Enhancer SINEs Link Pol III to Pol II Transcription in Neurons

Spatiotemporal regulation of gene expression depends on the cooperation of multiple mechanisms, including the functional interaction of promoters with distally located enhancers. Here, we show that, in cortical neurons, a subset of short interspersed nuclear elements (SINEs) located in the proximity of activity-regulated genes bears features of enhancers. Enhancer SINEs (eSINEs) recruit the Pol III cofactor complex TFIIIC in a stimulus-dependent manner and are transcribed by Pol III in response to neuronal depolarization. Characterization of an eSINE located in proximity to the Fos gene (FosRSINE1) indicated that the FosRSINE1-encoded transcript interacts with Pol II at the Fos promoter and mediates Fos relocation to Pol II factories, providing an unprecedented molecular link between Pol III and Pol II transcription. Strikingly, knockdown of the FosRSINE1 transcript induces defects of both cortical radial migration in vivo and activity-dependent dendritogenesis in vitro, demonstrating that FosRSINE1 acts as a strong enhancer of Fos expression in diverse physiological contexts

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy

ABSTRACT The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant \u3b22-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters\u2019 expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing \u3b22-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying \u3b22-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing \u3b22-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of \u3b22-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits

Receptive field properties of long-range projections in mouse visual cortex

Neuronal circuits in the neocortex are characterized by a precise architecture of connections between cellular subtypes in different layers and specialized areas. These complex networks provide the substrate for functional operations that are responsible for perception and other cognitive functions. The primary visual cortex (V1) provides a well-suited model to study how sensory stimuli are represented and processed by cortical circuits and passed on to other brain areas in order to generate behaviourally relevant outputs in response to environmental inputs. Cortical circuits are characterized by a variety of projection pathways and especially in the superficial layers of V1 layer 2/3 neurons have an extensive network of intra-areal long-range projections. Previous studies have provided some indications of a possible functional role of long-range projections in sensory processing but so far techniques to answer further questions were lacking. Two-photon calcium imaging and the use of genetically-encoded calcium sensors make it possible to record responses from both the cell bodies of neurons and their axonal projections. In order to shed light on the functional role of long-range projections in mouse V1 we compared receptive field properties of local populations of cell bodies and of axonal boutons of sub-populations of these cells that projected to different sites within V1. Receptive fields were of similar size when measured in boutons of long-range projections and in cell bodies. However, receptive fields of long-range projection boutons had more elongated subfields, showed more ON subfields, and a larger separation of ON and OFF subfields than the general population of cell bodies. In summary this work presents an experimental approach to study the functional properties of lateral long-range connections and their role in sensory processing in mouse V1

Experience-dependent expression of miR-132 regulates ocular dominance plasticity

miR-132 is a CREB-induced microRNA that is involved in dendritic spine plasticity. We found that visual experience regulated histone post-translational modifications at a CRE locus that is important for miR-212 and miR-132 cluster transcription, and regulated miR-132 expression in the visual cortex of juvenile mice. Monocular deprivation reduced miR-132 expression in the cortex contralateral to the deprived eye. Counteracting this miR-132 reduction with an infusion of chemically modified miR-132 mimic oligonucleotides completely blocked ocular dominance plasticity

Crowdfunding for cancer research: the TRACe campaign as an example

Over the past 7 years, research funding has witnessed a changing trend. Besides available competitive research funding such as Horizon 2020 (Europe), National Institutes of Health (USA), National Health Service (UK) grants, and other public research funding programmes around the world, including those from charitable organisations, researchers have started to engage in crowdfunding activities to look for alternative sources of funding. Managemen

The role of neuronal nicotinic receptors (nAChRs) in the pathogenesis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE): a study on wild-type and conditional transgenic mice expressing the beta2-V287L subunit

