Development of Novel Stem Cell Based Neurite Outgrowth Models and their Application to Study Neurite Inhibition in Neurological Disorders

Abstract

The inability of central nervous system (CNS) neurons to regenerate results in lost neural connectivity and is common to many neurological disorders. This loss of connectivity results in functional deficit following trauma such as spinal cord injury (SCI) and has been implicated in many neurodegenerative diseases including Alzheimer’s disease (AD). Throughout this project a robust, reliable and physiologically relevant 3D model of human neuritogenesis was developed and applied to investigate the process of neurite inhibition, along with its underlying signalling mechanisms.

SCI results in the formation of a glial scar; involving the activation of astrocytes and secretion of inhibitory molecules that act through receptors to inhibit neurite growth thereby reducing neural connectivity. This project describes several approaches used to investigate this process including growth substrate coating, biomimetic 2D/3D functionalised growth surfaces and the development of a co-culture model to study reactive astrocyte-neuron interactions. Each approach has successfully led to neurite inhibition, and we have also demonstrated the ability of small molecules to restore neurite outgrowth despite the presence of inhibitory stimuli.

Inhibition of neurite growth is also associated with β-amyloid (Aβ) deposition, a hallmark of AD. Neurites that pass through deposits become dystrophic and their ability to form connections is reduced. This study has also focussed on elucidating the molecular mechanisms that underpin Aβ-mediated inhibition, through growth substrate coating, exogenous Aβ application and the development of a disease-specific neurite outgrowth model based on iPSC technology, all of which successfully led to inhibition of neurite outgrowth. Small molecules that target specific downstream signalling events were used to restore neurite growth in the presence of inhibitory Aβ. This has helped identify common signalling pathways involved in neurite inhibition in both SCI and AD.

Activation of the Rho A and ROCK signalling pathway is common to neurite inhibition in the glial scar and AD. This thesis provides compelling evidence as to the common role of Rho A activation in neurite inhibition. A ROCK inhibitor, Y-27632 and ibuprofen, an inhibitor of Rho A, were used to restore neurite outgrowth in the inhibitory models described throughout this project. These data suggest a common role for Rho A and ROCK activation in neurite inhibition, inhibition of which may provide a useful therapeutic strategy to promote regeneration in the CNS and enhance neuronal connectivity in many neurological disorders.