Illuminating the mechanisms of human synaptic and neural network dysfunction in Down Syndrome

Abstract

Human cortical circuit assembly is critical to neurodevelopment, but our understanding of this process in Down Syndrome (DS) remains incomplete. Previously, a model for studying DS neurodevelopment was created by xenotransplanting induced pluripotent stem cell (iPSC) derived neurons, made from DS individual fibroblasts, into the somatosensory cortex of an immunodeficient mouse. The transplanted graft cells were then imaged longitudinally using multiphoton microscopy. In this thesis, I develop an alternate graft type within the framework of the previous model, in order to study synaptic structural stability and spontaneous and oscillatory calcium activity in a model of the early DS brain. In addition to utilizing cells derived from an individual whose genetics have not been studied using this model-type, I have also demonstrated the ability to utilize this model for extended long-term imaging, with a pilot study of animals imaged at 8 months post-transplantation. In this study, 2-photon images were taken of axonal en passant boutons (EPB) and of somatic calcium signals over time. EPB turnover rate (TOR) and EPB size (strength) changes were quantified using EPBscore software. Calcium imaging was analyzed via MATLAB/Fiji scripts to determine event frequency, integral, and amplitude. I found no significant difference in EPB TOR, EPB strength, spontaneous somatic calcium activity, or oscillatory calcium activity between DS and isogenic control grafts. Therefore, differences in synaptic stability and somatic activity outcomes have been normalized across genotypes. Correlative analysis was used to determine whether normalization of SATB2+ and GFAP+ cell levels contributed to this rescue. GFAP+ cell numbers significantly correlate with synaptic stability outcomes, but no others. Overall, this study concludes that trisomy 21-related dysregulation in this graft is not sufficient to alter axonal bouton stability and neuronal activity compared to control neural networks of similar scale and cellular composition. I hypothesize that the lack of effect on synaptic structural stability and spontaneous and oscillatory activity may be correlated to an increased rate of astrogliogenesis in my grafts. As such, this graft model of DS neurodevelopment provides a foundation upon which to study the mechanisms behind early fetal changes in DS neurodevelopment, specifically related to the role of astrocytes in DS. Additionally, this model could be used as an excellent in vivo astrocyte research model, in WT or diseased cells, due to the high volume of developed astrocytes. Future research will include comparative transcriptomic analysis of this DS graft type to identify mechanisms of early DS neurodevelopmental dysfunction.