Paul Brehm - US grants

DESCRIPTION (provided by applicant): The long term interests of my lab have centered on establishing the respective roles of presynaptic calcium channels and postsynaptic cholinergic receptors in neuromuscular transmission. My lab has traditionally relied on use of developing Xenopus neuromuscular junction where, among other advantages, direct cellular analysis of synapse function can be assessed through voltage-clamp of pre and postsynaptic cells. During the past funding period we initiated study of Zebrafish neuromuscular junction, which is functionally similar to Xenopus, but has the additional advantage that behavioral consequences of synaptic dysfunction are directly ascertained. We have identified Zebrafish mutant lines that exhibit locomotory dysfunction resulting from defects in neuromuscular transmission. Two lines exhibit myasthenia gravis-like symptoms in the form of use-dependent fatigue, and the symptoms can be partially rescued by inhibitors of cholinesterase function. Recently, we have shown the defect in one mutant line results from a defective rapsyn gene, and we have succeeded in rescuing the behavioral defect in the mutant fish. Another line with defective synaptic receptor density shows similar progressive weakness but outgrows the behavioral phenotype with age. Both lines exhibit profound synaptic depression at the nerve-muscle junction when compared to wild type animals. We will use these two lines to establish the mechanisms through which alterations in postsynaptic receptor densityleads to the observed synaptic depression. A similar depression can be recorded at Xenopus nerve-muscle synapses where both pre and postsynaptic cells can be voltage clamped. Using this preparation we will determine whether alterations in presynaptic release or postsynaptic receptor desensitization are responsible for synaptic depression. The four specific aims that are proposed merge the collective strengths of in vitro use of Xenopus with behavioral mutants of zebrafish, in order to investigate the mechanisms underlying synaptic plasticity at the neuromuscular junction.

The goal of the tissue culture core is to facilitate the use of tissue culture by all of the project members. The members relying on this core facility have a wide range of expertise in tissue culture technology and the tissue culture core will serve as a resource for disseminating this collective knowledge amongst investigators. The general reliance on this methodology is reflected in the dependence of all 6 projects.on cell culture. The core will provide training and support for all aspects of cell culture, from primary dissociated cells to transfected cell lines. To best serve the members, this tissue culture core will occupy a single central laboratory space which is dedicated solely to core members. This will alleviate duplication costs for equipment, supplies, and salaries. By nature of its central location, the core facility will also enhance scientific interaction among project members. The philosophy of the core facility is that the director (Brehm) and research assistant (Kennedy) provide the central resources common to all projects and specific training for individual projects. The specific services provided by the core are as follows: a) Training in cell culture techniques b) Maintaining inventory of supplies and anticipating specific project requirements c) Providing quality control for media and Nerve Growth Factor. d) Optimizing serum conditions by testing lots on individual cell types e) Maintaining cell lines, re-establishing frozen lines, mailing transfected lines, and assisting in primary dissociations. f) Regular maintenance of all tissue culture equipment such as hoods, incubators, and baths.

Propagation of fast electrical signals in the nervous system is mediated by the activation of voltage-dependent sodium channels. Molecular studies have indicated the existence of structurally distinct forms of sodium channels. The structural diversity can arise from 3 sources: distinct alpha subunit genes, alternative splicing of the alpha subunit mRNAs, and different associations between alpha and accessory subunits. Little information is available on the structural basis of functionally diverse sodium channels in neurons. Project 1 addresses this gap in our knowledge. We have isolated cDNA coding for a new alpha subunit type expressed exclusively in the PNS, termed PN1. Using both macroscopic current and single channel measurements we will determine the complete repertoire of functional properties of the PN1 and brain type II/IIA alpha subunit channels in Xenopus oocytes, in the presence and absence of beta1 subunit RNA. The effects of addition of poly A+ mRNA will also be examined to determine possible contributions of as yet unidentified accessory subunits or proteins which may be important in vivo. Functional properties of the type II/IIA and PN1 sodium channels will be examined in situ in mutant lines of PC12 cells engineered to express either type II/IIA or PN1 sodium channels. The properties of the endogenous channels in neurons will be compared to identified sodium channel types expressed in exogenous cells using the different alpha and beta1 subunit RNAs. Antibodies and toxins will be tested for specific effects on either Type II/IIA or PN1 functioning in an effort to identify specific probes for peripheral sodium channel types. The molecular and electrophysiological data will provide the framework for understanding sodium channel regulation in the peripheral (PNS) and central (CNS) nervous system (PNS) relying, in part, on the ability to discriminate between CNS- and PNS-type sodium channels.

