Kelsey C. Martin - US grants

The specific aim of this proposal is to create cDNA libraries from neuronal processes in order to study the localization of mRNAs to dendrites and/or axons. A number of studies have indicated that localization of mRNAs to neuronal processes and regulated translation of these mRNAs serves as an important mechanism of gene expression in neurons. To begin to study this form of gene expression, we will create preparations of isolated neuronal processes, develop methodologies for creating cDNA libraries from the severely limited amount of mRNA collectable from these preparations, and develop methodologies for analyzing the representation and accumulation of mRNA species in neuronal processes in both basal and stimulated states. We will use cultured Aplysia sensory neurons and cultured mouse hippocampal neurons. The unique advantage of Aplysia neurons is that cell bodies and glia can be manually removed from cultured neurons, leaving pure neuronal processes as a starting material for cDNA library construction. Rodent hippocampal neurons can be cultured on membranes such that the neuronal axons and dendrites can penetrate through the pores in the membrane and can therefore be separated from cell bodies. Using these preparations we will: 1. Develop preparations of pure neuronal processes 2. Develop methodologies for creating cDNA libraries from these neuronal processes 3. Develop methodologies to test the representation and localized accumulation of transcripts in unstimulated and stimulated neuronal processes.

DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the mechanisms whereby long-lasting, transcription-dependent neuronal plasticity can be regulated in a synapse-specific manner. A number of studies have indicated that localization and regulated translation of mRNAs at synapses serves as an important mechanism of gene expression in neurons during learning-related synaptic plasticity. We will use two model systems of learning-related plasticity, cultured Aplysia sensory-motor synapses and cultured mouse hippocampal neurons, to identify the population of mRNAs that are localized to synapses, and to investigate the mechanisms underlying the localization and regulated translation of these mRNAs. The specific aims are as follows: 1) Identify synaptically localized mRNAs. This aim is directed toward identifying, in an unbiased manner, the population of mRNAs present in Aplysia sensory neurites and in mouse hippocampal dendrites, by cDNA library construction and microarray analysis. 2) Characterize the effect of synaptic stimulation on mRNA localization. These experiments will examine the effect of synaptic stimulation on mRNA localization in cultured Aplysia neurons, in cultured mouse hippocampal neurons, and in adult mouse hippocampus. 3) Characterize the effect of synapse formation on mRNA localization. The aim of these experiments is to study how synapse formation alters mRNA localization in cultured Aplysia neurons. 4) Investigate the translational regulation of synaptically localized mRNAs and the function of local protein synthesis during plasticity. The aim of these experiments is to determine how synaptic stimulation regulates translation of localized mRNAs and to use gene specific silencing (RNA interference) to examine the function of local translation in synaptic plasticity. These studies should elucidate mechanisms underlying activity-dependent regulation of gene expression in neurons. In addition, they should elucidate the function of local translation during synaptic plasticity. From a larger perspective, they are likely to advance our understanding of the many physiological and pathological phenomena in the brain involving neuronal plasticity, including learning and memory and diseases in which learning and memory is altered.

DESCRIPTION (provided by applicant): Synaptic plasticity, the modulation of synaptic strength and structure with experience, is central to many physiological and pathological states, including learning and memory and a variety of neuropsychiatric diseases. While much research has focused on the contributions of transcription and translation to long-lasting forms of synaptic plasticity, regulated protein degradation through the ubiquitin proteasome pathway (UPP) provides another common means of controlling the protein composition of cells. Because the UPP can regulate protein concentration with exquisite spatial and temporal control, it could readily contribute to aspects of plasticity over variable time domains and in a synapse-specific manner. The aim of this R21 exploratory grant is to test the hypothesis that the UPP functions locally at the synapse to regulate synaptic strength and growth. We have found that inhibition of the UPP with several bath applied proteasome inhibitors in Aplysia sensory-motor cultures produces long-lasting increases in synaptic strength, enhances serotonin-induced plasticity, and leads to the growth of new synaptic contacts between sensory and motor neurons. We now propose to determine whether the UPP at the synapse functions locally to modulate synaptic efficacy and structure. In specific aim 1, we will differentiate between the role of the UPP at the synapse and at the cell body by locally perfusing proteasome inhibitors at the synapse or the soma of cultured Aplysia sensory-motor neurons and measuring the effect on synaptic strength and on serotonin-induced longterm plasticity. The spatial restriction of proteasome inhibition will be monitored using a GFP reporter construct that is normally degraded by the proteasome. In specific aim 2, we will inhibit the proteasome at the synapse and determine the effect on synaptic structure by time-lapse microscopy. The experiments outlined in this proposal will allow us to elucidate a role for the UPP at the synapse during neuronal plasticity. They are consistent with the purpose of the R21 mechanism in that they will also allow us to obtain a sufficient body of data to write an R01 application, whose aims we anticipate will involve identification of the substrates of the UPP that regulate synaptic efficacy and growth, and analysis of whether and how these substrates are modulated by stimuli that produce long-lasting synaptic plasticity.

