Michela Fagiolini - US grants

DESCRIPTION (provided by applicant): The first symptoms of neurodevelopmental disorder such as Rett Syndrome appear during early childhood when sensory experience is sculpting neuronal circuits in what it will become the mature brain6. Mutations in MeCP2 gene account for 80% of RTT cases. MeCP2 regulates the expression of a wide range of genes and can coordinate either transcriptional repression or activation depending on the molecular and/or cellular context. This directly implicates an epigenetic pathway in neurodevelopmental disorders. Interestingly, perturbation of MeCP2 expression shifts the dynamic cortical excitatory/inhibitory balance in favor of inhibition in visual cortex18. Moreover, sensory experience can selectively induce MeCP2 phosphorylation, regulating dendritic patterning, spine morphogenesis, and BDNF transcription101. Surprisingly, RTT-like neurological defects can be rescued by delayed restoration of MeCP2 gene31,32,54 as well as overexpression of BDNF9. By establishing the principle of reversibility in mice, these studies suggest that RTT and related disorders are also reversible, even in the late stages of the disease. Our research has revealed that excitatory/inhibitory balance dictates the timing of critical periods of visual cortical maturation23. Direct manipulation of this balance can accelerate or delayed activity-dependent processes and can be used successfully to rescue plasticity defects23-25,46,47 . Perturbing neuronal activity causes aberrant gene-expression patterns, many of which are linked to misregulated epigenetic systems. Our central hypothesis is that the excitatory/inhibitory balance drives MeCP2 regulation of gene translation and repression in an activity-dependent manner during critical periods of heightened cortical plasticity in infancy. Their disruption leads to the complex behavioral phenotype of neurodevelopmental disorders such as Rett Syndrome. Hence, manipulation of Excitatory/Inhibitory balance will be used to rescue cortical impairments in animal models of Rett syndrome. By applying in vivo electrophysiological techniques, we aim to reveal role of MeCP2 function in excitatory/inhibitory circuit balance during cortical circuit refinement. The results will provide potential therapeutic strategies for reactivating brain plasticity in neurodevelopmental disorders. PUBLIC HEALTH RELEVANCE: In the present grant, we propose to test the hypothesis that an excitatory/inhibitory balance drives a complex epigenetic state of gene translation and repression in an activity-dependent manner during critical periods of heightened cortical plasticity in infancy. Their disruption leads to the complex behavioral phenotype of neurodevelopmental disorders such as Rett Syndrome and ASDs. By applying in vivo electrophysiological techniques, we aim to reveal MeCP2 function in relation to excitatory/inhibitory circuit balance in cortical circuit refinement. The results will provide potential therapeutic strategies for reactivating brain plasticity in animal models of Rett Syndrome.

DESCRIPTION (provided by applicant): Autism Spectrum Disorders (ASD) represents a group of severe, highly heritable, neurobehavioral syndromes with heterogeneous phenotype. The clinical features of autistic children are notable for an unawareness of their surrounding environment, impaired language and social interactions, and repetitive behaviors. They often exhibit relatively normal initial maturation followed by stagnation or regression. The underlying cause of this clinical course is unknown. Here, we propose to test a novel idea-- that disrupted interactions between the thalamus and the cortex during their circuit maturation underlie this developmental sequel. Recently, there has been increasing interest in the idea that ASD might involve dysfunction of experience- dependent circuit maturation and refinement. Sensory systems, such as the visual system, are thought to develop sequentially in a feed forward manner during sensitive periods in early development. However, this model for development has been challenged by our recent findings demonstrating overlap in the timing of refinement of thalamic and cortical critical periods. Our hypothesis is that cortical feedback to the thalamus drives the refinement of thalamic synaptic circuits, and the resulting thalamic function influences cortical development. Disruption of this interaction could result in the late developmental abnormalities observed in ASD. To test this hypothesis, we will take advantage of MeCP2 deficient mouse, an animal model of Rett Syndrome (RTT). RTT is a neurodevelopment disorder associated with ASD. The visual system will be used as an experimental system for understanding the developmental relationship between thalamus and cortex. MeCP2 null mice exhibit impaired development of visual function both at the thalamic and cortical level. In this proposal, we will selectively disrupt the expression of the MeCP2 gene either cortically or in the retino-thalamic circuitry and assess the functional maturation of retinogeniculate or cortical circuits respectively. If our hypothesis is true, selective cortical defect should affect the experience-dependent sensitive period for thalamic circuit plasticity and a focal deficit in the retino-thalamic circuitry will ultimately affect cortical development. Our results would transform the fundamental thinking of the mammalian central nervous system development. A feed forward and feedback interaction between the CNS structures would mean that defects in one area could affect the other and amplify over time. In addition, interaction between the two structures raises the possibility that changes in one structure can compensate for defects in the other. Thus, a deeper understanding of the developmental relationship between the thalamus and cortex could have implications in neurodevelopment disorders such as autism spectrum disorders. PUBLIC HEALTH RELEVANCE: Sensory information (such as vision, hearing and touch) is transmitted through various stations in the brain as it is relayed to the cortex. In this proposal we test a novel hypothesis that during development, the cortex sends information to guide the development of these stations. A feed-forward and feedback communication between different regions of the brain could result in a spreading of an initially focal abnormality. This could be the underlying cause of neurodevelopment diseases such as Rett Syndrome and Autism Spectrum Disorders. Thus it is important to understand the importance of communication between different areas of the brain during development.

