Browsing by Author "West, Anne E"

Olfaction, the sense of smell, is one of the least well understood sensory systems. Central to the lack of understanding has been an inability to reliably control the stimulus, odorants. Here I developed analytical frameworks to further our understandings of how to quantify odor molecules based on responses elicited from olfactory receptors. I also identified a novel subset of olfactory receptors which displayed sexual dimorphism, plasticity, and served to respond to semiochemicals in mice. Finally, I identified a single candidate rabbit olfactory receptor to respond to a rabbit mammary pheromone.

During brain development, chromatin regulation determines how and when genes are transcribed, making sure that the right genes are turned on and the wrong genes are turned off. Previously, we have identified a role for the Jumonji-C lysine-specific histone demethylase Kdm6b in cerebellar granule neurons (CGNs) in mediating the induction of a mature gene expression program. More specifically, this program of gene expression contains a variety of synaptic genes, crucial for the post-synaptic differentiation of CGNs and the overall cerebellar circuit. Consistent with this, we report that neurons without Kdm6b have smaller and less complex dendritic claws, structures that contain most of the synaptic inputs onto the cells. Additionally, we show that proper claw formation is dependent on the enzyme’s catalytic activity. CGNs lacking Kdm6b have increased distribution of excitatory synapses on their smaller claws, suggesting that normally, Kdm6b works to develop these claws so the cells can properly distribute synapses. Interestingly, mice lacking Kdm6b activity show deficits in social novelty and discrimination, highlighting a possible behavioral consequence linked to chromatin misregulation. Whether this aberrant phenotype is specifically due to synaptic defects associated with loss of demethylase activity remains to be resolved. However, these results defined a novel role for Kdm6b in regulating synapse formation and have expanded our knowledge on how chromatin regulation contributes to neuronal maturation and ultimately, social behavior.

Robo receptor is a central component in multiple stages of neural development. Accumulating evidence also suggests that it functions in organogenesis, regulation of stem cells, and cancer. Despite its diverse functions, all of the reported functions of Robo rely on binding with its canonical ligand, Slit. However, it has long been known that in C. elegans, which has only one Slit (slt-1) and one Robo (sax-3) gene, mutants of Robo exhibit more severe phenotypes than Slit mutants, suggesting that Robo has Slit-independent functions. This proposal aims to explain some of the Slit-independent Robo mutantphenotypes: 1) defects in formation of the RME circuit, a head motor neural circuit, and 2) notched head in C. elegans. Preliminary results suggest that SAX-3 acts as its own ligand in mediating RME circuit formation. More surprisingly, the extracellular domain of SAX-3 can rescue the notched head phenotype. The proposed work is an essential step towards comprehensive understanding of Robo receptors and may provide insights in understanding

human genetic diseases.

