Robert Spitale - US grants

? DESCRIPTION (provided by applicant): The study of the factors that control RNA expression to give rise to diverse cell types, from the same genome, has occupied scientists for more than 50 years. However, transcriptional regulation is just the beginning. Each expressed RNA can potentially adopt a new purpose as a function of its spatial position within a cell. RNA localization has been analyzed one RNA at a time because assaying RNA spatial organization on a systems-level is currently not possible. This Innovator proposal is focused on developing the methods to produce the first ever view of RNA localization on a transcriptome-wide level within cells. We will develop a novel methodology to seek and find the cellular localization of every RNA within specific cellular locations. These methods are designed to be applicable to any cell type. By merging our technology with RNA sequencing we will construct RNA Localization Heatmaps to identify sites where certain RNAs are localized to. We will then integrate our data with existing datasets to understand how RNA-binding proteins direct RNAs to their cellular destinations. Our ultimate goal is to not only find where RNAs are, but also how they get there. Deciphering the molecular code for RNA organization will have broad impact on the biomedical community. Such knowledge could be used to better understand RNA-guided cellular reprogramming, design better RNA therapeutics, and further our understanding of how RNA localization contributes to normal and diseased phenotypes. This proposal is highly suitable for the New Innovator Award mechanism. Planned experiments will develop novel tools and methods to radically transform our understanding of cellular and RNA organization, while at the same time discovering an extended set of molecular codes that researchers could employ for RNA design.

Many trans and cis acting factors that control alternative splicing have been identified. The explosion of next-generation sequencing approaches have identified thousands of regulated splicing events (RNA-seq), and binding sites of many proteins which regulate alternative splicing have been identified via CLIPseq. An important question remaining for the field is: What are the rules governing the behavior of alternative splicing decisions? For example, what are the RNA elements (sequence and structure) that determine if alternatively regulated exons will respond at low concentrations or at high concentrations to a splicing factor, and will the splicing responses exhibit cooperative behavior or not? Addressing these questions is important for providing a framework for understanding how changes in splicing factor concentration can lead to disease. To address these questions, we have created cellular models that allow us to precisely titrate the level of an alternative splicing regulator, the Muscleblind-like 1 (MBNL1) protein. MBNL1 has been shown to regulate thousands of alternative splicing events and is important for the development of skeletal muscle, heart and the central nervous system. This regulation is highlighted by the primary role that MBNL proteins play in the disease myotonic dystrophy (DM), in which MBNL1 and its paralogs (MBNL2 and MBNL3) are sequestered by expanded CUG or CCUG repeat RNAs, resulting in aberrant RNA processing. The mis- splicing of MBNL targets has been shown to be responsible for causing some of the symptoms associated with DM, including the hallmark symptom myotonia. .  

Project Summary. A grand challenge of the 21st century is to gain a complete understanding of the brain's most fundamental characteristics, which include its potential for plasticity and its ability to learn from experience. Activity- dependent gene expression is central to neural plasticity, learning, and memory; however, efforts to elucidate the underlying mechanisms in the brain have remained elusive due to conceptual limitations in appreciating how our genome rapidly adapts to environmental changes and technical limitations with regard to molecular tools to interrogate this process. For example, the transcriptional programs of individual neurons are highly specific, and this is obscured by the massive diversity of cell-types in the adult brain that are also organized in a region specific manner. Therefore, a major hurdle has been the lack of tools to study dynamic gene expression programs in the brain, especially in real-time. The primary goal of this R21 proposal is to develop a novel experimental program to track nascent transcription in two cell types (neurons and astrocytes), simultaneously within a living brain. Our strong preliminary data demonstrates that we have identified orthogonal neucleoside/nucleobase-enzyme pairs that can be exploited for nascent metabolic labeling of RNA. Our specific aims are to optimize this system within cultured neuronal cells in vitro, and to transfer our optimized protocols into a living mouse brain. Successful completion of our aims is sure to empower us with tools and preliminary data to tackle RNA transcription in a living brain during learning and memory formation.

