DRAFT: This module has unpublished changes.

In this laboratory I had the opportunity to investigate endosomal signaling related to retrograde neurotrophin transport in PC12 cells, a neuronal mimic. 

 

I performed confocal microscopy, immunocytochemistry, tissue culture, recombinant DNA work, and DNA purification. 

 

I worked to map the activation of endosomal signaling proteins Erk-p and Akt-p with respect to Trk in response to NGF treatment and investigate relationships between mutant LRRK2, linked to Parkinson’s Disease, and Erk-p, Akt-p, and Trk.  

 

During the summer of 2013 my research was funded by Undergraduate Research & Creative Activities (URECA). Below is a culmination of that body of work in an abstract.

 

 

Mapping of Activated Akt With Respect To Neuronal Endosomal Signaling

 

Jamie Leonard and Simon Halegoua, Department of Neurobiology and Behavior

      Involved in the survival and phenotype control of neurons, Neurotrophins such as Nerve Growth Factor (NGF) are released onto nerve endings from peripheral neuronal target tissues.  After binding to its receptor, TrkA, NGF is then retrogradely transported via signaling endosomes up the axon to the soma.  It has been shown that activation of the signaling protein kinases Erk and Akt mediate neuronal phenotype and survival, respectively.  Activated Erk (P-Erk) has been well mapped with signaling endosomes in both time and space.  Activated Akt (P-Akt), has not been well mapped with respect to endosomal signaling.  Akt has two activating phosphorylation sites, at serine473 and threonine308.  Serine473 is phosphorylated by the mTOR complex (the mammalian target of rapamycin).  Threonine504 is phosphorylated by PDK, downstream of PI3 Kinase signaling from TrkA.  Akt is fully activated when Akt is phosphorylated at both sites.  Using phospho-specific antibodies in immunocytochemical staining of neuronal PC12 cells, we mapped the distributions of both P-Erk and P-Akt (at P-ser473 and P-thr308) over time and space after NGF treatment. We found that while P-Erk is clearly seen in endosomes, P-Akt was surprisingly not in endosomes, but rather at the plasma membrane.  In addition, we saw that the Akt phosphorylation sites were differentially distributed on the membrane.  P-serine473-Akt was seen in a regional membrane distribution, while P-threonine308-Akt was limited to focal adhesions and/or sites of membrane ruffling.  These findings suggest that the different activating pathways for Akt were differentially localized.  Future investigation will include studies on how P-Akt localization relates to its pro-survival and other actions. 

 

Acknowledgments: This work was supported with funding from Undergraduate Research & Creative Activities (URECA).

 

 

DRAFT: This module has unpublished changes.