In the following document, I will describe three distinct projects which together provide information to better understand the genetic basis of autism spectrum disorder (ASD) as well as the genetic and molecular basis of Rahman syndrome. Specifically, the majority of my thesis focuses on how the gene H1-4 can contribute to intellectual disability (ID). First, in Chapter 2, I will provide an early project in my graduate career, which provides an illustration of the need to better understand the relationship between epigenetics and ASD. My work studying the DNA demethylase, TET1, highlights the effects that epigenetic dysregulation can have on ASD-like behaviors. Utilizing a novel Tet1 knockout mouse, we have shown that lack of TET1 results in reduction of the Oxytocin receptor (Oxtr) gene transcript. These Tet1 knockout animals display abnormal splicing of the Oxtr transcript, increased aggressive behaviors, decreased maternal care, and altered firing in neurons. This work sheds light on how two ASD implicated genes (TET1 and OXTR) interact to generate abnormal gene products and functions that relate to ASD-like behaviors. Second, in Chapters 3 and 4, I will present evidence to better understand how frameshift mutations in the histone linker, H1-4, contribute to pathogenicity of Rahman syndrome (RMNS), a syndromic form of ID. My work here utilized a combination of multiple approaches including review of clinical data, use of RMNS patient derived cells, and ex vivo neuronal cellular cultures to determine the effects of frameshift H1.4 (H1.4fsRMNS) on cellular transcription and function. I investigated transcriptional dysregulation in RMNS patient derived cells, as well as the effect of exogenous expression of H1.4fsRMNS on neuronal structure and function. My work in these chapters provide the first evidence for aberrant function of H1.4fsRMNS in post mitotic neurons of the brain. These data also help to better understand the role this mutant protein plays in creating an ID related phenotype. Of specific interest to this thesis, literature searches for these experiments highlight a gap in the literature with regards to what is known about the function of wild-type (WT) H1.4 in the brain. Finally, in Chapter 5, I characterize the expression of H1.4 as well as H1.2 and .3 throughout murine brain development. I show that these “replication-dependent” histones are actually expressed and present in mature post-mitotic neurons. I utilize a combination of transcriptional investigation, microscopy, top-down proteomics, and endogenously tagged H1f4 mouse model to systemically track expression of H1.4 throughout development, in multiple brain regions, and within a single, specific cell type (the CGN). My characterization of H1s during murine brain development together affirm that there is little known about distinct expression and function of individual somatic replication-dependent H1 proteins. Specifically, in a spatiotemporal specific manner in the brain. My work is the first to systemically interrogate the expression and binding of H1.4 in vivo in mature neurons. Further, I show that expression and post-translational modification of individual H1 subtypes is developmentally dependent and may have an effect on gene expression. My thesis sheds light on the molecular function of H1.4 within the brain. My work is useful for not only laying the foundation of how frameshift mutant H1.4fsRMNS leads to RMNS. But also, in understanding the function of the WT endogenous H1.4 protein. Together, this data lays the foundation to better understand the relationship between H1.4 function and brain development. Better understanding the expression of both the WT and frameshift mutant H1.4fsRMNS, will be useful in providing a basis for future studies and potential gene therapy strategies for RMNS.

At excitatory synapses, NMDAR-mediated synaptic plasticity occurs in response to activity inputs by modifying synaptic strength. While comprehensive studies have been focused on the induction and expression mechanisms underlying synaptic plasticity, it is less clear whether and how synaptic plasticity itself can be subjected to regulations. The presence of "plasticity of plasticity", or meta-plasticity, has been proposed as an essential mechanism to ensure a proper working range of plasticity, which may also offer an additional layer of information storage capacity. However, it remains elusive whether and how meta-plasticity occurs at single synapses and what molecular substrates are locally utilized. Here, I develop systems allowing for sustained alterations of individual synaptic inputs. By implementing a history of inactivity at single synapses, I demonstrate that individual synaptic inputs control synaptic molecular composition homosynaptically, while allowing heterosynaptic integration along dendrites. Furthermore, I report that subunit-specific regulation of NMDARs at single synapses mediates a novel form of input-specific metaplasticity. Prolonged suppression of synaptic releases at single synapses enhances synaptic NMDAR-mediated currents and increases the number of functional NMDARs containing NR2B. Interestingly, synaptic NMDAR composition is adjusted by spontaneous glutamate release rather than evoked activity. I also demonstrate that inactivated synapses with more NMDARs containing NR2B acquire a lower induction threshold for long-term synaptic potentiation. Together, these results suggest that at single synapses, spontaneous release primes the synapse by modifying its synaptic state with specific molecular compositions, which in turn determine the synaptic gain in an input-specific manner.