? DESCRIPTION (provided by applicant): RNA modification, and N6 methyladenosine (m6A) in particular, is a newly discovered epigenetic mechanism in the adult brain that is has recently been shown to be highly dynamic and, as indicated by our preliminary evidence, appears to be involved in fear-related learning and memory. The overarching goal of this research program is to establish, for the first time, a causal relationship between the epitranscriptomic regulation of gene expression and the formation and maintenance of memory in a preclinical model of fear-related anxiety disorder. We can then capitalize on this information to design better treatments for neuropsychiatric disorders characterized by impairments in cognitive function. Successful completion of these experiments also has the potential to dramatically change the way we think about mechanisms of adaptive plasticity by shedding new light on how the qualitative nature of RNA, rather than its overall abundance, is involved in a key learning process with implications for our understanding of neuropsychiatric disorders characterized by abnormally intense memories. This will be achieved through a potent combination of advance high-throughput sequencing approaches, robust behavioral paradigms and viral-mediated manipulation of gene activity in the adult brain.

Uncovering the context-dependent complexity of neural RNA modifications Brain development requires a delicate balance between neural progenitor proliferation and differentiation. Loss of this balance has been implicated in autism, anxiety and depression. Regulation of neural progenitor proliferation and differentiation occurs at all levels of gene expression, including messenger RNA decay and translation. The molecular mechanisms that control mRNA decay and translation are varied, but recent discoveries demonstrate that nucleotide modifications, the ?epitranscriptome?, are a common determinant of mRNA fates. The most abundant modification in human mRNA, N6-methyladenosine (m6A), affects mRNA decay and translation. While progress has been made in determining the functional significance of m6A, a major question remains unanswered: how do m6A modifications and their effects on metabolism vary according to cell type in vivo? Our team is uniquely positioned to address this question. We recently developed a method to purify cell type-specific RNAs from the Drosophila brain and this method can be paired with techniques that map m6A at nucleotide resolution (miCLIP), measure mRNA decay (EC-tag pulse/chase) and measure ribosome dynamics (5PSeq). We will use these novel experimental approaches to uncover the complexities of the m6A epitranscriptome in the Drosophila brain. The m6A methyltransferase complex is enriched throughout the Drosophila nervous system, but the distribution of m6A across mRNAs and the effects of m6A on mRNA decay and translation are unknown. First we will use EC-tagging coupled with miCLIP (EC- miCLIP) to generate m6A mRNA maps from whole larvae and compare these data to m6A maps generated from neural progenitors and differentiated neurons. The accuracy of our m6A mapping will be confirmed by performing EC-miCLIP in Drosophila that lack the m6A methyltransferase Ime4. These experiments will reveal neural cell type-specific patterns of m6A modification. Next we will measure mRNA decay and ribosome dynamics in wildtype and Ime4 mutant neural progenitors and pair these data with the site-specific m6A maps. These experiments will identify the metabolic consequences of different types of m6A modification. This work will significantly advance our understanding of the brain epitranscriptome and establish a powerful new method for identifying cell type-specific RNA modifications. This will lay the foundation for wide-ranging future epitranscriptome studies since EC-tagging may be applied to other model organisms and all types of RNA.

Abstract Microglia are the primary immune cells of the CNS and are critical to maintaining neuron health and responding to neuropathology. However, current methods for studying and harnessing the unique biology of human microglia is currently severely limited. Xenotransplantation of human microglia into immunodeficient mice is a promising new approach that provides extensive functional and visual access to human microglia in vitro. Xenotransplanted microglia (XMGs), generated from induced pluripotent stem cells (iPSCs) derived from human patients, can be modified to generate reporter and effector lines for unique microglial active states that activate in response to specific forms of neuropathology. Once transplanted into humanized MITRG mice these XMGs colonize the CNS while maintaining human expression patterns, but it remains to be verified if they exhibit the same response patterns and activity as in human brain tissue. The purpose of this supplement is to contribute to establishing the methodology for using XMGs as a proxy for studying human microglia in vivo and validation of reporter lines for both microglial activity and responses to neuropathology. Functional and morphological XMG responses will be observed in vivo using multiphoton imaging of a reporter for calcium activity, Salsa6f. Calcium signaling patterns will be compared between XMGs and endogenous mouse microglia reacting to laser-induced microlesions in brain tissue to verify that the XMGs retain their human response characteristics. Localized responses of XMGs to ?-amyloid plaques will be evaluated using brain- wide histology of a reporter for CD9, which has implicated as an indicator for the microglial ?-amyloid response state. Using an optical clearing technique, iDISCO+, it is possible to render complete intact mouse brains transparent. These cleared brains can be used to produce highly detailed three-dimensional renders of all XMGs and ?-amyloid plaques throughout the whole brain. From these renders, the distribution of CD9- expressing XMGs will be compared between control and ?-amyloid-expressing brains in order to validate them as a reliable reporter for proximity to ?-amyloid plaques. Validation of these imaging methods and XMG reporter lines may provide for unprecedented access into studying human microglial activity. Exploitation of targeted microglial active states also gives XMGs promising potential as vectors for targeted delivery of effectors localized to neuropathology afflicted regions which may have applications for drug discovery and therapeutics.

