1985 — 1990 |
Benowitz, Larry Ira |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. S07Activity Code Description: To strengthen, balance, and stabilize Public Health Service supported biomedical and behavioral research programs at qualifying institutions through flexible funds, awarded on a formula basis, that permit grantee institutions to respond quickly and effectively to emerging needs and opportunities, to enhance creativity and innovation, to support pilot studies, and to improve research resources, both physical and human. |
Molecular Bases of Neural Connectivity @ Mc Lean Hospital (Belmont, Ma)
In the goldfish, a severed optic nerve will regenerate within a few months of being cut, reforming the original pattern of connections between the eye and the brain and restoring visually guided behaviors. The present study is aimed at identifying specific proteins which are involved in such aspects of this process as neurite outgrowth, contact guidance, intercellular recognition and synaptogenesis. Double labeling and electrophoretic separation will be used to contrast the protein synthesis patterns of a regenerating and the contralateral intact eyes from individual goldfish. The studies will examine proteins in various phases of axoplasmic transport, using the axon as a column to separate different functional groups of proteins from each other according to their transport velocities. The first studies will focus on the rapidly transported proteins, since it is this phase which contains vesiculated membranous material that becomes incorporated in the growing tips and, later on, the axon terminals. Our preliminary studies have already identified several proteins transported in this phase whose labeling is greatly increased (greater than 40%) during regeneration (85 and 110,000 daltons), and others whose labeling is greatly decreased (20-37,000) from day 29 of regeneration to day 62. Two-dimensional electrophoretic methods will be used to better separate and characterize these proteins and, using preparative methods, to purify them. Eventually, we will raise antibodies against the species of interest to use for immunochemical mapping and for examining the physiological effects of manipulating the proteins. Isotope incorporation methods will be further used to determine which particular carbohydrate groups characterize the proteins and to contrast proteins differentially transported to different topographic regions of the tectum (i.e., chemo-affinity markers). An in vivo procedure will also be developed for studying the binding characteristics of retinally synthesized proteins to cells of the optic tectum.
|
0.933 |
1987 — 1989 |
Benowitz, Larry Ira |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gap/B50: Molecular Marker Neuronal Growth @ Mc Lean Hospital (Belmont, Ma)
neuropeptides; neurogenesis;
|
0.933 |
1990 — 2012 |
Benowitz, Larry Ira |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. S07Activity Code Description: To strengthen, balance, and stabilize Public Health Service supported biomedical and behavioral research programs at qualifying institutions through flexible funds, awarded on a formula basis, that permit grantee institutions to respond quickly and effectively to emerging needs and opportunities, to enhance creativity and innovation, to support pilot studies, and to improve research resources, both physical and human. |
Molecular Bases of Neuronal Connectivity @ Children's Hospital Boston
In order for damaged pathways to regenerate after injury, nerve cells must begin to express gene products required for the reconstruction of their axons; this in turn is influenced by the extracellular milieu provided by non-neuronal support cells. Lower vertebrates are able to regenerate their optic nerve after injury and recover vision fully, a phenomenon that may enable us to understand the molecular and cellular changes that underlie growth and plasticity in the vertebrate central nervous system. We will utilize biochemistry and gene cloning methods to identify neuronal and non-neuronal molecules that are critical for the regeneration of the goldfish optic nerve. Aim 1 will identify portions of the molecular cascade involved in the expression of the best characterized growth-associated protein, GAP-43. Our preliminary evidence indicates that the expression of GAP-43 is controlled largely at the post-transcriptional level, and involves the binding of specific proteins to sequences in the 3'untranslated end of the messenger RNA that may serve to protect it from degradation by nucleases. We will isolate these proteins and identify the nucleotide sequences to which they bind. Aim 2 will isolate and sequence a less well-characterized growth-associated protein of the optic pathway, GAP-24. We will compare the mechanisms that regulate its expression with those found in Aim 1 to gain a broader understanding of the cascade of events occurring within the neuron as it regenerates its axon. Aim 3 focuses on a protein secreted from the non-neuronal sheath cells of the nerve that may serve as a trigger for the molecular changes studied in the other two sections. We will combine classical protein purification methods, monoclonal antibody technology partial amino acid sequencing, cDNA library screening, antibodies and probes to identified growth factors, and several bioassay systems to isolate and sequence this trophic factor. Together, these studies represent an integrated approach to understanding the cellular and molecular biology underlying regeneration in the visual system, knowledge that may eventually be applied for inducing visual recovery in man.
