Mesquita-Ribeiro, Raquel
(2019)
Axonal microRNAs in cortical neuronal development and network connectivity.
PhD thesis, University of Nottingham.
Abstract
Cognitive brain function requires the establishment of neuronal networks, which rely on the formation and elongation and branching of axonal projections and the differentiation of presynaptic terminals during development. The cellular events involved in these processes are dependent on protein translation locally in the axon compartment, which enables rapid changes of the axonal proteome in response to neurotrophic cues to regulate axon growth and patterning. How these dynamic changes of the axonal proteome are regulated locally in the axon has been a topic of intensive investigation in the past decade. MicroRNAs are small RNAs known to regulate protein expression by controlling mRNA translation repression/degradation. Recently, these regulatory RNAs have emerged as key players in the modulation of several molecular pathways underlying neuronal differentiation in early stages of development, making this class of non-coding RNAs interesting candidate regulators of local protein translation in the axon during development. However, the role of microRNAs in the axon, in particular axonal outgrowth and presynaptic differentiation, is only beginning to be unravelled.
The work described here used next generation sequencing to identify a set of microRNAs enriched in the axonal fraction of primary cortical neurons cultured in compartmentalised microfluidic devices. Following the characterization of axonal microRNA expression levels, two microRNAs, miR-3470b and miR-99a, were selected for subsequent functional studies.
The miR-3470 family are mouse-specific repeat-derived microRNAs originating from the B1-element Mus1. Repetitive elements have a major role in shaping the structure and function of the genome, but evidence on the functional relevance of lineage-specific repeat-derived microRNAs is still limited. Inhibition of miR-3470b during early development of primary cortical neurons produced a significant decrease in axonal growth. Moreover, we discovered a significant association between miR-3470b targets and proteins involved in cell-to-cell contact/synaptic pathways. To investigate the role of miR-3470b in the formation of neuronal networks we used a microelectrode array cortical culture model, in which spontaneous electrical activity shows a progressive increase from day 9 in vitro, reflecting the establishment of functional synaptic connections. Inhibition of miR-3470b from the beginning of the third week in culture, when its endogenous levels are high, produced a marked decrease in network activity, suggesting its role in neuron connectivity during cortical neuron development.
Functional studies in cortical cultures showed that inhibition of miR-99a in primary cortical neurons produced a significant decrease in axonal growth, with overexpression of a miR-99a mimic increasing axonal length. By using bioinformatics, luciferase reporter assays and functional rescue experiments we could identify a new target for miR-99a in axonal development, heparan sulphate 3-O-sulphotransferase 2 (Hs3st2), an enzyme part of the biosynthesis of heparan sulphate proteoglycans, key players in axon-extracellular matrix interactions. To investigate the effect of axonal miR-99a in the emergence of neuron connectivity and the functioning of neuronal networks we used imaging of calcium transients to assess the spontaneous rhythmic activity of developing neuronal cultures, demonstrating how inhibition of miR-99a alters the patterns of calcium oscillation frequency and synchronicity, suggesting its relevance for network development and maturation.
Overall, this work identified two microRNAs capable of regulating axonal outgrowth in the development of mouse cortical neurons in vitro. Both microRNAs were found to exert growth promoting actions in developing axons. Furthermore, this work demonstrated the ability of mR-99a and miR-3470b to act as regulators of neuronal network formation in vitro, raising potential implications to the development of neuronal connectivity in vivo.
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