Vitaly Klyachko - US grants

DESCRIPTION (provided by applicant): Loss of Fragile X mental retardation protein (FMRP) due to mutations in the Fmr1 gene causes Fragile X syndrome (FXS), the most common form of inherited mental disability and the leading genetic cause of autism. Despite two decades of intensive studies characterizing FMRP functions at synapses, the molecular basis of FXS remains poorly understood. FMRP is thought to function primarily as a regulator of protein synthesis in dendrites, and research to date on FXS has concentrated on the postsynaptic effects of FMRP loss leading to altered long-term synaptic plasticity (LTP). While LTP is thought to play important roles in learning and memory, short-term plasticity (STP) is widely believed to control other essential neural functions such as information processing, working memory and decision making. STP dysregulation may thus play a significant role in the cognitive impairments in FXS. However, STP dysregulation in FXS has received little attention and is poorly understood. Moreover, whether FMRP plays a role in synaptic mechanisms controlling STP remains largely unknown. Our recent studies revealed that loss of FMRP causes marked STP defects and abnormal information processing in excitatory hippocampal synapses. We further demonstrated that FMRP loss causes abnormal increase of a major calcium-dependent form of rapid presynaptic enhancement, known as augmentation, and that the calcium influx in presynaptic neurons is also increased. We therefore hypothesize that altered presynaptic calcium dynamics represents a major underlying cause of STP defects in the absence of FMRP. Importantly, our results indicate that at least some of the underlying mechanisms of these defects have a cell-autonomous presynaptic origin and arise from a novel FMRP function that is not related to its traditional role in protein translation. We propose to combine electrophysiological and imaging approaches with pharmacology and molecular biological tools to (i) determine how loss of FMRP alters calcium dynamics and STP; (ii) Examine the functions of FMRP mediating these defects; and (iii) Determine the impact of synaptic abnormalities associated with FMRP loss on computations performed by canonical neural circuits. We anticipate that these studies will provide fundamental new insights into the function of FMRP in synapses and a novel way to approach synaptic dysfunction in FXS. PUBLIC HEALTH RELEVANCE: Fragile X syndrome, which is attributed to loss of Fragile X mental retardation protein (FMRP), represents the most common inherited form of mental retardation and the leading genetic cause of autism, yet the mechanisms underlying the cognitive impairments in Fragile X syndrome remain poorly understood. We propose to investigate novel functions of FMRP leading to dysregulation in short-term plasticity, which is widely believed to play important roles in the brain's ability to analyze information. Our studies will provide fundamental new insights into the function of FMRP in synapses and will help elucidate the molecular mechanisms of FXS.

DESCRIPTION (provided by applicant): Transcriptional silencing of the Fmr1 gene encoding Fragile X mental retardation protein (FMRP) is the most common inheritable cause of mental disability, known as Fragile X syndrome (FXS). FXS is also characterized by a high co-morbidity with autism and epilepsy. Extensive research efforts have thus focused on elucidating the functions of FMRP at synapses to uncover the molecular basis of FXS. FMRP was identified as an RNA-binding protein regulating local protein synthesis in dendrites, and research to date on FXS has concentrated extensively on the translation-dependent roles of FMRP in dendritic function. Yet despite two decades of intensive studies the molecular basis of FXS remains incompletely understood. We have recently identified important functions of FMRP beyond the dendritic compartment; most notably the critical presynaptic roles of FMRP in regulating action potential (AP) waveform and synaptic transmission. Specifically, our results demonstrate that FMRP regulates neurotransmitter release in excitatory hippocampal neurons via modulation of AP duration in a cell-autonomous presynaptic manner. These presynaptic actions of FMRP are translation-independent and are mediated specifically by FMRP interaction with the large-conductance calcium-activated K+ (BK) channels. Loss of FMRP causes reduced BK channel activity and excessive AP broadening, leading in turn to elevated presynaptic calcium influx, increased neurotransmitter release and short-term plasticity (STP) during repetitive activity. Information theory-based analysis indicates that these defects associated with FMRP loss cause marked abnormalities in the ability of synapses to transmit information. Our observation that FMRP modulates AP duration both in the hippocampal and cortical pyramidal neurons suggests that BK channel-dependent regulation of presynaptic function by FMRP may be a widespread phenomenon that could play a role in the pathophysiology of FXS. Indeed, our current studies suggest that pharmacological or genetic manipulations targeting BK channel function can reduce seizure susceptibility and rescue several synaptic and behavioral deficits caused by FMRP loss. Yet the mechanisms by which FMRP modulates the activity of BK channels and the roles of FMRP-BK channel interaction in cognitive and behavioral abnormalities in FXS remain unknown. The proposed studies will span from single-channel to behavioral analyses to address these critical questions. Our results are expected to provide major new insights into pathophysiology of FXS and open new avenues for drug development and intervention.

