Jerry Silver - US grants

The primary goal of this proposal is to identify factors that guide retinal ganglion cell axons out of the eye and along their stereotyped routes to the brain during development and regeneration. The focus of this research is the neural cell adhesion molecule (NCAM) which appears to be directly involved in the attraction and guidance of pioneering optic axons within specialized regions of their non-neuronal environment. In our initial studies on chick embryos we have found a preformed array of NCAM on neuroepithelial cell endfeet that temporally and spatially foreshadows the route of optic axons (Silver and Rutishauser in press, and appendix 1). Following on these observations, the specific aims of this proposal are designed to further explore (1) the developmental events that lead to the formation and maintenance of the NCAM pathway as well as boundary regions that lack NCAM; (2) how abnormalities of endfeet or NCAM expression can lead to congenital axonal malformations in the retina and optic nerve of ocular retardation and albino mutants, (3) how the modulation of NCAM on endfeet could influence the success or failure of retinal ganglion cell axon regeneration and (4) why intraocular injection of anti-NCAM Fab in the living embryo causes dramatic retinal and optic nerve malfunctions in addition to altering axon pathways. It is hoped that these studies will lead to a better understanding of the role of adhesion mediated cell-cell interactions in normal and abnormal development as well as in regeneration of the vertebrate optic system.

[unreadable] DESCRIPTION (provided by applicant): We have developed a new in vitro model of the glial scar in which inhibitory CS-PG in a crude gradient more closely resembles that which occurs in vivo after injury. Classic dystrophic endballs form at the tips of adult sensory axons that become trapped within the gradient. We can now ask, for the first time, a variety of basic questions concerning the biology of the dystrophic growth cone. Three important questions we wish to pursue in aim 1 are: (A) whether the dystrophic state is a peculiarity of adult neurons, (B) whether CNS neurons would respond similarly to the PG gradient environment and, (C) whether dendrites respond differently to the inhibitory gradient than do axons. Finally, (D) we would like to know what molecular changes occur in would- be dystrophic axons that are induced to regrow across the potently inhibitory rim of the gradient via a new combinatorial regeneration stimulating strategy. Our ultimate goal is to devise an optimal strategy in vitro that can be used to overcome growth cone dystrophy and stimulate axon regeneration past the glial scar in vivo. In aim 2, preliminary in vivo results based on a successful regeneration promoting strategy using our in vitro gradient model, have shown evidence that a combination of chronic sterile inflammation induced in the DRG prior to root crush, plus chondroitinase application to the root entry zone at the time of crush, can foster robust regeneration of sensory axons into the spinal cord. We hypothesize that a similar strategy may foster sensory fiber regeneration in a more clinically relevant post-injury model. We propose to study the functional efficacy of these fibers. In aim 3, we will utilize a novel microlesion model of the cingulum in which one can make the smallest lesion possible but still clearly identify only those axons that have been severed and have potentially regenerated. With the microlesion model, we can use the micropipette, that cuts the axons, to inject bridge building cells or other factors upon withdrawal of the pipette from the brain. Preliminary evidence shows that injection of chondroitinase combined with immature astroglia can stimulate regeneration clearly past the lesion. None-the-less, once past the lesion the fibers only grow short distances. Using this model we hypothesize that by also driving the intrinsic growth potential of the regenerating neurons at the vicinity of their cell body, regeneration will be significantly enhanced. [unreadable] [unreadable]