The ability to convert sensory stimuli into long-lasting changes in brain function is essential for animals to interact with and learn from their environment. This process is achieved by encoding sensory stimuli into temporal patterns of neuronal activity, which in turn modulate the connectivity and strength of neural circuits in the brain. These long-term plastic changes in the brain are known to depend on the neuronal activity-regulated transcription of new gene products. My dissertation research sought to elucidate how the timing and level of transcriptional responses following neuronal activity can be precisely regulated to form proper neuronal connections. In the first part of this dissertation, I investigated the role of the developmentally regulated GluN3A subunit in NMDAR-induced transcription. I observed that neurons lacking the transcription factor CaRF showed enhanced NMDAR-induced expression of Bdnf and Arc both in cultured neurons and following sensory stimulation in the developing brain in vivo. I identified GluN3A as a regulatory target of CaRF and found that neurons lacking GluN3A showed selective enhancement of NMDAR-induced transcription. GluN3A limited synaptic activity-induced transcription by inhibiting both NMDAR-induced nuclear translocation of the p38 MAP kinase and activation of the transcription factor MEF2C. These data demonstrate that GluN3A negatively regulates NMDAR-dependent activation of gene transcription and reveal a novel mechanism that regulates the level of NMDAR-induced transcriptional response in the developing brain. In the second part of my dissertation, I examined the role of enhancer histone acetylation in neuronal activity-regulated gene transcription. I applied quantitative single-molecule fluorescence in situ hybridization to measure neuronal activity-induced gene transcription at the single neuron level, taking advantage of the intrinsic stochasticity of transcription to quantify the effects of enhancer regulation on the dynamics of promoter state transitions. Locally-induced enhancer histone acetylation by CRISPR-mediated epigenome editing was sufficient to increase Fos mRNA expression both under basal conditions and following membrane depolarization in primary hippocampal neurons, via a mechanism that involves enhancer recruitment of Brd4, increased transcriptional elongation by the release of paused polymerase, and prolonged activation of Fos promoters. These data indicate that enhancer histone acetylation plays a causative role in the induction of neuronal activity-regulated gene transcription and open up the possibility to specifically control the level and timing of the neuronal activity-induced transcriptional response. Taken together my dissertation works elucidate mechanisms that control the specificity, timing, and amplitude of transcriptional responses to neuronal activity, revealing novel information about the dynamic range of this fundamental cellular process.

Activation of monoaminergic receptors is essential to the mechanism by which psychostimulants and antidepressants induce changes in behavior. Although these drugs rapidly increase monoaminergic transmission, they need to be administered for several weeks or months in order to produce long-lasting alterations in behavior. This observation suggests that it is likely that molecular mechanisms downstream of receptor activation contribute to the effects of psychostimulants and antidepressants on behavior.

Recently, we and others have demonstrated that the methyl-CpG-binding protein 2 (MeCP2) contributes to both neural and behavioral adaptations induced by repeated psychostimulant exposure (Deng et al, 2010, Im et al, 2010). Psychostimulants induce rapid and robust phosphorylation of MeCP2 at Ser421 (pMeCP2), a site that is thought to modulate MeCP2-dependent chromatin regulation (Cohen et al, 2011), and this phosphorylation event is selectively induced in the GABAergic interneurons of the nucleus accumbens (NAc). In order to understand the signaling pathways that contribute to the pattern of pMeCP2 we observe, I characterized the monoaminergic signaling pathways that regulate pMeCP2. I found that activation of dopamine (DA) and serotonin (5-HT) transmission is sufficient to induce pMeCP2. The novel finding that drugs that activate serotonergic signaling induce pMeCP2 suggests that pMeCP2 may be involved in serotonergic mediated behaviors.

To determine the requirement of pMeCP2 in serotonergic mediated behaviors, I utilized mice that bear a knockin (KI) mutation that converts serine to alanine at 421 (S421A) (Cohen et al, 2011). After characterizing the behavioral phenotype of these mice, I conducted tests to assess anxiety- and depression-like behavior. I found that the KI mice do not display heightened anxiety in several assays. However, the KI mice exhibit depression-like behavior in the forced swim and tail suspension but show no differences compared to wild-type (WT) littermates in the sucrose preference test, suggesting that pMeCP2 may be implicated in the behavioral response to stressful stimuli.

Because we are interested in examining the role of pMeCP2 in the behavioral adaptations to chronic monoaminergic signaling, I then put the KI mice and their WT littermates through chronic social defeat stress, a behavioral paradigm in which repeated exposure to aggressive mice causes social avoidance that is reversed by chronic but not acute antidepressant treatment. Although the WT mice show an increase in social interaction following chronic imipramine treatment, the KI mice fail to show a behavioral response to chronic treatment. These data suggest that pMeCP2 may be implicated in the antidepressant action of chronic imipramine. Finally, investigation of the brain regions in which pMeCP2 may be contributing to the behavioral response to chronic imipramine treatment revealed that chronic but not acute imipramine treatment induces pMeCP2 in the lateral habenula (LHb), a brain region involved in the behavioral response to stress and reward. Together, these data implicate a novel role for pMeCP2 in depression-like behavior and the behavioral response to chronic antidepressant treatment.