DESCRIPTION (provided by applicant): Despite the great advances made in our understanding of synapse development at the neuromuscular junction, the roles of several key proteins remain unclear. The proposed experiments will use the unique advantages offered by zebrafish genetics and development to identify the roles of rapsyn, acetylcholine receptor, MuSK and beta-dystroglycan in synapse formation in vivo. Zebrafish offers tremendous advantages over mammalian in vitro and in vivo systems. Studies using in vitro mammalian expression systems and cultured myotubes have both been hampered by the inability to study bona-fide synapses. The in vivo studies have been limited by the inability of the mouse knock-outs to survive through the period of synapse formation. In the case of receptor knock-out, for example, the consequences are so severe that no studies have been able to address the consequences of receptor-less development. By contrast, functional knock-outs of acetylcholine receptor, rapsyn, and MuSK have been identified in mutant lines of zebrafish. These fish were originally identified on the basis of swimming abnormalities that reflect direct consequences of knock-outs of each of these key synaptic proteins. This analysis is possible in zebrafish because, unlike their mammalian counterparts, these mutant animals die well after synapse formation is completed and the animal behavior can be assessed. To date our findings have revealed most unexpected roles for the acetylcholine receptor and for rapsyn in governing synapse development and function. In particular, we have found that the receptor likely plays a key role in localizing rapsyn to the synapse and rapsyn plays a critical role in regulating receptor function. Additionally, our studies have provided new predictions for human neuromuscular diseases, one of which has been confirmed on patients afflicted with rare forms of myasthenia gravis. We are confident that this model system will, through its many unique advantages, resolve some of the outstanding paradoxes involving the roles of signaling molecules in synapse formation.

DESCRIPTION (provided by applicant): We are cloning a gene from an echinoderm that has the potential of representing the first long-term marker of calcium and neuronal activity. The gene encodes a protein that is responsible for the bioluminescence in certain species of brittlestars and has remarkable fluorescence properties. Only after calcium-triggered bioluminescence does this protein adopt a bright green fluorescence that persists indefinitely! The protein is present in nerves and it marks the active neurons in vivo for days after stimulated to produce luminescence and indefinitely if the tissue is fixed for histology. Like GFP, the fluorescence of this protein is intense and slow to bleach so it does not require low light level measurements. It has spectral properties very similar to GFP so that all of the resources developed for this standard can be used for ophiopsilin. Once cloned, we should be able to target the protein in any cell type of interest and examine the history of activity. Because virtually all cells use calcium signaling pathways it would serve as a universal indicator. Ideally suited for this type of approach are nerve, muscle and secretory cell types. It may be possible to use this indicator in the central nervous system to trace the circuitry at a single cell level. Thus, this calcium latch protein has the potential to revolutionize the manner in which we image the history of cellular activity in healthy and diseased tissue. Project Narrative We are exploiting the unique features of a naturally occurring calcium dependent fluorescence indicator in the neurons of echinoderms. The protein responsible for bioluminescence latches into a persistent GFP like fluorescence after binding calcium. As such it represents the first long-term marker of calcium and membrane activity. Because it is genetically coded it can be targeted for expression to any cell type and the fluorescence even persists through histology. As such it could be serve as a read out history of membrane activity and calcium signaling in healthy or diseased tissue. PUBLIC HEALTH RELEVANCE: We are exploiting the unique features of a naturally occurring calcium dependent fluorescence indicator in the neurons of echinoderms. The protein responsible for bioluminescence latches into a persistent GFP like fluorescence after binding calcium. As such it represents the first long-term marker of calcium and membrane activity. Because it is genetically coded it can be targeted for expression to any cell type and the fluorescence even persists through histology. As such it could be serve as a read out history of membrane activity and calcium signaling in healthy or diseased tissue.

DESCRIPTION (provided by applicant): There is currently no way to correct disease-causing mutations in the nervous system without altering the physiological level of the endogenous mRNA. This is a serious challenge because haplo-insufficiency or two-fold over-expression is often sufficient to cause neurological disorders. An example is Rett Syndrome, caused by mutations in the Mecp2 gene. Mecp2 gene duplication, as well as loss-of-function, results in severe disease. We propose to meet the challenge by harnessing the natural ability of RNA editing enzymes to site-specifically fix mutations in endogenous mRNAs. As a target for gene therapy, mRNA offers advantages over DNA. Messenger RNA is cytoplasmic, a readily available substrate, and unlike DNA in which 'mistakes' will be maintained, mRNAs turnover, replenishing the therapeutic target. Our new approach, Site Directed RNA Editing (SDRE), offers enormous untapped potential for correcting mutations, particularly those affecting the nervous system, and for exploring fundamental biological questions. RNA editing, which occurs through adenosine or cytidine deamination, is a natural process. When it occurs within the coding sequence of an mRNA specific codons can be re-coded to produce an altered amino acid sequence. For example, excitatory neurotransmission absolutely depends on the editing of a single adenosine within AMPA-type glutamate receptor mRNAs. Recognizing the power of this activity, we engineered a hybrid modular adenosine deaminase. When used in combination with a small antisense guide RNA we can site-specifically target any chosen adenosine. A similar strategy will be employed to create a site-directed cytidine deaminase. Unlike established therapies that focus strictly on regulating gene expression, SDRE can also fine-tune protein function. Inherited mutations that underlie diseases due to amino acid substitutions or premature stop codons can be corrected, and second-site suppressor mutations that restore function can be selectively introduced. We will demonstrate the power of SDRE within the context of neurobiology, but importantly, it applies to any biological system.

