Oswald Steward - US grants

The experiments of this proposal seek to define some of the cellular and molecular mechanisms of synapse regeneration following denervation and synapse formation during development. The central hypothesis is based on our discovery of protein synthetic machinery (comprised of polyribosomes and associated membranes) beneath synapses on CNS neurons; this machinery is particularly prominent during the initial formation of synaptic connections during normal development and increasing under synaptic sites during reinnervation. We believe that the polyribosomes produce proteins that are required for the construction or consolidation of the synaptic junction and that the local synthesis of protein at the postsynaptic site plays a role in regulating innervation. This 5 year project has 5 Specific Aims: 1) We will determine at what stage in the mutation of the synaptic junction the polyribosomes are most prominent and evaluate whether the positioning of the polyribosomes at postsynaptic sites is determined by a program of differentiation within the postsynaptic cell, by the presence or activity of the presynaptic terminal, by an interaction between pre- and postsynaptic elements during synaptogenesis, or by factors that are extrinsic to the pre- and postsynaptic neurons. Quantitative electron microscopic techniques will be used to evaluate polyribosome distribution on neurons in vivo and in vitro while varying the extent of afferent innervation and the rate of synaptogenesis. 2) We will evaluate whether synapse replacement following lesions depends on local synthesis of protein at the postsynaptic site; we will evaluate whether synaptogenesis is blocked when synthesis in dendritic laminae is inhibited during the period of synapse construction. 3) We will evaluate whether afferent activity affects protein synthesis in dendrites. 4) We will use pulse-labeling techniques in vitro in conjunction with subcellular fractionation to determine whether some of the proteins that are produced at the postsynaptic site are constituents of the synaptic junctional complex. 5) We will determine what types of mRNA are present at the postsynaptic site by isolating mRNA from dendrites and evaluating whether the message is capable of directing the synthesis of functional neurotransmitter receptors or channels dendrite specific cytoskeletal proteins or constituents of the psd. 6) We will define how the arborization pattern of individual axons of the crossed temporodentate pathway changes during sprouting using the PHA- L method.

An extensive body of research indicates that following central nervous system injury, there is often a naturally- occurring compensatory reorganization of surviving neural pathways. The behavioral consequences of such reorganization are largely unknown, although some types of reorganization seem to contribute to functional recovery. In these cases, however, it is not clear exactly how the reorganization contributes to the recovery that occurs. Dr. Steward's research program, which has been supported by the National Science Foundation for the past 13 years, has evaluated the functional consequences of the reorganization which occurs in the hippocampus of the rat after damage to the entorhinal cortex, one of the principle inputs to that brain region. The currently proposed experiments will evaluate three hypotheses: (1) that fibers reinnervating the hippocampus contribute to behavioral recovery as a result of the information they convey, (2) that behavioral recovery results from a return of excitatory driving and the consequent increase in activity of the denervated cells, and (3) that behavioral recovery depends on both the restoration of excitatory drive and on the information conveyed by the reinnervating fibers. This research will determine whether neural activity plays a role in inducing synaptic reorganization. It represents an important step toward understanding how to manipulate the response of the nervous system to injury.

