Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture

Abstract

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive
metabolic labeling of proteins, and therefore is difficult to apply to cells that do not divide or are
unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for
straightforward SILAC quantitation using partially labeled cells because the two cell populations
are always equally labeled. Here we report the application of this labeling strategy to primary
cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete
labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling
in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major
component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement
of proteins such as VAMP2, Scamp1 and Scamp3 suggest NT-3 may lead to enhanced exocytosis
of synaptic vesicles.

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Identifiers

ORCID for Katrin Deinhardt: orcid.org/0000-0002-6473-5298

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