Genevieve Konopka - US grants

[unreadable] DESCRIPTION (provided by applicant): The requirement for HNF4 in hepatic development has been previously documented through the generation of mice lacking HNF4. The series of experiments outlined in this proposal aim to determine which transcriptional targets of HNF4 contribute to regulation of differentiation and epithelial formation of the liver. Initial experiments have identified 20 genes with known roles in epithelial formation arid cell adhesion functions downregulated in the liver upon HNF4 loss. The importance of these targets in regulating hepatic differentiation will be tested by viral-mediated knockdown of expression in a culture system that mimics many hepatic features. To determine whether these targets are sufficient to rescue the phenotype of HNF4 null livers, expression of the targets will be reconstituted in progenitor cells from the livers of HNF4 loxP/loxP mice treated with adenoviral-Cre. Finally, the in vivo developmental function of the target or targets with the most profound affect on hepatic differentiation will be studied through the generation of knockout and/or liver-conditional knockout mice. These studies will provide understanding of the HNF4-mediated signaling cascade mediating development of the liver, and the molecular and cellular processes these signals control. [unreadable] [unreadable]

Impaired cognition, and in particular language, is a hallmark of common neuropsychiatric diseases such as autism and schizophrenia; however, the molecular mechanisms underlying higher cognitive function development and evolution in humans remain unknown. The elucidation of signaling pathways that are important for language and cognition will provide targets for future therapeutics. Frontal-striatal circuitry is critical for normal cognitive function and is frequently disrupted in neuropsychiatric disease. The transcription factor F0XP2 is the only gene currently identified that is mutated in patients with isolated language disturbances, and it has high expression in both frontal and striata) regions of developing human brain. Current data supports a role for both F0XP2 and its regulation of genes involved in autism and schizophrenia, The research in this proposal will focus on the developmentally regulated signaling pathways downstream of FbXP2 and how perturbations to these pathways result in cognitive defects in both ASD and schizophrenia, the specific aims include: 1) Identify the signaling pathways regulated by F0XP2 in human neurons, and which of these pathways are important for neuronal differentiation and/or maintenance, 2) Determine evolutionarily conserved and human-specific F0XP2 targets by conducting comparative whole gene transcriptome sequencing and F0XP2 promoter binding in fetal human, rhesus macaque, and mouse brain, and 3) Ascertain how Fdxp2 and Foxpl cooperatively regulate gene expression during CNS development by generating Foxpl conditional knockout mice and conducting genome wide Foxp2 promoter binding analysis.

How we learn from social experiences and during social interactions is poorly understood, but it is thought to involve intricate changes to nerve cells in the brain and the connections between these cells. Cells involved in social learning are intermingled and intertwined with cells that may have completely different functions. Because of this complexity, identifying and studying the specific cells and networks involved in social learning remain a major challenge, and new methods are required to address this needle-in-a-hay-stack problem. This research will build a new set of genetic tools that allow researchers to mark cells in the brains of mice and zebra finches that are specifically involved in learning during social interactions, and will apply cutting-edge imaging, physiological, and genetic methods to dissect how the marked cells change during learning. This research is of fundamental importance because it will shed light on the brain mechanisms involved in social learning and build a new set of genetic tools that can be used by the scientific community to study brain mechanisms involved in learning and memory. The research also is of importance because developmental disorders and head injuries can severely compromise circuits in the brain and individuals' ability to learn from social encounters and navigate complex social interactions. The tools and methodologies developed in this research will be made freely available to other scientists through the world-wide web (http://www.utsouthwestern.edu/education/medical-school/departments/neuroscience/index.html) and through the Addgene public repository (http://www.addgene.org/). Funding for this research will also be used to educate and train young scientists in novel genetic, molecular, imaging and behavioral methodologies.

The proposed research will identify neuronal mechanisms involved in social learning from olfactory and auditory cues in mice and zebra finches, respectively. The proposal takes a highly interdisciplinary, collaborative approach involving four independent laboratories. The researchers will fluorescently "tag" neurons in mice and zebra finches that are selectively activated by olfactory and auditory social experiences using novel genetic strategies and viral tools that leverage the immediate-early gene c-Fos. Within brain regions of interest (olfactory and vocal learning circuits), these viral tools will differentially label neuronal populations depending on cellular activity and the specific social cues animals experience. In vivo Ca2+ imaging will be used to identify and map populations of neurons involved in processing and learning from social encounters. Novel optical methods will be used to map synaptic connectivity among tagged neuronal populations in vivo. Electrophysiological and transcriptomic analyses will be used to identify physiological and genetic factors unique to each tagged population, and identify neural subtypes and subpopulations responsible for social learning. These combined approaches will help reveal the network-level plasticity induced by social experiences. This collaborative, high-risk/high-impact research will generate novel in vivo molecular tools that allow fine and selective dissection of the network components of social learning.

