Axon Development in Health and Disease

Abstract

During embryonic development neurons migrate and grow their protrusions (neurites) over long distances in a strictly orchestrated manner to form complex neuronal networks. Subtle changes in neuronal network formation may lead to various neurological disorders ranging from congenital mirror movements, to autism spectrum disorders and Parkinson’s disease. Apart from canonical axon guidance proteins, cell adhesion molecules (CAMs) are indispensable for proper neuron migration and the formation of correct synaptic connections. Interestingly, the expression and function of these proteins is spatiotemporally regulated, resulting in the potential to control a disproportionally large number of guidance decisions. In this thesis some of these regulatory strategies, including post-transcriptional regulation by microRNAs (miRNAs) and membrane targeting of CAMs by Molecules interacting with Casl (MICALs), are highlighted, and combined with a detailed description of dopamine axon bundle development and a review on the role of canonical axon guidance proteins in nervous system diseases.

MiRNAs are small, non-coding RNA molecules that generally downregulate translation of their target mRNAs. In the human brain, over thousand miRNAs have been identified, however functional roles are unknown for most of them. In this thesis, two different strategies were used to clarify roles of miRNAs during axon development. First, a profiling study identified groups of miRNAs that are involved in specific neurodevelopmental stages. Second, a high-throughput whole-miRNA-nome cellomics screen lead to the identification of the miRNA-135-family as regulators of neurite outgrowth by targeting Krüppel-like factor 4 (KLF4). This provides interesting new opportunities for axon regeneration therapy development, since it is long known that KLF4 is an important downregulator of axon growth after injury.

In many brain nuclei, including the hippocampus, synaptic connections are organized in specific laminae. CAMs play an important role directing the formation of these lamina-specific synapses, but the mechanisms behind these processes are still unclear. We discovered that the redox protein MICAL-1 is involved in membrane-targeting of specific CAMs (NCAM and CHL1) by regulating actin cytoskeleton rearrangements in the growth cone. By that, it controls lamina-specific targeting of dentate gyrus mossy fiber synapses in the hippocampus in two ways: 1) it regulates the fasciculation of mossy fibers and confines them to the supra- and infrapyramidal axon bundles, and 2) it regulates subcellular-specific synapse formation of dentate gyrus mossy fibers with proximal dendrites of CA3 pyramidal neurons and restricts synaptic formation to the stratum oriens and the stratum lucidum of the hippocampus.

Using a novel genetic mouse model called ITC, we were able to distinguish developing midbrain dopaminergic neurons and axons from the substantia nigra compacta (SNc) by fluorescent labeling. We performed a detailed analysis of their specific migration-, axon growth- and striatal innervation patterns from early on in embryonic development, and discovered that SNc axons are segregated dorsally in the medial forebrain bundle and project directly in a dorsal-to-dorsal fashion to the striatum. This suggests a specific axon guidance mechanism, which we will be able to investigate further using these novel transgenic mice. Further, these mice provide the opportunity investigating pre-symptomatic alterations in dopaminergic connectivity in Parkinson’s disease.