Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to upregulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor. A xon regeneration generally fails after injury to the CNS, preventing substantial recovery. CNS regeneration is limited in part by the presence of extrinsic inhibitory molecules at the injury site, and also because adult CNS neurons possess an intrinsically low capacity for axon growth compared with embryonic or peripheral nervous system (PNS) neurons (1-3). Intrinsic regenerative capacity appears to be particularly low in the corticospinal tract (CST), an essential motor control pathway and important therapeutic target in humans (4). For instance, CST axons have shown mixed responses when inhibitory signals are neutralized (5, 6), and regenerate minimally or not at all into growth permissive tissue grafts that support some regeneration from propriospinal and brainstem neurons (7-10).The intrinsic molecular mechanisms that limit CNS axon growth remain unclear, but likely reflect a suboptimal pattern of regenerative gene expression (1, 11). In sensory neurons, overexpression of GAP43 and CAP23 or transcription factors, including ATF3, STAT3, or ID2, have produced modest gains in spinal regeneration (12-15). In the visual system, overexpression of p300 modestly increases growth (16), and knockdown of PTEN and SOCS3 results in substantial regeneration (17,18). In the CST, overexpression of the neurotrophin receptor TrkB enables regeneration into subcortical BDNF-secreting tissue grafts, and, notably, knockout of PTEN evokes regeneration in the spinal cord (19,20). The regenerative response evoked by these gene manipulations, including PTEN knockout, remains incomplete in terms of both the distance traveled by regenerating axons and the percent of neurons that respond to treatment.These findings illustrate the critical need to develop additional tools to enhance the intrinsic growth state of CNS neurons.We...

Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.Cre recombinase ͉ pet-1 ͉ transgenic ͉ yellow fluorescent protein ͉ monoamine S erotonin (5HT) is a transmitter of broad relevance to nervous system development and function (1-4). Serotonergic pathways innervate most cytoarchitectonic structures of the CNS, and accordingly they have been implicated in the modulation of circuitry involved in nearly all behaviors and physiological processes (3, 5-7). Additionally, 5HT neurotransmission is modulated through abundant afferent information arising from, for example, other monoaminergic systems (8, 9) and from orexinergic, glutamatergic, and GABAergic pathways (10-12). The remarkably expansive neuromodulatory influence of 5HT is the product of a complex transcriptional cascade that generates 5HT-synthesizing neurons in the ventral hindbrain (13-18). 5HT also figures prominently in mental health disorders as a number of lines of evidence provide strong support for the hypothesis that altered serotonergic signaling contributes to neurological and psychiatric pathogenesis (19)(20)(21)(22). Despite considerable progress in understanding the importance of 5HT neurotransmission, however, the mechanisms governing 5HT neuron development and the precise physiological roles of 5HT in the modulation of CNS circuitry are not yet clear.Studies of 5HT neurons are hind...

A number of genes regulate regeneration of peripheral axons, but their ability to drive axon growth and regeneration in the central nervous system (CNS) remains largely untested. To address this question we overexpressed eight transcription factors and one small GTPase alone and in pairwise combinations to test whether combinatorial overexpression would have a synergistic impact on CNS neuron neurite growth. The Jun oncogene/signal transducer and activator of transcription 6 (JUN/STAT6) combination increased neurite growth in dissociated cortical neurons and in injured cortical slices. In injured cortical slices, JUN overexpression increased axon growth to a similar extent as JUN and STAT6 together. Interestingly, JUN overexpression was not associated with increased growth associated protein 43 (GAP43) or integrin alpha 7 (ITGA7) expression, though these are predicted transcriptional targets. This study demonstrates that JUN overexpression in cortical neurons stimulates axon growth, but does so independently of changes in expression of genes thought to be critical for JUN’s effects on axon growth. We conclude that JUN activity underlies this CNS axonal growth response, and that it is mechanistically distinct from peripheral regeneration responses, in which increases in JUN expression coincide with increases in GAP43 expression.

To fully understand cell type identity and function in the nervous system there is a need to understand neuronal gene expression at the level of isoform diversity. Here we applied Next Generation Sequencing of the transcriptome (RNA-Seq) to purified sensory neurons and cerebellar granular neurons (CGNs) grown on an axonal growth permissive substrate. The goal of the analysis was to uncover neuronal type specific isoforms as a prelude to understanding patterns of gene expression underlying their intrinsic growth abilities. Global gene expression patterns were comparable to those found for other cell types, in that a vast majority of genes were expressed at low abundance. Nearly 18% of gene loci produced more than one transcript. More than 8000 isoforms were differentially expressed, either to different degrees in different neuronal types or uniquely expressed in one or the other. Sensory neurons expressed a larger number of genes and gene isoforms than did CGNs. To begin to understand the mechanisms responsible for the differential gene/isoform expression we identified transcription factor binding sites present specifically in the upstream genomic sequences of differentially expressed isoforms, and analyzed the 3′ untranslated regions (3′ UTRs) for microRNA (miRNA) target sites. Our analysis defines isoform diversity for two neuronal types with diverse axon growth capabilities and begins to elucidate the complex transcriptional landscape in two neuronal populations.