Erin M. Schuman - US grants

nitric oxide; long term potentiation; synapses; calcium; neural plasticity; neural transmission; citrulline; hippocampus; arginine; membrane potentials; tetany; ADP ribosylation; neurons; laboratory rat;

Understanding the cellular basis of learning and memory formation in the brain will lead to drug interventions that can ameliorate the memory loss associated with many modern diseases. Long-term potentiation (LTP) in the hippocampus, a brain structure known to be important for human memory formation, involves long-lasting increases in synaptic efficacy which are thought to form the basis of memory storage in the brain. The cellular events that initiate LTP occur in the postsynaptic neuron. In contrast, the longer lasting phases of LTP appear to involve alterations in presynaptic neurotransmitter release, leading to the requirement for a retrograde signal which travels from postsynaptic to presynaptic cells during the initiation of LTP. The main objective of this proposal is to further our understanding of how 3 different putative retrograde molecules function in synaptic transmission and plasticity in the hippocampus. One goal of this study is to characterize the mechanisms by which the recently identified neural messenger nitric oxide (NO), and 2 other diffusible signals, carbon monoxide and arachidonic acid, regulate synaptic transmission and plasticity in the hippocampus. Another aim is to determine whether LTP induction results in the activity of a putative NO target, an ADP-ribosyltransferase. Studies are proposed to characterize NO-stimulated ADP-ribosylated proteins in the hippocampus and to assess their contributions to synaptic transmission and long-term potentiation. Finally, the existence of diffusible signals in the hippocampus predicts that many synapses near the site of signal generation may be influenced. Following from this idea, studies are proposed to characterize the spreading of synaptic potentiation that has been observed between synapses into neighboring CA1 pyramidal neurons.

The ability of the brain to alter information processing by changing the structure and strength of synaptic connections is essential for the successful development and survival of organisms. There is increasing evidence that the central nervous system utilizes some of the same molecular mechanisms during both developmental and adult plasticity. The neurotrophins are a class of signalling molecules that promote the growth and survival of distinct neuronal populations during the development of the nervous system. Our previous work demonstrated that two neurotrophins, BDNF and NT-3, but not NGF, can rapidly enhance synaptic transmission in the CA1 region of the adult rat hippocampus. An as yet unanswered question is whether the rapid changes in hippocampal synaptic transmission are accompanied or followed by structural synaptic changes in the adult animal. The results of many developmental studies highlight the enormous potential of the neurotrophins as signals which promote morphological changes during neuro- and synaptogenesis. We now propose to examine the effects of acute and long-term neurotrophin exposure on neuronal and synaptic morphology in living adult rat hippocampal neurons. To date, most studies have relied on retrospective examination of tissue treated with a neurotrophin or a pharmacological agent known to alter synaptic transmission. We will use two-photon scanning laser microscopy to examine dynamic changes in synaptic structure during and following the period of neurotrophin exposure. To examine the long-term effects of neurotrophin or Trk receptor overexpression on synaptic plasticity, we will use adenovirus(Ad) vectors containing cDNAs for these proteins. We will inject Ad-neurotrophin vectors into hippocampal tissue in vitro (slices) and in vivo. We will examine a variety of measures of synaptic transmission, synaptic structure and animal behavior. To further address the involvement of the neurotrophins in plasticity, we will also use dominant negative Trk constructs to test for effects on synaptic or behavioral plasticity.

DESCRIPTION (provided by applicant): The Gordon Research Conference on Neural Plasticity has been held in alternate years since 1977. We are requesting partial support for the Neural Plasticity Conference planned for July 15 to 20, 2001 at the Salve Regina University in Newport, RI. The Gordon Research Conferences were established to stimulate scientific interchange in an informal setting. Interchange is promoted by the informal nature of the conference, by the large amount of time committed to question and answer periods in the scientific sessions, and by the many opportunities for discussion in other settings, such as meals, poster sessions and structured and unstructured social activities. The Gordon Research Conference rule prohibiting publication or citation of the meetings and presentations promotes open discussion of the latest results and current ideas. This format has been particularly useful for the Conference on Neural Plasticity, a highly interdisciplinary meeting in which the subject of modifiability of the nervous system is explored at molecular, cellular, systems, and computational levels. The 2001 Conference will have 9 scientific sessions and a keynote talk by Dr. Richard Morris, a scientist at the forefront of learning and memory. The scientific sessions will cover a range of topics of current interest in Neural Plasticity, from protein trafficking during plasticity to brain circuits modified by reward systems. The speakers and session chairs are all world leaders in their fields. Full participation by conference attendees is encouraged in several ways. Afternoon poster sessions provide an opportunity for all interested participants to present and discuss their work. Each scientific session will include, in addition to the scheduled speakers, one or 2 short talks by junior participants, selected from the submitted abstracts. This is something that worked very well, and was highly rated by conference participants in 1999. Question and answer periods are generously scheduled. Social events permit new participants to meet speakers and chairs informally. The beautiful environment provided by the Salve Regina campus is an attractant for speakers and participants to linger long after the end of each session. Past participants have found these informal interactions to be the distinct advantage of the Gordon Conference, giving rise to a stimulating and productive meeting.

