MicroRNAs (miRNAs) are implicated in both tissue differentiation and maintenance of tissue identity. In most cases, however, the mechanisms underlying their regulation are not known. One brainspecific miRNA, miR-124a, decreases the levels of hundreds of nonneuronal transcripts, such that its introduction into HeLa cells promotes a neuronal-like mRNA profile. The transcriptional repressor, RE1 silencing transcription factor (REST), has a reciprocal activity, inhibiting the expression of neuronal genes in nonneuronal cells. Here, we show that REST regulates the expression of a family of miRNAs, including brain-specific miR-124a. In nonneuronal cells and neural progenitors, REST inhibits miR-124a expression, allowing the persistence of nonneuronal transcripts. As progenitors differentiate into mature neurons, REST leaves miR-124a gene loci, and nonneuronal transcripts are degraded selectively. Thus, the combined transcriptional and posttranscriptional consequences of REST action maximize the contrast between neuronal and nonneuronal cell phenotypes.noncoding RNA ͉ repression ͉ neuronal phenotype

Summary We identify the leucine-rich repeat transmembrane protein LRRTM2 as a key regulator of excitatory synapse development and function. LRRTM2 localizes to excitatory synapses in transfected hippocampal neurons, and shRNA-mediated knockdown of LRRTM2 leads to a decrease in excitatory synapses without affecting inhibitory synapses. LRRTM2 interacts with PSD-95 and regulates surface expression of AMPA receptors, and lentivirus-mediated knockdown of LRRTM2 in vivo decreases the strength of evoked excitatory synaptic currents. Structure-function studies indicate that LRRTM2 induces presynaptic differentiation via the extracellular LRR domain. We identify Neurexin1 as a receptor for LRRTM2 based on affinity chromatography. LRRTM2 binds to both Neurexin 1α and Neurexin 1β, and shRNA-mediated knockdown of Neurexin1 abrogates LRRTM2-induced presynaptic differentiation. These observations indicate that an LRRTM2-Neurexin1 interaction plays a critical role in regulating excitatory synapse development.

The repressor element 1 (RE1) silencing transcription factor (REST) helps preserve the identity of nervous tissue by silencing neuronal genes in non-neural tissues. Moreover, in an epithelial model of tumorigenesis, loss of REST function is associated with loss of adhesion, suggesting the aberrant expression of REST-controlled genes encoding this property. To date, no adhesion molecules under REST control have been identified. Here, we used serial analysis of chromatin occupancy to perform genome-wide identification of REST-occupied target sequences (RE1 sites) in a kidney cell line. We discovered novel REST-binding motifs and found that the number of RE1 sites far exceeded previous estimates. A large family of targets encoding adhesion proteins was identified, as were genes encoding signature proteins of neuroendocrine tumors. Unexpectedly, genes considered exclusively non-neuronal also contained an RE1 motif and were expressed in neurons. This supports the model that REST binding is a critical determinant of neuronal phenotype.

SUMMARY Our understanding of mechanisms that regulate the differentiation of specific classes of synapses is limited. Here, we investigate the formation of synapses between hippocampal dentate gyrus (DG) neurons and their target CA3 neurons and find that DG neurons preferentially form synapses with CA3 rather than DG or CA1 neurons in culture, suggesting that specific interactions between DG and CA3 neurons drive synapse formation. Cadherin-9 is expressed selectively in DG and CA3 neurons, and downregulation of cadherin-9 in CA3 neurons leads to a selective decrease in the number and size of DG synapses onto CA3 neurons. In addition, loss of cadherin-9 from DG or CA3 neurons in vivo leads to striking defects in the formation and differentiation of the DG-CA3 mossy fiber synapse. These observations indicate that cadherin-9 bidirectionally regulates DG-CA3 synapse development and highlight the critical role of differentially expressed molecular cues in establishing specific connections in the mammalian brain.

Neurons in the human central nervous system (CNS) are unable to regenerate, as a result of both an inhibitory environment and their inherent inability to regrow. In contrast, the CNS environment in fish is permissive for growth, yet some neurons still cannot regenerate. Fish thus offer an opportunity to study molecules that might surmount the intrinsic limitations they share with mammals, without the complication of an inhibitory environment. We show by in vivo imaging in zebrafish that post-injury application of cyclic adenosine monophosphate can transform severed CNS neurons into ones that regenerate and restore function, thus overcoming intrinsic limitations to regeneration in a vertebrate.

BackgroundThe assembly of neural circuits requires the concerted action of both genetically determined and activity-dependent mechanisms. Calcium-regulated transcription may link these processes, but the influence of specific transcription factors on the differentiation of synapse-specific properties is poorly understood. Here we characterize the influence of NeuroD2, a calcium-dependent transcription factor, in regulating the structural and functional maturation of the hippocampal mossy fiber (MF) synapse.ResultsUsing NeuroD2 null mice and in vivo lentivirus-mediated gene knockdown, we demonstrate a critical role for NeuroD2 in the formation of CA3 dendritic spines receiving MF inputs. We also use electrophysiological recordings from CA3 neurons while stimulating MF axons to show that NeuroD2 regulates the differentiation of functional properties at the MF synapse. Finally, we find that NeuroD2 regulates PSD95 expression in hippocampal neurons and that PSD95 loss of function in vivo reproduces CA3 neuron spine defects observed in NeuroD2 null mice.ConclusionThese experiments identify NeuroD2 as a key transcription factor that regulates the structural and functional differentiation of MF synapses in vivo.

Transcription factor binding sites (TFBSs) are short DNA sequences interacting with transcription factors (TFs), which regulate gene expression. Due to the relatively short length of such binding sites, it is largely unclear how the specificity of protein–DNA interaction is achieved. Here, we have performed a genome-wide analysis of TFBS-like sequences for the transcriptional repressor, RE1 Silencing Transcription Factor (REST), as well as for several other representative mammalian TFs (c-myc, p53, HNF-1 and CREB). We find a nonrandom distribution of inexact sites for these TFs, referred to as highly-degenerate TFBSs, that are enriched around the cognate binding sites. Comparisons among human, mouse and rat orthologous promoters reveal that these highly-degenerate sites are conserved significantly more than expected by random chance, suggesting their positive selection during evolution. We propose that this arrangement provides a favorable genomic landscape for functional target site selection.

The relatively simple neural circuit driving the escape response in zebrafish offers an excellent opportunity to study properties of neural circuit formation. The hindbrain Mauthner cell is an essential component of this circuit. Mutations in the zebrafish deadly seven/notch1a (des) gene result in supernumerary Mauthner cells. We addressed whether and how these extra cells are incorporated into the escape-response circuit. Calcium imaging revealed that all Mauthner cells in desb420 mutants were active during an elicited escape response. However, the kinematic performance of the escape response in mutant larvae was very similar to wild-type fish. Analysis of the relationship between Mauthner axon collaterals and spinal neurons revealed that there was a decrease in the number of axon collaterals per Mauthner axon in mutant larvae compared with wild-type larvae, indicative of a decrease in the number of synapses formed with target spinal neurons. Moreover, we show that Mauthner axons projecting on the same side of the nervous system have primarily nonoverlapping collaterals. These data support the hypothesis that excess Mauthner cells are incorporated into the escape-response circuit, but they divide their target territory to maintain a normal response, thus demonstrating plasticity in the formation of the escape-response circuit. Such plasticity may be key to the evolution of the startle responses in mammals, which use larger populations of neurons in circuits similar to those in the fish escape response.