Semaphorin 5B mediates synapse elimination in hippocampal neurons.
BACKGROUND
Semaphorins are known to play an important role in axon guidance and growth by triggering dynamic rearrangements of the actin cytoskeleton in the neuronal growth cone. Intriguingly, some of these guidance molecules are persistently expressed after axonal pathfinding and target recognition are completed. Although their function at these later stages is poorly understood, recent findings suggest a role for these proteins in regulating synaptic connections.


RESULTS
Here we demonstrate that semaphorin 5B (Sema5B) regulates the elimination of synaptic connections in cultured hippocampal neurons. We show that Sema5B is proteolytically processed in neonatal brains and primary hippocampal cultures, resulting in the secretion of Sema5B fragments that include the biologically active semaphorin domain. Overexpression of full-length Sema5B in hippocampal neurons reduces synapse number while expression of a Sema5B construct lacking the semaphorin domain has no effect. Moreover, bath application with the proteolytically processed, secreted fragments containing the semaphorin domain of Sema5B, results in a rapid elimination of synaptic connections as demonstrated by time-lapse imaging. Conversely, depletion of endogenous Sema5B using RNA interference results in a significant increase in synapse number as well as a significant increase in the size of presynaptic and postsynaptic compartments.


CONCLUSIONS
Our results demonstrate that in addition to its role as a guidance cue, Sema5B regulates the development and maintenance of synapse size and number in hippocampal neurons. In addition, proteolytic cleavage of Sema5B results in the release of a potentially diffusible semaphorin domain that is a necessary component for its biological function in the regulation of synapse morphology.
O'Connor
Timothy P
TP
Department of Cellular & Physiological Sciences & the Brain Research Centre, University of British Columbia, Vancouver, Canada. jimo@interchange.ubc.ca
Cockburn
Katie
K
Wang
Wenyan
W
Tapia
Lucia
L
Currie
Erin
E
Bamji
Shernaz X
SX
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D000076122:Disks Large Homolog 4 Protein; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D051528:Guanylate Kinases; D006624:Hippocampus / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D039961:Semaphorins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D016708:Synaptophysin / Q000378:metabolism; D014162:Transfection
D017077:Culture Media, Conditioned; D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D039961:Semaphorins; D016708:Synaptophysin; D049452:Green Fluorescent Proteins; D051528:Guanylate Kinases