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Structural and functional consequences of PTEN deletion in mature circuits of the adult central nervous system

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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.

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