Activity-dependent Gene Expression Research Articles

Activity-dependent aberrations in gene expression and alternative splicing in a mouse model of Rett syndrome

Rett syndrome (RTT) is a severe neurodevelopmental disorder that affects about 1 in 10,000 female live births. The underlying cause of RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2); however, the molecular mechanism by which these mutations mediate the RTT neuropathology remains enigmatic. Specifically, although MeCP2 is known to act as a transcriptional repressor, analyses of the RTT brain at steady-state conditions detected numerous differentially expressed genes, while the changes in transcript levels were mostly subtle. Here we reveal an aberrant global pattern of gene expression, characterized predominantly by higher levels of expression of activity-dependent genes, and anomalous alternative splicing events, specifically in response to neuronal activity in a mouse model for RTT. Notably, the specific splicing modalities of intron retention and exon skipping displayed a significant bias toward increased retained introns and skipped exons, respectively, in the RTT brain compared with the WT brain. Furthermore, these aberrations occur in conjunction with higher seizure susceptibility in response to neuronal activity in RTT mice. Our findings advance the concept that normal MeCP2 functioning is required for fine-tuning the robust and immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation.

Mild Maternal Hypothyroxinemia During Pregnancy Induces Persistent DNA Hypermethylation in the Hippocampal Brain-Derived Neurotrophic Factor Gene in Mouse Offspring.

Thyroid hormones are essential for normal development of the central nervous system (CNS). Experimental rodents have shown that even a subtle thyroid hormone insufficiency in circulating maternal thyroid hormones during pregnancy may adversely affect neurodevelopment in offspring, resulting in irreversible cognitive deficits. This may be due to the persistent reduced expression of the hippocampal brain-derived neurotrophic factor gene Bdnf, which plays a crucial role in CNS development. However, the underlying molecular mechanisms remain unclear. Thiamazole (MMI; 0.025% [w/v]) was administered to dams from two weeks prior to conception until delivery, which succeeded in inducing mild maternal hypothyroxinemia during pregnancy. Serum thyroid hormone and thyrotropin levels of the offspring derived from dams with mild maternal hypothyroxinemia (M offspring) and the control offspring (C offspring) were measured. At 70 days after birth, several behavior tests were performed on the offspring. Gene expression and DNA methylation status were also evaluated in the promoter region of Bdnf exon IV, which is largely responsible for neural activity-dependent Bdnf gene expression, in the hippocampus of the offspring at day 28 and day 70. No significant differences in serum thyroid hormone or thyrotropin levels were found between M and C offspring at day 28 and day 70. M offspring showed an impaired learning capacity in the behavior tests. Hippocampal steady-state Bdnf exon IV expression was significantly weaker in M offspring than it was in C offspring at day 28. At day 70, hippocampal Bdnf exon IV expression at the basal level was comparable between M and C offspring. However, it was significantly weaker in M offspring than in C offspring after the behavior tests. Persistent DNA hypermethylation was also found in the promoter region of Bdnf exon IV in the hippocampus of M offspring compared to that of C offspring, which may cause the attenuation of Bdnf exon IV expression in M offspring. Mild maternal hypothyroxinemia induces persistent DNA hypermethylation in Bdnf exon IV in offspring as epigenetic memory, which may result in long-term cognitive disorders.

Cdk7 Is Required for Activity-Dependent Neuronal Gene Expression, Long-Lasting Synaptic Plasticity and Long-Term Memory.

In the brain, de novo gene expression driven by learning-associated neuronal activities is critical for the formation of long-term memories. However, the signaling machinery mediating neuronal activity-induced gene expression, especially the rapid transcription of immediate-early genes (IEGs) remains unclear. Cyclin-dependent kinases (Cdks) are a family of serine/threonine kinases that have been firmly established as key regulators of transcription processes underling coordinated cell cycle entry and sequential progression in nearly all types of proliferative cells. Cdk7 is a subunit of transcriptional initiation factor II-H (TFIIH) and the only known Cdk-activating kinase (CAK) in metazoans. Recent studies using a novel Cdk7 specific covalent inhibitor, THZ1, revealed important roles of Cdk7 in transcription regulation in cancer cells. However, whether Cdk7 plays a role in the regulation of transcription in neurons remains unknown. In this study, we present evidence demonstrating that, in post-mitotic neurons, Cdk7 activity is positively correlated with neuronal activities in cultured primary neurons, acute hippocampal slices and in the brain. Cdk7 inhibition by THZ1 significantly suppressed mRNA levels of IEGs, selectively impaired long-lasting synaptic plasticity induced by 4 trains of high frequency stimulation (HFS) and prevented the formation of long-term memories.

