Amphiphysin I antisense oligonucleotides inhibit neurite outgrowth in cultured hippocampal neurons.
Amphiphysin I is an SH3 domain-containing neuronal protein, enriched in axon terminals, which was reported to act as a physiological binding partner for dynamin I in synaptic vesicle endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I, have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a role in actin dynamics and cell polarity by testing the effect of amphiphysin I suppression on neurite outgrowth. Freshly plated hippocampal neurons were exposed to antisense oligonucleotides via a new delivery system based on a polycationic amphipathic polymer, PS980. Western blot analysis revealed that amphiphysin I levels steadily increased with neuronal differentiation, whereas in antisense-treated cultures amphiphysin I levels were reduced to approximately 10% of control levels at 48 hr. Concomitantly, a collapse of growth cones and a severe inhibition of neurite outgrowth and axon formation were observed. A similar effect was observed previously after dynamin I suppression in the same culture system (). We also have found that amphiphysin I and dynamin I colocalize in developing neurons at all developmental stages and that a pool of both proteins is colocalized with actin patches at the leading edge of growth cones. Our findings suggest a conserved role of the amphiphysin protein family in the dynamics of the cortical cell cytoskeleton and provide new evidence for a close functional link between amphiphysin I and dynamin I.
Mundigl
O
O
Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06510, USA.
Ochoa
G C
GC
David
C
C
Slepnev
V I
VI
Kabanov
A
A
De Camilli
P
P
D000199:Actins / Q000032:analysis; D000818:Animals; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000032:analysis; D015870:Gene Expression / Q000502:physiology; D006624:Hippocampus / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016501:Neurites / Q000737:chemistry / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D012333:RNA, Messenger / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D013572:Synaptic Vesicles / Q000737:chemistry / Q000201:enzymology; D050857:Synaptotagmins; D014404:Tubulin / Q000032:analysis
D000199:Actins; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014404:Tubulin; D050857:Synaptotagmins; C075637:amphiphysin; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins