Michael E. Greenberg - US grants

The electrical stimulation of neurons during the development and maintenance of the nervous system has both immediate and longer term effects on cellular physiology. The long term neuronal responses include alterations in neuronal sprotting, and changes in neurotransmitter, transmitter receptor and ion channel protein production. These cellular changes appear to require the activation of new gene expression, and may be fundamental to processes such as neural development, and information storage. The long-range objectives of the proposed research are to understand the mechanisms by which electrical stimulation controls the expression of genes in neurons, and the function of these electrical stimulation controls the expression of genes in neurons, and the function of these electrically-regulated genes during the development, and maintenance of the nervous system. Recent studies have identified a class of "immediate early genes" that encode mRNAs whose transcription is activated rapidly as a response to electrical stimulation. Many of these genes encode transcription factors that have been hypothesized to control the neuronal cell response to trans-synaptic stimulation. Experiments are proposed that will elucidate the biochemical pathway by which membrane depolarizing agents induce the transcription of two members of the immediate early gene family c-fos and nur/77. Activation of these genes in the pheochromocytoma cell line PC12 by electrical stimulation requires an influx of calcium from the extracellular medium. Depolarization-activation of c-fos is controlled by a calcium response element (CaRE) within the c- fos promoter that binds the transcription factor CREB. Membrane depolarization activates the phosphorylation of CREB raising the possibility that electrical stimulation of gene expression in neurons is mediated by the phosphorylation-activation of a specific transcription factor. The specific aims of the proposed research are: (1) to determine whether the sites of phosphorylation on CREB are critical for transcriptional activation; (2) to characterize the protein kinases that mediate the depolarization response; (3) to identify novel CaREs within the immediate early gene promoters, and CaRE binding proteins, that control alternative pathways for electrical stimulation of gene expression; (4) to test the hypothesis that varying the duration, strength, or repetitive nature of the electrical stimulation alters the pattern of activation of immediate early genes. Given the function of these genes in the process of normal cell growth and differentiation it is likely that alterations in the developmental process that lead to cancer, neurodegenerative diseases, or seizure disorders will involve changes in the regulation of function of the immediate early genes.

DESCRIPTION (provided by applicant): The Eph subfamily of receptor tyrosine kinases mediates multiple aspects of neural development, including long-range axonal pathfinding, synaptogenesis, and synaptic plasticity. In addition, a disruption of Eph function has been linked to neurological disorders such as autism and Alzheimer's disease. In preliminary studies we identified a ligand-independent EphB signaling mechanism whereby the guanine nucleotide exchange factor Ephexin5 associates with the cytoplasmic domain of EphBs and suppresses excitatory synapse development through activation of the small GTPase RhoA. This brake on excitatory synapse development is relieved by binding of EphBs to their ephrinB ligands, which triggers rapid Ephexin5 tyrosine phosphorylation, ubiquitination, and degradation. Knockout studies in mice indicate that EphB-Ephexin5 signaling functions as a synaptogenesis checkpoint so that excitatory synapses form at the right time and place and in correct numbers during brain development. Ephexin5 loss-of-function mutations have been detected in several cases of idiopathic infantile epilepsy. We have also identified Ephexin5 as a novel neuronal substrate of the E3 ubiquitin ligase Ube3a. Loss-of-function mutations in UBE3A give rise to Angelman syndrome (AS), a neurodevelopmental disorder characterized by motor dysfunction, severe mental retardation, speech impairment, and seizures; and our preliminary findings suggest that elevated levels of Ephexin5 contribute to at least some aspects of the neurological and cognitive dysfunction associated with AS. In this proposal we address specific gaps in our understanding of the importance of the EphB-Ephexin5 complex in nervous system development and disease. Our specific aims are: (1) to investigate the mechanisms by which EphB- Ephexin5 signaling controls synapse development, (2) to investigate the contribution of Ephexin5 mutations to human infantile epilepsy, and (3) to determine the contribution of elevated Ephexin5 levels to phenotypic defects observed in a mouse model of AS. It is our hope that the proposed experiments will advance our understanding of the molecular mechanisms controlling synapse development and ultimately provide new opportunities for the development of therapeutic strategies to combat neurodevelopmental disorders such as infantile epilepsy and autism.

