). Values are normalized to untreated (control) slices. n represents number of independent experiments. Statistical significance is calculated according to the Wilcoxon test for slices treated in parallel. (C and D) Input resistance (C) and whole-cell capacitance (D) from hippocampal slices treated as indicated. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (E) Paired-pulse facilitation (PPF) ratios from whole-cell synaptic responses evoked by stimulation with different interstimulus intervals (50, 100, 200, and 400 ms). Hippocampal slices were treated as indicated. Representative traces at 50-ms interstimulus interval are shown above the graph. Bars: (vertical) 50 pA; (horizontal) 50 ms. (F) Mean ratio of synaptic responses mediated by AMPARs and GABAARs (AMPA/GABA ratio). Representative traces are shown above the histogram. Bars: (vertical) 50 pA; (horizontal) 10 ms. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (G) Cumulative distribution of mEPSC amplitude from dissociated neuronal cultures treated with Chol Ox or untreated controls, as indicated. Mean mEPSC amplitude from the same data is plotted as a histogram in the inset. n represents number of miniature responses recorded from four cells for each condition. Representative traces are shown above the graphs. Bars: (horizontal) 1 s; (vertical) 10 pA. (H) Similar to G for mIPSC amplitudes. Bars: (horizontal) 1 s; (vertical) 50 pA. Error bars show means ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362467&req=5

fig2: Effect of cholesterol removal on synaptic function. (A and B) Quantification of cholesterol content of hippocampal slices treated with cholesterol oxidase (Chol Ox) or methyl-β-cyclodextrin (MBCD) from plasma membrane fraction (A) or from microsomal fraction (B). Values are normalized to untreated (control) slices. n represents number of independent experiments. Statistical significance is calculated according to the Wilcoxon test for slices treated in parallel. (C and D) Input resistance (C) and whole-cell capacitance (D) from hippocampal slices treated as indicated. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (E) Paired-pulse facilitation (PPF) ratios from whole-cell synaptic responses evoked by stimulation with different interstimulus intervals (50, 100, 200, and 400 ms). Hippocampal slices were treated as indicated. Representative traces at 50-ms interstimulus interval are shown above the graph. Bars: (vertical) 50 pA; (horizontal) 50 ms. (F) Mean ratio of synaptic responses mediated by AMPARs and GABAARs (AMPA/GABA ratio). Representative traces are shown above the histogram. Bars: (vertical) 50 pA; (horizontal) 10 ms. n represents the number of cells. Statistical significance is calculated according to the Mann–Whitney test. (G) Cumulative distribution of mEPSC amplitude from dissociated neuronal cultures treated with Chol Ox or untreated controls, as indicated. Mean mEPSC amplitude from the same data is plotted as a histogram in the inset. n represents number of miniature responses recorded from four cells for each condition. Representative traces are shown above the graphs. Bars: (horizontal) 1 s; (vertical) 10 pA. (H) Similar to G for mIPSC amplitudes. Bars: (horizontal) 1 s; (vertical) 50 pA. Error bars show means ± SEM.
Mentions: After observing that LTP induction leads to a rapid decrease in cholesterol content in CA1 neurons, we decided to test whether an acute cholesterol loss may in turn affect synaptic function. To this end, we used pharmacological approaches, rather than slower genetic interventions or inhibitors of enzymatic activities, to bypass compensatory effects and/or accumulation of intermediary products (Kotti et al., 2006). Endogenous cholesterol was reduced by short time applications (30 min) of MBCD (10 mM) or cholesterol oxidase (Chol Ox) enzyme (10 U/ml). These two manipulations offer complementary information to control for potential nonspecific effects because MBCD is only partially specific for cholesterol (Ohtani et al., 1989) but does not generate additional metabolites, whereas Chol Ox is very specific for cholesterol but generates H2O2 and a ketone derivative of cholesterol as byproducts. As shown in Fig. 2 A, both protocols produced a moderate but significant decrease of cholesterol levels in hippocampal slices, as probed by a fluorescence enzymatic assay. This decrease was stable 1 h after the end of the treatment (Fig. S2 A). Of note, these manipulations initially target plasma membrane cholesterol, but they produced a similar reduction of cholesterol from intracellular membranes (Fig. 2 B), probably because of the extensive exchange between these two pools of cholesterol (Zidovetzki and Levitan, 2007). Importantly, this pharmacological reduction in cholesterol from intracellular membranes was quantitatively similar to the one observed upon cLTP induction (compare Fig. 1 A and Fig. 2 B).

Bottom Line: However, little is known about the contribution of lipid metabolism during these processes.We found that N-methyl-d-aspartate-type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron.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