Neurons are remarkably long-lived cells that are born early on in development and maintained over the lifespan of an organism. Their birth is followed by their iterative maturation into functional neurons that can participate in CNS circuitry. This involves the temporally regulated programs of gene transcription enabling them to migrate, generate axons, dendrites, synapses, and spatial connections with other cells within their niche. This is mediated at least in part by dynamics in chromatin biology, as mutations in chromatin regulators are strongly implicated in the advent of neurodevelopmental and neuropsychiatric disorders. The mechanisms by which chromatin dynamics orchestrate neuronal maturation remain poorly understood. We find that the postnatal maturation of cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a causal function for this chromatin modification in gene regulation beyond its canonical role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes that turn on as CGNs mature is facilitated by the lysine demethylase, and ASD-risk gene, Kdm6b, through its catalytic activity. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. We also find dynamics in regulatory chromatin topology to facilitate the interaction between cerebellar enhancers and their cognate genes during cerebellar maturation, that appears to be poised by H3K27me3. These data demonstrate that dynamics at the level of chromatin primary, secondary, and tertiary structures in developing postmitotic neurons regulate the temporal coordination of gene expression programs that underlie functional neuronal maturation.

Exposure to psychostimulant drugs of abuse exerts lasting influences on brain function via the regulation of immediate and persistent gene transcription. These changes in gene transcription drive the development of addictive-like behavior by inducing cellular and synaptic plasticities in neurons within brain reward circuits

including the nucleus accumbens (NAc). The long-lasting nature of addictive-like behaviors suggests they may be mediated by equally persistent mechanisms of transcriptional regulation such as epigenetic modifications of chromatin. However, an important limitation to testing this model using traditional methods for studying

chromatin is the fact that brain regions like the NAc are comprised of a variety of interacting cell types that differentially shape the region’s impact on the circuit, that are not resolved by biochemical methods. In this dissertation I will overcome this barrier by using varied targeted methods to study cell-type specific changes in chromatin

induced in the NAc by chronic amphetamine.

We have data showing that silencing of PV+ interneurons significantly diminishes the locomotor sensitization to chronic psychostimulant exposure, revealing for the first time a function for these interneurons in addictive-like behaviors. PV+ interneurons of the NAc show amphetamine-induced transcription of genes like Fos and with repeated drug exposure display transcriptional desensitization of Fos, a process that is thought to be epigenetically mediated. This and other cellular adaptations occurring in PV+ interneurons persist through extensive withdrawal periods, suggesting a specific and lasting means by which these regulatory shifts underlying the behavioral response are imprinted on this cell type. Taking these data together, I hypothesize that changes to chromatin structure and gene expression in PV+ interneurons of the NAc contribute to the addictive-like behavioral changes and cellular adaptations seen following psychostimulant use. To test this, I will make use of a novel isolation method to specifically purify the nuclei of PV+ interneurons from the NAc and assay the chromatin and gene expression changes that persist following psychostimulant exposure

and correlate with addictive-like behaviors. In this proposal I will identify the program of persistent amphetamine-regulated gene expression and chromatin remodeling genome-wide in PV+ interneurons of the NAc. I will subsequently use a variety of targeted bioinformatic analyses to survey the relationships between changes to the chromatin landscape genome-wide and lasting alterations in transcriptional regulation and cellular function. These studies will greatly propel our understanding of how epigenetic regulation of gene transcription can contribute to lasting changes in circuit function and behavioral output.