Summary/Abstract: Mutations in MeCP2, a methyl-CpG-binding protein that functions as a regulator of gene expression, are a major cause of Rett Syndrome (RTT), an X-linked progressive autism spectrum disorder that is among the most common causes of profound cognitive impairment in girls and women. While the selective inactivation of MeCP2 in neurons has been suggested to be sufficient to confer a Rett-like phenotype in mice, the specific mechanisms by which the loss of MeCP2 function in postimitotic neurons contributes to RTT phenotypes remain unclear. We have identified serine 421 (S421) on MeCP2 as a site of neuronal activity-dependent phosphorylation that is induced selectively in the brain in response to physiological stimuli. Significantly, we have found that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription in both cultured neurons and slice preparations. To further explore the role of this regulatory mechanism in neural development in vivo, we have generated a knock-in mouse in which S421 of MeCP2 is mutated to an alanine residue (S421A KI), preventing the phosphorylation of MeCP2 at this site. Intriguingly, whereas the abrogation of MeCP2 S421 phosphorylation in vivo does not result in the motor and survival phenotypes seen with complete loss of MeCP2 expression, our preliminary studies have revealed a deficit in cortical inhibitory synaptic development in these S421A KI mice, suggesting that activity-dependent phosphorylation may be involved in a specific subset of MeCP2 functions relevant to the synaptic and cognitive defects observed in RTT. To begin to test this hypothesis and determine the extent to which MeCP2 functions as a general regulator of neuronal activity- dependent gene expression, we propose the following specific aims: (1) to investigate the contribution of MeCP2 S421 phosphorylation to experience-dependent synaptic development in vivo; (2) to assess the role of MeCP2 S421 phosphorylation in the regulation of activity-dependent neuronal gene expression; and (3) to characterize additional sites of activity-dependent MeCP2 phosphorylation. It is our hope that the proposed experiments will provide a better understanding of MeCP2 function, give insight into the mechanisms of activity-dependent gene expression, and provide new opportunities for the development of therapeutic strategies to alleviate RTT pathology.

2.a.2. Overall Objective The Proteomics Core aims to provide MRDDRC investigators with access to state of the art technologies in mass spectrometry and microscale capillary high performance liquid chromatography (proteomics) required for advanced studies in the cell and molecular biology of neurological developmental disabilities. Dr Greenberg (the Director of this MRDDRC) will be the Director of the Core. Dr. Jebanathirajah, a new Assistant Professor in the Neurobiology Program at Children's Hospital, and an expert in the field of mass spectrometry, will be the Manager responsible for supervising all Core activities. Major goals of the Core will be to use the techniques of mass spectrometry to assist MRDDRC investigators in the characterization of protein complexes that are relevant to neuronal function, to characterize changes in protein composition that occur in the nervous system during development and under conditions that lead to developmental disabilities, and to identify and characterize the function of post-translational modification of neuronal proteins. The Proteomics Core is actively involved in research in the area of mass spectrometry and any newly developed tools and methods will be made available to the MRDDRC investigators. The Core will also provide and develop proteomics related statistical and data handling tools.

DESCRIPTION (provided by applicant): In this Institutional Center Core Grant to Support Neuroscience Research, we propose to establish an innovative Neural Imaging Center composed of four Core facilities that will serve NINDS-funded Harvard Medical School (HMS) and Children's Hospital Boston (CHB) investigators. These state-of-the-art facilities will provide important new resources to the HMS and CHB neuroscience community, and will perform essential services that are difficult and impractical for individual laboratories to provide on ther own. The Imaging Center will be composed of an Administrative Core, a High-Content Cellular Imaging Core, an immunohistochemistry-based Array Tomography Core, and a Super-resolution Imaging Core. The experimental opportunities and innovative services provided by the Imaging Center will give area neuroscientists access to unique equipment and training in several new cutting-edge methodologies, greatly benefiting the research programs of NINDS-funded investigators at these institutions. Moreover, the Center will function as the centerpiece of a concerted effort to strengthen ties between the neuroscience communities at HMS and CHB. Thus, a major focus of this Center will be to serve as a nexus for collaborative interactions. To this end, we propose not only to establish a set of core facilities, but also to adopt several strategies that reduce the barriers to their widespread utilization, including provisions for informal education regarding core methodologies and significant technical support. Through the development of this Center, we hope to shift from a complete reliance on individual laboratory-centered research to a more cost-effective and productive use of extraordinary cores while further deepening existing ties between these two vibrant neuroscience communities. RELEVANCE: We propose to establish a Neural Imaging Center to serve NINDS-funded Harvard Medical School and Children's Hospital Boston investigators. These facilities will provide important new resources and perform essential services that are difficult and impractical for individual laboratories to perform on their own, thereby accelerating the pace of neuroscience research at these institutions.

DESCRIPTION (provided by applicant): Early-life experiential and environmental conditions, particularly those occurring during heightened periods of brain plasticity, are known to promote long-term changes in physiology and behavior that act independently of changes in the DNA code. Accumulating evidence suggests an important role for epigenomic processes in these epigenetic phenomena. In particular, recent data from a variety of sources suggest that experience-dependent changes in DNA methylation can have a long-lasting impact on neural function through sustained effects on neuronal gene expression. However, the fact that these modifications occur in vivo in a relatively small number of cells within highly heterogeneous neural tissue limits the study of these modifications with existing genomic approaches and greatly complicates investigation of the underlying molecular mechanisms. We propose a two-pronged approach to begin to address these issues. First, we will pursue a reductionist approach to the study of experience-driven changes in DNA methylation, employing a dissociated neuronal culture system that shows activity-induced changes in DNA methylation and in which a large number of cells can be synchronously activated with robust stimuli. Moreover, to complement and address limitations inherent in this reductionist approach, we have also developed a general genetic strategy to specifically isolate chromatin from defined cell types in vivo, enabling the analysis of DNA methylation changes induced in specific neuronal cell populations in response to early-life experiences using massively parallel sequencing techniques. Thus, we propose: 1) To employ a dissociated neuronal culture system to characterize neuronal activity-induced changes in DNA methylation, and 2) To investigate long-lasting neuronal epigenomic correlates to experience-driven behavioral and physiological changes in vivo. It is our hope that the proposed experiments will establish new approaches for the analysis of neuronal epigenomic modifications, advance our understanding of the regulation of DNA methylation in the developing central nervous system, and ultimately provide new insights into the importance of these mechanisms for neurodevelopment, cognitive behavior, and disease. PUBLIC HEALTH RELEVANCE: Adverse early life events are known to influence risk for neurodevelopmental and psychiatric disorders, triggering long-lasting changes in physiology and behavior that act independent of changes to the DNA code. In an effort to gain insight into the underlying molecular basis of these effects, the proposed study will explore the role of regulated DNA methylation in the persistent alteration of neuronal gene expression.

No abstract provided

Project Summary: Our limited ability to genetically access specific cell types within the nervous system constitutes a fundamental impediment in our efforts to probe brain function and intervene therapeutically, particularly in species lacking the well-developed genetic resources available in the mouse. Targeted payload delivery using recombinant viral approaches possesses a number of potential advantages, including anatomical specificity, ease of experimental implementation, and utility in a broad range of mammalian species. Moreover, the incorporation of endogenous cell-type-selective promoter elements has shown significant promise in targeting viral payload expression to distinct neuronal subsets. To date, however, this approach has not been pursued systematically or at sufficient scale, and the stand-alone cell-type-specificity of existing viral reagents remains relatively limited. Here, we propose to establish and validate a general strategy for the identification of cell-type-specific adeno-associated viral (AAV) drivers by exploiting recent epigenomic advances in conjunction with a novel application of single-cell transcriptome analysis. First, comparative epigenomic profiling between several major neuronal subclasses in the target tissue will be used to generate a library of over 1,000 gene regulatory elements (GREs) that is enriched for highly cell-type-restricted enhancers. This barcoded GRE library will be packaged into AAV and screened en masse in vivo for cell-type-specific activity through the use of single-cell RNA sequencing. This novel approach will allow us to simultaneously assess the specificity of individual GREs against the full complement of cell types present in the target tissue. Focusing initially on mouse cortex, we will seek to validate this approach by identifying viral drivers that are specific for six excitatory and four inhibitory neuronal subtypes. To this end, we propose the following specific aims: 1) Generation of a library of GREs enriched for neuronal cell-type-specific enhancers; 2) High-throughput screening for cell-type-restricted GRE-driven AAV reporters; and 3) Validation and in vivo characterization of newly isolated GRE-driven cell- type-restricted AAV vectors. The viral drivers developed for the ten neuronal cell types in the proposed study should prove of immediate utility for a variety of cell-type-specific applications, including monitoring neuronal activity, optogenetic and chemogenetic manipulation, axonal tracing, gene delivery, and genome editing. Moreover, it is likely that these AAV vectors will retain their cell-type-specificity across brain regions and in other mammalian species. Finally, it is our hope that the proposed studies will demonstrate the feasibility of this general approach as a portable methodology, applicable in a host of brain regions, for identification of cell- type-restricted viral drivers that will be useful in a variety mammalian species.

