Post Transcriptional Regulation Of Neuronal Identity And Plasticity

Neurons have the difficult task of persisting throughout the life of the animal. This requires a rigid adherence to their highly specialized identity. Paradoxically, they must be flexible, able to modulate their properties in the face of a constantly shifting activity landscape. The work presented here suggests that these two critical processes depend (at least partially) on regulation through microRNAs. I have shown that proprioceptive sensory neurons display a decline in celltype specific gene expression as well as a loss of their specialized muscle afferents upon conditional ablation of the microRNA biogenesis enzyme Dicer. Also, demonstrated here is the fact that parvalbumin-positive fast-spiking cells employ mir-7 to modulate their intrinsic excitability during activity deprivation in culture, as well as sensory deprivation in vivo. These studies support a role for microRNAs in the maintenance of neuronal identity and the regulation of plasticity.

Regulation of gene transcription by neuronal activity is evident in a large number of neuronal processes ranging from neural development and refinement of neuronal connections to learning and response to injury. In the field of activity-dependent gene expression, rapid progress is being made that can impact these, and many other areas of neuroscience. This book offers an up-to-date picture of the field.

The central nervous system is the most complex and highly organized tissue in animals; composed of thousands of neurites connected in specific and highly reproducible ways. My thesis research has focused on the generation of neuronal diversity: specifically how neurons adopt individual, often unique, identities. Work in many labs has revealed that a large set of transcription factors act in combinatorial manner to specify the fate of individual neurons or small groups of neurons. However, in most cases, it remains unclear how individual or specific combinations of transcription factors directly control the terminal differentiation of neurons via the regulation of different genes, such as neurotransmitters. My thesis work has focused on the identification and characterization of new members of the combinatorial code of transcription factor and on initial attempts to link these transcription factors to the expression and activity of genes that contribute directly to neuronal differentiation. In chapter 2, I describe the identification and characterization of Dbx, a homedomain-containing transcription factor, expressed in a mixture of progenitor cells and a subset of GABAergic interneurons. I show that Dbx is expressed in many interneurons that are sibling to motor neurons, and that Dbx is required to promote the development of these interneurons via cross-repressive interactions with Eve and Hb9, which are expressed in the sibling motor neurons. In chapter 3, I detail the identification of FoxD, a transcription factor that is positively regulated by the homeodomain-containing transcription factor Hb9 in the Drosophila CNS. FoxD is expressed in a subset of Hb9 positive neurons and also in all octopaminergic neurons in the Drosophila embryonic CNS. I have identified the enhancers that drive expression in these neurons and have recently generated two mutant alleles of foxD. Loss of foxD appears to result in hyperactivity, which is most pronounced in males. As octopamine is the fly equivalent of norepinephrine, these results suggest that FoxD may function in specific cells to regulate the synthesis and release of octopmaine. Thus, my thesis has identified two members of the combinatorial code of transcription factors that govern neuronal identity. In addition, it has begun to place the functions of these genes within the genetic regulatory hierarchy of this code and started to link the function of individual transcription factors to the regulation of terminal differentiation genes and animal behavior.

The results from this study suggest that long-lasting synaptic plasticity requires transcriptional activation of the F1/GAP-43 gene. This view was supported by observation that facial neurons after nerve crush induced F1/GAP-43 primary transcript. Since axonal re-growth is seen in facial neurons after nerve crush, increases in F1/GAP-43 mRNA expression through the activation of transcriptional mechanism would be a key molecular event associated with the role of F1/GAP-43 in axonal outgrowth. However, both qualitative and quantitative assessment of F1/GAP-43 gene expression suggested different patterns of transcriptional regulation in potentiated hilar neurons and regenerating facial neurons, probably recruiting transcription factors specific to each neuronal system.

The dynamic complexity of synaptic function is matched by extensive multidimensional regulation of neuronal mRNA translation which is achieved by a number of post-transcriptional mechanisms. The first key aspect of this regulatory capacity is mRNA distal trafficking through RNA-binding proteins, which governs the transcriptomic composition of post-synaptic compartments. Small non-coding microRNA and associated machinery have the capacity to precisely coordinate neural gene networks in space and time by providing a flexible specificity dimension to translational regulation. This RNA-guided subcellular fine-tuning of protein synthesis is an exquisite mechanism used in neurons to exert control of synaptic properties. Emerging evidence also implicates brain-enriched long non-coding RNA and novel circular RNA in posttranscriptional regulation of gene expression through the modulation of both mRNA and miRNA functions, thereby exemplifying the complex nature of neuronal translation. Herein, we review current knowledge of these regulatory systems and analyse how these mechanisms of transcriptomic regulation may be linked together to achieve high-order spatiotemporal control of post-synaptic translation.

The mammalian nervous system is comprised of an unknown, but recognizably large, number of diverse neuronal subtypes. Recently, direct reprogramming (also known as transdifferentiation) has become an established method to rapidly produce "induced" neurons of numerous different subtypes directly from fibroblasts by overexpressing specific combinations of transcription factors and/or microRNAs. This technique not only provides the means to study various neuronal subtype populations that are not easily accessible, particularly in humans, but it also serves as a tool to interrogate the transcriptional codes that regulate neuronal subtype identity and maintenance. Both in vivo studies and direct reprogramming protocols have demonstrated that basic helix-loop-helix (bHLH) and Pit-Oct-Unc (POU) transcription factors can aid in the specification of distinct neuronal subtypes. Therefore, we set out to comprehensively and systematically address whether first, additional bHLH and POU factor pairings could reprogram fibroblasts into functional neurons and second, dissect out the discrete and synergistic roles of these factors in neuronal subtype specification. We discovered over 70 novel pairs of bHLH and POU (and non-POU) transcription factors sufficient to generate candidate induced neurons (iNs) from mouse embryonic fibroblasts. Transcriptomic analysis of 35 of these candidate iN populations revealed gene expression profiles similar to those of endogenous neuronal populations. Additionally, differences between iN populations were observed at both a transcriptional and functional level.

Reelin glycoprotein is a serine protease with important roles in embryogenesis and during adult life. This comprehensive and integrative book examines the role that reelin plays in the etiology of various neuropsychiatric disorders, including schizophrenia and autism. The book provides an unprecedented analysis of this emerging and novel protein by examining evidence from genetic, neuroanatomic, biochemical, and behavioral studies.

Since the 1996 publication of Translational Control, there has been fresh interest in protein synthesis and recognition of the key role of translation control mechanisms in regulating gene expression. This new monograph updates and expands the scope of the earlier book but it also takes a fresh look at the field. In a new format, the first eight chapters provide broad overviews, while each of the additional twenty-eight has a focus on a research topic of more specific interest. The result is a thoroughly up-to-date account of initiation, elongation, and termination of translation, control mechanisms in development in response to extracellular stimuli, and the effects on the translation machinery of virus infection and disease. This book is essential reading for students entering the field and an invaluable resource for investigators of gene expression and its control.