DESCRIPTION: This application seeks funds for the continuation of a project aimed at studying the biological signals that induce a reactive state in astrocytes in vivo. The model system that has been exploited during the current funding period is primarily the hippocampal formation of the rat. Astrocytic responses are induced by lesions, either surgical (aspiration of entorhinal cortex) or electrical (seizures or spreading depression). The response that has been measured is the up-regulation of GFAP and GFAP mRNA. During the current funding period, the principal investigator has determined that electrolytic brain lesions lead to an astrocytic response, measured as a marked increase in GFAP.This increase is not attenuated by blocking all neuronal activity in the affected area (by injection of TTX), and the TTX block alone has no effect. At the same time "intense stimulation" of entorhinal cortex (i.e., electrical activity) does lead to a modest increase in GFAP. The timing of the increase has been studied in detail as has its unilateral vs. bilateral nature. The role of "diffusible gliotrophic substances" has been studied by placing gel plegets directly on the cerebral cortex of experimental animals. Pledgets that have been pretreated with saline (controls) induce no response. By contrast, the experimental gels that had undergone a 24 hr incubation in a lesion induced cavity in a donor rat induced a substantial GFAP response. The role of degeneration debris in stimulating astrocytic responses was measured in a clever system, the Ola mouse strain (an inbred strain in which Wallerian degeneration occurs on a much delayed time course). The GFAP response in the denervated region was delayed by the same degree as the degeneration. The role of high [K+] in the GFAP induction was investigated by putting K+-gelfoam on the cortex and getting a huge response. There was an increase in cerebral cortex as well, but on a slower time base. In each, the extent of the GFAP rise was related to whether the resulting seizures were accompanied by spreading depression.The sprouting of cholinergic afferents, a normal part of the hippocampal response to entorhinal cortex lesions was blocked by seizures induced in the early post-lesion period.

9310367 Steward Neurons in the brain are interconnected with one-another forming many complex circuits. When a neuronal circuit is activated by certain patterns of activity, there is a change in the strength of the connections between nerve cells in that circuit. The most typical change is that it subsequently requires less input to activate the circuit. This is called long-term potentiation (LTP). LTP is especially prominent in brain regions that are important for learning and memory and is, therefore, believed to be involved in the mechanism for information storage in the nervous system. Because the changes induced during LTP endure for weeks or more, it has been proposed that this change results from the growth of new connections between neurons. This project will test this hypothesis directly. Quantitative electron microscopy will be used to determine if the induction of LTP leads to the formation of new connections between neurons. The results of this project will potentially offer new insights into the mechanisms of learning and memory. *** connected with one-another forming many complex circuits. When a neuronal cir ! ! F 2 2 8 Times New Roman Symbol & Arial " Univers (WN) " h 9 e c % c % + 8 Steve McLoon, IBN William Proctor, IBN

9415255 Steward This is an award to support a FASEB Summer Research Conference entitled "Sorting and Intracellular Transport of RNA," to be held on the campus of the University of California at Santa Cruz, July 16-21, 1994. This is, to the best of our knowledge, the first conference on this topic to be held anywhere. This is a timely conference on an important and emerging area in cell biology. ***

DESCRIPTION (applicants' abstract) The experiments of this proposal continue studies by the applicants of the mechanism through which particular mRNAs come to be localized in dendrites. This project is the outgrowth of their discovery of a selective positioning of protein synthetic machinery beneath synaptic sites on CNS neurons. They now call these elements synapse-associated polyribosome complexes. The central hypotheses that have guided this work are: 1) that SPRCs synthesize certain key protein constituents of the postsynaptic site, including some of the important functional molecules of the synapse; 2) that this local synthesis is critical for the construction of the synaptic site and for modifying existing synapses; 3) that the synthetic activity of synapse associated polyribosomes is regulated in part by synaptic activity. There is evidence that these processes play a key role in synapse function. Indeed there is emerging evidence that a common human genetic disorder (Fragile X mental retardation syndrome) may cause neuronal dysfunction as a result of disrupting gene expression at the synapse. The current experiments are based on studies of the selective targeting of the transcript of a unique immediate early gene (IEG) to dendrites. This gene, termed activity related cytoskeleton associated protein (Arc) is strongly induced by physiological activity like other IEGs, yet is unique because its mRNA is rapidly delivered throughout dendrites. Even more important, activation of particular synapses causes the mRNA to localize selectively in activated dendritic domains. The Specific Aims are: 1) To characterize the process through which synaptic activity causes mRNAs to localize selectively in activated dendritic segments; 2) To define the synaptic mechanisms (i.e. the receptors that play a role) and the post-receptor signal transduction pathways that underlie the selective targeting of Arc mRNA to particular dendritic domains; 3) To define the address markers within the mRNA that determine the selective targeting; 4) To develop strategies to disrupt the selective localization of mRNA in neurons in vivo so as to allow an assessment of the role of mRNA sorting in neuronal function; 5) To define the RNA Transport Packets (RTPs) in which Arc mRNA is conveyed from the cell body into dendrites and determine whether mRNAs that are targeted to active dendritic domains come to be localized at synapse-associated polyribosome complexes.

