Abstract
The spinal cord is composed of bundles of nerve fibres called axons, which connect the neurons in the brain with the rest of the body. Damage to these axons disrupts communication between neurons in the brain and their cellular targets below the injury. In the adult central nervous system, injured axons fail to regenerate effectively and cannot overcome the inhibitory environment created by molecules within the lesion. As a result, lost connections are rarely re-established and spinal cord injury (SCI) often leads to permanent impairments of motor, sensory and autonomic functions. Both limited intrinsic growth capacity and inhibitory cues at the injury site restrict axonal regrowth and represent major barriers to recovery. Identifying molecular regulators of axon regeneration is therefore critical for developing targeted therapeutic strategies to promote regeneration after injury. The present thesis investigates the role of microtubule-associated protein 2 (MAP2), a neuron-specific regulator of the microtubule cytoskeleton and axonal transport, in axon growth and regeneration after injury, using adult sensory neurons as a model system. We first evaluate multiple RNA-targeting CRISPR-Cas systems for their ability to efficiently knockdown MAP2 expression in adult sensory neurons, identifying the high-fidelity Cas13d effector as the most effective. This establishes a tool for investigating the role of MAP2 in axon growth and regeneration. We then demonstrate that MAP2 depletion enhances axon elongation under permissive and inhibitory conditions that mimic the lesioned spinal cord using sensory neurons from adult rats and MAP2 transgenic mice. Specifically, we show that MAP2 depletion remodels the axonal proteome, including altered localisation of the adhesion molecule L1CAM, and increases microtubule dynamics in the distal axon and the growth cone. These coordinated changes facilitate axon extension through inhibitory environments and contribute to increased axon regeneration following injury. Altogether, CRISPR-hfCas13d-mediated MAP2 knockdown provides a neuron-specific strategy to enhance axon growth and regeneration, establishing a framework for future in vivo studies. Collectively, this work highlights MAP2 as a unique neuron-specific protein capable of regulating axon growth and regeneration under both permissive and inhibitory conditions, providing a foundation for developing strategies to enhance recovery after SCI.