Naren L. Banik - US grants

This project explores the hypothesis that a calcium-activated neutral proteinase(s) (CANP) is associated with myelin and/or localized in axons and glial cells. This thesis has been developed from our demonstration of a calcium-mediated degradation of cytoskeletal as well as myelin proteins by a proteinase(s) present in spinal cord and brain white matter. Thus our detailed studies will be 1) to establish that CANP is associated with myelin: we plan to examine the enzyme (CANP) activity in different regions of brain, in myelin and subcellular fractions of brain during development and myelin from CNS and PNS of different species, and 2) to examine the localization of CANP by immunocytochemical technique after antisera is raised from purified CANP (which we have recently purified from bovine brain). The presence of this enzyme in myelin and related structures or cells may play a crucial role in the breakdown of myelin in demyelinating diseases (e.g. MS).

DESCRIPTION (Adapted from Investigator's Abstract): Spinal cord injury (SCI), a major health-care problem, warrants pathophysiological and therapeutic study to minimize damage for functional recovery. Increased calpain activity and expression concomitant with structural protein loss in SCI lesions indicate a pivotal role for this protease in the irreversible cell damage. Partially damaged cells in the area surrounding a necrotic lesion (traumatic penumbral) and areas adjacent to the traumatic penumbra may be rescued or protected by calpain inhibitors, facilitated by additional drugs, synergistic for tissue protection. Since many pathways participate in secondary tissue destruction in SCI, calpain inhibitor inclusion with other agents that block degradative reactions could offer more potent neuroprotection. Their specific aims are to: (1) Define calpain's expression in both the SCI necrotic lesion and the surrounding traumatic penumbra and its adjacent areas which have a potentially reversible apoptotic component; (2) Examine strategies to improve neuroprotection in SCI that emphasize exploration and delineation of synergistic agents affecting reactions in the secondary injury cascade; (3) Study calpain's role in the induction of apoptosis in vitro and how this is related to events and their constraint by specific calpain inhibitors in the traumatic penumbra. To support these aims, they will: (1) Determine activity and expression (mRNA and protein levels) of u and mcalpain and their inhibitor, calpastatin; ascertain their cellular localization by double-label immunofluorescence; define the extent and nature of cell death in defined areas of the cord following injury; (2) Study the therapeutic effects and synergy of calpain-specific cell permeable inhibitors (calpeptin, E64-d, MDL-28170) with methylprednisolone, indomethacin and brain-derived neutrotrophic factors in acute and chronic SCI, examining parameters indicated previously; correlate changes with behavioral outcome; (3) Determine the effect of cytokines on calpain activation in neural cell culture, examining apoptotic characteristics (biochemical and morphological) effected by calpain inhibitors.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Multiple Sclerosis (MS) is a debilitating autoimmune demyelinating disease, which leads to paralysis and other functional disabilities. The factors responsible for myelin destruction in MS are not clearly understood. Thus, a pathophysiological/therapeutic study to minimize or prevent myelin damage and maximize functional recovery is warranted. Findings from animals with experimental allergic encephalomyelitis (EAE), an animal model for MS, demonstrate increased calpain activity and expression concomitant with myelin protein degradation in the CNS signifying a pivotal role for calpain in the mechanism of myelin breakdown. Using calpain inhibitors and methylprednisolone (MP) alone and in combination as therapeutic agents could inhibit myelin protein degradation, protect oligodendrocytes, and delay or prevent development of the disease. In this project, we will focus our studies on the following specific aims: 1. Define the time-course of calpain expression and activity in the spinal cord and spleen/lymph nodes in animals with EAE. 2. Examine the effects of calpain inhibitors and anti-inflammatory agents affecting myelin protein degradation which could delay or prevent the development of EAE. 3. Study the role of calpain in the induction of apoptosis in vitro in neural cells and how calpain inhibitors affect apoptotic death in glial/inflammatory cells in EAE. To support these specific aims, the following experiments are proposed: 1) Determine calpain/calpapstatin activity, expression (mRNA, protein), and the specific cellular localization (by double immunofluorescence staining) in spinal cord and spleen/lymph nodes at various times after challenge; 2) Examine the effects of cell-permeable calpain inhibitors alone and in combination with methylprednisolone in EAE development; 3) Determine calpain/calpapstatin activity, expression, and cellular localization in treated verses untreated animals and correlate these findings with the extent and type of cell death in the CNS; 4) Determine the effects of cytokines on intracellular calcium levels, calpain activation, and apoptosis in glial cell cultures; 5) Examine calpain expression, activity, and secretion from activated MPP-specific T cells and determine immunogenicity of MBP peptides generated by these cells.