The Functioning of a Cortex without Layers.

A major hallmark of cortical organization is the existence of a variable number of layers, i.e., sheets of neurons stacked on top of each other, in which neurons have certain commonalities. However, even for the neocortex, variable numbers of layers have been described and it is just a convention to distinguish six layers from each other. Whether cortical layers are a structural epiphenomenon caused by developmental dynamics or represent a functionally important modularization of cortical computation is still unknown. Here we present our insights from the reeler mutant mouse, a model for a developmental, “molecular lesion”-induced loss of cortical layering that could serve as ground truth of what an intact layering adds to the cortex in terms of functionality. We could demonstrate that the reeler neocortex shows no inversion of cortical layers but rather a severe disorganization that in the primary somatosensory cortex leads to the complete loss of layers. Nevertheless, the somatosensory system is well organized. When exploring an enriched environment with specific sets of whiskers, activity-dependent gene expression takes place in the corresponding modules. Precise whisker stimuli lead to the functional activation of somatotopically organized barrel columns as visualized by intrinsic signal optical imaging. Similar results were obtained in the reeler visual system. When analyzing pathways that could be responsible for preservation of tactile perception, lemniscal thalamic projections were found to be largely intact, despite the smearing of target neurons across the cortical mantle. However, with optogenetic experiments we found evidence for a mild dispersion of thalamic synapse targeting on layer IV-spiny stellate cells, together with a general weakening in thalamocortical input strength. This weakening of thalamic inputs was compensated by intracortical mechanisms involving increased recurrent excitation and/or reduced feedforward inhibition. In conclusion, a layer loss so far only led to the detection of subtle defects in sensory processing by reeler mice. This argues in favor of a view in which cortical layers are not an essential component for basic perception and cognition. A view also supported by recent studies in birds, which can have remarkable cognitive capacities despite the lack of a neocortex with multiple cortical layers. In conclusion, we suggest that future studies directed toward understanding cortical functions should rather focus on circuits specified by functional cell type composition than mere laminar location.

Mapping Gene Expression in Excitatory Neurons during Hippocampal Late-Phase Long-Term Potentiation.

The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.

A neural basis for password-based species recognition in an avian brood parasite.

Obligate avian brood parasites are raised by heterospecific hosts and, therefore, lack crucial early exposure to relatives and other conspecifics. Yet, young brood parasites readily recognize and affiliate with others of their own species upon independence. One solution to this social recognition paradox is the ontogenetic 'password' mechanism used by obligate parasitic brown-headed cowbirds (Molothrus ater), whereby conspecific identification is initially mediated through the cowbird chatter: a non-learned vocal cue. We explored the neural basis of such password-based species recognition in juvenile and adult male cowbirds. We found that cowbird auditory forebrain regions express greater densities of the protein product of the immediate-early gene ZENK in response to the password chatter call relative to control sounds of mourning dove (Zenaida macroura) coos. The chatter-selective induction of ZENK expression occurs in both the caudal medial nidopallium (NCM) and the caudal medial mesopallium (CMM) in adults, but only within the NCM in juveniles. In contrast, we discovered that juvenile cowbirds exhibit neural selectivity to presentations of either conspecific or heterospecific songs, but only in CMM and only after recent experience. Juvenile cowbirds that did not have previous experience with the song type they were exposed to during the test period exhibited significantly lower activity-dependent gene expression. Thus, in juvenile male cowbirds, there is early onset of species-specific selective neural representation of non-learned calls in NCM and recently experienced song in CMM. These results suggest that NCM is evolutionarily co-opted in parasitic cowbirds to selectively recognize the password chatter, allowing juvenile cowbirds to identify adult conspecifics and avoid mis-imprinting upon unrelated host species. These ontogenetic comparisons reveal novel insights into the neural basis of species recognition in brood parasitic species.

