TSC Haploinsufficiency Influences PS6 and GFAP activity in the PFC and Hippocampus

• Angelina Pezzuto, Neuroscience, University of Delaware

• Amanda Hernan, Neuroscience Research, Nemours

Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder that results in a variety of symptoms across multiple organ systems, particularly the brain. Previously, research in our lab showed that female BXD87 mice with a heterozygous loss of the Tsc1 gene had significant seizure activity, and male and female BXD87 mice had gross structural changes in the brain. This study sought to examine these anatomical changes utilizing a PS6 and GFAP stain, looking for mTOR pathway activation and astrocytosis respectively. Cells were sliced at 30µM into wells, washed in PBS-TX, and blocked for an hour. Slices were then incubated in primary for 48 hours at -4 degrees celsius. Tissue was then washed and fluorophore-conjugated secondary was applied and incubated for one hour at room temperature. Tissue was mounted onto slides and cover slipped. Tissue was then scanned via a lionheart microscope at 10x in areas of interest stains. Using ImageJ software the images were assessed for cell count as well as intensity of stain uptake. In conclusion of this phase of the project, BXD87 TSC and WT mice were evaluated to assess the intensity of immunolabeling and counts of numbers of positive cells labeling for both PS6 and GFAP proteins in the hippocampus and PFC. Cell counts showed no significant differences. However the higher levels of GFAP in the Hippocampus of TSC+/- mice point toward the increased activation and possible reactive astrocytosis in TSC heterozygous mice. Future directions include the addition of more animals.