|
1 |
2001 — 2003 |
Benowitz, Larry Ira |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Mechanisms of Corticospinal Tract Regeneration @ Children's Hospital Boston
DESCRIPTION (provided by applicant): The corticospinal tract (CST) is essential for voluntary control of the body's distal musculature. The absence of effective treatments to restore this pathway after injury has devastating consequences for victims of spinal cord damage. In animal studies, progress has been made in getting injured CST axons to regenerate by altering the extracellular environment, though to date, the number of fibers that grow past an injury site remains small. Recent experiments in our lab show that the purine nucleoside inosine activates an intracellular mechanism in neurons that leads to extensive axon growth. In mature rats with unilateral transections of the corticospinal tract, inosine infused into the normal sensorimotor, cortex induced uninjured cortical pyramidal cells to sprout axon collaterals that crossed over to the denervated half of the spinal cord, and in some instances formed anatomically appropriate synapses. Following up on these observations, Aim 1 will examine whether transected corticospinal tract axons can be induced to regenerate by treating their cell bodies with inosine, while at the same time using neural stem cells and/or olfactory ensheathing cells to provide a cellular environment at the lesion site conducive to axonal growth. In Aim 2 we will retrogradely label the pyramidal cells of the rat's sensorimotor cortex prior to surgery and then, after various experimental treatments, use fluorescence-activated cell sorting to isolate the neurons that give rise to the corticospinal tract. Through the use of microarrays (gene chips), we will identify genes associated with axon growth, growth cone guidance, and other aspects of corticospinal tract regeneration. These studies will provide basic information on the biological mechanisms that regulate CST axon growth and will help us develop novel approaches to restoring, function after injury to this pathway.
|
1 |
2004 — 2008 |
Benowitz, Larry Ira |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Adaptive Rewiring of the Mature Brain After Injury @ Children's Hospital Boston
DESCRIPTION (provided by applicant): Recovery from stroke or traumatic brain injury is limited in part by the inability of mature neurons to reorganize their connections significantly. Such reorganization may require both positive stimuli to activate neurons' intrinsic growth state and methods to overcome inhibitory signals that normally suppress growth. Inosine, a purine nucleoside, activates a cellular signaling pathway that regulates the expression of genes important for axon outgrowth. In mature rats, inosine stimulates the reorganization of major cortical pathways after transecting a specific fiber tract or after a unilateral stroke. This reorganization allows projections from the intact hemisphere to partially reinnervate brainstem and spinal cord areas that have lost their normal inputs, and results in improved behavioral outcome. Aim 1 will investigate whether agents that help overcome inhibitory influences on axon growth can enhance the effects of inosine on neural reorganization and behavioral outcome. Aim 2 will investigate whether intensive training after unilateral brain damage itself promotes axonal reorganization, and whether such training will augment the effects of inosine treatment. Finally, there is a limited time window after brain injury in which inosine treatment must begin in order to achieve long-lasting benefits. Aim 3 will attempt to identify genes whose expression enables neurons to respond to inosine during this "window of opportunity", as well as genes that are expressed as a result of inosine treatment during this period. This will be done using laser-capture microdissection to isolate cortical pyramidal cells and microarrays to identify genes whose expression is selectively altered. The second part of Aim 3 will use a gene therapy approach to investigate the effects of altering the expression of genes related to axon growth in cortical pyramidal cells. These studies will increase our understanding of plasticity in the mature brain and potentially enable us to improve functional outcome after injury.
|
1 |
2014 — 2015 |
Benowitz, Larry Ira |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Optic Nerve Regeneration: Translational Studies @ Children's Hospital Corporation
Summary The optic nerve cannot regenerate if injured, leaving victims of traumatic or ischemic nerve damage or degen- erative diseases such as glaucoma with permanent visual losses. We have recently identified ways to partially reverse this situation by activating the intrinsic growth state of retinal ganglion cells (RGCs), the projection neu- rons of the eye. When exposed to oncomodulin (Ocm), a growth factors produced by inflammatory cells, RGCs lacking the pten gene and having elevated levels of cAMP are able to regenerate injured axons through the en- tire optic nerve, across the optic chiasm, and into appropriate target areas, where they form synapses and par- tially restore visual responses. We now propose to develop ways to translate these findings into methods that are suitable for clinical application. We will develop and test adeno-associated viruses to express Ocm, elevate intracellular levels of cAMP, and counteract cell-intrinsic and cell-extrinsic inhibitors of axon growth. These studies will increase our ability to augment regeneration in the optic nerve, potentially helping many victims of traumatic or degenerative visual disorders. The methods and reagents developed here may also be useful for restoring neural circuits in other parts of the CNS. !! ! !