DESCRIPTION (adapted from applicant's abstract): Although the cause of multiple Sclerosis (MS) remains unknown, there is strong evidence that demyelination and inflammation are mediated by a 7 cell autoimmune mechanism. Since CD4 7 ceils are important in the etiology and pathogenesis of MS, the mechanisms by which these cells become activated are crucial to understanding and treating demyelinating diseases. This proposal examines the role of calpain, a calcium-activated neutral proteinase, in activation of peripheral blood mononuclear cells (PBMC) cultures. One important factors involved in T cell activation is a transcription factor, nuclear kappa B (NFkB). The activation of NFicB promotes interleukin-2 (IL-2) synthesis, CD25 expression, T cell proliferation, and T cell survival, It has been suggested that NFB is activated by neutral proteinase(s) -calpain may be one such participant. Calpain has been shown to be involved in T cell proliferation and integrinmediated cell migration. Also, in demyelinating diseases (i.e., MS), the content and activity in inflammatory cells, such as CD4+ T cells, is significantly increased. From these findings, we propose the following hypotheses: (I) Calpain has a central mle in the activation of PBMCs, degradation of IkBa, and activation of NFkB promoting IL-2 synthesis; (II) Calpain activity and expression may be altered in PBMC and myelin basic protein (MBP) -specific T cells of MS patients during the course (relapse and remission) of the disease; (HI) Released calpam from activated MBP-specific T cells may contribute to epitope spreading and demyelination. These specific aims will be used to investigate these goals: (1) Examine and characterize the role that calpain plays in IL-2 synthesis and CD25 expression; (2) Examine 1KB degradation and NFkB activation and characterization of calpain interaction with 1KB in normal PBMCs; (3) Measure the expression and activity of calpain in PBMCs of MS patients and the susceptibility of these cells to calpain inhibition; (4) Measure the expression and activity of calpain in MBP-specific T cells of MS patients and the ability of calpain to produce immunogenic peptides from intact human MBP. Understanding the mechanisms involved in T cell activation and proliferation and the progression of demyelination may help to develop therapeutic strategies for the treatment of MS.

DESCRIPTION (Adapted from applicant's abstract): Every year in the United States, more than 12,000 people suffer debilitating spinal cord injuries (SCI) - the result of numerous causes including accidents involving motor vehicle, sports, work, etc. Although investigation of SCI has progressed, an effective therapy to restore function remains the goal. Examining factors involved in central nervous system (CNS) cell death, fiber damage, neuron protection and neural tissue stability over time is important to the genesis of potent injury treatments. While in SCI, damaged neurons die of necrosis, a significant number of neurons in the penumbra later die of apoptosis or programmed cell death. Since calpain has been previously implicated as crucial in tissue destruction and cell damage following SCI, the long-term objective of this study is to investigate its role in the cell death mechanism in the lesion and potentially salvageable penumbra following injury and develop means for cell rescue and/or repair with preservation or restoration of function. Our preliminary results have shown mitochondrial damage, increased intracellular Ca2+ and calpain activity, increase pro-apoptotic mediators (e.g., cytochrome c release) and neuronal cell death in the lesion and penumbra following injury. From these findings, we propose the following broad spectrum hypothesis: In SCI, increased intracellular Ca2+ overload will cause calpain activation and loss of calpastatin regulation, leading to cell death while Ca2+ will damage mitochondria, increase mitochondrial permeability transition pore, induce cytochrome c release for caspase activation and cell death. Both calpain and caspase inhibitors, together with other agents, may protect neurons and preserve function. The following specific aim outline the investigational approach: (1) Investigate the contributions that increased intracellular calcium and calpain, los of calpastatin regulation to neuronal death and mitochondrial damage in SCI; (2) Study the role of pro-apoptotic factors, Bax/Bcl-2, cytochrome c and caspase-3 in mediating neuron and glial cell dysfunction/death in SCI; (3) Examine the therapeutic effect of calpain/caspase inhibitors, together or in combination with agents, affecting other destructive pathways in S( and assess cellular protection and function; (4) Study the role of oxidative stress and glutamate toxicity in primary culture cortical neurons, motor neuron cell line and glial cells and determine cell function before and after treatment with calpain/ caspase inhibitors and how this relates to events in trauma. Studies will use relevant and novel techniques to determine Ca2+ levels in cultured cells and SCI tissue slices, cell death by combined TUNEL/immunofluorescent labeling and cell function by electrophysiology. Understanding the mechanisms c cell death and cell protection in relation to calpain/caspase will establish inhibition of protease(s) as a viable SCI treatment.