D1. OBJECTIVES The capacity to generate transgenic and knockout mice that model human neurodevelopmental disorders has revolutionized research in this field. Neurodevelopmental disorders include many behavioral and cognitive syndromes that have onset during childhood, including autism spectrum disorders (ASD), attention-deficit hyperactivity disorder (ADHD), Tourette syndrome, Tuberous Sclerosis Complex (TSC), Neurofibromatosis type I (NF1), Fragile X (FXS), and Rett syndrome, many of which can be modeled in mice. Recent data suggest that early deficits that impact attention, learning, and social interaction are not only impairing in themselves, but may also alter the beneficial influence of normal environmental experience by perturbing experience-dependent brain development. There is, therefore, growing interest not only in addressing the modeling of symptoms of these developmental disorders in mice, but also in studying their underlying neurobiological causes, their impact on normal developmental changes and in testing new treatments. A neurodevelopmental behavioral core focused specifically on neurobiological and cognitive disorders will have a major role in addressing these priorities. Manipulation of gene expression (knock-out, knock-in, conditional, site-specific viral vector delivery, siRNA silencing, etc.) provides exciting opportunities for understanding gene function in relation to many different neurodevelopmental disorders. Dissecting the function of a specific gene or pathway requires molecular, biochemical, anatomical, physiological, imaging and pathological studies. However, since behavior is the final output of the nervous system, measurement of behavior is absolutely integral to revealing the processes responsible for the normal development of the nervous system and for determining the bases for and new treatments of neurodevelopmental diseases. Measuring the behavioral phenotypic outcome(s) of any given gene manipulation in mice is, however, a challenging task, particularly for high level brain function. While several laboratories in the IDDRC at Children's Hospital Boston have experience in a few particular behavioral phenotype protocols, no single lab has the requisite experience, capacity or technology to fully, comprehensively assess neurodevelopmental models across the whole range of relevant behavioral outcomes related to the full spectrum of neurodevelopmental disorders. We plan to address this problem by doing the following: a) Developing a state-of-the-art infrastructure to enable IDDRC investigators to comprehensively characterize nervous system function and complex behaviors in mouse models of neurodevelopmental disorders along their developmental trajectories. b) Exploiting mouse surrogate models o f human neurodevelopmental disorders to test novel therapeutic agents and therapies. c) Training new and established investigators and their students in how to use behavioral assays in a reliable, reproducible, and, accurate manner for understanding and measuring neurodevelopmental disorders. The Neurodevelopmental Behavioral Core is designed to raise the quality and breadth of mouse model behavioral testing/phenotyping at this IDDRC by providing a wide array of protocols, training, and equipment to all our investigators. Comprehensive characterization of a new mutant line will typically include an initial battery of basic observational tests for general health, neurological reflexes, sensory abilities and motor function, followed by more specific measures focused on careful evaluation of cognitive, perceptual, and mood-related behaviors (social interaction, vocalization, emotion and anxiety). Phenotype will typically be followed from birth until adulthood. This will extend our understanding of behavioral changes during normal development as well as of neurodevelopmental disorders and their clinical impact. Early stage drug development will be advanced by providing a neuro-focused preclinical drug testing service that will help investigators generate proof of principle (animal efficacy) data and early stage safety and preliminary toxicity assessments. Collaboration will be encouraged, duplication reduced, and the pooling of data sets generated by multiple PIs studying the same mouse model will create a valuable data source. Collectively these activities will contribute to a deeper and more complete understanding of mouse models of neurodevelopmental disorders and their behavioral phenotype than individual investigators can achieve on their own. Currently, for every mouse of interest, the scope of studies that can be performed is limited by the resources available to an individual researcher. It is not cost-effective for most laboratories to purchase and maintain the equipment needed for many specialized types of studies, and investigators must turn to collaboration with other laboratories or commercial vendors to obtain such resources, or simply not explore all phenotypes. We are confident that a shared behavioral facility will offer the advantage to investigators of access to a wide range of tests at a lower cost, with the necessary expertise, giving the investigators freedom to expand their analyses beyond their original goals. We will also be able to follow the development of each phenotype from birth until adulthood and the core will facilitate development of new models and outcome measures. Successful development of new approaches to testing and refining mouse models will greatly benefit, we believe, the national and international neurodevelopmental disorders community.

