Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells.
A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.
Valaperta
Rea
R
Department of Medical Chemistry, Biochemistry and Biotechnology, and Center of Excellence on Neurodegenerative Diseases, University of Milan, Segrate, Italy.
Valsecchi
Manuela
M
Rocchetta
Federica
F
Aureli
Massimo
M
Prioni
Simona
S
Prinetti
Alessandro
A
Chigorno
Vanna
V
Sonnino
Sandro
S
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D005732:Gangliosides / Q000378:metabolism; D020868:Gene Silencing / Q000502:physiology; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D009420:Nervous System / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D009439:Neuraminidase / Q000235:genetics*; D009447:Neuroblastoma; D012313:RNA / Q000235:genetics; D013107:Sphingolipids / Q000378:metabolism; D014161:Transduction, Genetic; D019061:src-Family Kinases / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism
D002518:Ceramides; D005732:Gangliosides; D008565:Membrane Proteins; D013107:Sphingolipids; D016875:tau Proteins; D012313:RNA; D019061:src-Family Kinases; D000110:Acetylcholinesterase; C511796:Neu3 protein, mouse; D009439:Neuraminidase