1995 — 1999 |
Marshall, John |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Expression and Regulation of Kainate Receptors
Glutamate, the major excitatory neurotransmitter in the mammalian CNS, gates three major types of ionotropic receptors, named NMDA, AMPA and kainate, based on their pharmacology and molecular properties of the receptor subunits. This proposal will focus on the kainate receptor of which five subunits have been cloned so far: GluR5, GluR6, GluR7, KA1 and KA2. Although these subunits are expressed throughout the brain, fast- desensitizing kainate receptors have only been found in the dorsal root ganglion (Huettner, 1990). In this study, we will investigate the subunit composition of native kainate receptors, how the biophysical properties can be regulated by different subunit forms, and the role of phosphorylation in the modulation of channel properties. Kainate receptors are believed to play a key role in mediating fast synaptic transmission and may regulate Ca2+ entry during development. Therefore, a better understanding of their structure, function and modulation will provide insight into their role in synaptic plasticity and cell death. The developmental expression of kainate receptor subunit RNA and protein will be studied in cerebellar granule cells to identify which isoforms are present in this single neuronal type. Patchclamp experiments will determine the functional properties of these channels recombinantly expressed in HEK293 cells. For this project, we will primarily use cloned kainate receptor cDNAs stably integrated into the genome or transiently transfected into HEK293 cells. The stable expression system provides additional and necessary information to cerebellar granule cells because a single population of identical channels can be studied in the absence of other glutamate-receptor gene products. Biochemical studies on the effect of phosphorylation on kainate receptor expression will be performed on the stable cell lines, as well as cerebellar granule cells to identify functional differences. The advantage of stable cells and cerebellar granule cells is that large quantities of kainate receptors can be obtained for biochemical analysis of the regulation of the level of surface expression and phosphorylation. Working together, the two approaches can provide powerful insights into the molecular mechanisms that underlie kainate receptor function in the CNS.
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1 |
2001 — 2004 |
Marshall, John |
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. |
Mechanism of L Channel-Mediated Neuronal Survival
DESCRIPTION (provided by applicant): The cellular mechanisms regulating survival are complex and comprise parallel and potentially interactive pathways. In neurons, one pathway effective in serum-deprivation is known to commence with increased L calcium channel activity. This application focuses on establishing the intracellular signaling processes by which L channel activity is neuroprotective. Because our recent work has demonstrated that insulin-like growth factor-l (IGF-l) is protective through, in part, the rapid regulation of L channels via an IGF-l receptor-PT 3-kinase-Akt and src signal transduction cascade, we will also determine whether physiological levels of IGF-1 govern particular survival pathways via potentiating L channel-mediated influx. Although the mechanisms remain largely unknown, studies on L channel-induced neuroprotection are now underway. Several groups have shown that calcium influx promotes survival via calmodulin and that survival is associated with a rise in nuclear calcium levels. Because nuclear calcium and calmodulin-dependent kinases (CaMK) promote transcription, calcium-dependent transcription of anti-apoptotic genes has been suggested to mediate IGF-l-neuroprotection in ischemia. Our data indicate that CaMKIV is protective in serum withdrawal via L channel activity and that IGF-1 is protective in hypoglycemia, partially through an L channel-dependent mechanism. Conversely, preliminary data suggest that the transcription factor, C/EBPb, may be pro-apoptotic, antagonizing L channel-dependent survival. Here, we will establish the means by which L channel-mediated influx protects neurons from toxic insults, determining: (1) if L channel activity, IGF- 1 or IGF- i/L channel-modulation are neuroprotective in hypoxia, hyper- or hypoglycemia, (2) if ser/thr phosphorylation of neuronal a1C, the primary subunit of the neuronal L channel modulated by IGF-1, is essential for IGF-1-potentiation, and (3) if L channel-mediated influx, either by direct stimulation or via L channel-potentiation, activates specific nuclear signaling cascades, leading to survival. Together, the proposed experiments will significantly advance our understanding of the mechanisms that regulate neuronal survival in the central nervous system, with particular relevance to diabetic neuropathies and traumatic disorders such as stroke.