? DESCRIPTION (provided by applicant): Rett syndrome (RTT), a devastating pediatric disorder, is caused by de-novo mutations in the MECP2 gene. The key feature and basic requirement for Rett syndrome diagnoses is the loss of acquired skills or regression, occurring between the ages of 1.5 and 4-5 years after an apparent initial normal development. Once regression is complete, it was thought that the symptoms were irreversible in adults. Mice deficient in Mecp2 recapitulate many of the symptomatic features of RTT. Recent groundbreaking studies in these mouse models have demonstrated that some symptoms of the disorder, such as general health conditions, defects in mobility, coordination and breathing are reversible. These results raise the question of whether other RTT symptoms, such as impairments in sensory modalities, cognition, social interaction and communication can also be rescued. These features of human behavioral repertoire are all acquired in an experience-dependent manner during well-defined critical periods of plasticity in early postnatal life. As adulthood is reached, neuronal circuits consolidate and plasticity diminishes. To begin to delineate the possibilities and limitations of RTT reversibility, it is necessary to have a better understanding of the underlying mechanism of regression. Neuronal circuits in Mecp2 deficient mice are disrupted and exhibit aberrant excitatory-inhibitory (E/I) balance well before the onset of symptomatic regression. Whether these abnormalities lead to regression is still not clear. Using a sensory circuit model for regression established by the Chen and Fagiolini laboratories, we will address these important questions and test the hypothesis that reversal of sensory circuit defects in Mecp2 KO mice requires the correction of such E/I circuit imbalance. The Chen and Fagiolini Laboratories have independently demonstrated that after initial normal development in Mecp2 KO mice a progressive disruption of thalamic and cortical visual circuits occurs both at the anatomical and functional level. The time course of this regression tracks very closely with onset of RTT phenotypic symptoms. Notably a specific inhibitory circuit, involving the fast-spiking parvalbumin-positive cells (PV), is abnormally connected very early in development prior to the onset of visual function abnormalities and may contribute to silencing of cortical circuits. These results suggest that early abnormalities in the PV inhibitory circuit could drive gradual regression of visual function. PV cells not only regulate critical developmental periods in multiple cortical systems, but also constantly and dynamically adjust brain activity. Here, we will test whether recovery can occur in sensory systems by either globally re-expressing Mecp2 or by manipulating E/I balance selectively in cortex. Taken together, the results of our proposal will provide insight into underlying neuronal circuit dysfunction and regression and, importantly, into novel approaches for treatment.

