Gamma Secretase Activity Is Necessary For BMP‐7‐Induced Dendritic Growth In Embryonic Sympathetic Neurons

Abstract

Dendrites are the primary sites of synapse formation and accurate patterning of the dendritic arbor is crucial for the normal functioning of the nervous system. However, the mechanisms underlying the regulation of dendritic growth have not been fully elucidated. Previous studies have shown that members of the bone morphogenetic protein (BMP) family promote dendritic growth in central and peripheral neurons through the nuclear translocation of proteins known as SMADs. The goal of this study was to identify other signaling pathways that interact with BMP signaling to regulate dendritic arborization in peripheral neurons. Mutations in presenilin‐1 (PSEN‐1), the catalytic subunit of the gamma secretase (γ‐secretase) complex, have been shown to regulate dendritic growth in hippocampal neurons. However, the importance of γ‐secretase and its interplay with BMP signaling during the dendritogenesis in the peripheral nervous system is not known. Therefore, in this study, we examined the interaction between γ‐secretase activity and BMP signaling during dendritic growth in sympathetic neurons. Sympathetic neurons cultured from E20/E21 rat pups were grown in the absence or presence of a maximally effective concentration of BMP‐7 (50 ng/mL) and subjected to morphological and biochemical analyses. Using immunostaining and immunoblotting, we showed that members of the γ‐secretase complex ‐ PSEN‐1, PSEN‐2, Nicastrin and APH1 ‐ are present in cultured embryonic sympathetic neurons grown in the absence of BMP‐7. Treatment with BMP‐7 did not alter the intensity of staining or localization of these components. We next examined whether inhibiting γ‐secretase activity interfered with BMP‐7 induced dendritic growth. The γ‐secretase activity was inhibited in cultured sympathetic neurons either pharmacologically using two different γ‐secretase inhibitors ‐ 10 mM γ‐secretase inhibitor IX (DAPT) or 10 mM LY‐411575 ‐ or genetically by transfecting with an siRNA for PSEN‐1. The cultures were maintained in the presence or absence of BMP‐7 (50 ng/mL) for 5 days at which point cultures were fixed and dendrites visualized using MAP2 immunocytochemistry. Morphometric analyses of MAP2‐immunopositive processes indicated that BMP‐7 induced dendritic growth was significantly inhibited by both pharmacological inhibitors and by the PSEN‐1 siRNA. The effects of DAPT and LY‐411575 were found to be concentration‐dependent with no adverse effects on axonal growth and cellular metabolism. Furthermore, our data suggest that inhibition of γ‐secretase activity did not alter BMP‐mediated nuclear translocation of SMAD indicating that γ‐secretase inhibition did not alter early stages of BMP signaling. In summary, our data show that γ‐secretase activity is necessary for BMP‐7 induced dendritic growth in sympathetic neurons.Support or Funding InformationNIH (grant # R01 ES014901), Saint Mary's College Summer Research Program, Saint Mary's College Faculty Development Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.