Project Summary: While traditionally conceived as passive support elements for neuronal networks, non-neuronal brain cells are now appreciated as dynamic integral components of central nervous system (CNS) circuitry. Astrocytes, for example, serve as powerful regulators of neuronal spiking, synaptic plasticity, and brain blood flow. Similarly, microglia not only respond to a wide range of CNS perturbations, but also participate in circuit development and plasticity through the active elimination of synaptic connections. Dynamic oligodendrocyte and brain endothelial cell responses to neuronal signaling also play key roles in shaping and homeostatically stabilizing neuronal circuit activity. While dysfunction of these glial and vascular cell types is implicated in a wide range of neurological disorders, study of the diversity and function of glial and vascular cells has suffered from an historical underinvestment in basic tool development relative to that focused on neuronal cell types. In this regard, one major persistent impediment to ongoing investigations stems from our limited ability to genetically access specific glial or vascular cell types, with many of the existing reagents displaying significant shortcomings. Capitalizing on recent advances in epigenomic analysis and single-cell profiling, we recently developed and validated a scalable strategy for the generation of cell-type-specific adeno-associated viral (AAV) drivers incorporating cell-type-restricted gene regulatory elements (GREs). Here we propose to apply this novel approach to isolate viral drivers that are specific for four distinct classes of non-neuronal cell types: astrocytes, oligodendrocytes, microglia, and endothelial cells. Moreover, these viral libraries will be designed to enrich for candidate drivers specific for distinct fine-grained subtypes within each of these broad cell type classifications, including subtypes not specifically accessible with existing transgenic lines. To this end, we propose the following specific aims: 1) Single-cell profiling of AAV serotype tropism; 2) High-throughput screening for cell-type-restricted GRE-driven AAV reporters; and 3) Validation and in vivo characterization of newly isolated GRE-driven cell-type-restricted AAVs. These studies should significantly expand genetic access to a broad array of functionally relevant non-neuronal brain cell types. The viral vectors developed in this study will possess immediate utility for a variety of cell-type-specific applications. Furthermore, given that previous viral drivers have been found to largely retain their specificity in other species, this strategy should provide important new tools for future investigations in additional experimental contexts, particularly in genetically inaccessible model organisms such as primates. !

The mature central nervous system (CNS) is sculpted by the combined effects of intrinsic genetic programs and dynamic environmental input, yet the precise manner by which these two processes collaborate to give rise to the functional diversity of the mature nervous system remains to be fully explored. Using transgenic approaches that allow for the purification of sparse neuronal subtypes, we find that lineage-committed cortical interneurons undergo dynamic changes in gene expression in the early postnatal period, including the downregulation of genes governing cell proliferation and migration as well as the concomitant upregulation of subtype-specific genes important for mature neuronal function. We recently discovered that this postnatal transition in transcription state is mediated by the licensing and decommissioning of thousands of cis-regulatory enhancer elements across the genome. In the course of defining the regulatory elements that orchestrate these transcriptional changes, we have uncovered a possible role for the AP-1 (Fos/Jun) family of stimulus-inducible transcription factors (TFs) in promoting neuronal maturation through the de novo selection of sets of neuronal subtype-specific enhancer elements, suggesting that external cues from the environment in early life have an instructive role in shaping mature neuronal identities. To gain further insight into the mechanisms mediating early postnatal enhancer selection and its contribution to neural circuit maturation and function, we propose (1) to assess the role of sensory-driven activity in postnatal neuronal enhancer selection, (2) to characterize the molecular mediators of postnatal enhancer selection, and (3) to test the contribution of enhancer remodeling to postnatal neuronal maturation. It is our hope that the proposed experiments will yield a better understanding of the molecular mechanisms underlying enhancer selection in the developing CNS, further illuminate how cell-intrinsic and -extrinsic mechanisms coordinate to drive mature circuit function, and ultimately provide new opportunities for the development of therapeutic strategies to combat a subset of neurodevelopmental disorders.