Learning and memory are mediated by changes in synaptic and neuronal function within brain circuits, and is supported by dynamic waves of stimulus-dependent transcription in the nucleus of neurons. Stimulus-dependent transcription relies heavily on the epigenetic landscape of a given neuron, which is highly cell-type specific and can be further tuned by experience. Developments in genetic tools and methods to survey the entire transcriptome and epigenome have increased our ability to study stimulus-dependent transcription in diverse cell-types, including rare populations of interneurons. Application of these tools to addiction models, where long-lasting changes in behavior depend on stimulus-dependent transcription in diverse cell-types in multiple areas of the corticomesolimbic reward circuit, presents a particularly potent opportunity to increase our understanding of the functional consequences of stimulus-dependent transcription in diverse cell-types in a system that is highly relevant to human health. In the nucleus accumbens (NAc), parvalbumin-expressing (PV+) interneurons exert strong control over local circuit output and downstream behavior, including behavioral responses to drugs of abuse. Recent data from our lab suggest that perineuronal net (PNN) genes are a promising target for behaviorally-relevant, drug-dependent functional adaptations in this rare cell-type. In this dissertation, I use histological techniques in mouse tissue to validate in situ the heterogeneous transcriptional regulation of NAc PV+ interneurons and specific cell adhesion gene targets in response to psychostimulants, and explore the developmental and drug-dependent regulation of PNNs and the PNN gene Bcan. I use genetic and viral tools to specifically knockdown Bcan expression in NAc PV+ cells, and demonstrate that Bcan stabilizes their excitatory synaptic inputs even in adulthood and restricts the development of cocaine-context associations, showing that NAc PV+ interneurons and their PNNs play an important role in limiting the development of addiction-related behaviors. Finally, I use dCas9-mediated epigenetic editing to tune activity-dependent transcription of select rapid primary response genes, which are thought to interact with cell-type and cell-state dependent chromatin to coordinate later waves of stimulus-dependent transcription. I show that the fine details of activity-dependent transcription of rapid primary response genes resulting from chromatin state can lead to physiological changes in protein, and downstream neural physiology and behavior.

The epigenome underlies cell type and state and in post-mitotic neurons, and it regulates the ability for rapid response to activity. Since neurons exit the cell cycle early in development and are long lived, remodeling of brain function requires that neurons show transcriptional plasticity to let then change in function in response to stimuli including psychostimulants and developmental cues. This response is driven by the epigenomic regulation in a cell-type-specific manner. Many studies assessing experience driven genomic responses have been carried out in bulk tissues so cell-type-specific genomic responses to stimuli that drive neuronal plasticity remains poorly understood. To understand the epigenomic and transcriptomic mechanisms driving neuronal plasticity, here we study multi-omic genomic data from two contexts in the mouse brain: 1) psychostimulant responses in the nucleus accumbens and 2)the postnatal and postmitotic maturation of developing cerebellar granule neurons. In both systems, I implemented integrative bioinformatic approaches to predict transcription factor (TF) activity in regulating the transcriptome. I elucidated cell-type-specific amphetamine induced transcriptomic responses, identified canonical activity regulated transcription factors regulating those responses, and determined collaborators and developmental targets of the Zic family TFs, revealing novel roles of Zics regulating migration and synaptic maturation in CGN development. The studies reveal novel mechanistic insights into neuronal plasticity in different neuronal cell types by using integrative computational approaches to model chromatin topology, chromatin accessibility, gene expression, and TF binding.

Changes in gene transcription driven by the activation of intracellular calcium signaling pathways play an important role in neural development and plasticity. A growing body of evidence suggests that stimulus-driven modulation of histone modifications play an important role in the regulation of neuronal activity-regulated gene transcription. However, the histone modifying enzymes that are targets of activity-regulated signaling cascades in neurons remain to be identified. The histone demethylases (HDMs) are a large family of enzymes that have selective catalytic activity against specific sites of histone methylation. To identify HDMs that may be important for activity-regulated gene transcription in neurons, we induced seizures in mice and screened for HDMs whose expression is induced in the hippocampus. Among the few HDMs that changed expression, Kdm6b showed the highest induction. Kdm6B is a histone H3K27-specific HDM whose enzymatic activity leads to transcriptionally permissive chromatin environments. In situ hybridization analysis revealed that Kdm6b is highly induced in post-mitotic neurons of the dentate gyrus region of the hippocampus. We can recapitulate the activity-dependent induction of Kdm6b expression in cultured hippocampal neurons by application of Bicuculline, a GABAA receptor antagonist that leads to synaptic NMDA receptor activation and calcium influx. Kdm6b expression is also induced following application of BDNF, a neurotrophic factor that is upregulated in the seized hippocampus. To investigate possible functions of Kdm6b in neuronal development, we performed in situ hybridization analysis that allows for the identification of regions with high Kdm6b expression that could be sites of potential function in the developing mouse brain. We found high levels of Kdm6b expression in the inner layer of the external granule layer of the cerebellum, a region where pre-migratory immature neurons reside and a site of significant apoptosis. On the basis of this data and the fact that intracellular calcium signaling arising from synaptic firing supports neuronal survival, we explored the necessity for Kdm6b in the survival of cultured cerebellar granule cells. Knock down of Kdm6b by RNAi increases cell death, demonstrating that Kdm6b contributes to neuronal survival. Ongoing experiments are addressing the role of Kdm6b in neuronal differentiation. Overall these data raise the possibility that stimulus-dependent regulation of Kdm6b, and perhaps regulation of H3K27 methylation mediated by Kdm6b, may contribute to the regulation of gene expression in neurons and thus to their proper development and plasticity.