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1 |
2001 — 2004 |
Marshall, John |
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. |
Modulation and Targeting of Kainate Receptors
DESCRIPTION: Glutamate gates NMDA, AMPA and kainate ionotropic receptors. For kainate receptors, comparatively little is known about how their function is regulated by subunit composition or anchoring molecules such as postsynaptic density proteins (PSD/SAP). Our recent work demonstrates that PSD-95/SAP9O colocalizes with kainate receptors at synapses, binds the kainate receptor KA2 and GIuR6 subunits, and significantly modifies GluR6 and KA2/G1uR6 physiology (Garcia et al., 1998). We find that tyrosine phoshorylation correlates with the modification of physiological response and that potential regulatory molecules, src and mss4 (a guanine nucleotide exchange factor) preferentially associate with, respectively, KA2 and PSD-95/SAP9O. Furthermore, we also find that both associations are regulated by the KA2-PSD-95/SAP9O interaction, and additionally that SAP97 is unable to bind KA2-containing receptors. Together, the results imply that PSD proteins may function not only in anchoring receptors at synaptic Sites, but also in regulating receptor activity and which receptor subtypes are present in a given site. This proposal will establish mechanisms by which PSD-95/SAP9O regulates kainate receptor function and targeting. We will assess how it regulates receptor activity and if regulation depends on receptor subunit composition, determining agonist affinities, desensitization properties and sensitivity to inhibitors. For potential mechanisms, we will examine how binding of ligands, such as GKAP, to the GK domain may promote the association of the SH3 domain of PSD-95 with KA2, the molecular sites on KA2 and PSD-95 that determine tyrosine phosphorylation, as well as the sites on mss4 and PSD-95/SAP9O that regulate mss4-PSD-95/SAP9O-KA2 interactions will be identified. The physiological effects of these interactions and those of src on KA2 and PSD-95, will be established by expressing active and inactive enzymes with receptor subunits (wild-type vs. mutated). To determine potential mss4 regulation of receptor targeting, the surface expression of receptors and receptor distribution (clustered vs. unclustered) will be evaluated. These studies are expected to provide novel information on how functional properties can be determined through mechanisms controlling the clustering/targeting of receptors of specific subunit composition.
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1 |
2008 — 2009 |
Marshall, John |
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.) |
Development of Neuroprotective Pdz-Domain Inhibitors For the Treatment of Ms
[unreadable] DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is characterized by immunological and inflammatory changes that contribute to demyelination of nerve fibers and neuronal cell death. Although MS is a prototypic primary demyelinating disease, increasing evidence indicates that neuronal degeneration also occurs in MS, contributing to the acquisition of nonremitting clinical deficits. The main features of MS can be reproduced in an animal model, experimental autoimmune encephalomyelitis (EAE). Drugs that reduce the activity of the NMDA or Kainate subtypes of glutamate receptors (NMDAR, KAR), which are expressed both on neurons and oligodendrocytes, suppress EAE. In this proposal, a novel cyclic peptide compound, CN2097, will be tested for its ability to ameliorate MS-like symptoms and pathology. This compound binds the PDZ-domain of the postsynaptic density protein, PSD-95, disrupting its interaction with KAR and NMDAR subtypes. PSD-95 plays a critical role in kainate-mediated excitotoxicity by acting as a molecular scaffold to link KARs to JNK activation. NMDA-induced cell death is linked to the association of PSD-95 with CaMKII, nNos and other primary cell death signaling proteins. In an in vivo retinal toxicity model CN2097 completely blocked NMDA-induced cell death. Aim 1 will examine the signaling pathways and the effects of this compound on attenuating kainate-induced cell death in an in vivo retinal toxicity model, as well as in cortical neurons and retinal oligodendrocytes in vitro. Aim 2 will determine whether CN2097 can increase the survival and function of optic and spinal cord nerves using a MS animal model, myelin oligodendrocyte glycoprotein-experimental autoimmune encephalomyelitis (MOG-EAE), that emulates MS. Testing the neuroprotective effects of CN2097 in EAE rats is of clinical relevance in slowing or reversing MS-related neurological disability. We hypothesize that this PDZ-domain inhibitor will improve the tolerance for the treatment because it does not appear to interfere with normal NMDA or AMPA receptor activity, while ameliorating the disease related excitotoxic damage. PUBLIC HEALTH RELEVANCE: Recent studies show that glutamate receptors, which are important in transmitting signals from one nerve cell to another, also increase sensitivity to the death of neurons and myelinating oligodendrocytes in multiple sclerosis. We have synthesized a series of high affinity compounds that completely prevent neuronal death and are predicted to improve the tolerance for treatment because they do not affect the ability of glutamate receptors to signal between nerves, while preventing the disease related damage. The central goal of this research grant is to better understand the action of our most promising compound (CN2097) and to determine its potential as a new therapeutic approach for treating multiple sclerosis. [unreadable] [unreadable] [unreadable]
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1 |
2011 |
Garcia, Elizabeth Marshall, John (co-PI) Parang, Keykavous (co-PI) [⬀] Slitt, Angela L (co-PI) [⬀] |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
Treatment of Stroke Using a Novel Pdz Binding Peptidomimetic Drug.
