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Approaches to promoting mTOR-mediated growth and regeneration in the mature central nervous system

  • Author(s): Gallent, Erin Ann
  • Advisor(s): Steward, Oswald
  • et al.
Abstract

Regeneration of the injured adult central nervous system is limited. The phosphatase and tensin homolog on chromosome ten (PTEN) gene has been shown to enable axonal regeneration in optic nerve crush and spinal cord injury models following neonatal conditional genetic deletion of PTEN (Park et al., 2008; Liu et al., 2010). As a follow-up to these experiments, I assessed the long-term histological and behavioral consequences of neonatal PTEN deletion (Gutilla et al., 2016). In this study, I observed massive cell body enlargement of the neurons that give rise to the major descending voluntary motor tract in the spinal cord (called cortical motoneurons, or CMNs).

This discovery led us to hypothesize that deletion of PTEN during adulthood would also result in neuronal growth. Using two-photon microscopy and electron microscopy I found that adult PTEN deletion in neurons results in significant cell body and axonal growth, and that these enlarged neurons continue to express markers of growth-promoting pathways throughout life.

To gain a better understanding of the morphological consequences of PTEN deletion in adult neurons, I imaged thick sections (200µm) containing fluorescently labeled PTEN deleted and control neurons in 3D using a modified CLARITY protocol and two-photon microscopy (Chung and Deisseroth, 2013). 3D images were then used to generate 2D projections that were processed using Sholl analysis, or concentric circle analysis (Sholl, 1953). We found that at both 6 and 12 months post-PTEN deletion, pyramidal neurons have a significantly higher density of dendrites, and that this increase is significant at the largest number of radii away from the soma at 12 months after deletion. The progressive changes in dendritic morphology were also seen in the number of basal dendrites projecting off of the soma number, maximal dendritic projections, and nodes, with the highest values for all three measurements seen at 12 months, not 6 months, post-deletion. What’s more, the radius of maximal intersection for pyramidal neurons is normally between 60-80 µm away from the soma. Only at 12 months post-deletion did pyramidal neurons exhibit a shift in the radius of maximal intersection, indicating that as PTEN deletion-induced changes in morphology progress over time, neurons becomes increasingly abnormal compared to controls.

To assess the effect of adult PTEN deletion on neuronal function and behavior, I performed behavioral assessments of forelimb motor function at 2 month intervals following induction of adult PTEN deletion. Using the rotarod task to measure baseline coordination and motor learning, and the cylinder task to identify asymmetry in spontaneous forelimb exploration, I found significant improvement in baseline coordination and motor learning beginning at 8 months after PTEN deletion, and spared symmetry of forelimb exploration at all assessed time points. Pulling our structural and functional data together, the timing of functional improvement onset correlates with the progressive development of increased dendritic density, implicating a role for PTEN deletion-induced dendritic changes in mediating the functional gains.

The regenerative effect of PTEN deletion is largely due to increased activity of the mammalian target of rapamycin (mTOR) pathway, and a commonly used biomarker for mTOR is phosphorylated ribosomal S6 (pS6, (Sawyers, 2008). Using a high frequency stimulation paradigm, I found that stimulation of the motor cortex results in marked induction of pS6 and therefore mTOR activation. It remains to be determined whether pathways other than mTOR contribute to stimulation-induced S6 phosphorylation, or if high frequency stimulation of the motor cortex could be used to promote regeneration after spinal cord injury. Altogether, the findings reported here highlight the ability for PTEN deletion to induce a robust, life-long mTOR-mediated growth program even in adult neurons, and that PTEN loss in the adult motor cortex results in functional improvements.

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