DESCRIPTION: (Adapted from the Applicant's Abstract.) The experiments in this proposal seek to compare and contrast the cellular and molecular mechanisms by which astroglia may inhibit the growth of axons in two regions of the spinal cord: 1) the roof plate, which is a transient boundary to dorsal column axons during development and 2) the glial dome of the dorsal root entry zone, which forms a doorway for afferents into the CNS during development but becomes a boundary to dorsal root afferent regeneration after birth. The applicant and his colleagues plan to correlate the development of the dorsal columns and the later forming dorsal commissure with morphogenesis of the midline glia and with the initial presence and subsequent disappearance of keratin sulfate/chondroitin sulfate glycosaminoglycans, putative boundary molecules that they have localized in this region. Enzyme digestion of the roof plate GAG's in an in vitro slice preparation will test whether dorsal column axons aberrantly cross the midline. For the studies on the DREZ, they plan first to describe the normal cellular and basal lamina elements at the CNS/PNS interface and the molecules that are expressed during the period of axonal ingrowth into the spinal cord. They will then compare these constituents with those of the DREZ in animals with crush injuries of the roots to determine if mechanical or chemical factors may contribute to regenerative failure in this region. Preliminary studies have shown that GAG's found in normal glial barriers such as the roof plate appear in reactive glia following root lesions. The putative astroglial boundary molecules of the roof plate and lesioned DREZ will be investigated in an in vitro assay system where the growing axons will be challenged with combination of putative pathway and boundary cells or molecules in various geometric patterns.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The dorsal column white matter tracts of the adult rat spinal cord have been tested in a novel way to learn whether the purportedly inhibitory glial scar or myelin can promote or hinder axonal regeneration of adult DRG's. This has been done by utilizing a microtransplantation technique which can introduce a small bolus of DRG cell bodies into either the unlesioned or prelesioned dorsal column white matter distal to the site of injury. This procedure allows for introduction of axotomized neurons without causing further inflammation and glial scarring at the site of implantation. The exciting results show that both normal as well as lesioned white matter away from an area of trauma are robustly permissive for long distance axon regrowth, at least for adult sensory axons. However, upon reaching the area of the forming scar, the rapidly regenerating growth cones halt abruptly and become dystrophic within a field of reactive glial matrix. It is suggested that these observations constitute compelling evidence that the glial scar and, hence, inhibitory factors such as proteoglycans at his locale, constitute the major environmental impediment to regeneration in the adult CNS. We propose to utilize the microtransplantation technique in a variety of interesting permutations of the preliminary experiments in order to explore the following questions. (1) Does a critical period exist for regeneration of adult DRG's into pre-degenerated dorsal column white matter? (2) What is the extent of reinnervation of the dorsal column nuclei or the dorsal horn grey matter by microtransplanted DRG's with increasing time after tract injury? (3) Can we develop a combinational strategy for stimulating dystrophic, transplanted DRG axons trapped within a scar, to regenerate through and beyond the glial scar? The long term goals of these experiments are to understand the basic biology that underlies the mechanisms of axon regrowth or its failure within adult white matter and to develop effective bridging strategies that allow adult axons to utilize the massive potential for regeneration which we now know exists beyond the glial scar.

DESCRIPTION (provided by applicant): C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. The paralysis is a result of disruption of bulbospinal inputs from medullary respiratory centers to the phrenic nucleus. However, there exists a very small, latent pathway that descends contralateral to the hemisection and crosses the midline innervating phrenic neurons, essentially bypassing the lesion. Normal plasticity and activation of this so-called "crossed phrenic pathway" (CPP) can slowly restore partial function to the initially paralyzed hemidiaphragm. Motor neurons, including phrenic motor neurons, are enveloped in a perineuronal net that is composed of chondroitin sulfate proteoglycans (CSPGs), extracellular matrix molecules whose glycosaminoglycan sugar side chains create an unfavorable environment for neuronal sprouting and synaptic plasticity. CSPGs in the forming scar also block overt regeneration through the lesion site. Recent studies have demonstrated that the enzyme chondroitinase ABC (ChABC) by cleaving the sugar side chains has the ability to abrogate axon growth inhibition of both the perineuronal net and scar-associated matrix resulting in regeneration and/or sprouting after spinal cord injury. We will test the hypothesis that, by enzymatically modifying inhibitory extracellular matrices in the perineuronal net surrounding phrenic motor neurons ipsilateral to a high cervical hemicordotomy, the sprouting capacity of such remaining fibers from the contralateral side, especially those of the serotonergic system, will be maximized. In additional experiments we will attempt to drive activity in the sprouted fibers using intermittent hypoxia or pharmacological manipulation of cAMP. Finally, we will construct a PNS bridge across the lesion to promote long distance regeneration but also allow regenerated axons to exit the bridge via degradation of inhibitory ECM at the PNS/CNS interface. This multipartite strategy has the potential to lead to an unprecedented amount of functional respiratory plasticity/regeneration and recovery after SCI. PUBLIC HEALTH RELEVANCE: Greater than 50% of all spinal cord injuries (SCI) occur at the cervical level. Respiratory complications following SCI are some of the leading causes of despair and death in the SCI population. In this proposal, we plan to utilize a C2 spinal cord hemisection model of SCI in adult rodents to investigate potential therapies to modulate inhibitory extracellular matrix molecules in an attempt to promote regeneration and plasticity of damaged respiratory pathways. Our strategy has the potential to restore breathing in animals lesioned at high cervical levels.