[unreadable] DESCRIPTION (provided by applicant): This program will provide research-training opportunities for medical students who may elect to pursue careers in basic or clinical neurological sciences research. The program will provide summer research fellowships to potential clinician scientists early in their education. In addition to providing hands-on laboratory experience, the program will inform students of clinical neuroscience careers, and educate them in patterns of research support. Training will be on a full-time basis for 2 months in the summer. The program is designed to encourage highly competitive students to enter the Medical Student Scholars Program or MD/PhD program by pursuing a Ph.D. during their medical training. Trainees will be selected based on an application filed initially at the end of their first year of medical school. Trainees will be assigned to a laboratory of one of the participants in the program, where they will carry out mentored research. Preceptors have been selected based on their active research programs in areas related to the clinical neurosciences, especially stroke, trauma, and neurodegenerative disorders. Together, the laboratories provide a broad range of research opportunities ranging from basic molecular biological/biochemical approaches through community-based human research programs. At the end of the first summer's research program, the students will present the results of their research at a fall medical student research symposium, which will also involve research presentations by students in the MSTP program. Students selected for appointment will be encouraged to obtain multiple periods of short-term research training during the years leading to their clinical degree. Such appointments may be consecutive or during "off-quarter" periods. Where appropriate, trainees will be encouraged to present their results at national meetings, and support will be provided for students to attend such meetings. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We have recently discovered a form of regenerative sprouting of cortico-spinal tract (CST) axons following spinal cord injury in mice. This regenerative sprouting, and perhaps other forms of axon regeneration, are enhanced in certain lines of mice in which genes encoding axon growth inhibitory molecules (Nogo) have been deleted. The local regenerative growth in normal mice and the enhanced growth in genetically modified mice is of the sort that could restore descending input to neuron pools mediating motor function near the site of injury. This form of growth could be especially important following lesions at the cervical level, where growth over a even single segment could restore function to motoneurons supplying critical muscle groups of the forelimb. These mice provide the opportunity to ask a key question that was previously impossible to address-whether limited CST regeneration is sufficient to restore voluntary (CST-mediated) motor function. In the present project, we will define the nature of the local axonal growth responses of CST axons that occur after spinal cord injury in normal mice, and test the hypothesis that this growth mediates recovery of function in segmental motor circuitry. We will quantify the time course and extent of regenerative CST sprouting following injuries at both the thoracic and cervical levels and in the same animals, assess whether recovery of hindlimb and forelimb motor function respectively occurs during the period of regenerative growth. These analyses will allow us to correlate the nature and extent of regenerative growth with functional outcome. To test the hypothesis that recovery is due to reinnervation, we will carry out parallel experiments in mice carrying a mutation that delays Wallerian degeneration of synapses (WldS), in which reinnervation is also delayed, and use genetically modified mice that exhibit enhanced regenerative sprouting and bona fide long-tract regeneration (lines of mice that lack Nogo) to precisely define the relationship between CST regeneration recovery of motor function below the level of the injury. Together, these studies will provide a critical data base regarding the growth capacity of cortico-spinal tract axons in mice, which will be essential for future studies involving genetically modified mice.

This contract is to identify one of two NINDS [unreadable]Facility of Research Excellence in Spinal Cord Injury (FORE-SCI) sites to conduct research to replicate promising studies that could lead to new and effective treatments for SCI. The objective of this contract is to independently review and replicate novel treatments for SCI and to compare the efficacy of different treatments in a standardized environment with a minimum of variability in surgery, animal care, outcome evaluation and cellular analyses.