The Role of Epigenetic Mechanisms in the Regulation of Gene Expression in the Nervous System.

Neuroepigenetics is a newly emerging field in neurobiology that addresses the epigenetic mechanism of gene expression regulation in various postmitotic neurons, both over time and in response to environmental stimuli. In addition to its fundamental contribution to our understanding of basic neuronal physiology, alterations in these neuroepigenetic mechanisms have been recently linked to numerous neurodevelopmental, psychiatric, and neurodegenerative disorders. This article provides a selective review of the role of DNA and histone modifications in neuronal signal-induced gene expression regulation, plasticity, and survival and how targeting these mechanisms could advance the development of future therapies. In addition, we discuss a recent discovery on how double-strand breaks of genomic DNA mediate the rapid induction of activity-dependent gene expression in neurons.

Primate Neurons Flex Their Musclin

Sensory experience evokes long-lasting changes in neural circuits through activity-dependent gene expression. Ataman etal. (2016) report in Nature that primates evolved novel transcriptional responses to neuronal activity, including induction of musclin/osteocrin (OSTN), which may regulate specialized aspects of primate neural circuits.

The O-GlcNAc Modification of CDK5 Involved in Neuronal Apoptosis FollowingIn Vitro Intracerebral Hemorrhage.

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in regulating mammalian CNS development. Studies of CDK5 have focused on its phosphorylation, although the diversity of CDK5 functions in the brain suggests additional forms of regulation. Here we expanded on the functional roles of CDK5 glycosylation in neurons. We showed that CDK5 was dynamically modified with O-GlcNAc in response to neuronal activity and that glycosylation represses CDK5-dependent apoptosis by impairing its association with p53 pathway. Blocking glycosylation of CDK5 alters cellular function and increases neuronal apoptosis in the cell model of the ICH. Our findings demonstrated a new role for O-glycosylation in neuronal apoptosis and provided a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identified a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and apoptosis.

Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons

Properly functional CNS circuits depend on inhibitoryinterneurons that in turn rely upon activity-dependent gene expression for morphological development, connectivity, and excitatory-inhibitory coordination.Despite its importance, excitation-transcription coupling in inhibitory interneurons is poorly understood. We report that PV+ interneurons employ a novel CaMK-dependent pathway to trigger CREB phosphorylation and gene expression. As in excitatory neurons, voltage-gated Ca(2+) influx through CaV1 channels triggers CaM nuclear translocation via local Ca(2+) signaling. However, PV+ interneurons are distinct in that nuclear signaling is mediated by γCaMKI, not γCaMKII. CREB phosphorylation also proceeds with slow, sigmoid kinetics, rate-limited by paucity of CaMKIV, protecting against saturation of phospho-CREB in the face of higher firing rates and bigger Ca(2+) transients. Our findings support the generality of CaM shuttling to drive nuclear CaMK activity, and they are relevant to disease pathophysiology, insofar as dysfunction of PV+ interneurons and molecules underpinning their excitation-transcription coupling both relate to neuropsychiatric disease.

Histone turnover and chromatin accessibility: Critical mediators of neurological development, plasticity, and disease.

In postmitotic neurons, nucleosomal turnover was long considered to be a static process that is inconsequential to transcription. However, our recent studies in human and rodent brain indicate that replication-independent (RI) nucleosomal turnover, which requires the histone variant H3.3, is dynamic throughout life and is necessary for activity-dependent gene expression, synaptic connectivity, and cognition. H3.3 turnover also facilitates cellular lineage specification and plays a role in suppressing the expression of heterochromatic repetitive elements, including mutagenic transposable sequences, in mouse embryonic stem cells. In this essay, we review mechanisms and functions for RI nucleosomal turnover in brain and present the hypothesis that defects in histone dynamics may represent a common mechanism underlying neurological aging and disease.

