Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

Biochim Biophys Acta. 2013 Dec;1831(12):1665-78. doi: 10.1016/j.bbalip.2013.08.007. Epub 2013 Aug 22.

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.

Keywords: Caveolar uptake; Endocytosis; High density lipoprotein; Sphingomyelinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Boron Compounds
  • Caveolins / genetics
  • Caveolins / metabolism
  • Cell Line
  • Endocytosis
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism*
  • Fluorescent Dyes
  • Gene Expression Regulation
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism*
  • Hydrolysis
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Lipoproteins, HDL / metabolism
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism*
  • Mice
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / enzymology*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction
  • Sphingomyelin Phosphodiesterase / antagonists & inhibitors
  • Sphingomyelin Phosphodiesterase / genetics
  • Sphingomyelin Phosphodiesterase / metabolism*
  • Sphingomyelins / metabolism*
  • Sphingomyelins / pharmacology
  • Temperature

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Caveolins
  • Fluorescent Dyes
  • Isoenzymes
  • Lipoproteins, HDL
  • RNA, Small Interfering
  • Sphingomyelins
  • Sphingomyelin Phosphodiesterase