DESCRIPTION (provided by applicant): Abstract Stroke is the third leading pathological cause of death, behind heart and cancer (NCHS mortality data.). We are developing a new drug, CN 2097, that has demonstrated therapeutic potential for stroke. CN 2097 obstructs the propagation of the cell death signal whilst not interfering with normal neuronal transmission, thus reducing the incidence of side effects and toxicity but allowing the titration of a therapeutic dose. This Phase I SBIR study will involve confirming the therapeutic potential of this drug by confirming its favorable pharmacokinetic profile and proving efficacy in the middle cerebral artery occlusion (MCAo) model. The first milestone will be producing sufficient additional quantities (500mg) of CN2097 to complete the pre-clinical in-vivo studies proposed in Aim 2. The second milestone will be the demonstration that CN 2097 has sufficient bioavailability and stability to cross the blood brain barrier and prevent MCAo-induced ischemic stroke neuronal damage and the significant decrease of disabling injury. Based upon our previous results we anticipate that CN 2097 will be an effective treatment in the MCAo model of stroke and will attain therapeutic brain concentrations. Completion of these reliable animal model studies will allow us to enter Phase II development of CN 2097, focusing on toxicology and dosage form design in preparation for Clinical trials. SBIR provides a bridge that will translate our laboratory studies into a Clinical treatment for stroke. PUBLIC HEALTH RELEVANCE: Narrative This project is designed to undertake pre-clinical trials that will provide compelling evidence that this innovative drug, CN 2097, has the potential to prevent or treat the damage caused by stroke. Stroke is simultaneously one of the deadliest diseases and also one of the most undertreated;the emergence of this drug as a viable treatment will eliminate stroke as a major threat to humans. This will result in a drastically changed public health climate, resulting in less disability and longer, more productive life spans amongst the victims of stroke.
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0.306 |
2012 — 2015 |
Marshall, John Joseph [⬀] Marshall, John Joseph [⬀] Marshall, John Joseph [⬀] |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Kainate Receptor Signaling in Striatal Synaptic Function and Plasticity @ Northwestern University At Chicago
DESCRIPTION (provided by applicant): Obsessive Compulsive Disorder (OCD) is a chronic anxiety disorder characterized by maladaptive compulsive behavior such as excessive grooming, hoarding or checking. OCD is a debilitating disease, estimated to affect 1-2% of the population, making it the fourth most common mental illness after depression, alcoholism and social anxiety disorder. Despite innovations in pharmacological, behavioral, and surgical treatments at least 30% of cases prove intractable. Recent advances in understanding the neurobiological underpinnings of OCD have highlighted a central role for dysfunction of the glutamate system, the brain's major excitatory neurotransmitter, in the subcortical region of the striatum. In addition to clinical literature linking OCD to alterations in striatal activity, anima studies have indicated that the synapses formed by cortical neurons onto striatal spiny projection neurons (SPNs) are crucial in regulating habit formation. This convergent evidence indicates that perturbations in normal striatal activity will be crucial to understanding the emergence of maladaptive compulsive, habitual behavior seen in patients with OCD. Mice lacking expression of the kainate receptor, a subtype of glutamate receptor, have a dramatic compulsive grooming and anxiety phenotype reminiscent of that seen in OCD. Despite being highly expressed in the striatum, it is not known how kainate receptors contribute to striatal synaptic transmission and plasticity, and therefore to the overall regulation of the striatal circut. Preliminary work has determined that, in the striatum, activating kainate receptors facilitates the release of endocannabinoids. This is a novel interaction that has not been previously demonstrated. Endocannabinoids are common neuromodulators that serve to down-regulate transmitter release at synapses throughout the brain. I hypothesize that this coupling of kainate receptors to endocannabinoids provides a physiologically relevant method of regulating glutamate release in the striatum that is highly important for the behavioral phenotype of kainate receptor knockout mice. The following aims propose to determine the biochemical mechanism by which kainate receptors couple to endocannabinoids and the contribution of this interaction to normal striatal synaptic function and plasticity. PUBLIC HEALTH RELEVANCE: Obsessive-compulsive Disorder (OCD) is a debilitating disease characterized by maladaptive compulsive or habitual behavior that is estimated to affect 1-2% of the population. We have developed a novel mouse model of OCD to help elucidate how changes in synaptic transmission in specific brain regions give rise to habitual behavior. While currently 30% of OCD cases are intractable, understanding these biochemical and synaptic abnormalities will lead to treatments that are more effective, specific, and give rise to fewer complications.