DESCRIPTION (provided by applicant): Although the understanding of spinal cord injury (SCI) and its mechanisms have increased over the years, effective therapy to minimize tissue damage and maximize functional recovery remains a primary goal. The only treatment, methylprednisolone, has limited clinical efficacy. Emphasis is needed on early pharmacological intervention of secondary damage involving Ca2+ overload, free radicals, glutamate toxicity, and enzyme activation. Investigations into cell and fiber damage, tissue destruction, neuron protection, and maintenance of structural integrity are essential to effective treatments. After SCI, intracellular Ca2+ influx initiates secondary pathological events (cell damage, tissue destruction). Our goal is to protect CNS cells from secondary damage by using agents that preserve and restore function. Since several pathways cause cell damage and tissue destruction, multiple action drugs or a combination therapy will likely be more effective. One group of potential agents, steroid hormones, estrogen, and progesterone, are neuroprotective in cultured neurons and in CNS disorders. We hypothesize that estrogen/progesterone will control inflammatory processes, suppress intracellular Ca2+ levels, and inhibit Ca2+-dependent events. Suppression of intracellular Ca2+ will inhibit activation of lipases and proteinases (e.g., calpain) and protect cells from secondary damage. Estrogen decreases infiltration of inflammatory cells (e.g., macrophages), reduces Ca2+ levels and Ca2+-dependent events, and restores mitochondrial function in SCI lesion and penumbra (compared to vehicle-treated animals). Our preliminary data reveals that the effects seen with estrogen treatment may be superior to those seen with methylprednisolone. Cells undergoing apoptosis due to H202 and glutamate toxicity have increased intracellular Ca2+. Estrogen treatment reduced Ca2+, protected these cells from damage, and led to functional recovery. Thus, the following Specific Aims are proposed: . Examine the effects of estrogen treatment on inflammation following acute SCI . Investigate estrogen effects on intracellular Ca2+ influx and Ca2+ -dependent events in cell and tissue damage in SCI . Investigate whether estrogen treatment will preserve motor function in chronic SCI . Examine whether estrogen treatment will protect and preserve the function of neurons (cortical and motor) and glial cells subjected to oxidative stress and glutamate toxicity in culture. Novel and relevant techniques, i.e., determining Ca levels in cultured cells and SCI slices (fura-2), cell damage (TUNEL), cell function (electrophysiology), and motor recovery ("BBB" scale), will be used. Understanding estrogen's mechanisms in cell protection and recovery as it relates to multidestructive pathways will establish it as a viable SCI treatment agent.

The irreversible neurological deficits in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are due primarily to axonal and neuronal degeneration brought on by a pro-inflammatory assault on the central nervous system (CNS) by autoreactive T cells and other immune cells. Previous studies in our laboratory and others have indicated that the calcium (Ca2+)-activated protease, calpain, plays a role in both the immune arm and the neurodegenerative arm of EAE develop- ment. The goal of this project is to understand the precise timing and molecular mechanisms involved in axonal and neuronal damage in acute and chronic EAE as these events correlate with increased calpain expression and activity and to examine if inhibiting calpain activity will attenuate disease progression by blocking components of the immune arm and/or neurodegenerative arm. Thus, we hypothesize that changes in Ca2* influx and calpain activation will promote infiltration of autoreactive T cells and macrophages, leading to axonal and neuronal degeneration. A corollary hypothesis is that calpain inhibition will prevent migration of activated T cells and macrophages, reduce the production of antigenic myelin basic protein (MBP) peptides, and delay or prevent axonal and neuronal degener- ation;which will lead to reduced disability (paralysis, limp tail) in acute and relapsing/remitting (R/R) EAE models. Preliminary data indicate that Ca2+ influx, calpain activation, T cell activation, axonal degeneration, and apoptotic proteins are increased in EAE spinal cord at the onset of acute disease. Additionally, treatment with the calpain inhibitor SJA6017 (SJA) reduced immune cell activation and infiltration, as well as calpain activity and expression, and prevented cell death in EAE spinal cord, as compared to vehicle-treated animals. Calpain inhibition also reduced MBP degradation by MBP-specific T cells in vitro and greatly attenuated disease development in a R/R adoptive transfer EAE mouse model. To further investigate the roles of calpain in EAE, the following Specific Aims will be addressed: (1) Characterize the timing of Ca2+ influx and calpain activation as these events correlate with increased mechanisms of axonal damage and apoptosis in neurons and glial cells in spinal cord from Lewis rats with acute EAE. (2) Investigate whether treatment with the calpain inhibitor SJA will attenuate EAE development in the acute EAE Lewis rat model. (3) Examine whether calpain inhibition by SJA treatment will delay or block disease development and attenuate neurodegeneration in the R/R model of EAE in SJL/J mice. The proposed studies targeting calpain as therapeutic strategy for attenuating clinical disability in EAE/MS by blocking immune dysfunction and/or neurodegeneration will further development of novel treatments for these debilitating diseases.

