Noriyuki Sugo

Nucleocytoplasmic translocation of HDAC9 regulates gene expression and dendritic growth in developing cortical neurons

Transcriptional regulation of gene expression is thought to play a pivotal role in activity‐dependent neuronal differentiation and circuit formation. Here, we investigated the role of histone deacetylase 9 (HDAC9), which regulates transcription by histone modification, in the development of neocortical neurons. The translocation of HDAC9 from nucleus to cytoplasm was induced by an increase of spontaneous firing activity in cultured mouse cortical neurons. This nucleocytoplasmic translocation was also observed in postnatal development in vivo. The translocation‐induced gene expression and cellular morphology was further examined by introducing an HDAC9 mutant that disrupts the nucleocytoplasmic translocation. Expression of c‐fos, an immediately‐early gene, was suppressed in the mutant‐transfected cells regardless of neural activity. Moreover, the introduction of the mutant decreased the total length of dendritic branches, whereas knockdown of HDAC9 promoted dendritic growth. These findings indicate that chromatin remodeling with nucleocytoplasmic translocation of HDAC9 regulates activity‐dependent gene expression and dendritic growth in developing cortical neurons.

Development of thermotolerance requires interaction between polymerase-β and heat shock proteins

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat‐induced DNA double‐strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat‐labile repair proteins such as DNA polymerase‐β (Polβ). Here the authors show that thermotolerance was partially suppressed in Polβ−/– mouse embryonic fibroblasts (MEF) when compared to the wild‐type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat‐induced γH2AX was suppressed in the thermotolerant cells. These results suggest that Polβ at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat‐induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat‐labile proteins besides Polβ. The present novel findings provide strong evidence that Polβ functions as a critical element involved in thermotolerance and exerts an important role in heat‐induced DSB. (Cancer Sci 2008; 99: 973–978)

Single-Molecule Imaging Reveals Dynamics of CREB Transcription Factor Bound to Its Target Sequence

Proper spatiotemporal gene expression is achieved by selective DNA binding of transcription factors in the genome. The most intriguing question is how dynamic interactions between transcription factors and their target sites contribute to gene regulation by recruiting the basal transcriptional machinery. Here we demonstrate individual binding and dissociation events of the transcription factor cAMP response element-binding protein (CREB), both in vitro and in living cells, using single-molecule imaging. Fluorescent–tagged CREB bound to its target sequence cAMP-response element (CRE) for a remarkably longer period (dissociation rate constant: 0.21 s-1) than to an unrelated sequence (2.74 s-1). Moreover, CREB resided at restricted positions in the living cell nucleus for a comparable period. These results suggest that CREB stimulates transcription by binding transiently to CRE in the time range of several seconds.

A molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing neocortex.

During development, cortical neurons extend axons to their targets based on their laminar locations and cell types. Here we studied the molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing mouse cortex. Upper layer neurons were labeled with enhanced yellow fluorescent protein (EYFP) by in utero electroporation at E15.5. Cortical slices containing EYFP‐labeled cells were dissected at E16, when axonal outgrowth from upper layer neurons is not initiated, and were cultured in an organotypic manner. After 3 days in culture, most labeled cells were found to extend axons medially in the same fashion as those observed in vivo. This oriented growth was disrupted when the lateral side of the cortical slice was removed, indicating that a laterally located repellent is involved in the medially oriented growth. Strikingly, the medially directed growth within the slices was reduced in the medium containing Semaphorin3A (Sema3A) or soluble form of Neuropilin‐1 (Npn1), a receptor for Sema3A. Importantly, we found that Sema3A was expressed in a gradient from lateral‐high to medial‐low within the cortex, and callosal axons originating from upper layer neurons uniquely expressed Npn1. Consistent with these findings, ectopically expressed Sema3A repelled medially oriented elongation of upper layer cell axons in vivo. These results therefore suggest the operation of a repulsive mechanism for medially oriented axon growth of upper layer neurons, and further point to a role for a gradient expression of Sema3A in this directional axon growth along the mediolateral axis within the neocortex. J. Comp. Neurol. 519:834–848, 2011.

Regulation of thalamocortical axon branching by BDNF and synaptic vesicle cycling.

During development, axons form branches in response to extracellular molecules. Little is known about the underlying molecular mechanisms. Here, we investigate how neurotrophin-induced axon branching is related to synaptic vesicle cycling for thalamocortical axons. The exogenous application of brain-derived neurotrophic factor (BDNF) markedly increased axon branching in thalamocortical co-cultures, while removal of endogenous BDNF reduced branching. Over-expression of a C-terminal fragment of AP180 that inhibits clathrin-mediated endocytosis affected the laminar distribution and the number of branch points. A dominant-negative synaptotagmin mutant that selectively targets synaptic vesicle cycling, strongly suppressed axon branching. Moreover, axons expressing the mutant synaptotagmin were resistant to the branch-promoting effect of BDNF. These results suggest that synaptic vesicle cycling might regulate BDNF induced branching during the development of the axonal arbor.