|
0.964 |
2015 — 2019 |
Benowitz, Larry Ira Rosenberg, Paul Allen [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Zinc Is a Critical Regulator of Cell Death and Axon Regeneration After Cns Injury @ Children's Hospital Corporation
DESCRIPTION (provided by applicant): Zinc has been shown to have multiple important and distinct effects on synaptic transmission and has been implicated as a critical mediator of neuronal injury. We have now discovered a previously unrecognized role for zinc as a major suppressor of axon regeneration and cell survival following axonal injury in the central nervous system (CNS). Under normal conditions, neurons in the adult CNS cannot regenerate damaged axons, placing severe limitations on the amount of recovery that can occur after spinal cord injury, stroke, and other types of neurological damage. The optic nerve is an integral part of the central nervous system (CNS) that has been widely used to investigate CNS regeneration due to its accessibility, anatomical simplicity, and functional importance. Although the projection neurons of the eye, the retinal ganglion cells (RGCs), are normally unable to regenerate injured axons, this inability can be partially reversed in mice by treatments that activate RGCs' intrinsic growth state and by counteracting cell-extrinsic inhibitors of axon growth. However, these manipulations result in only limited regeneration, suggesting that our current understanding of the factors that regulate neurons' regenerative potential in the CNS is incomplete. Our preliminary data show that within 6 hours after injuring the optic nerve, there is a dramatic elevation of Zn2+ in the inner plexiform layer (IPL) of the retina, which contains synaptic contacts from amacrine and bipolar cells onto the dendrites of RGCs. This increase represents a very early event following optic nerve damage. Over the next few days, Zn2+ accumulates in RGC somata. Importantly, agents that chelate extracellular Zn2+ provide enduring protection against RGC death and have a dramatic effect on these cells' ability to regenerate injured axons through the optic nerve. We therefore hypothesize that Zn2+ is a major suppressor of the regenerative potential of axons after nerve injury as well as a cause of neuronal death. The specific aims are to: 1) Characterize the timing, localization, and mechanism of Zn2+ accumulation following optic nerve crush; 2) Determine whether Zn2+ regulates axon regeneration via histone deacetylases; and 3) Characterize the pathways by which Zn2+ suppresses, and chelation enhances, RGC survival. These studies will add greatly to our understanding of the role that Zn2+ plays in the normal and injured nervous system, and may lead to treatments to help improve outcome after CNS injury.
|
1 |
2018 — 2020 |
Benowitz, Larry Ira Rosenberg, Paul Allen |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Interneuronal Signaling Network Governs the Fate of Retinal Ganglion Cells After Optic Nerve Injury @ Boston Children's Hospital
Alzheimer's disease (AD) is a common devastating neurodegenerative disease. As with other chronic neurodegenerative diseases, the pathogenesis remains unknown, even though gene mutations causing familial AD were discovered over 20 years ago. The neuropathology of AD is characterized by extracellular amyloid plaques, comprised primarily of fibrillar Abeta 1-40 and Abeta 1-42 proteins derived from amyloid precursor protein (APP) and intracellular neurofibrillary tangles comprised primarily of tau protein. Abeta protein deposition is promoted by interaction with zinc. Abundant evidence supports the hypothesis that amyloid plaques themselves are not toxic but rather the toxicity is primarily due to the soluble oligomeric Abeta 1-42 fragment of APP (oA?). Several of the mutations that cause AD affect the processing of APP to increase the production of oA?, consistent with the amyloid hypothesis. The eye is part of the central nervous system (CNS), and an extension of the amyloid hypothesis has developed over the last 15 years with the recognition that the retina is affected by the deposition of Abeta in AD, and that Abeta may play a role in retinal diseases involving degeneration of retinal neurons such as glaucoma and age-related macular degeneration. Optic nerve injury (ONI) is another important retinal disorder, and the consequences of transection of axons in the optic nerve is death of retinal ganglion cells and failure of axon regeneration. There has been little or no study of the role of Abeta production and deposition in the neurodegeneration and failure of regeneration following ONI, or how ONI might affect APP processing. In preliminary experiments, using an antibody against Abeta proteins, we have found Abeta aggregates at the site of injury to the optic nerve forming weeks after injury in normal animals but not in animals with a knockout of the zinc transporter ZnT3. We hypothesize that APP processing is highly influenced in the retina by ONI and, that APP processing may play important roles in the neuronal loss and regenerative failure following ONI. To test this hypothesis, we propose the following aims: Aim 1. Characterize APP expression and Abeta deposition in the normal mouse retina and following ONI. Aim 2. Assess the effects of altering zinc homeostasis and tetanus toxin on the processing of APP after ONI. Aim 3. Assess the effect of blocking Abeta production on neuronal survival and regeneration failure after ONI. RELEVANCE TO ALZHEIMER'S DISEASE: Our understanding of the processing of APP is incomplete. The cause of abnormal production of Abeta in sporadic AD is unknown as is the basis for the toxicity of oA? to neurons. APP processing, Abeta production, and Abeta aggregation are influenced by oxidative stress, zinc and copper homeostasis, neuronal activity, axonal transport, and growth state of axons all of which are important in the pathophysiology of ONI. The ONI model offers a uniquely accessible in vivo model of the CNS for the study of APP processing, the factors modulating this processing, and the toxicity of oA?. It is hoped that the work outlined in this proposal will ultimately lead to new approaches for treating AD.
|
1 |