MOUSE NEURODEVELOPMENTAL BEHAVIOR CORE (CORE D) ABSTRACT The Mouse Neurodevelopmental Behavior Core (NBC) at Boston Children's Hospital (BCH) is designed to enable the comprehensive identification and quantification of complex behavioural phenotypes in mouse models of neurodevelopmental disorders. As well as providing cutting edge equipment, we continually validate the best protocols and generate base-line data for quality control management. Having such capabilities for in vivo analysis of mouse models of human disorders facilitates efficacy testing of novel therapeutic compounds and interventions, to provide evidence for transitioning into the clinic. The Core is equipped to perform extensive batteries of tests that phenotype specific social, emotional and cognitive behaviors, as well as motor, auditory and visual function, together with the general health of the animals. In addition, the NBC provides complementary technologies for evaluating the neurobiological mechanisms behind changes in behaviour, such as EEG, ECG and lasers for optogenetic studies. The core also provides a unique opportunity for training fellows, graduate and undergraduate students, as well as PIs, in the in vivo analysis of mouse models of human disorders. Looking ahead, we aim to keep the NBC at the forefront of in vivo analysis of genetic models of human disorders. One  major new initiative will be the establishment of a rat behavioral facility to exploit the increasing ability to efficiently modify the genome of rats to create genetic models of tuberosclerosis, Rett syndrome and other neurodevelopmental disorders. This will occupy ~1500 sq.ft of new space for the NBC and we have the required equipment for measuring cognition, anxiety, exploration and motor function in rats as well as EEG and in vivo imaging capacity. Another initiative is to offer reverse light housing for up to 300 mouse cages, so that investigators can study mouse behavior over the full diurnal cycle. We are also developing synergistic partnerships with the Cellular Imaging Core that has a two photon microscope to image neurons in conscious behaving mice, to bring together cutting edge imaging and behavioral technologies to the enable the mechanistic study of neurodevelopmental diseases. Finally, we recognize that the IDDRC network of behavioral Core facilities in the US provides a unique opportunity to establish a set of standards for behavioral assessment and reporting of rodent models of neurodevelopmental disorders.  We will run with Jackie Crawley (Director, UC Davis, IDDRC Behavior Core Facility) a series of comparative studies to establish standardized protocols for execution and analysis of neurodevelopmental disorders.

? DESCRIPTION (provided by applicant): Rett syndrome (RTT), a devastating pediatric disorder, is caused by de-novo mutations in the MECP2 gene. The key feature and basic requirement for Rett syndrome diagnoses is the loss of acquired skills or regression, occurring between the ages of 1.5 and 4-5 years after an apparent initial normal development. Once regression is complete, it was thought that the symptoms were irreversible in adults. Mice deficient in Mecp2 recapitulate many of the symptomatic features of RTT. Recent groundbreaking studies in these mouse models have demonstrated that some symptoms of the disorder, such as general health conditions, defects in mobility, coordination and breathing are reversible. These results raise the question of whether other RTT symptoms, such as impairments in sensory modalities, cognition, social interaction and communication can also be rescued. These features of human behavioral repertoire are all acquired in an experience-dependent manner during well-defined critical periods of plasticity in early postnatal life. As adulthood is reached, neuronal circuits consolidate and plasticity diminishes. To begin to delineate the possibilities and limitations of RTT reversibility, it is necessary to have a better understanding of the underlying mechanism of regression. Neuronal circuits in Mecp2 deficient mice are disrupted and exhibit aberrant excitatory-inhibitory (E/I) balance well before the onset of symptomatic regression. Whether these abnormalities lead to regression is still not clear. Using a sensory circuit model for regression established by the Chen and Fagiolini laboratories, we will address these important questions and test the hypothesis that reversal of sensory circuit defects in Mecp2 KO mice requires the correction of such E/I circuit imbalance. The Chen and Fagiolini Laboratories have independently demonstrated that after initial normal development in Mecp2 KO mice a progressive disruption of thalamic and cortical visual circuits occurs both at the anatomical and functional level. The time course of this regression tracks very closely with onset of RTT phenotypic symptoms. Notably a specific inhibitory circuit, involving the fast-spiking parvalbumin-positive cells (PV), is abnormally connected very early in development prior to the onset of visual function abnormalities and may contribute to silencing of cortical circuits. These results suggest that early abnormalities in the PV inhibitory circuit could drive gradual regression of visual function. PV cells not only regulate critical developmental periods in multiple cortical systems, but also constantly and dynamically adjust brain activity. Here, we will test whether recovery can occur in sensory systems by either globally re-expressing Mecp2 or by manipulating E/I balance selectively in cortex. Taken together, the results of our proposal will provide insight into underlying neuronal circuit dysfunction and regression and, importantly, into novel approaches for treatment.

