For centuries regeneration was thought to be unattainable in the spinal cord, but recent tools and advancements imbue optimism. This dissertation serves to guide the field in using a genetically-labeled mouse for studying CST regeneration, and to assess wiring of CST regeneration that results from PTEN deletion.

Mice designed to have yellow-fluorescent protein (YFP) expressed by the CST (CST-YFP mice) were proposed to be useful for studying CST regeneration. We show by anterograde labeling, anterograde degeneration, and retrograde labeling that some YFP-labeled axons in the spinal cord are not of the CST. We further demonstrate that dense YFP-labeled arbors impede tracing individual YFP-labeled axons such as could be used to confirm CST origins. We also found that YFP fluorescence in these mice is faint for clearing-based 3D imaging as could be used for screening regenerative responses. These results overall limit the utility of CST-YFP mice for studying CST regeneration.

Deletion of PTEN has been demonstrated to enhance CST regeneration after spinal cord injury. For regeneration to be functionally meaningful, it is critical that the axons form synapses. We assessed whether CST axons that regenerated due to PTEN deletion form synapses at the level of electron microscopy. In PTEN-floxed mice that were injected with AAV-Cre near birth to delete PTEN and as adults given a complete thoracic crush lesion, we identified synaptic structures of regenerated CST axons caudal to the lesion. This result indicates that manipulation of the PTEN pathway can produce a regenerative response that may contribute to caudal circuitry.

Toward understanding regeneration that may contribute to circuitry of motor function, the laterality of CST regenerative growth that results from PTEN deletion was assessed. PTEN-floxed mice were injected with AAV-Cre near birth and as adults given a dorsal hemisection lesion in the low thoracic spinal cord. By anterograde tracing, CST laterality in control mice was found to be predominantly contralateral to the injected cortex. Laterality of regenerative growth caudal to the lesion was found to be variable and sometimes predominantly on the ipsilateral side. These results indicate that regenerative growth does not follow the contralateral rule of the non-injured CST.

Phosphatase and tensin homolog (PTEN) is an important negative regulator of the mechanistic target of rapamycin (mTOR) pathway responsible for cell growth during development. Previous studies have documented that both embryonic and early postnatal PTEN deletion results in neuronal hypertrophy, formation of aberrant circuitry, seizures, and high mortality rates. We have recently documented that intracortical PTEN deletion in uninjured adult neurons triggers progressive growth of cell bodies and dendrites, without any obvious adverse consequences. In Chapter 2, we expand these results to include increases in cell and nuclear sizes following targeted PTEN deletion in thousands of mature cortical motoneurons utilizing a novel retrograde vector that, when injected into the spinal cord, is transported back to the cell bodies of neurons that give rise to the corticospinal tract. Still, the consequences of PTEN deletion, persistent mTOR activation, and growth of mature neurons within established circuits of the adult brain are not fully understood.

The present study explores the structural, circuit, and functional consequences of PTEN deletion in mature granule cells of the dentate gyrus in adult mice. The dentate gyrus and hippocampus are an ideal model to study such effects due to its precise organization and highly specific laminar pattern of its inputs and outputs. PTEN was deleted in adult PTEN- floxed, ROSA-reporter mice through unilateral, intra-dentate injections of AAV-Cre. In this model, Cre transduces granule cells focally, resulting in PTEN deletion and tdTomato expression in the same cells. Focal PTEN deletion in this model is advantageous as it allows for targeted PTEN deletion in granule cells, while preserving PTEN expression in granule cell input neurons and target cells.

In Chapter 3, we show that cell body size of PTEN deleted granule cells increased gradually by 2 months after injection and progressed out to 6 months, with significant growth occurring sooner in more densely transduced regions. Increases in soma size was accompanied by increases in molecular layer thickness by 4 and 6 months after AAV-Cre injection. Quantitative assessment of Golgi-stained granule cells at 6 months post deletion revealed increases in dendritic length, dendrite caliber, and spine densities in the inner, middle, and outer molecular layers, with many spines exhibiting an immature morphology. Thus, postsynaptic dendritic growth was sufficient to trigger the formation of new synapses by input pathways in which PTEN expression was preserved. Despite substantial growth of apical dendrites, tract tracing of perforant path and commissural projections from the entorhinal cortex and contralateral hippocampus to PTEN deleted granule cells revealed that the laminated pattern of input to the inner and middle molecular layers was maintained. Additionally, axonal projections from PTEN-deleted granule cells appropriately projected to, but expanded their terminal field in the CA3, suggesting increased connectivity onto PTEN expressing target neurons. Supra-granular mossy fibers were seen in some mice at long post- injection time points.

Chapter 4 present experiments that assess if the initiation and maintenance of progressive growth in mature neurons is dependent on sustained mTOR activity. Prolonged rapamycin treatment reduced the phosphorylation of ribosomal protein S6 in PTEN deleted granule cells, suggesting effective inhibition of downstream mTOR activity. Rapamycin administration during the acute period after PTEN deletion (0-2 months) prevented growth of cell bodies, while delayed rapamycin administration from 2 to 4 months after AAV-Cre injection prevented dendritic and axonal growth, prevented the occurrence of supragranular mossy fibers, and reversed increases in soma size.

To assess for altered function in mice with Cre-mediated PTEN deletion in adulthood, in Chapter 5 mice underwent continuous video electroencephalogram (EEG) recordings. Monitoring revealed onset of electrographic and behavioral seizures beyond 2 months after PTEN deletion, suggesting a critical period for circuit modification, with evidence of occasional death during a seizure. Our results indicate that PTEN deletion in mature granule cells triggers a dramatic growth phenotype that is dependent on mTOR activation, that growth of granule cell processes is sufficient to increase connectivity with PTEN expressing inputs and target cells, that the fundamental specificity of hippocampal connectivity is largely maintained, and that even unilateral focal deletion of PTEN in the dentate gyrus of adult mice is sufficient to result in the development of spontaneous seizures. Together, these results reveal the potential of PTEN deletion to induce plasticity in mature neuronal populations and established brain circuits, and that loss of PTEN can influence functional outcomes in the adult central nervous system.

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.