Cholesterol reduction impairs exocytosis of synaptic vesicles.
Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.
Linetti
Anna
A
CNR-Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.
Fratangeli
Alessandra
A
Taverna
Elena
E
Valnegri
Pamela
P
Francolini
Maura
M
Cappello
Valentina
V
Matteoli
Michela
M
Passafaro
Maria
M
Rosa
Patrizia
P
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D037341:Fumonisins / Q000494:pharmacology; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D021962:Membrane Microdomains / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008959:Models, Neurological; D009474:Neurons / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D050863:Synaptotagmin I / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D014233:Tricarboxylic Acids / Q000494:pharmacology
D000924:Anticholesteremic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D020864:Calcium Channels, N-Type; D002784:Cholesterol; D037341:Fumonisins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007074:Immunoglobulin G; D008148:Lovastatin; D050600:SNARE Proteins; D013107:Sphingolipids; D050863:Synaptotagmin I; D014233:Tricarboxylic Acids; C494312:Syt1 protein, rat; C075117:squalestatin 1; C012258:mevastatin