Abstract There is a growing interest to effectively combine optical approaches with electrophysiology at large scale and with great precision to fully leverage the complementary spatial and temporal resolution advantages of both techniques. It is also widely recognized that device softness and compliance are important attributes to dramatically lower tissue injury and irritation and maintain signal quality over time. Our long-term goals are (i) to converge electrophysiology with optical brain recording/stimulation seamlessly at the large scale to achieve high-spatiotemporal-resolution brain activity mapping which captures both the finest spatial intricacies of the neuronal circuit and fastest temporal dynamics of neuronal communication and (ii) to integrate electrode arrays seamlessly with the brain tissue. The objective of this R01 application, which is the first step in achieving these goals, is to develop and validate a novel neuroelectronic tool which provides state-of-the-art electrophysiological capabilities while allowing at the same time, optical and chronic-bio- compatibilities, realized critically through the optical transparency and mechanical ultra-softness of the entire MEA, along with other engineering efforts. We are very ambitious about tackling both of these two big challenges because of a unified technical concept, nanomeshing conventional electrode materials. In our prior work, we have proposed this novel electrode concept, which has led to the demonstration of transparent, flexible electrodes with high performance of sizes down to 15×15µm2, and with the ability to record single-unit spikes. In this application, we aim to prove: this nanomeshing concept can lead to 100s-electrode- scale, high-density, transparent and ultra-soft electrode arrays that simultaneously allow both the capability of (i) effectively integrating electrical recordings/stimulation with optical imaging in vivo, and (ii) chronic stability of single-unit recordings. The proof of this concept will readily enable stable, concurrent electrical/optical investigations of the brain at the mm-to-cm scale with further scalability, while also providing unique opportunities for next-generation therapeutic interventions via sustainable neural prosthetics. In three inter- related aims, we will develop and validate proof-of-concept, nanomesh-microelectrode-based, transparent, ultra-soft, high-density (NANOMESH) array with at least 256 high-performance nanomesh microelectrodes and artifact rejecting wireless data link through an interdisciplinary 3-year plan integrating innovative technological developments with basic neuroscience testing. We will benchmark our devices to industry standards in vivo, and integrate neural engineering feedback throughout the design, testing and validation phases of the project. This project leverages a vibrant and successful collaboration between material scientists, neuro-engineers, electrical engineers, and neuroscientists at Northeastern University (NU), the University of California Los Angeles (UCLA), and Boston Children?s Hospital (BCH), to translate transparent nanomesh technology into large-scale brain-mapping tools and implantable devices.

ABSTRACT The mission of the Animal Behavior and Physiology (AB&P) Core is to enable deep animal model phenotyping and early-phase CNS drug and gene therapy discovery by providing cutting-edge behavioral and physiological assays and related scientific expertise to the IDD research community. In line with the mission, the AB&P Core is therefore designed to synergize cutting-edge behavior and in vivo physiology analysis. As a facility focused on in vivo IDD models, the AB&P Core will provide unified services to IDDRC investigators at the interface between the molecular/cellular and clinical/translational IDDRC components. The Core enables (1) the standardization and validation of experimental procedures with appropriate rigor and reproducibility, (2) quantitative and in-depth mammalian IDD model phenotyping, and (3) the development of translational biomarkers, particularly for purposes of detecting target engagement by experimental therapeutics. The integration of behavioral endpoints with quantitative physiological biomarkers provides a means for assessing both preclinical proof of concept and clinical trial readiness of proposed IDD treatments. As new technologies become available, the Core rapidly introduces them into its tool portfolio and makes them available to Core users. In addition to providing the experimental platforms, the Core provides deep expertise in in vivo experimental design, signal analysis and overall study interpretation.

ABSTRACT Rett Syndrome (RTT) is a rare X-linked developmental brain disorder due to de novo pathogenic variants in MECP2 and mainly affecting girls. Notably, RTT individuals reach typical developmental milestones in the first 6-18 months of life, followed by stagnation and then regression of acquired skills. The severe cognitive delays, deficits in sensory processing and dysregulated behavioral states profoundly impact both patient and family quality of life. We still do not know when and how autonomic and central brain networks begin to derail from the neurotypical developmental trajectory, nor we have effective treatments targeting these impairments. Hence, there is an urgent need for objective, quantitative, non-invasive, and translational biomarkers for early assessment of cognition and behavioral states in RTT, their progression over time and response to therapeutic interventions. Our goals are to establish 1) spontaneous pupil and heart rate (HR) fluctuations as new biomarkers for RTT, 2) how arousal impacts the progression of RTT cortical pathophysiology and 3) develop targeted interventions. We will address these challenges using a multi-level circuit approach both in RTT girls and awake Mecp2 female heterozygote mice during the progression of the disorder. The proposed work will refine and establish spontaneous pupil and HR fluctuations as highly translational biomarkers to track autonomic nervous system function, while dissecting how and when neuromodulation impacts sensory processes in RTT. Together these approaches will allow the development of new circuit-based therapies in patients. Our results will be pave the way to future studies of RTT related disorders such as MECP2 duplication, CDKL5 deficiency disorder and FOXG1 syndrome.