? DESCRIPTION (provided by applicant): One out of every fifty people living in the United States suffers from some form of paralysis, a total of almost 6 million Americans. This proposal outlines the translational development of a cellular therapeutic already in clinical trials with a novel, regenerative peptide to be used in combination for treatment of spinal cord injury (SCI) and associated dysfunction. Preclinical data from Athersys has demonstrated that acute intravenous administration of bone marrow-derived multipotent adult progenitor cells (MAPC) provides immediate and durable neuroprotection after SCI. Benefit to locomotor recovery was seen beginning at one week post injury, as well as significant sparing of white matter tracts. Dr. Silver and his colleagues have identified the glial scar as a significant impediment to long distance regeneration, specifically inhibitory molecules known as chondroitin sulfate proteoglycans (CSPGs). After discovering the receptor and signaling pathways associated with this pathway, Dr. Silver's laboratory has developed a novel peptide, Intracellular Sigma Peptide (ISP), which modulates the key receptor in this pathway. In a preclinical study, long term subcutaneous treatment with this peptide initiated in the acute phase following spinal cord injury allowed for delayed recovery of locomotion and bladder behaviors following SCI. Interestingly, while ISP treatment resulted in recovery it had no neuroprotective effects. In the 6 month time frame of this Phase I project, we propose to combine the use of MAPC with ISP treatment following a translationally relevant rodent model of contusive SCI with the therapeutic aims of i) promoting enhanced neuroprotection, and ii) enhancing sprouting/regeneration by altering the response of regenerating axons to inhibitory extracellular matrix. We will evaluate the synergistic effects of treatment by analyzing improvements in motor hind limb recovery, return of coordinated control of the lower urinary tract and quantification of neuronal sprouting/regeneration. Dr. Silver and collaborators have been investigating the ability of ISP peptide to induce sympathetic neural regeneration and alleviate arrhythmia following myocardial ischemia/reperfusion injury. This data suggests that ISP could also be utilized in other disorders in which CSPGs play a critical inhibitory role, such as traumatic brain injury, multiple sclerosis and stroke. The fact that MAPC has already proven efficacious in preclinical models of acute myocardial infarction, multiple sclerosis, traumatic brain injury and stroke dramatically expands the impact that this combinatorial therapy could have in the field of regenerative medicine. If MAPC and ISP administration can be shown to be effective in ameliorating SCI associated deficits and dysfunction, this therapy could relieve some of the economic burden on the direct and indirect costs associated with SCI related care, and more importantly provide meaningful improvement to SCI patients. Both treatments are non- invasive and systemic, making them highly attractive therapeutic options for clinical use.

