High Throughput Interrogation of Neuronal Chemotaxis and Its Application in Brain Regeneration from Injuries

應用高通量的方法來研究神經細胞遷移及在腦損傷再生中的應用

Student thesis: Doctoral Thesis

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Award date30 Apr 2018

Abstract

Molecular gradients played instrumental roles in the developing brain. Through graded distribution, signaling molecules, especially members of the netrin, semaphorin, and slit families, as well as growth factors and cell adhesion molecules, guided neurons into different layer and organized neurites forming accurate connections. Although current in vitro and in vivo study proved that molecular gradients were greatly involved in neuronal chemotaxis, they did not systematically investigate the effects of gradients with detailed parameters, especially different steepness, on neuronal chemotaxis, let alone exploring the underlying mechanism. Moreover, molecular gradients have also been implicated in participating regeneration process in the injured brain. Chemoattractive signals in terms of gradients will recruit neural stem/progenitor cells (NSPCs) to the injured area. In the neocortex formation, semaphorin3A (Sema3A) also exhibited a descending gradient across the cortical layers with the highest concentration at the pial surface, which induced progenitor neurons to migrate to the appropriate destination. However, there has been far more than successes in the application of Sema3A to treat brain injury perhaps due to the fact that they did not adopt the right delivering strategy: molecular gradients. So whether it is effective to treat brain injury by recreating a Sema3A gradient existed in the developing brain also remains an outstanding question.

In the thesis, we developed a novel microfluidic device to allow high-throughput generation of a large-scale library of molecular gradients with distinct steepness and investigated the corresponding neuronal response and related molecular targets. Our results suggested that the great complexity existed in neuronal response to gradients with different steepness. Neurons exhibited similar growth pattern over a large variation of netrin-1 gradient steepness; while Sema3A exhibited steepness-dependent regulation for both neuronal migration as well as neurite repellence. Exposure to NGF gradient with different steepness, neurons demonstrated steepness-dependent and steepness-independent response. Moreover, we found that serine/threonine kinase 11 (STK11) and glycogen synthase kinase-3 (GSK3) signaling pathways were differentially involved in the Sema3A gradient steepness dependent regulation of neurite guidance and neuronal migration, and GSK3 activity was especially critical for sensing the Sema3A steepness in neuronal migration.

To reproduce a developing environment in the injured brain, Sema3A gradient hydrogel was implanted in the injured area. We found that Sema3A gradient hydrogel can recruit NSPCs to the injured area and promote the migrated NSPCs differentiation into mature neurons. Enhanced neurogenesis was also discovered in the surrounding tissue. To explore the underlying mechanism, we compared expression profiles of regenerated tissues in Sema3A gradient hydrogel, tissues in blank hydrogel and normal brain tissue. In general, tissues in Sema3A gradient hydrogel exhibited high similarity to the normal brain tissue. Genes related to neuron migration and differentiation were particularly upregulated in comparison to tissues in blank hydrogel. Moreover, the crosstalk between Sema3A and Wnt/β-catenin signaling pathways may be responsible for Sema3A induced brain regeneration.

In summary, the thesis integrated microfabrication, microfluidics with microscale three-dimensional (3D) culture to explore the importance of gradient steepness in neuronal chemotaxis and demonstrated the successful application of Sema3A gradient to treat brain injury from gross morphology, immunohistochemistry and transcriptome analysis. These data provided novel insights about the role of gradient steepness in neuronal chemotaxis and revealed the feasibility of application Sema3A gradient in the treatment of brain injury. The discovery may also have potential implications for future therapeutic strategies for regenerative medicine.