We studied mice conditionally expressing the beta2-V287L nAChR subunit, linked to ADNFLE. Expression of beta2-V287L during the first two postnatal weeks (a critical phase of synaptic maturation) is necessary for the epileptic phenotype to manifest. MATURE NEOCORTEX. Compared to the control littermates (CTRL), adult mice (P60) expressing beta2-V287L (TG) display no alteration in neocortex thickness, neuron count, nAChR density, and expression of GABAergic markers. Densitometric analyses of KCC2 (the major Cl- extruder) displayed increased expression in prefrontal (PFC) layer V and a decrease in the thalamic reticular nucleus, in TG mice. In pyramidal neurons of PFC layer V, we tested the effect of 10 microM nicotine on spontaneous excitatory (EPSC) and inhibitory (IPSC) postsynaptic currents. The basal EPSC/IPSC frequency ratio was similar in TG and CTRL, but nicotine tripled the EPSC frequency in TG mice, an effect stronger than observed in CTRL. Hence, beta2-V287L mainly augmented the glutamatergic activity. Pharmacological tests confirmed the role of alpha4beta2 nAChRs. The miniature postsynaptic events and the EPSC amplitudes show that most of the effect occurred on synaptic terminals. TG mice were also more susceptible to develop epileptiform activity in the presence of low Mg2+ and 4AP. We are now investigating how ACh regulates the basal hyperexcitability. DEVELOPMENT. Because of its role in network maturation, we studied the developmental balance between the expression of KCC2 and NKCC1 (which absorbes Cl-). The kinetics of transporters\u2019 expression was not altered by expression of beta2-V287L. Consistently, the timing of developmental switch of the GABAAR reversal potential was identical in TG and CTRL mice. However, studying the EPSCs revealed an approximate triplication of the basal glutamatergic activity in TG mice, in the second postnatal week. We conclude that the beta2-V287L is not directly implicated in the maturation of the GABAergic system, but causes an early increase in the efficacy of the glutamatergic transmission, in agreement with the results obtained in the mature PFC. IN VITRO MODEL OF ADNFLE. By multi-electrode array (MEA) methods, we found that longterm TG neocortical cultures develop long (10\u201332 s) global bursting events, whose duration is similar to the in vivo seizures. No pretreatment with convulsants was necessary, thus reproducing the features of genetic epilepsies. The long bursts were blocked by carbamazepine, a drug of choice in ADNFLE. CONCLUSIONS. Beta2-V287L increased the background excitability and the sensitivity to nAChR agonists, which we attribute to increase of glutamatergic transmission during early postnatal stages, as well as intrinsic hyperfunctionality of alpha4beta2-V287L. Our chronic model of hyperexcitability opens the way to in vitro studies on the role of beta2-V287L on excitatory synapse formation and its modulation by antiepileptics

Expression of the K+/CL- cotransporter KCC2 in a conditional murine model of autosomal dominant nocturnal frontal lobe epilepsy

The K+/Cl- cotransporter KCC2 is the main chloride extruder in neurons and it exerts an essential role in determining the polarity of GABAA receptor-mediated chloride currents. The increase of KCC2 expression after birth is responsible of the \u201cswitch of GABA\u201d effect from excitatory to inhibitory. KCC2 also covers a critical role in dendritic spine morphogenesis and in maintenance of glutamatergic synapses. Alteration in its expression could lead to an imbalance between excitation and inhibition and to the pathogenesis of diseases such as epilepsy. We studied KCC2 expression and distribution in a murine model of Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE), conditionally expressing the ADNFLE-linked nicotinic \u3b22V287L mutation (TG), for comparison with control mice (CTRL) in prefrontal cortex (PFC), somatosensory cortex (SS) and thalamus (TH). By immunocytochemical approaches, we carried out densitometric analyses of KCC2 expression at different postnatal ages (P8, P21, P60) and we observed a significant increase of KCC2 expression in PFC layer V and a decrease of this cotransporter in the thalamic reticular nucleus (RT) in TG P60 mice compared to age-matched controls. Further analyses of some GABAergic markers showed no substantial alterations. Next, we estimated GABAA reversal potential (EGABA) by perforated patch-clamp recordings on acute dissociated neurons from TG and CTRL mice during postnatal development. EGABA progressively hyperpolarized up to the second postnatal week, but no difference was found between TG and CTRL mice. On the other hand, in mature (older than P28) cortical slices, a potentiation of both EPSCs and IPSCs stimulation was detected in response to nicotine in TG compared to control neurons and, especially in the case of EPSCs, leading towards excitation. Our results suggest that KCC2 and the GABAergic system could be implicated in the pathogenesis of ADNFLE and that \u3b22V287L mutation could alter the excitatory/inhibitory balance in murine forebrain

Acetylated SINEs are a distinctive feature of NEE-induced genes.

