Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons.
Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4°C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55-67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain.
['Nusshold|Christoph|C|', 'Uellen|Andreas|A|', 'Bernhart|Eva|E|', 'Hammer|Astrid|A|', 'Damm|Sabine|S|', 'Wintersperger|Andrea|A|', 'Reicher|Helga|H|', 'Hermetter|Albin|A|', 'Malle|Ernst|E|', 'Sattler|Wolfgang|W|']
D000818:Animals D001896:Boron Compounds D022461:Caveolins D002460:Cell Line D004705:Endocytosis D004721:Endoplasmic Reticulum D005456:Fluorescent Dyes D005786:Gene Expression Regulation D006056:Golgi Apparatus D006868:Hydrolysis D007527:Isoenzymes D008075:Lipoproteins, HDL D021962:Membrane Microdomains D051379:Mice D009474:Neurons D034741:RNA, Small Interfering D015398:Signal Transduction D013108:Sphingomyelin Phosphodiesterase D013109:Sphingomyelins D013696:Temperature
Endocytosis Sphingomyelinase Caveolar uptake High density lipoprotein