Differential Arc expression in the hippocampus and striatum during the transition from attentive to automatic navigation on a plus maze

The strategies utilized to effectively perform a given task change with practice and experience. During a spatial navigation task, with relatively little training, performance is typically attentive enabling an individual to locate the position of a goal by relying on spatial landmarks. These (place) strategies require an intact hippocampus. With task repetition, performance becomes automatic; the same goal is reached using a fixed response or sequence of actions. These (response) strategies require an intact striatum. The current work aims to understand the activation patterns across these neural structures during this experience-dependent strategy transition. This was accomplished by region-specific measurement of activity-dependent immediate early gene expression among rats trained to different degrees on a dual-solution task (i.e., a task that can be solved using either place or response navigation). As expected, rats increased their reliance on response navigation with extended task experience. In addition, dorsal hippocampal expression of the immediate early gene Arc was considerably reduced in rats that used a response strategy late in training (as compared with hippocampal expression in rats that used a place strategy early in training). In line with these data, vicarious trial and error, a behavior linked to hippocampal function, also decreased with task repetition. Although Arc mRNA expression in dorsal medial or lateral striatum alone did not correlate with training stage, the ratio of expression in the medial striatum to that in the lateral striatum was relatively high among rats that used a place strategy early in training as compared with the ratio among over-trained response rats. Altogether, these results identify specific changes in the activation of dissociated neural systems that may underlie the experience-dependent emergence of response-based automatic navigation.

Protein expression pattern in cerebellum of Cav2.1 mutant, tottering-6j mice.

Neuronal voltage-gated Cav2.1 channel controls a broad array of functions, including neurotransmitter release, neuronal excitability, activity-dependent gene expression, and neuronal survival. The Cav2.1 channel is molecular complexes consisting of several subunits: α1, α2/δ, β, and γ. The pore-forming subunit, α1, is encoded by the Cacna1a gene. Tottering-6j mice, generated by the Neuroscience Mutagenesis Facility at The Jackson Laboratory, are a recessive mutant strain in which the mutation has been chemically induced by ethylnitrosourea. In tottering-6j mice, mutation in the Cacna1a gene results in a base substitution (C-to-A) in the consensus splice acceptor sequence, which results in deletion of a part of the S4-S5 linker, S5, and a part of S5-S6 linker domain I in the α1 subunit of Cav2.1 channel. The mice display motor dysfunctions and absence-like seizures. However, protein expression in the cerebellum of tottering-6j mice has not been investigated. Real-time quantitative reverse transcription polymerase chain reaction and histological analyses of the cerebellum of tottering-6j mice revealed high expression levels of tyrosine hydroxylase, zebrin II, and ryanodine receptor 3 compared with those of wild-type mice. Conversely, a low level of calretinin expression was found compared with wild-type mice. These results indicate that Cacna1a mutation plays a significant role in protein expression patterns and that the tottering-6j mouse is a useful model for understanding protein expression mechanisms.

What do we learn from the murine Jacob/Nsmf gene knockout for human disease?

ABSTRACTMutations in the NSMF gene have been related to Kallmann syndrome. Conflicting results have been reported on the subcellular localization of Jacob/NELF, the protein encoded by the NSMF gene. Some reports indicate an extracellular localization and a function as a guidance molecule for migration of GnRH-positive neurons from the olfactory placode to the hypothalamus. Other studies have shown protein transport of Jacob from synapse-to-nucleus and indicate a role of the protein in neuronal activity-dependent gene expression. A recent publication casts doubts on a major role of Jacob/NELF in Kallmann syndrome and neuronal migration of GnRH-positive neurons during early development. Instead a murine NSMF gene knockout results in hippocampal dysplasia, impaired BDNF-signaling during dendritogenesis, and phenotypes related to the lack of BDNF-induced nuclear import of Jacob in early postnatal development.

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.