<p>(A and C) Average H3K9K14ac ChIPseq tag density of constitutively silent (CS, gray dashed line), expressed (CE, black dashed line), NEE-induced (NI, black solid line) and repressed (NR, gray solid line) genes in somatosensory cortex of control mice (A) and mice exposed to NEE for 45 minutes (C). For each gene group, the average density of ChIPseq reads per 100 bp window is plotted relative to the TSS. (B and D) Box and whiskers plots summarizing the distribution of ChIPseq tag density at +0/+0.5 kbp from TSS, highlighted in yellow in (A) and (C), for each of the four gene groups, in control (B) and NEE-stimulated (D) mouse cortex. Lower and upper whiskers indicate the minimum and maximum value of the distribution, respectively. The lower and upper limits of the box indicate the 25^th and 75^th percentile, respectively. The solid line denotes the median. The red lines below each box plot indicate whether the difference between the medians of two data sets is statistically significant (P<0.0001, two-tailed Mann-Whitney test). The tag density values for control (A, B) and NEE-stimulated (C, D) cortex are not directly comparable, as they have not been normalized across samples. (E) Ac1 and Ac2 motifs were found by applying motif inference on +Δac regions. (F) Average density of <i>de novo</i> acetylated SINEs distribution for constitutively silent (CS, gray dashed line), constitutively expressed (CE, black dashed line), NEE-induced (NI, black solid line) or NEE-repressed (NE, gray solid line) genes. For each gene group, the average density of <i>de novo</i> acetylated SINEs per 50 kbp window (at 5 kbp intervals) is plotted. Horizontal red bars represent regions where the SINE density of NI genes is significantly higher than that of CE genes (P<0.05, two-tailed Mann-Whitney test). Red arrowheads under the graph indicate the <i>de novo</i> acetylated SINEs detected within 100 kbp of the <i>c-Fos</i> and <i>Gadd45b</i> TSSs.</p

Binding of TFIIIC to SINE Elements Controls the Relocation of Activity-Dependent Neuronal Genes to Transcription Factories

<div><p>In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation <i>in vivo</i>, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes.</p></div

Inducible genes relocate to TFs in response to depolarization.

<p>(A) Representative images of single confocal sections of immuno-DNA FISH experiments showing nuclear localization of <i>c-Fos</i>, <i>Gadd45b</i>, <i>Csn2</i>, <i>Gapdh</i>, <i>Bdnf</i> and <i>Fcf1</i> loci (green) relative to TFs. TFs were detected by RNAPII-ser5P immunostaining (red), in cortical neurons either stimulated with 50 mM KCl for 45 minutes or left untreated. Nuclei were stained with DAPI (blue). For each image series, the distance between the centre of the FISH signal and the nearest TF is indicated (top right inset). Scale bars, 2 µm (images on the left) and 0.5 µm (magnified images). (B) Box and whiskers plot of the distribution of the distance between <i>c-Fos</i>, <i>Gadd45b</i>, <i>Csn2</i>, <i>Gapdh</i>, <i>Bdnf</i> and <i>Fcf1</i> loci and the nearest TF. Neurons were maintained either in basal conditions or exposed to 50 mM KCl for 45 minutes. Whiskers denote the 90th and 10th percentiles, box edges denote the 75th and 25th percentiles, solid lines denote medians, dashed lines denote averages. (C) Percentage of co-localization with TFs of <i>c-Fos</i>, <i>Gadd45b</i>, <i>Csn2</i>, <i>Gapdh</i>, <i>Bdnf</i> and <i>Fcf1</i> gene loci both in basal conditions an in response to KCl (*, P<0.05, Fisher's exact test). For each condition shown in (B) and (C), 31 to 64 FISH signals were analysed. (D) DIV10 cortical primary neurons were stimulated with 50 µM bicuculline for 45 minutes, either in the presence of 1 µM tetrodotoxin (TTX) or alone, and analysed by immuno-DNA FISH. Histograms show the percentage of co-localization with TFs of <i>c-Fos</i> gene locus (*, P<0.05, Fisher's exact test; n = 36 to 41 FISH signals per condition). (E) Cortical primary neurons were stimulated with 50 mM KCl for 45 minutes, either in the presence of 5 µM nifedipine, 50 µg/ml DRB or alone, and analysed by immuno-DNA FISH. Histograms show the percentage of co-localization with TFs of <i>c-Fos</i> gene locus (*, P<0.05, Fisher's exact test; n = 30 to 41 FISH signals per condition).</p