DESCRIPTION (provided by applicant): It is now clear that protein synthesis is required for animals to establish long-term memories. Misregulation of synaptic tranmission and protein synthesis plays an important role in many diseases. Until recently, it was assumed that all of the proteins required for all neuronal function were made in the cell body. The discovery of polyribosomes at the base of neuronal synapses suggested the possibility that proteins might be synthesized in dendrites in response to synaptic activity. In the previous grant period, we focussed our attention on the development of imaging techniques to visualize protein synthesis in dendrites and discovered several forms of plasticity that are implemented by local protein synthesis. In this proposal we will examine the signaling mechanisms that couple miniature synaptic transmission (minis) to the protein translation machinery. We previously discovered that minis tonically inhibit the dendritic protein synthesis machinery. Loss of minis leads to an upregulation of translation and a rapid homeostatic response. We wish to examine which intracellular signaling pathways couple neurotransmitter receptor activation to the protein synthesis machinery. We will also determine whether there is stimulation-dependent assembly and trafficking of ribosomes in dendrites. Previous observations of ribosomes in dendrites of hippocampal neurons suggest that the translational capacity of synapses in spines is limited by the number of ribosomes available in the dendritic pool. Using biochemical approaches and dynamic time-lapse imaging, we will examine whether polyribosomes might be assembled locally in spines or trafficked to spines. One of big unanswered questions concerns the relative contributions of somatically vs. dendritic synthesized proteins to synaptic function and plasticity. Recently we have developed a technique that can be used to identify the constituents of the dendritically proteome. Building on this technology, we will modify our procedure for labelling proteins in lysates to include fluorescent labelling of proteins in intact cells and tissue slices. We will develop multiple fluorescent tags to separately track proteins made in the cell body and the dendrites.The fate of proteins synthesized in these two compartments will be analyzed over time to address the fractional contribution of somatic vs. dendritic protein synthesis to the synaptic protein population and how these contributions change with synaptic activity and plasticity.

