Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons.

Neuronal activity-dependent transcription plays a key role in plasticity and pathology in the brain. An intriguing question is how neuronal activity controls gene expression via interactions of transcription factors with DNA and chromatin modifiers in the nucleus. By utilizing single-molecule imaging in human embryonic stem cell (ESC)-derived cortical neurons, we demonstrate that neuronal activity increases repetitive emergence of cAMP response element-binding protein (CREB) at histone acetylation sites in the nucleus, where RNA polymerase II (RNAPII) accumulation and FOS expression occur rapidly. Neuronal activity also enhances co-localization of CREB and CREB-binding protein (CBP). Increased binding of a constitutively active CREB to CBP efficiently induces CREB repetitive emergence. On the other hand, the formation of histone acetylation sites is dependent on CBP histone modification via acetyltransferase (HAT) activity but is not affected by neuronal activity. Taken together, our results suggest that neuronal activity promotes repetitive CREB-CRE and CREB-CBP interactions at predetermined histone acetylation sites, leading to rapid gene expression.

Suppression of DNA Double-Strand Break Formation by DNA Polymerase ß in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons.

Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase ß (Polß) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polß fl/fl mice of either sex, in which forebrain postmitotic excitatory neurons lack Polß expression. Polß deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polß maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENT Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polß, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polß deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

BACKGROUND: CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. RESULTS: Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. CONCLUSIONS: Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

Genome Stability by DNA Polymerase ß 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 ß (Polß), a key enzyme in DNA base excision repair pathway, in the developing cortex using distinct forebrain-specific conditional knock-out mice, Emx1-Cre/Polß fl/fl and Nex-Cre/Polß fl/fl mice. Polß expression was absent in both neural progenitors and postmitotic neurons in Emx1-Cre/Polß fl/fl mice, whereas only postmitotic neurons lacked Polß expression in Nex-Cre/Polß fl/fl mice. We found that DNA double-strand breaks (DSBs) were frequently detected during replication in cortical progenitors of Emx1-Cre/Polß fl/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/Polß fl/fl mice. Moreover, cultured Polß-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 Polß-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 ß (Polß)-dependent DNA base excision repair pathway contributes to the process is still unknown. We found that loss of Polß 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 Polß-deficient neural progenitors. Thus, genome stability by Polß-dependent base excision repair in neural progenitors is required for the viability and differentiation of daughter neurons in the developing nervous system.

Nucleocytoplasmic Shuttling of Histone Deacetylase 9 Controls Activity-Dependent Thalamocortical Axon Branching.

During development, thalamocortical (TC) axons form branches in an activity-dependent fashion. Here we investigated how neuronal activity is converted to molecular signals, focusing on an epigenetic mechanism involving histone deacetylases (HDACs). Immunohistochemistry demonstrated that HDAC9 was translocated from the nucleus to the cytoplasm of thalamic cells during the first postnatal week in rats. In organotypic co-cultures of the thalamus and cortex, fluorescent protein-tagged HDAC9 also exhibited nuclueocytoplasmic translocation in thalamic cells during culturing, which was reversed by tetrodotoxin treatment. Transfection with a mutant HDAC9 that interferes with the translocation markedly decreased TC axon branching in the culture. Similarly, TC axon branching was significantly decreased by the mutant HDAC9 gene transfer in vivo. However, axonal branching was restored by disrupting the interaction between HDAC9 and myocyte-specific enhancer factor 2 (MEF2). Taken together, the present results demonstrate that the nucleocytoplasmic translocation of HDAC9 plays a critical role in activity-dependent TC axon branching by affecting transcriptional regulation and downstream signaling pathways.

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.

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.

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.

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-beta 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-beta (Pol beta). Here the authors show that thermotolerance was partially suppressed in Pol beta(-/-) 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 gamma H2AX was suppressed in the thermotolerant cells. These results suggest that Pol beta 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 Pol beta. The present novel findings provide strong evidence that Pol beta functions as a critical element involved in thermotolerance and exerts an important role in heat-induced DSB.

Decreased PARP-1 levels accelerate embryonic lethality but attenuate neuronal apoptosis in DNA polymerase beta-deficient mice.

In mammalian cells, DNA polymerase beta (Polbeta) and poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in base excision repair (BER) and single-strand break repair. Polbeta knockout mice exhibit extensive neuronal apoptosis during neurogenesis and die immediately after birth, while PARP-1 knockout mice are viable and display hypersensitivity to genotoxic agents and genomic instability. Although accumulating biochemical data show functional interactions between Polbeta and PARP-1, such interactions in the whole animal have not yet been explored. To study this, we generate Polbeta(-/-)PARP-1(-/-) double mutant mice. Here, we show that the double mutant mice exhibit a profound developmental delay and embryonic lethality at mid-gestation. Importantly, the degree of the neuronal apoptosis was dramatically reduced in PARP-1 heterozygous mice in a Polbeta null background. The reduction was well correlated with decreased levels of p53 phosphorylation at serine-18, suggesting that the apoptosis depends on the p53-mediated apoptosis pathway that is positively regulated by PARP-1. These results indicate that functional interactions between Polbeta and PARP-1 play important roles in embryonic development and neurogenesis.

Decreased mutant frequency in embryonic brain of DNA polymerase beta null mice.

DNA polymerase beta (Polbeta) knockout mouse embryos exhibit extensive apoptosis in postmitotic neuronal cells and die immediately after birth. In contrast, no apoptosis has been observed in other tissues as well as liver in the mutant embryos. To study the relationship of Polbeta deficiency and mutagenesis during development and neurogenesis, we examined spontaneous mutations in Polbeta null (Polbeta-/-) and wild-type (Polbeta+/+) mouse embryos, by using the transgenic mutation detection system consisting of a pSSW shuttle vector with the Escherichia coli rpsL reporter gene. Unexpectedly, we found a significant decrease in the mutant frequency of Polbeta-/- brain (1.63+/-0.67x10(-5)) compared with wild-type controls (3.12+/-0.83x10(-5)) (P<0.001). In contrast, no such difference was found between livers from Polbeta-/- (0.92+/-0.38x10(-5)) and wild-type (0.71+/-0.31x10(-5)) embryos. Analysis of mutation spectra revealed that mutations in brains from the two genotypes were almost exclusively single-base deletions and that these sites fell within runs of 2-6 identical bases and a two base repeat in the rpsL sequence, while mutations in the corresponding livers contained base substitutions as well as single-base deletions. Taken together with the extensive neuronal apoptosis associated with Polbeta deficiency, we suggest that the lower mutant frequency observed in Polbeta-/- embryonic brain may be caused by the elimination of neuronal cells with unrepaired DNA damage through apoptosis.

p53 Deficiency rescues neuronal apoptosis but not differentiation in DNA polymerase beta-deficient mice.

In mammalian cells, DNA polymerase beta (Polbeta) functions in base excision repair. We have previously shown that Polbeta-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Polbeta(-/-) p53(-/-) double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Polbeta deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Polbeta(-/-) p53(-/-) mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Polbeta(-/-) p53(+/+) mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Polbeta in the differentiation of specific neuronal cell types.