DESCRIPTION (provided by applicant): This project centers on our recent discovery of separate sources of intracellular calcium for synchronous and asynchronous modes of synaptic transmission at the zebrafish neuromuscular junction. Asynchronous release, in particular, has received much attention, as a result of its newly appreciated role in synaptic plasticity. However, the mechanisms causal to preferential release through the asynchronous mode is presently a hotly contested subject. Our lab was the first to identify a calcium sensor specific to asynchronous release, and we now find that, additionally, synchronous versus asynchronous release is mediated by two distinct voltage- activated calcium channel isoforms. The synchronous release utilizes a P/Q type calcium channel and the asynchronous release utilizes a voltage dependent calcium channel isoform that awaits molecular identification through Aim 1 experiments. In this proposal, we present much unpublished data in support of differential locations of these two channel isoforms, with the synchronous channel in the synaptic bouton and the asynchronous isoform located extrasynaptically at axonal branch points. Establishing its location in the cell forms the basis of aim 2 experiments. Additionally, the combined technologies of in vivo calcium imaging, exocytosis indicator lines, and paired recordings have pointed to activation of a calcium wave that is activated by the asynchronous calcium channel isoform and propagates through active release of internal calcium to reach the synapse. This is causal to the signature delayed onset and persistence of asynchronous release at nearly all studied synapses. Numerous reports of calcium waves exist for both the neuromuscular junction and central neurons but, until now, the physiological significance vis-a-vis synaptic transmission has remained obscure. In aim 2 we will determine the molecular basis of calcium release through activation of the extrasynaptic calcium current establish the links to the asynchronous release process. Finally, in Aim 3 we will use the collective advantages of zebrafish to test the requirement for each proposed signaling molecule in the physiology of spontaneous, synchronous and asynchronous release modes.

? DESCRIPTION (provided by applicant): Despite advances in identifying defective genes underlying neuropathologies, how these defects underlie symptoms is not known, and this gap is a formidable stumbling block for therapeutics. A case in point is Rett Syndrome (RTT), a severe neurological disease in girls. The disease is due to sporadic mutations in the transcription factor, MeCP2, but why loss of MeCP2 causes neuropathology is enigmatic. Further, RTT holds a unique place in neurological disease because key symptoms are reversible in mice by expressing MeCP2 throughout the brain or just in astrocytes, the prominent glial cell type in brain. The rescue opens the door to therapeutic approaches, but requires a better understanding of what is deficient in RTT and precisely what is rescued upon MeCP2 restoration. Traditional approaches, such as microarray analysis, have focused almost exclusively on individual gene transcript changes, primarily in neurons. This approach has not led to clear answers about the functions of MeCP2 or the cellular basis of the disease, in part due to cellular heterogeneity. It also ignores work indicating a role for astrocytes in contributin to symptoms. In no case is there a molecular benchmark for extent of rescue. Our goal is to attack these issues head on by focusing specifically on rescue of RTT symptoms by astrocytes. Here, we perform a co-expression network analysis, using RNA seq combined with membrane proteomics, on brain and on pure populations of cells sorted from murine brain (aim 1). With an eye towards human-specific therapies, we identify the molecular and cellular consequences of loss and gain of MeCP2 in neural cells from RTT patient IPSCs, and test predictions from these studies in human/mouse xenografts (aim 2). Finally, we test a new hypothesis (aim 3), based on recent preliminary results, that reduced excitatory signaling between astrocytes and neurons may be a functional outcome of the alterations in molecular and membrane properties of these cells (aims 1 and 2).

The advantages provided by the zebrafish neuromuscular synapse have led to discoveries that have greatly advanced our basic understanding of neurotransmission and neuromuscular diseases. Driving all our studies has been our development of paired motoneuron muscle patch clamp recordings, which has only been possible in zebrafish. We recently used this methodology to explore frequency-dependent depression, one of the most prevalent forms of synaptic plasticity, and obtained evidence for heterogeneity among release sites. The existence of functionally distinct classes of release sites remains an unexplored territory among vertebrates, calling for development of probes adequate to detect their function. Also unique to zebrafish neuromuscular junction is a small number of release sites that are well separated from one another. To probe each of these release sites independently we created a transgenic line wherein the calcium indicator GCaMP6f is fused to postsynaptic rapsyn. Due to the high calcium permeability of the nicotinic receptor, we have been able to optically track release of single quanta from individual motoneuron release sites. With this powerful detector, we now outline a series of experiments that will test for release site heterogeneity and its role in synaptic depression. We also utilize optical quantal analysis to further address an outstanding question asking whether the synchronous, asynchronous and spontaneous modes of synaptic transmission work through the same or different release sites. Finally, to identify the structural counterparts of functional heterogeneity, we are collaborating with Christian Stigloher, an expert in high-resolution tomographic analysis of zebrafish neuromuscular synapses. Individual fast and slow synapses will be identified on the basis of optical quantal analysis and subjected to tomographic reconstruction to test for differences in cytomatrix components of the active release site. Only through the many unique features offered by this vertebrate cholinergic synapse is it possible to perform the proposed experiments to decipher synaptic depression and associated use dependent fatigue, common to almost all myasthenias.