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0.923 |
2016 — 2017 |
Colwill, Ruth M (co-PI) [⬀] Marshall, John |
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.) |
A Novel Bdnf Potentiator For Treating Cognitive Dysfunction in Angelman Syndrome
Angelman syndrome (AS) is a severe cognitive disorder caused by loss of expression of the maternally inherited allele of the Ube3A gene that encodes the Ube3A ubiquitin ligase. A mouse model of AS shows impairment in long-term potentiation (LTP) and behavior. Recent studies suggest that the deficits in synaptic plasticity stem from inappropriate expression of Arc/Arg3.1 (Arc), which associates with the synaptic scaffolding protein postsynaptic density-95 (PSD-95), to impair brain-derived neurotrophic factor (BDNF)-mediated PLC?/CaMKII and Akt/mTORC1 signaling. We have designed a novel water-soluble cyclic-peptide drug (MW 2376), CN2097, that crosses the BBB to selectively target PSD-95 to reinstate BDNF-signaling in AS mice. Critically, CN2097 produces a prolonged activation of enzymes involved in memory processes, to lower the threshold for synapse strengthening (long term potentiation, LTP). This property makes this compound suited to treat the memory and executive functioning deficits in AS. A unique feature of CN2097 is that it has no effect in the absence of BDNF, making CN2097 highly specific for ?active? synapses involved in learning, while avoiding adverse effects associated with widespread BDNF enhancement. The objective of this project is to evaluate the efficacy of CN2097 to mitigate the cognitive deficits in the AS mouse model. Assessment of restored function will be made by examining performance of CN2097 treated AS mice on tasks measuring simple associative learning and executive functions. In Aim 1, we will investigate the pharmacokinetics of CN2097, examining the duration of action and optimal dose required to rescue signaling and LTP. In Aim 2 we will examine effects on learning and memory using standard control procedures absent in prior studies and an expanded repertoire of learning tasks to characterize more fully the range of cognitive deficits expressed by AS mice. This project has the potential to lead to the first effective therapy for AS and autism spectrum disorders.
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1 |
2016 — 2020 |
Chodobski, Adam Marshall, John (co-PI) |
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. |
A Novel Cyclic Peptide-Based Treatment For Tbi
PROJECT SUMMARY Traumatic brain injury (TBI) is a devastating medical condition that affects more than 1.7 million civilians in the US and represents an unmet clinical need. Effective therapies are urgently needed, as TBI is associated with high rates of hospitalization, mortality, and disability. Previous clinical trials have failed to demonstrate therapeutic efficacy in patients with TBI. Due to the complex nature of the pathophysiological events accompanying TBI, an effective treatment necessitates the use of drugs with complementary therapeutic effects directed against multiple pathological mechanisms. We have developed a new cyclic peptide-based drug?CN2097?that possesses such properties. When administered peripherally, CN2097 crosses the blood-brain barrier to selectively target PSD-95, an intracellular scaffolding protein required for synapse strengthening (long term potentiation, LTP). CN2097 acts to potentiate brain-derived neurotrophic factor (BDNF) signaling pathways required for neuroprotection and learning. CN2097 produces a prolonged activation of the enzymes involved in memory processes to lower the threshold for LTP, making this compound suited to treat the memory and executive functioning deficits in TBI patients. In addition, CN2097 interferes with glutamate-mediated excitotoxic cell death and is the first in this class of compounds to have anti-neuroinflammatory properties. This suggests that CN2097, by simultaneously redu- cing excitotoxic damage and neuroinflammation, and facilitating synaptic plasticity, is a new multifunctional drug with a significant therapeutic potential to curtail neuronal death and facilitate functional recovery after TBI. There are three major goals of this project that focus on translational (preclinical), mechanistic, and neuro- behavioral aspects of post-traumatic treatment with CN2097. In Aim 1, we will assess the efficacy of CN2097 in mitigating secondary injury and reducing the loss of neural tissue resulting from TBI. The experiments will also be conducted to establish the potential therapeutic time-window for delayed treatment with CN2097, and to quantitatively assess the ability of CN2097 to cross the BBB and determine its distribution in the injured brain. In Aim 2, we will assess the efficacy and mechanism by which CN2097 attenuates synaptic and neuronal loss in the hippocampus and restores synaptic plasticity after TBI. Lastly (Aim 3), we will assess the efficacy of CN2097 in improving neurobehavioral outcome. The multifunctionality of CN2097 together with our initial preclinical data obtained in a rodent model of TBI strongly suggest that this new drug will be highly efficacious in targeting complex secondary injury processes resulting from neurotrauma.
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0.194 |