? DESCRIPTION (provided by applicant): The molecular mechanisms underlying the significant co-morbidity of sleep and circadian disturbances in patients with autism spectrum disorder remain unknown. There is mounting evidence for genetic and genomic alteration to core circadian factors in autism. The transcription factor CLOCK has been well-studied for its role in circadian function within the suprachiasmatic nucleus, but its role in other parts of the brain are not well known. We have observed that the human neocortex has a significant increase in CLOCK expression and altered CLOCK coexpression compared to other primates. We therefore hypothesize that CLOCK-mediated transcriptional networks specific to the human neocortex are involved in circadian and cognitive function and are disrupted in cognitive disorders such as ASD. To test this hypothesis, we propose to identify the transcriptional networks regulated by CLOCK specifically in human neurons. We will determine transcriptional targets of human CLOCK using three systems: primary human neural progenitors and their differentiated neurons, human neocortical post-mortem tissue, and mice expressing human CLOCK together with its regulatory elements. This novel approach of using evolutionary data to inform cognitive disease directed functional studies should provide important new insights into aspects of human brain evolution that are most at risk in cognitive disorders with a circadian component.

Abstract: Not applicable for this application.

? DESCRIPTION (provided by applicant): The neural underpinnings of vocal communication remain mostly unknown. The long-term goal of our laboratory is to elucidate the molecular signaling pathways important in vocal communication that are disrupted in neurodevelopmental disorders. Mutations in the gene encoding FOXP2 have previously been identified in individuals with speech and language disorders. In addition, FOXP2 transcriptionally regulates many genes involved in neurodevelopmental disorders such as autism and schizophrenia. Our preliminary studies have shown that expression of FOXP2 in the cerebellum is important for normal vocalizations and motor function. Moreover, we have uncovered a conserved site of post-translational modification of FOXP2 that it is important for gene expression regulation and motor function. Based on these data, the central hypothesis driving this proposal is that post-translational modification of FOXP2 is critical for regulating vocalizations and motor function. We propose to identify the role of post-transcriptional modification of FOXP2 on cerebellar-specific gene expression and motor function by manipulating Foxp2 expression in the mouse cerebellum through four specific aims: 1) Determine whether Foxp2 expression in the developing cerebellum is important for vocalizations and gene expression; 2) Assess the role of post-translational modification of Foxp2 on vocalizations; 3) Determine the role of post-translational modification of Foxp2 on gene expression; and 4) Assess the role of post-translational modification of Foxp2 on motor function. Together, these aims will determine the transcriptional program regulated by Foxp2 in the cerebellum and how Foxp2 gene regulation may be related to vocalizations and other motor-relevant behaviors. Completion of the proposed aims will provide increased knowledge as to the molecular pathways that can be targeted for treatment in individuals with communication disorders, cerebellar based motor disorders, and autism, which involves disrupted cerebellar function. These data will also provide insight into the basic molecular mechanisms governing normal brain development.

PROJECT SUMMARY The molecular signaling pathways important to speech and language remain mostly unknown. FoxP2 is one of a few genes that has been directly linked to both speech and language in humans and learning and production of vocalizations in animal models (songbirds and mice). In humans and songbirds, striatal expression of FoxP2 is a key factor in the accurate development of learned vocalizations. This multi-PI grant will synthesize expertise in genomics and behavior to examine the role of FoxP2 during the sensitive period for vocal learning. We will examine the function of FoxP2 during vocal learning, and in adult animals, using a combination of novel viral methods for the reversible knockdown of FoxP2, and functional genomics, through two specific aims. In the first aim, we will use novel viral methods to test whether restoring normal FoxP2 expression levels in adults is sufficient to rescue vocal deficits caused by knockdown of FoxP2 expression during the sensitive period for vocal learning. In the second aim, we will use functional genomics to identify the expression and regulation of FoxP2 target genes during the sensitive period for vocal learning. The results of these studies will provide new insights into neurodevelopmental genetic disorders, including the identification of novel signaling pathways involved in learning of vocal behaviors, and how therapeutic intervention in adults can impact behavioral disorders acquired during developmental sensitive periods. These data can also be used for the future development of targeted therapeutics for the treatment of a range of neurodevelopmental disorders with speech and language phenotypes.

PROJECT SUMMARY Our proposal seeks to unite two powerful tools in contemporary neuroscience: brain recordings from awake, behaving individuals and large-scale gene expression from human brain tissue. We hope to understand more about cognitive function in epilepsy and other disorders by studying gene expression profiles linked with brain oscillation information. The key innovation of this proposal is the collection of these two types of data from the same patients at different stages of their evaluation and treatment for epilepsy. In our first aim, we will build upon our recent findings examining gene/oscillation correlations through the generation of a proposed new large dataset of brain oscillations and by extending our analysis to include item retrieval during episodic memory. In our second aim, we will build an entirely new dataset by collecting intracranial EEG oscillatory data as subjects perform an episodic memory task and then also collect temporal lobe tissue specimens from the same patients. This is because the patients first undergo seizure mapping with intracranial electrodes, and then undergo resection of the temporal lobe. This offers the possibility of capturing oscillatory and gene expression data from the same tissue across subjects. In our third aim, we will generate gene expression data from surgical epilepsy patients and compare these profiles to datasets derived from cadaveric tissue samples. These comparisons will serve as a control to help interpret our correlation results and as a substantial contribution to the literature in its own right. Although this ambitious plan requires close integration between labs with expertise in gene expression analysis and signal processing, and requires a pipeline for processing tissue obtained in the operating room as well as specific surgical techniques to produce good specimens, the methodological groundwork we have established and preliminary data we show demonstrate that our plans have a high chance of success. The genes we identify as being highly correlated with mnemonic processing will be strong candidates for further animal experimentation and potential therapeutic targeting. We believe this line of investigation is a novel way to address the problem of cognitive decline in epilepsy and potentially other disorders such as mild cognitive impairment.

