Pharmacological Regulation of Neural Circuit Formation in hIPSC-Derived Neurons and ‘Mini-Brains’

Abstract

Emerging evidence suggests that altered neural connectivity, particularly at the level of synaptic connections, contributes to the pathology of many neurodevelopmental and neurodegenerative diseases. For instance, post-mortem Autistic patient brain samples have increased numbers of excitatory to inhibitory synaptic connections, referred to as an E/I imbalance [42]. Contrastingly, post-mortem brain samples from patients diagnosed with Alzheimer's disease have decreased numbers of synaptic connections [42]. In order to understand the mechanisms that underlie the formation of these synaptic circuits, we develop 3-D human cortical organoids ('mini-brains') from human-induced pluripotent stem cells (hIPSCs). Previous research demonstrates that rearrangements of the actomyosin cytoskeleton drive neural circuit formation, in particular the development and maturation of actin-enriched spines at excitatory synapses. This thesis work investigates how pharmacological regulation of actomyosin activity affects neuronal connectivity during neurite formation in 2-D and excitatory synapse formation in 3-D 'mini-brains'. The Rho-Kinase (ROCK) inhibitor, Y-27632, both inhibits non-muscle myosin II (NM-II) and leads to a corresponding increase in Rac-driven actin polymerization. In 2-D, Y-27632 promotes neurite formation. Specifically, Y-27632 increases the number, length, and branching of neurites in hIPSC-derived neurons. Furthermore, Y-27632 increases neurite persistence, while decreasing neurite protrusion and retraction rates. However, in 3-D, acute Y-27632 treatment increases excitatory synapse area, consistent with an increase in Rac-driven actin polymerization [39]. Thus, Y-27632 increases both neurite outgrowth and excitatory synapse formation and may serve as a potential therapeutic for neurodegenerative diseases associated with synapse loss such as Alzheimer's disease. This study demonstrates the need for physiologically-relevant brain models, such as 3-D cortical organoids, to assess the impact of drug therapies on developing neural circuits to potentially treat neurodevelopmental and neurodegenerative disorders.