title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons.
abstract: 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.
authors: Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.
authors: Christoph
authors: C
authors: Nusshold
authors: Andreas
authors: A
authors: Uellen
authors: Eva
authors: E
authors: Bernhart
authors: Astrid
authors: A
authors: Hammer
authors: Sabine
authors: S
authors: Damm
authors: Andrea
authors: A
authors: Wintersperger
authors: Helga
authors: H
authors: Reicher
authors: Albin
authors: A
authors: Hermetter
authors: Ernst
authors: E
authors: Malle
authors: Wolfgang
authors: W
authors: Sattler
mesh_terms: D000818:Animals; D001896:Boron Compounds; D022461:Caveolins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D022461:Caveolins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D008075:Lipoproteins, HDL; D034741:RNA, Small Interfering; D013109:Sphingomyelins; D013108:Sphingomyelin Phosphodiesterase
keywords: Caveolar uptake; Endocytosis; High density lipoprotein; Sphingomyelinase
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