Subcellular localization impacts PTEN activity in the murine dentate gyrus

One out of 36 children is diagnosed annually with Autism Spectrum Disorder in the United States. Loss of function mutations in PTEN characterize PTEN Hamartoma Tumor Syndrome, a multi-system group of syndromes associated with increased risk of Autism Spectrum Disorder and increased risk of breast, thyroid, and renal cancer. One possibility to support development of targeted therapies for those with PTEN mutations is to better understand the mechanisms within commonly mutated pathways. In this study, we examine subcellular localization of PTEN and how it may affect the neuronal morphology of cells. Mechanistically, PTEN dephosphorylates PIP3 to PIP2,thereby lowering downstream AKT activation and downregulating the mTOR complexes in the P13K / AKT / mTOR pathway in the cytosol, post-synaptic density, and cytoskeleton. Within the nucleus, PTEN functions as transcriptional regulator of p53 and various checkpoints in the cell cycle, operating independently of its phosphatase activity. The P13K / AKT / mTOR pathway is largely implicated in the growth and division of cells and is highly conserved in healthy cells to regulate neuronal soma size and other growth characteristics. As demonstrated by our lab’s previous research, loss of PTEN as a negative regulator of the P13K / AKT / mTOR pathway results in increased soma size and dendritic branching in hippocampal granule neurons. This work will expand on previous research which demonstrates in mice that PTEN knockout results in neuronal overgrowth by investigating how the subcellular localization of PTEN affects its regulation of neuronal morphology via retroviral-mediated recombination of four PTEN fusion proteins (NES-PTEN, NLS-PTEN, PTEN-FBAR, and PTEN-Homer) in order to achieve spatial control over PTEN. PTEN subcellular localization was measured using PTEN fluorescence intensity ratios in various subcellular compartments relative to other subcellular compartments and the background. In this thesis, we demonstrate success at optimizing PTEN immunostaining methodology in somas for all constructs except for PTEN-FBAR and show that PTEN-Homer and NLS-PTEN have respectively localized PTEN to the cytosol and the nucleus. Only PTEN-Homer out of all four fusion proteins achieved detectable PTEN immunostaining greater than background in dendrites and spines. Because control PTEN over-expression could not be detected above background in dendrites we were unable to determine if subcellular localization of PTEN in dendrites and spines was altered by our fusion proteins. In addition, we uncovered unexpected PTEN immunostaining in NES-PTEN mimicking that of PTEN knockout while maintaining a wildtype (WT) morphology. This poster will ponder these conclusions and brainstorm the next steps in examining other techniques for subcellular localization of PTEN.https://digitalcommons.dartmouth.edu/wetterhahn_2023/1002/thumbnail.jp