Sunday, May 18, 2025
5/18/2025
Mechanisms of axonal pathfinding during nervous system development - Despite the prevalence of genetically heritable forms of intellectual disability (ID), only a handful of genes have been causally linked to ID. Previous studies identified inactivating recessive mutations in the human gene ZC3H14 that are linked to intellectual disability. ZC3H14 binds to polyadenosine RNA and controls the length of the poly(A) tail, suggesting that ZC3H14 functions in the post-transcriptional control of gene expression. To better understand the molecular and cellular consequences of ZC3H14 loss we created a ZC3H14 ID model using the fruit fly, Drosophila melanogaster. The D. melanogaster ortholog of ZC3H14, called Nab2, also binds polyadenosine RNA, is required for control of poly(A) tail length, and is essential for viability and nervous system development. Importantly, expression of either fly Nab2 or human ZC3H14 in the neurons of Nab2 null flies can rescue poly(A) tail length and viability defects, suggesting that Nab2 and ZC3H14 are functional orthologs. This trans-species rescue also validates our use of flies to study the overall cellular function of ZC3H14. We have also recently demonstrated that alterations in the level of fly Nab2 cause axon mis- projection defects in both the mushroom bodies, a central region of the fly brain required for associative memory formation, and photoreceptor neurons in the fly eye. Presumably, these mis-targeting phenotypes are the result of defects in the post-transcriptional regulation of specific transcripts required for axonal pathfinding. Interestingly, loss of Nab2 causes alterations in the expression of several enzymes responsible for control of cyclic nucleotide levels. Previous studies have demonstrated that cAMP and cGMP play critical roles in mediating the strength of attractive and repulsive navigational cues during axon pathfinding. Mathematical modeling from other labs corroborates these experimental findings and suggests that maintenance of precise cyclic nucleotide levels is necessary for the dynamic control of pathfinding decisions. Importantly, we show preliminary data here in support of that hypothesis and seek to extend these studies using an in vivo Drosophila model of axon guidance. Specifically, it is our overall model that cyclic nucleotide levels are precisely regulated during pathfinding in order that extending growth cones can dynamically respond to extracellular guidance cues. In the current proposal we aim to examine the overall necessity of cyclic nucleotide second messengers in axonal guidance. Aim 1 of this proposal will use a combination of genetic, cell biology, and biochemical techniques to uncover the molecular mechanism by which Nab2 controls expression of enzymes responsible for maintaining intracellular levels of cAMP and/or cGMP during axon pathfinding. In Aim 2 we will extend these studies to test whether directly disrupting cyclic nucleotide homeostasis results in pathfinding defects in vivo. Given the conservation between many of the enzymes involved in cyclic nucleotide biosynthesis, these studies have the potential to greatly increase our understanding of how cyclic nucleotide second messengers control axonal growth and synapse selection.
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