[unreadable] DESCRIPTION (provided by applicant): The goal of this R21 exploratory grant is to systematically develop the use of RNA interference (RNAi) in Aplysia neurons. Electrophysiological and behavioral studies in Aplysia have delineated the circuitry mediating simple forms of learning and memory in the animal. Cultured sensory-motor neurons from Aplysia have provided a model system for elucidating many of the molecular and cell biological mechanisms underlying learning-related synaptic plasticity. These mechanisms have been found to be generalizable to learning-related neuronal plasticity across species. While Aplysia offers many experimental advantages for cell biological and electrophysiological studies, it has not been suitable for genetic analyses. RNAi technology promises to transform Aplysia into a system in which genetic, behavioral, electrophysiological and cell biological analyses can be performed both in the animal and at the level of single cells and synapses. The experiments outlined in this proposal are aimed at developing methodologies for the use of RNAi in Aplysia. We will focus on investigating and optimizing 1) the type of RNA used for RNAi-long double stranded RNA (dsRNA) or small interfering RNAs (siRNAs)-- and 2) the method of delivery of the RNAi. To do this, we will target four endogenous Aplysia genes as well as exogenously overexpressed destabilized eGFP. We will determine whether the RNAi effectively silences target genes and whether or not this silencing is specific to the target gene. In addition, our experiments will identify the most efficient means of delivering RNA for RNAi and the best techniques for assaying the efficacy and specificity of RNAi-mediated gene silencing in Aplysia neurons. The results of the proposed experiments will be invaluable to researchers working in the Aplysia model system. From a broader perspective, the ability to use RNAi in Aplysia will generate valuable information about the molecular mechanisms underlying synapse formation, synaptic transmission and synaptic plasticity. This information likely will lead to the identification of potential therapeutic targets for the many neurological and psychiatric diseases in which these fundamental processes are perturbed. We propose to improve methods to study the molecular basis of learning and memory. The technologies we propose to develop will identify genes that are required for learning and in so doing will lead to potential therapies for the many diseases in which learning and memory are altered. Such diseases include mental retardation, age-related memory loss, Alzheimer's disease, drug addiction as well as many neuropsychiatric diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Synaptic plasticity, changes in the strength of connections between neurons with experience, provides a mechanism for information storage in the brain. Long-lasting forms of plasticity have been shown to require RNA and protein synthesis, indicating that signals can be transported from the synapse, where they are generated, to the nucleus, where they are converted into changes in gene expression. The extreme polarity of neurons and the significant distances that can exist between distal synapses and cell soma present a unique set of challenges to nucleocytoplasmic trafficking. The aim of this proposal is to delineate the role of the active nuclear import pathway in transporting signals from synapse to nucleus during long-lasting forms of learning-related synaptic plasticity. In this pathway, proteins bearing nuclear localization signals (NLSs) are recognized by a nuclear transport adaptor, called importin alpha, which then binds a nuclear transporter called importin betal. Importin betal docks the heterotrimeric complex at the nuclear pore and mediates its translocation into the nucleus. We plan to study importin-mediated nuclear transport, using both dissociated mouse hippocampal cultures and acute hippocampal slices to study various aspects of synaptic plasticity. In our first aim, we will determine whether importins are localized to the synapse and subsequently translocate following stimuli that lead to transcription-dependent plasticity. In the second aim, we propose to identify synaptically localized proteins that are transported to the nucleus following synaptic stimulation. In the final aim, we will determine how the importin-cargo complex is assembled at the synapse and what cell biological pathways are involved in the translocation of this complex to the nucleus. Relevance to public health: Understanding the mechanisms whereby synaptically generated signals trigger changes in gene expression in the nucleus during memory formation provides a means of identifying therapeutic targets for a variety of disorders including mental retardation, age-related memory loss, Alzheimer's disease, epilepsy, drug addiction as well as many neuropsychiatric diseases.