DESCRIPTION (provided by applicant): Chronic orofacial pain is a common clinical syndrome lacking specific and effective therapeutic agents due to the fact that cellular mechanisms of chronic orofacial pain are poorly understood. Based on our preliminary data from a trigeminal nerve injury model and in non-orofacial pain models, we hypothesize that trigeminal nerve injury induced thrombospondin-4 (TSP4) expression in trigeminal ganglia (TG) and associated brainstem/upper cervical spinal cord (Vc/C2) that causes sensory neuron hyperexcitability, and abnormal synaptogenesis in the trigeminal complex in the spinal cord. These changes underlie the transition from trigeminal nerve injury to chronic pain development. In this proposal, we plan to identify the critical domain(s) of TSP4 in mediating behavioral hypersensitivity and spinal neuron hyperexcitability. Viral driven TSP4 expression in TG or Vc/C2, respectively, will be used to identify the site of the TSP4's action in chronic pain processing. We will perform confocal and electron microscopy to determine the extent of abnormal synaptogenesis in the nerve injury models. In addition, the influence of descending modulatory pathways and voltage-gated-calcium channels on TSP4-mediated behavioral hypersensitivity and dorsal horn neuron hyperexcitability will be studies using respective drugs. The influence of TSP4 on sensory neuron excitability, calcium channel activities, and intracellular calcium signaling will be studied in isolated neurons or intact TG from nerve injury models, or after TSP4 treatment. To determine if TSP4 induces behavioral hypersensitivity and dorsal horn neuron hyperexcitability through its interactions with its receptor, the calcium channel alpha-2-delta-1 subunit (Cava2d1), in a sensory neuron specific manner, Cava2d1 conditional knockout mice with selective deletion of Cava2d1 in subpopulation of sensory neurons will be used for these studies. The final goal of the proposed studies is to identify the peripheral and/or central mechanisms underlying TSP4-mediated transition to chronic pain states after trigeminal nerve injury. PUBLIC HEALTH RELEVANCE: Chronic orofacial neuropathic pain often evolves from a preceding injury of the peripheral nerves, which is accompanied by initial nociceptive pain. Identification of the mechanisms underlying this transition would be critically valuable in preventing or reversing it. We plan to study a novel pathway mediated by injury-induced expression of thrombospondin 4 in mediating orofacial neuropathic pain. Completion of this study will provide important information for identifying a novel mediator for the transition to chronic orofacial pain after nerve injury.

DESCRIPTION (provided by applicant): This project is dedicated to discovering ways to induce regeneration of the corticospinal tract (CST) and recovery of motor function after spinal cord injury (SCI). The CST is the pathway that is responsible for the ability to move voluntarily. Damage to the CST as a result of a spinal cord injury is the reason people are paralyzed. The present project is based on recent extraordinary discoveries that the CST can be induced to regenerate following spinal cord injury by targeting molecular pathways that control cell growth in development, specifically phosphatase and tensin inhibitor (PTEN). PTEN is responsible for shutting down the type of protein synthesis that is critical for cell growth during development. PTEN acts by blocking the mammalian target of rapamycin, (mTOR), so deletion of PTEN releases inhibition on mTOR, which in turn allows the cell to synthesize proteins that are critical for cell growth. Importantly, the same molecular pathways are also the key to allowing neurons to regenerate their axons following injury. Based on this, recent studies have shown that genetic deletion of PTEN in mice allows neurons to mount a robust regenerative response. Most critically, our studies demonstrate that when PTEN is deleted in neurons in the cerebral cortex, the neurons that give rise to the CST are able to robustly regenerate their axons after SCI. The fact that regeneration of the CST can be successfully induced provides us with an unprecedented opportunity to address a question that is central to regeneration research-whether it is possible to induce regeneration in a therapeutically-relevant time frame and whether inducing CST regeneration is enough to restore circuits of sufficient specificity to allow some degree of restoration of motor function. The project uses anatomical and physiological methods to assess the degree to which regenerated axons grow along normal tracts, to normal targets, form functional synapses, and contribute to motor function. Pre-clinical experiments will also assess whether it is possible to down-regulate PTEN in a therapeutically-relevant time frame and using non-genetic interventions.