Chronic Mild Stress Modulates Activity-Dependent Transcription of BDNF in Rat Hippocampal Slices

Although activity-dependent transcription represents a crucial mechanism for long-lasting experience-dependent changes in the hippocampus, limited data exist on its contribution to pathological conditions. We aim to investigate the influence of chronic stress on the activity-dependent transcription of brain-derived neurotrophic factor (BDNF). The ex vivo methodology of acute stimulation of hippocampal slices obtained from rats exposed to chronic mild stress (CMS) was used to evaluate whether the adverse experience may alter activity-dependent BDNF gene expression. CMS reduces BDNF expression and that acute depolarization significantly upregulates total BDNF mRNA levels only in control animals, showing that CMS exposure may alter BDNF transcription under basal conditions and during neuronal activation. Moreover, while the basal effect of CMS on total BDNF reflects parallel modulations of all the transcripts examined, isoform-specific changes were found after depolarization. This different effect was also observed in the activation of intracellular signaling pathways related to the neurotrophin. In conclusion, our study discloses a functional alteration of BDNF transcription as a consequence of stress. Being the activity-regulated transcription a critical process in synaptic and neuronal plasticity, the different regulation of individual BDNF promoters may contribute to long-lasting changes, which are fundamental for the vulnerability of the hippocampus to stress-related diseases.

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

Neural activity directs dendrite development through the regulation of gene transcription. However, how molecular signals link extracellular stimuli to the transcriptional program in the nucleus remains unclear. Here, we demonstrate that neuronal activity stimulates the translocation of the kinase Cdk5 from the cytoplasmic compartment into the nucleus; furthermore, the nuclear localization of Cdk5 is required for dendrite development in cultured neurons. Genome-wide transcriptome analysis shows that Cdk5 deficiency specifically disrupts activity-dependent gene transcription of bdnf. The action of Cdk5 is mediated through the modulation of the transcriptional repressor methyl-CpG-binding protein 2. Therefore, this study elucidates the role of nuclear Cdk5 in the regulation of activity-dependent gene transcription and dendritic growth.

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle.

ObjectivePhysical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle.MethodsWe compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression.ResultsWe found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes.ConclusionsWe report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.

Gene expression parallels synaptic excitability and plasticity changes in Alzheimer's disease.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal accumulation of β-amyloid and tau and synapse dysfunction in memory-related neural circuits. Pathological and functional changes in the medial temporal lobe, a region essential for explicit memory encoding, contribute to cognitive decline in AD. Surprisingly, functional imaging studies show increased activity of the hippocampus and associated cortical regions during memory tasks in presymptomatic and early AD stages, whereas brain activity declines as the disease progresses. These findings suggest an emerging scenario where early pathogenic events might increase neuronal excitability leading to enhanced brain activity before clinical manifestations of the disease, a stage that is followed by decreased brain activity as neurodegeneration progresses. The mechanisms linking pathology with synaptic excitability and plasticity changes leading to memory loss in AD remain largely unclear. Recent studies suggest that increased brain activity parallels enhanced expression of genes involved in synaptic transmission and plasticity in preclinical stages, whereas expression of synaptic and activity-dependent genes are reduced by the onset of pathological and cognitive symptoms. Here, we review recent evidences indicating a relationship between transcriptional deregulation of synaptic genes and neuronal activity and memory loss in AD and mouse models. These findings provide the basis for potential clinical applications of memory-related transcriptional programs and their regulatory mechanisms as novel biomarkers and therapeutic targets to restore brain function in AD and other cognitive disorders.

GABA-driven excitatory neurotransmission: gene regulation by excitatory GABA and its possible role in the developing brain

Gamma-aminobutyric acid (GABA) has opposing functions, it acts not only as an inhibitory neurotransmitter in the nervous systems, but also as an excitatory one in early developing neurons. Previous studies reported that the excitatory effects of GABA controlled neuronal activity-dependent gene expression, which contributes to long-lasting changes in neuronal functions such as cell survival, differentiation, and synaptic plasticity. We recently demonstrated that excitatory GABA activated multiple Ca 2+ signaling pathways, mainly via L-type voltage-dependent Ca 2+ channels (L-VDCCs), to activate the transcription of the gene encoding brain-derived neurotrophic factor (BDNF). Since BDNF plays a fundamental role in the expression of various functions by the nervous system, the excitatory GABA-induced expression of the BDNF gene may be important for the neural development of the brain. In this review, we summarized the mechanisms underlying the excitatory GABA-induced expression of the BDNF gene, and proposed the possible roles of its regulation in the early development of the brain, an impairment in which may cause the abnormal development of the brain.