DESCRIPTION (provided by applicant): Abnormalities in RNA processing and translation within neurons likely contribute to Autism Spectrum Disorders (ASD). For example, mutations in Fragile X Mental Retardation protein, an RNA binding protein involved in RNA trafficking and translational regulation at the synapse, represent the most common single gene cause of ASD. More recently, human genetic studies identified the RNA binding protein Rbfox1 (also known as A2BP1) as another candidate autism gene. Rbfox1 binds a well-defined RNA sequence, (U)GCAUG, and functions in the nucleus as a regulator of RNA splicing. Rbfox1 itself is alternatively spliced into nuclear and cytoplasmic forms. We show that cytoplasmic Rbfox1 localizes to dendrites and synapses in mouse hippocampal neurons. Many neuronal RNAs contain conserved (U)GCAUG stretches in their 3' untranslated regions (3'UTRs), and our data indicate that cytoplasmic Rbfox1 regulates the stability and/or translation of these mRNAs. In addition, our experiments suggest that Rbfox1 regulates translation by interfering with microRNA (miRNA)-mediated translational repression of some target mRNAs. Many of the mRNA targets of cytoplasmic Rbfox1 have been identified as targets of Rbfox1 in a module of genes that are down regulated in brains of autistic subjects. We propose that dysregulation of mRNA stability and translation in neurons is an important component of the pathophysiology of ASD. Our proposal is aimed at 1) identifying the cytoplasmically localized mRNA targets of Rbfox1 and at 2) determining the mechanisms whereby Rbfox1 regulates their stability and/or translation. The results of our proposed studies may reveal fundamental cell biological mechanisms and specific molecular targets that underlie neural circuit dysfunction in neurodevelopmental disorders, including Autism Spectrum Disorders.

DESCRIPTION (provided by applicant): The mission of the UCLA-Caltech MSTP is to promote the education of outstanding physician-scientists. To fulfill this mission, our current goals are to 1) recruit exceptionally bright and accomplished students who exhibit an unusual degree of passion for scientific knowledge and a life-long commitment to research and leadership, 2) help guide admitted students toward outstanding training environments that encourage individual thinking and provide students with the tools needed to develop into accomplished physician-scientists, 3) provide a comprehensive support system to meet the trainees' needs and 4) play an increasingly prominent role in guiding the career development of undergraduate students from under-represented ethnic groups and disadvantaged backgrounds. To accomplish these goals as effectively as possible, the UCLA-Caltech MSTP is run by two equal Co-Directors, three Associate Directors, and a strong administrative team, all of whom are deeply committed to the Program. The Program is structured for an average of eight years of study. An integrated, problem-based medical school curriculum is particularly well suited for MSTP students, due to increased time for independent exploration and increased emphasis on research advances that contributed to current knowledge of disease etiology, diagnosis, and treatment. For their Ph.D. research, students choose mentors from a wide array of science and engineering Ph.D. Programs. The MSTP's commitment to excellence was perhaps most apparent when UCLA and Caltech entered into an affiliation agreement fifteen years ago. This affiliation, which provides an opportunity for two students per year to perform their thesis research at Caltech, not only has increased the number of outstanding mentors available to students, but also appears to have increased the Program's visibility and recruitment success. Substantial institutional support from the David Geffen School of Medicine at UCLA and from Caltech has permitted an increase in the size of the MSTP, with 97 students currently enrolled in the program. The MSTP derives great benefit from recent dramatic improvements in physical facilities at both UCLA and Caltech, from the financial health of the universities, and from the recruitment of a large number of outstanding new faculty members to UCLA and Caltech.