Neural communication is governed by the release of neurotransmitter-containing vesicles at the synaptic active zone (AZ). Several fundamental forms of neurotransmitter release are present at synapses, including synchronous mono- and multi-vesicular release, asynchronous and spontaneous release. Each of these release mechanisms plays important and distinct roles in synaptic development and function. However, despite several decades of research, how these canonical forms or release are organized and regulated in central synapses is poorly understood. This includes some of the most fundamental features of release, including the number, spatial organization and reuse of release sites supporting different forms of release, all of which remain largely undetermined because of the extremely small size and relative inaccessibility of central synapses to conventional recording techniques. Moreover, how the spatiotemporal properties and reuse of the release sites are regulated by neural activity is largely unknown. To overcome these limitations, we developed a nanoscale-resolution imaging approach that in combination with a pH-sensitive fluorescent indicator genetically tagged to the vesicle lumen, allows us to resolve individual vesicle fusion events at the AZ with ~27 nm precision. With this approach we have uncovered the presence of multiple distinct release sites in central synapses and demonstrated that their spatiotemporal properties are regulated by neural activity. By complementing this approach with computational single-molecule tools we are also able to robustly detect all other canonical forms of release. Our approach also permits us to visualize and track translocation of individual synaptic vesicles to the AZ, a critical time-limiting step in the refilling of the release sites during neural activity. Here we propose to combine this nanoscale-resolution imaging approach with advanced computational, genetic and pharmacological tools to study, at a single-vesicle level, the mechanisms governing organization and regulation of the canonical forms of neurotransmitter release at individual central synapses. We will further define the mechanisms governing vesicle translocation to the release sites and their activity-dependent regulation. These studies will provide major new insights into fundamental mechanisms of synaptic function.

Direct examination of presynaptic processes has historically been limited by the resolution constraints of conventional light microscopy. As a result, much of what we know about vesicle movement, fusion, and recycling relies on inferences from indirect electrophysiological and/or biochemical assays, or from electron micrographs that reflect a single instant of a dynamic system. The long-term goal of my research program is to understand the fundamental mechanisms of synaptic transmission at central synapses, including details of spatiotemporal dynamics under normal conditions, and what disruptions lead to disease states. Current projects in the lab address two central knowledge gaps. First, we directly probe and track dynamic presynaptic processes in living tissue by applying our own novel, nanoscale resolution imaging technology. Using this approach, we will, for the first time, visualize these processes at the level of single synaptic vesicles within identified synapses. We have already made significant contributions using this approach, including the discovery that synaptic vesicle dynamics are active, not passive, and are controlled by actin cytoskeleton and myosin motors. The second major knowledge gap we address is the contribution of presynaptic deficits to pathophysiology of Fragile X syndrome (FXS). FXS is the most common known cause of heritable intellectual disability and autism. Our recent findings have triggered a necessary shift in the field towards considering the contributions of presynaptic mechanisms in addition to postsynaptic mechanisms, thus creating an entirely new array of diagnostic and therapeutic possibilities. Continuing work in this area will focus on linking presynaptic defects with abnormalities at the circuit level and the implications of these abnormalities for behavior and cognition. Sustained funding through this R35 mechanism will support a multipronged approach to these important neurobiological questions that will maximize the potential for synergy and translational impact.