[unreadable] DESCRIPTION (provided by applicant): The pre-and post-synaptic elements of synaptic junctions are linked together by cell adhesion molecules: proteins that play crucial roles in stabilizing, signaling and adjusting the strength of the synapse. The cadherins, 1 type of adhesion molecule, link together 2 adjacent cell membranes by forming homodimers in 1 membrane that interact across the junction (synaptic cleft) with homodimers formed in the opposing cell membrane. In the last decade, much effort has been devoted to understanding the structure and molecular associations of the classic cadherins. Crystallographic and biophysical studies have yielded somewhat conflicting results and, as yet an unclear, picture of the interactions of the cadherin extracellular domain during dimerization. To better understand the dynamics of cadherin interactions we are developing Fluorescence Resonance Energy Transfer (FRET) based sensors to monitor cadherin associations across cellular junctions. FRET is unique in its ability to provide signals that are sensitive to changes in intra-or intermolecular distances in the 1-10 nm range, well below the inherent diffraction limit of conventional fluorescence microscopy. Cadherins will be fluorescently tagged in their extracellular domains; FRET-donor and FRET-acceptors will be expressed in pre-and postjunctional cells and the strength of adhesion will be monitored. It has long been known that and the structural integrity of cadherins is dependent on the local Ca2+ concentration. In the absence of Ca2+, cadherins undergo a reversible loss of their rod-like structure and collapse. Experimental data as well as simulations predict that Ca2+ is dynamically regulated in the synaptic cleft. We predict that alterations in cleft Ca2+ have important ramifications for cadherin-cadherin adhesion and signaling. We will use FRET to monitor dynamically, in living cells, cadherin interactions and conformational changes induced by changes in extracellular calcium and synaptic activity. By virtue of their localization at synaptic cleft and their interactions with cytoplasmic proteins, like beta-catenin, the cadherins occupy a pivotal position that can contribute to the synaptic dysfunction associated with disease. For example, in the absence of presenilin 1, a protein mutated in some forms of Alzheimer's disease, cadherins become destabilized at adherent junctions and fail to localize properly. This mislocalization presumably leads to profound alterations in synaptic structure and function. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In neurons, there is increasing evidence that local dendritic protein synthesis is used to allow individual synapses to respond dynamically to the environmental changes that accompany the establishment, maintenance and plasticity of synaptic connections. Furthermore, it is now well established that the essential components of the translational machinery as well as mRNAs are present at or near synapses and that dendrites can synthesize new proteins. Despite this, only a limited number of locally translated proteins have been identified so far, mainly through candidate-based approaches inspired by a particular laboratory's interest in protein "x" or "y" [unreadable]r by mRNA generation from single neuritic process with subsequent PCR experiments. However, we believe that a focus on translated proteins is essential for an accurate picture of how the synapse and the dendrite can mount a response to local changes in the environment. Current available methods to evaluate changes in the protein composition of a cell or of a cellular compartment rely on the comparison of two proteomes with one another. These differential approaches are successful only when proteins are present in sufficient quantity to be detected and when there are large differences in protein expression levels between the compared proteomes. Here we describe the development of a new high-throughput proteomic profiling technique to isolate and identify the translation products in neuronal dendrites and to determine the quantitative translational capability of dendrites under both basal and stimulated conditions. Specifically, we propose a procedure that uses artificial amino acids for the labeling of newly synthesized dendritic proteins in situ, thereby circumventing problems of traditional approaches. The obtained proteomic datasets will help us to understand the logic of local translation. This technology can be easily applied to many proteomic profiling questions, regardless of tissue type or cellular context. The changes in dendritic proteins that underly the synaptic modifications associated with plasticity, including addiction, can be elucidated using this technique. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The pre-and post-synaptic elements of synaptic junctions are linked together by cell adhesion molecules: proteins that play crucial roles in stabilizing, signaling and adjusting the strength of the synapse. The cadherins, 1 type of adhesion molecule, link together 2 adjacent cell membranes by forming homodimers in 1 membrane that interact across the junction (synaptic cleft) with homodimers formed in the opposing cell membrane. In the last decade, much effort has been devoted to understanding the structure and molecular associations of the classic cadherins. Crystallographic and biophysical studies have yielded somewhat conflicting results and, as yet an unclear, picture of the interactions of the cadherin extracellular domain during dimerization. To better understand the dynamics of cadherin interactions we are developing Fluorescence Resonance Energy Transfer (FRET) based sensors to monitor cadherin associations across cellular junctions. FRET is unique in its ability to provide signals that are sensitive to changes in intra-or intermolecular distances in the 1-10 nm range, well below the inherent diffraction limit of conventional fluorescence microscopy. Cadherins will be fluorescently tagged in their extracellular domains; FRET-donor and FRET-acceptors will be expressed in pre-and postjunctional cells and the strength of adhesion will be monitored. It has long been known that and the structural integrity of cadherins is dependent on the local Ca2+ concentration. In the absence of Ca2+, cadherins undergo a reversible loss of their rod-like structure and collapse. Experimental data as well as simulations predict that Ca2+ is dynamically regulated in the synaptic cleft. We predict that alterations in cleft Ca2+ have important ramifications for cadherin-cadherin adhesion and signaling. We will use FRET to monitor dynamically, in living cells, cadherin interactions and conformational changes induced by changes in extracellular calcium and synaptic activity. By virtue of their localization at synaptic cleft and their interactions with cytoplasmic proteins, like beta-catenin, the cadherins occupy a pivotal position that can contribute to the synaptic dysfunction associated with disease. For example, in the absence of presenilin 1, a protein mutated in some forms of Alzheimer's disease, cadherins become destabilized at adherent junctions and fail to localize properly. This mislocalization presumably leads to profound alterations in synaptic structure and function. [unreadable] [unreadable]