Project Summary: Stopping Bleeding in the Spinal Cord after Injury There are approximately 250,000 people in the U.S. with spinal cord injury (SCI) and over 2 million worldwide. SCI leads to chronic functional deficits and significantly decreased quality of life. The human impact of the injury is enormous and the health care costs associated with the injury are the third highest in the U.S. to The impact of spinal cord injury is devastating to the patients, their families, and the health care system. Spinal cord injury, though, is only one of a group of traumas to the central nervous system (CNS) that includes strokes and traumatic brain injuries. In all of these CNS injuries, hemorrhaging is one of the first steps that occurs in the injury process. This is followed by a secondary injury cascade that causes further damage and includes the influx of inflammatory cells and cytokines that exacerbate injury. We have intravenously administered hemostatic nanoparticles that stop internal bleeding including in the spinal cord after injury. We hypothesize that stopping the bleeding will reduce secondary injury progression, limit inflammation, protect tissue and improve function after spinal cord injury. In this proposal, we will determine the optimal window of time for administering the hemostatic nanoparticles after injury to limit bleeding. We will determine the impact of limiting bleeding on the critical inflammatory cells after injury, particularly neutrophils and the polarization of macrophages to the M1 and M2 phenotypes since these have been shown to be highly altered by injury and most critical to the degenerative versus repair processes. We will then determine the short and long term impacts of limiting bleeding following spinal cord injury on the neural tissue preservation and functional recovery. Together this will give us the basis for a potential therapy as well as a mechanistic understanding of the role of bleeding following spinal cord injury. Ultimately, we will be able to combine this approach with drug therapies we are developing to improve outcomes following SCI. Spinal cord injury is not only a devastating trauma, but it is an excellent model for investigating the role of controlling bleeding on inflammation, neural tissue preservation, and functional recovery. The knowledge we gain from this work will be applicable to the treatment of CNS injuries more broadly including traumatic brain injuries and hemorrhagic strokes. The work proposed here will lead to a new approach to treating SCI and CNS injuries more broadly and has the potential to be a translatable technology that fundamentally alters patient outcomes.

Stroke is one of the leading causes of death and disability worldwide and places a heavy burden on the economy in our society. Current treatment strategies for stroke primarily focus on reducing the size of ischemic damage and on rescuing dying cells early after occurrence. Treatments, such as the use of thrombolytic agents, are often limited by a narrow therapeutic time window. However, the regeneration of the brain after damage is still active days, or even weeks after stroke occurs, which might provide a second window for treatment. Our preliminary data suggests that systemic in vivo delivery of a peptide that blocks a specific receptor mediated inhibitory action of sulphated proteoglycans in the glial scar in stroke animals 24 hours after stroke or 7 days after stroke both improve their functional recovery. We hypothesize that the CSPG signaling pathway is involved in the regulation of neuroregeneration and axonal sprouting after stroke and that modulating the CSPG signaling pathway will lead to better functional outcome in stroke recovery. We will test this hypothesis in both young and aged mice in the proximal transient middle cerebral artery occlusion (MCAo) animal model. Towards this goal, we have developed a proposal that consists of three specific aims. In specific aim 1 and 2, we will investigate the role of the CSPGs signaling pathway in functional recovery in young or aged stroke animals. In specific aim 3, we will examine the mechanisms of neurorepair in stroke animals by combination of genetic and pharmacological modulation with inducible cell type specific RPTP? knockout or ISP peptide treatment. Two main mechanisms of neurorepair including neurogenesis and axonal sprouting in stroke will be analyzed in genetically and pharmacologically modulated stroke animals. Together, the comprehensive analysis of molecular, cellular and behavioral measurements in stroke animals will generate data that will provide insights on the precise role of CSPG signaling in the process of injury-induced neurorepair. The data gained will be directly applicable to developing novel therapeutic interventions in treating cerebral ischemia through the manipulation of the cellular microenvironment in the CNS. We anticipate that the resources and results generated from our study will open new avenues in neuroregeneration research and lead to the identification of molecular therapeutic targets.