Hebbian and homeostatic forms of synaptic plasticity require new gene expression for their persistence. For stimulus-induced alterations in transcription to occur, signals must be relayed from sites of synaptic stimulation to the nucleus. Such long-distance retrograde transport poses a unique set of challenges in neurons, where synapses can be located at great distances from the cell soma and nucleus. Electrochemical and calcium- dependent processes allow for extremely rapid signaling between subcellular compartments in neurons. Studies in a number of systems have also indicated that soluble signaling molecules can be transported from the synapse to the nucleus to effect changes in transcription. This proposal is aimed at elucidating the cell biology of synapse to nuclear signaling during long-lasting, learning-related synaptic plasticity in mouse hippocampal neurons. During the past funding cycle, we characterized a role for importin-mediated active nuclear import of synaptically localized transcription during hippocampal long-term potentiation. Synapse to nuclear transport of transcription factors provides a direct means of coupling synaptic activity with changes in gene expression. We focus this continuation proposal on the synapse to nuclear transport of the CREB regulated transcriptional coactivator CRTC1 during activity-dependent plasticity. We have shown that CRTC1 tracks glutamatergic activity in excitatory neurons to inform the nucleus about synaptic events. It is actively transported into the nucleus from stimulated synapses, and undergoes profound changes in post-translational modification in response to stimulation. Moreover, while glutamatergic stimuli trigger CRTC1 nuclear import, neuromodulatory inputs that elevate intracellular cAMP regulate the persistence of CRTC1 in the nucleus. We have generated a number of reagents to study and manipulate CRTC1 in neurons and now propose to use these to perform an in-depth analysis of the cell biology and function of its synapse to nuclear signaling during long-term synaptic plasticity of mouse hippocampal neurons. Towards this end we propose three specific aims directed at answering three sets of questions: 1) How does CRTC1 travel from synapse to nucleus; 2) How does CRTC1 nuclear import alter gene expression? How do stimulus-induced change in CRTC1 phosphorylation alter its nuclear transport and downstream transcription? and 3) How does neuromodulation regulate CRTC1-mediated gene expression? The answers to these questions will provide insight into the cell biology of learning-related gene expression, and into the particular function of CRTC1. The results of our studies are relevant to a spectrum of neuropsychiatric disorders, and to cognitive disorders (such as mental retardation, Alzheimer's Disease and age-related memory loss) in which long-term memory is impaired.

Project Summary The release of catecholamines (including both norepinephrine and dopamine) in response to novelty/unexpected reward and during times of stress or strong emotion has long been known to enhance long-term memory. Excessive catecholamine release may lead to stress-related memories such as Post- Traumatic Stress Disorder or, in the case of stimulant use, to unusually strong explicit contextual memories that can hinder efforts to abstain by producing strong context-specific ?reminders? of drug use. Synaptic plasticity?activity-dependent changes in the strength of synaptic connections between neurons?underlies many forms of memory. In addition to activity-dependent synaptic plasticity, brain circuits undergo metaplasticity, a process that alters the ability of neural circuits to undergo subsequent forms of synaptic plasticity. Catecholamines are known to induce a form of hippocampal metaplasticity that enhances long-term potentiation (LTP) of synapses in the hippocampus, a brain structure essential for encoding long-lasting explicit memories. Recent studies have reported that optogenetic activation of catecholamine-producing neuronal terminals originating from the locus coeruleus (LC) enhances hippocampal LTP and memory. In our studies, optogenetic stimulation of LC afferents at a frequency that mimics LC firing during stress (5 Hz) is sufficient to enhance LTP of CA3 to CA1 synapses in acute hippocampal slices. Previous studies have shown that catecholamine-induced metaplasticity of hippocampal LTP is dependent on transcription, but the specific program of transcription that is induced by catecholamines has not been characterized. In this R21 exploratory grant, we propose to undertake an unbiased analysis of the transcriptional program that is induced by LC activation. Towards this end, we combine optogenetic activation of LC afferents to the hippocampus with next- generation RNA sequencing approaches to identify changes in transcription induced by release of endogenous catecholamines. In Aim 1, we will conduct nascent RNA sequencing in excitatory CA1 neurons in hippocampal slices in which LC afferents have been photostimulated at frequencies that mimic stress amd that enhance LTP. In Aim 2, we follow up on our findings that LC activation triggers nuclear accumulation of the CREB co- activator CRTC1, performing CRTC1 chromatin-immunoprecipitation sequencing (ChIP-seq) to determine whether and how release of endogenous catecholamines alters DNA binding by CRTC1. Together, the results of our experiments will provide insight into the molecular mechanisms underlying catecholamine-induced hippocampal metaplasticity, opening a path towards developing treatments for drug addiction, PTSD and other forms of maladaptive memories.