Autism spectrum disorders (ASDs) are increasingly prevalent, and the costs associated with caring for affected patients across the lifespan are immense. However, the pathophysiology and brain regions involved in characteristic behavioral impairments remain poorly defined, which hinders progress towards targeted therapeutic development. Different brain regions have been suggested from human neuroimaging studies but the circuit mechanism is not known and cannot be easily defined in human studies. Genetic studies indicate that SHANK3, a gene encoding a scaffolding protein at the postsynaptic density, is a strong ASD causative gene. Studies of various isoform-specific knockout mice support these mice as valid models to dissect the pathophysiology of ASDs and implicate differential involvement of brain regions such as hippocampus and striatum. However, none of these mice recapitulate the most frequent SHANK-related mutation found in ASD patients: a deletion of the entire SHANK3 gene.

For this reason, we have created conventional complete knockout mice by deleting almost the entire coding region of exons 4 to 22, Shank3 Δe4-22, and performed a thorough characterization of their behavioral phenotypes. Their abnormalities in complex social and communication behaviors in addition to their profound display of repetitive and restrictive behaviors in combination with comorbid anxiety, locomotor, and learning phenotypes support them as a mouse model for SHANK3-causing autism with good construct and face validity. Additional studies by collaborators identified a striatal-centered model of circuit and synaptic dysfunction. Manipulation of metabotropic glutamate receptor 5 (mGluR5) activity attenuated the excessive grooming and instrumental learning differentially in Δe4-22-/- mice. These findings show that deficiency of the autism-associated Shank3 gene can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities which underlie deficits in learning and ASD-like behaviors. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs.

However, because these and other existing Shank3 mutant mice are not region specific, causality between different brain regions and ASD-like behaviors cannot be firmly established. In order to define anatomic regions implicated in behavioral manifestations of ASD, conditional knockout mice lacking Shank3 proteins in different brain regions including forebrain excitatory neurons (NEX-Cre) and striatal inhibitory neurons (Dlx5/6-Cre), as well as distinct cell populations including direct (D1-Cre) and indirect (D2-Cre) medium spiny neurons, were generated and subjected to behavioral phenotyping. Different autism-relevant behaviors as well as comorbid behaviors were recapitulated by targeting Shank3 deletion to different brain regions or cell types. Electrophysiological and biochemical studies further identified synaptic defects resulting from region- or cell-autonomous loss of Shank3, with different biochemical pathways implicated when Shank3 deletion was targeted to the cortex and hippocampus versus the basal ganglia. This study demonstrates the impact of specific brain regions in modulating ASD-related behavior and identifies key molecular defects which are restricted to specific brain regions in SHANK3-deficient ASD, thus providing future therapeutic targets.

Lastly, as one of the major advantages of modeling ASDs in mice is their amenability for pre-clinical studies of interventions, we have tested two different cellular therapies hypothesized to modulate neuronal circuit function either through direct differentiation of stem cells into brain cells including neurons, glia, and microglia or through indirect effects on neuro-immune modulation. While neither perinatal nor young adulthood treatment with human umbilical cord blood derived stem cells affected significant improvements in the behaviors of Shank3 knockout mice, these experiments underscored the robust, reliable behavioral phenotypes of this animal model as well as supported the safety and tolerability of these treatments in a rodent pre-clinical model with a relevant genetic construct.