Project Summary. Alzheimer?s disease (AD) is the most prevalent neurodegenerative disease of the elderly. The complement cascade, a powerful effector mechanism of the innate immune system that can be directly activated by fibrillar A?, is implicated as a player in this inflammatory scenario. In brain, expression of most complement components increases during aging and further increases in AD patients and animal models of AD, consistent with a role for complement immune activation in progression of the disease. Complement activation fragment C5a has been a major focus, as inhibition of its proinflammatory receptor, C5aR1, leads to less activation of microglia and astrocytes, preservation of neuronal complexity and reduction of cognitive loss in AD models. These critical observations strongly suggest C5a binding to its receptor C5aR1 initiates cellular activation leading to changes in protein expression in microglia and astrocytes, which result in pathological phenotypes and disease progression. The primary goal of this proposal is to systemically understand alterations in cell-specific protein expression that result from signaling via C5a-C5aR1 in the context of Alzheimer?s disease. Specifically, this will extend our knowledge of induction of specific RNAs to production of the proteins, and the contribution of each cell type to those functional proteins, that ultimately accelerate pathogenesis and neuronal dysfunction in Alzheimer?s disease models.

Project Summary. Alzheimer?s disease (AD) is the most prevalent neurodegenerative disease of the elderly. The complement cascade, a powerful effector mechanism of the innate immune system that can be directly activated by fibrillar A?, is implicated as a player in this inflammatory scenario. In brain, expression of most complement components increases during aging and further increases in AD patients and animal models of AD, consistent with a role for complement immune activation in progression of the disease. Complement activation fragment C5a has been a major focus, as inhibition of its proinflammatory receptor, C5aR1, leads to less activation of microglia and astrocytes, preservation of neuronal complexity and reduction of cognitive loss in AD models. These critical observations strongly suggest C5a binding to its receptor C5aR1 initiates cellular activation leading to changes in protein expression in microglia and astrocytes, which result in pathological phenotypes and disease progression. The primary goal of this proposal is to systemically understand alterations in cell-specific protein expression that result from signaling via C5a-C5aR1 in the context of Alzheimer?s disease. Specifically, this will extend our knowledge of induction of specific RNAs to production of the proteins, and the contribution of each cell type to those functional proteins, that ultimately accelerate pathogenesis and neuronal dysfunction in Alzheimer?s disease models.

PROJECT SUMMARY AD is the most common cause of age-related dementia, and is characterized by a progressive impairment in cognitive function. Pathologically, AD patients exhibit A? plaques and neurofibrillary tangles with extensive neuronal and synaptic loss. Unfortunately, there is still no effective treatment for this devastating disease. Failures likely arise from amyloid accumulation 10-20 years prior to symptoms and the limited blood brain barrier (BBB) penetrance of many candidate therapies. Earlier diagnoses coupled with an effective and continuous source of in-brain therapy may therefore provide a powerful approach to ameliorate AD pathology. Herein we explore a transformative approach toward delivering therapeutic payloads into the brain to treat A? plaque load. We will test if engineered human microglia can be designed to safely and effectively deliver therapies into mouse models of AD. The first specific aim is to demonstrate the feasibility of the approach by determining the change in A? load with engineered microglia transplantation. The second aim will determine if reduced A? plaque load results in rescue of long-term potentiation defects. Overall, this R21 is poised to test the feasibility of novel and transformative approaches for safe and effective delivery of therapies to combat A? plaque buildup on AD. Successful demonstration of this approach could have far-reaching implications for the delivery of therapeutics into the brain.