Facilities of Research Excellence (FORE) in Spinal Cord Injury Replication Studies

This project is dedicated to discovering ways to induce regeneration of the corticospinal tract (CST and recovery of motor function after spinal cord injury (SCI). The CST is the pathway that is responsible for the ability to move voluntarily. Damage to the CST as a result of a spinal cord injury is the reason people are paralyzed. The project is based on discoveries that the CST can be induced to regenerate following spinal cord injury by targeting molecular pathways that control cell growth in development, specifically phosphatase and tensin homolog (PTEN). PTEN is responsible for shutting down the type of protein synthesis that is critical for cell growth during development. PTEN acts by blocking the mammalian target of Rapamycin, (mTOR), so deletion of PTEN releases inhibition on mTOR, which in turn allows the cell to synthesize proteins that are critical for cell growth. We have shown that genetic deletion of PTEN in mice and knockdown of PTEN in rats with AAVshRNA allows CST neurons to mount a robust regenerative response, which is accompanied by recovery of motor function. The new project is based on exciting and novel recent discoveries that targeting PTEN in adult nerve cells induces a state of youthful vigor in which there is perpetual growth in addition to an ability to regenerate after injury. The overall goal of the project is to determine the cellular and molecular mechanisms of this growth-enabled state and identify the genes that are turned on or shut off during growth and regeneration. Defining the pattern of gene expression that underlies the growth-enabled state in nerve cells will identify targets for future therapeutic interventions to promote regeneration and repair after injury and potentially protect nerve cells from degeneration in neurodegenerative disorders.

Foundations of Rigorous Neuroscience Research Project Abstract Scientists at all career stages must be well-trained in the foundations of rigorous and reproducible research. Institutions and individual laboratories play a critical role in training researchers in aspects of rigorous laboratory practices, such as experimental design, data analysis, and reporting. However, systemic issues inherent to research culture often hinder scientists? abilities to apply these principles to their work. Factors collectively referred to as the sociology of science include biases that can influence experimentation and interpretation; practices related to data collection, management, and sharing; and incentives that underlie career advancement and the stability of scientists? research programs. The Society for Neuroscience (SfN) proposes to develop resources that focus on the sociology of science through the multimodal training series ? Foundations of Rigorous Neuroscience Research. The proposed program will build on SfN?s previous training efforts in scientific rigor and pursue two specific aims: (1) Develop multimodal platforms that promote awareness of barriers and solutions related to practicing rigorous and reproducible neuroscience research at all career stages. Through in- person workshops, a virtual conference, and online programming (videos, podcast series, case studies, written materials, and online discussions), training will focus on topics such as sources of bias that can influence scientific judgments, data sharing practices, and career advancement incentives. (2) Develop exportable training modules ? digital toolkits ? with quickly digestible, accessible resources to inform and empower researchers at all career stages to enhance rigor and reproducibility in their laboratory practices and professional activities. With content derived from programming under Specific Aim 1, SfN will produce and disseminate four digital ?toolkits? tailored to scientists in distinct career stages: graduate students, postdoctoral trainees, early-career independent investigators, and established investigators. Resources in each toolkit will include formats such as discussion guides, tip sheets, videos, brief downloadable PowerPoint presentations from experts, and ?citable statements? on best practices and opinions that users can deploy at their home institutions to influence opinion. With more than 1,000 disorders of the brain and nervous system resulting in more hospitalizations and lost productivity than any other disease group, it is critical to develop a diverse and well-trained neuroscience research workforce to find new and innovative ways to prevent and treat neurological illnesses. The research in which this workforce engages must be rigorous and reproducible, as other areas of science and scientific discovery will benefit from new discoveries. SfN seeks to develop resources that promote awareness of barriers and solutions related to conducting rigorous research, and to empower researchers at all levels to enhance rigor in their laboratory and professional practices.