Project Summary Mechanistically linking network connectivity and the dynamics of neural networks to variation in the behavior of individuals is an overarching goal of neuroscience. Here we address this goal using techniques from network science to calculate functional networks that summarize pair-wise and higher order interactions between all recorded neurons. Network activity will be assessed using sophisticated two-photon (2P) imaging of activity- dependent Ca2+ signaling optimized to maximize the rate of recording and the numbers of neurons recorded. Multineuronal interactions within the networks will be identified, giving rise to encoding models to predict the network activity. Techniques from statistical physics will be used to optimally couple data from intracellular recordings to biologically realistic Hodgkin-Huxley (HH) models representing the contributions of ion currents and other free model parameters of the individual neurons. Networks of HH neurons using model synapses will replace pair-wise correlations to delinate the interrelationships between the ion currents of individual neurons and network interactions and dynamics. Taking advantage of the birdsong learning model, in the proposed experiments these approaches will be applied to the cortical song system HVC nucleus, allowing us to link these scales of investigation directly to behavior. Recent results demonstrate that changes in the intrinsic properties (IP) (ion current magnitudes) of HVC neurons is related to each individual's song, implicating changes within neurons as well as at synapses and networks that are related to learning. Aim 1: fast 2P imaging will be made in brain slices containing HVC that express spontaneous network activity. Model building will be supported by extensive efforts at 3-cell and 4-cell whole cell patch recordings, to better characterize HVC connectivity. The hypothesis that network structure depends on learning will be tested by examining how models vary between individual birds who sang similar or different songs. Models will be extended to in vivo observations by fast 2P imaging in sleeping birds while eliciting fictive singing using song playback, and in singing birds using 1P imaging. Results from the other Aims will further constrain the network and HH model building of Aim 1. Aim 2: the predictive power of the models will be further tested by using cellular resolution 2P optogenetic inhibition of selected neurons in in vivo and in vitro preparations. Aim 3: the role of neuronal IP in shaping network dynamics will be tested by using genetic and viral techniques to transiently modify specific ion channels in specific classes of HVC neurons. Changes in birds' singing behavior will be compared against a predictive HH model relating song structure and ion channel efficacy. Fast 2P imaging in slice and multisite extracellular recordings in singing birds will help to define how IP contribute to network models. Aim 4: single cell gene expression techniques will be used to identify all the HVC cell classes, the ion channels they express, and assess individual variation by examining cohorts of related birds or those singing the same songs. The overall goals and the four Aims are also designed to align with a subsequent U19 application.

Project Summary/Abstract The contribution of individual disease-relevant genes to brain development still remains unknown. The long-term goal of our laboratory is to elucidate the intersection of molecular signaling pathways that are disrupted in neurodevelopmental disorders with those pathways that are important for specific aspects of brain development. Two members of the FOXP family of transcription factors, FOXP1 and FOXP2, have been linked to monogenetic forms of intellectual disability, autism spectrum disorders, and specific speech and language deficits. Variants in FOXP1 or FOXP2 are among the most significant genes associated with autism spectrum disorders. We previously showed that Foxp1 and Foxp2 both have significant contributions to cortical and striatal development. We linked these developmental changes via studies of gene expression, electrophysiology, and behaviors. We further identified non-cell-autonomous changes in gene expression using newly available single-cell RNA- sequencing technology. Based on these data, the central hypothesis driving this proposal is that Foxp1 and Foxp2 are key orchestrators of transcriptional signaling cascades in a cell type-specific manner that are important for neuronal function and are at risk in neurodevelopmental disorders such as autism. We propose to identify these cell type-specific contributions in the developing cortex by using rodent models through three specific aims: 1) Determine the cell type-specific gene expression programs regulated by Foxp1 in the developing cortex; 2) Determine the cell type-specific gene expression programs regulated by Foxp2 in the developing cortex; and 3) Assess the role of Foxp1 and Foxp2 in cell type-specific activity-dependent neuronal function. Together, these aims will delineate the cell type contribution of both Foxp1 and Foxp2 to cortical development. The rodent models and cell-type specific genomic datasets will aprovide insight into the basic molecular mechanisms governing normal mammalian brain development.