Genome Stability by DNA Polymerase β in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development

DNA repair is crucial for genome stability in the developing cortex, as somatic de novo mutations cause neurological disorders. However, how DNA repair contributes to neuronal development is largely unknown. To address this issue, we studied the spatiotemporal roles of DNA polymerase β (Polβ), a key enzyme in DNA base excision repair pathway, in the developing cortex using distinct forebrain-specific conditional knock-out mice, Emx1-Cre/Polβfl/fl and Nex-Cre/Polβfl/fl mice. Polβ expression was absent in both neural progenitors and postmitotic neurons in Emx1-Cre/Polβfl/fl mice, whereas only postmitotic neurons lacked Polβ expression in Nex-Cre/Polβfl/fl mice. We found that DNA double-strand breaks (DSBs) were frequently detected during replication in cortical progenitors of Emx1-Cre/Polβfl/fl mice. Increased DSBs remained in postmitotic cells, which resulted in p53-mediated neuronal apoptosis. This neuronal apoptosis caused thinning of the cortical plate, although laminar structure was normal. In addition, accumulated DSBs also affected growth of corticofugal axons but not commissural axons. These phenotypes were not observed in Nex-Cre/Polβfl/fl mice. Moreover, cultured Polβ-deficient neural progenitors exhibited higher sensitivity to the base-damaging agent methylmethanesulfonate, resulting in enhanced DSB formation. Similar damage was found by vitamin C treatment, which induces TET1-mediated DNA demethylation via 5-hydroxymethylcytosine. Together, genome stability mediated by Polβ-dependent base excision repair is crucial for the competence of neural progenitors, thereby contributing to neuronal differentiation in cortical development. SIGNIFICANCE STATEMENT DNA repair is crucial for development of the nervous system. However, how DNA polymerase β (Polβ)-dependent DNA base excision repair pathway contributes to the process is still unknown. We found that loss of Polβ in cortical progenitors rather than postmitotic neurons led to catastrophic DNA double-strand breaks (DSBs) during replication and p53-mediated neuronal apoptosis, which resulted in thinning of the cortical plate. The DSBs also affected corticofugal axon growth in surviving neurons. Moreover, induction of base damage and DNA demethylation intermediates in the genome increased DSBs in cultured Polβ-deficient neural progenitors. Thus, genome stability by Polβ-dependent base excision repair in neural progenitors is required for the viability and differentiation of daughter neurons in the developing nervous system.

Visualization of HDAC9 Spatiotemporal Subcellular Localization in Primary Neuron Cultures

Histone deacetylase (HDAC) 9 is one of class IIa HDACs which are expressed in developing cortical neurons. The translocation of HDAC9 from the nucleus to the cytoplasm is induced by neuronal activity during postnatal development, and is involved in regulation of various gene expressions. Visualization of HDAC9 subcellular localization is a powerful tool for studying activity-dependent gene expression. Here, we describe a time-lapse imaging method using fluorescent protein-tagged HDAC9 in dissociated cortical neurons. This method reveals dynamic HDAC9-mediated gene expression in response to various signals.

Synapse-dependent and independent mechanisms of thalamocortical axon branching are regulated by neuronal activity

Axon branching and synapse formation are critical processes for establishing precise circuit connectivity. These processes are tightly regulated by neural activity, but the relationship between them remains largely unclear. We use organotypic coculture preparations to examine the role of synapse formation in the activity‐dependent axon branching of thalamocortical (TC) projections. To visualize TC axons and their presynaptic sites, two plasmids encoding DsRed and EGFP‐tagged synaptophysin (SYP‐EGFP) were cotransfected into a small number of thalamic neurons. Time‐lapse imaging of individual TC axons showed that most branches emerged from SYP‐EGFP puncta, indicating that synapse formation precedes emergences of axonal branches. We also investigated the effects of neuronal activity on axon branching and synapse formation by manipulating spontaneous firing activity of thalamic cells. An inward rectifying potassium channel, Kir2.1, and a bacterial voltage‐gated sodium channel, NaChBac, were used to suppress and promote firing activity, respectively. We found suppressing neural activity reduced both axon branching and synapse formation. In contrast, increasing neural activity promoted only axonal branch formation. Time‐lapse imaging of NaChBac‐expressing cells further revealed that new branches frequently appeared from the locations other than SYP‐EGFP puncta, indicating that enhancing activity promotes axonal branch formation due to an increase of branch emergence at nonsynaptic sites. These results suggest that presynaptic locations are hotspots for branch emergence, and that frequent firing activity can shift branch emergence to a synapse‐independent process.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s−1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. SIGNIFICANCE STATEMENT The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.

Activity-Dependent Dynamics of the Transcription Factor CREB in Cortical Neurons Revealed by Single-Molecule Imaging

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s−1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. SIGNIFICANCE STATEMENT The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.