DESCRIPTION (provided by applicant): Parkinson's disease (PD), a progressive degenerative movement disorder associated with loss of dopaminergic neurons in substantia nigra (SN), leads to dysfunction. The current therapy, L-dopa, does not block disease progression;therefore, new therapies must be developed. Thus, the aim is to investigate inflammatory events in brain and spinal cord (SC) and their degeneration in PD and characterize whether SC integrity and neurons are lost in PD, contributing to dysfunction. Understanding the mechanisms of damage may help develop new therapeutic strategies. While the etiology of PD is not fully understood, neurotoxins have been im- plicated in PD pathogenesis. Toxic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been extensively used as an experimental model. Since 1-methyl-4-phenylpyridinium ion (MPP+), the active toxic metabolite of MPTP, increases intracellular-free Ca2+ level and promotes mitochondrial dysfunction, a Ca2+-mediated pathology in PD has been hypothesized. Increased Ca2+ levels will promote calpain activation, increase inflammatory responses, and damage brain/SC neurons, axons, and myelin, ultimately leading to functional deficit. Our preliminary findings of direct detection of MPP+ in PD mouse SC, activation of astrocytes and microglia, and increased calpain activity and expression in neurons indicate that SC is also affected. These findings were corroborated by preliminary data showing motoneurons from SC of PD patients are also damaged. MPP+ treatment of ventral SC motor neuron cells (VSC4.1) showed increased intracellular [Ca2+] and calpain activity with loss of membrane potential and death while calpain inhibitors (calpeptin, SJA6017) protected and restored cell function. From these findings, we hypothesize that, since SC coordinates movement and sensation of the body, damage to SC neurons, axons, and myelin, in addition to SN, may be an important factor in PD, and calpain plays a crucial role in this dysfunction by promoting inflammation and cell death and may be a target for therapy. Three specific aims will test the hypotheses. Specific Aim 1 will investigate whether MPTP is directly converted into MPP+ in SC, enters through the degenerating axons from brain, or a combination of both;examine the effects of MPTP (MPP+) on SN and SC neurons and white matter in acute and chronic parkinsonism;assess calpain expression and activity and subsequent inflammation and cell damage;and examine the status of neurons, axons, and myelin in SC of postmortem PD patients. Specific Aim 2 will explore the effects of neurotoxic MPP+ in differentiated VSC4.1 cells and test the neuroprotective efficacy of calpain inhibitors in vitro employing electrophysiological technique. Specific Aim 3 will examine whether calpain inhibitor treatment will attenuate inflammation, prevent apoptosis of brain and SC neurons, protect cells, preserve axons and myelin, and improve function in MPTP-induced PD mice. These studies will delineate the role of calpain in inflammation and neurodegeneration in MPTP-induced PD and the probable neuroprotective efficacy of calpain inhibitors in PD as therapeutic agents. PUBLIC HEALTH RELEVANCE: Since the spinal cord coordinates movement and sensation of the body, damage to spinal cord neurons, in addition to the substantia nigra (brain), and alteration in white matter integrity (i.e., axonal and myelin degeneration) as well as loss of myelin-forming cells may be important factors in Parkinson's disease (PD), and calpain could play a crucial role in this dysfunction. This study will examine the role of calpain in cell and axon damage in the progression of disease in an animal model, the efficacy of calpain inhibitor as a therapeutic agent in vivo and in vitro, and the status of neurons in post-mortem PD tissue. Delineating a role for calpain in the progression of disease could potentially lead to new therapeutic targets since the most potent therapy, L-dopa, does not block the progression of Parkinson's disease.

DESCRIPTION (provided by applicant): Impaired vision due to optic nerve damage is one of the most common presenting symptoms of patients with multiple sclerosis (MS). MS-associated optic nerve damage is due to an influx of auto-reactive T cells and other immune cells. The inflammatory process is thought to contribute to demyelination, axonal degeneration, and loss of oligodendrocytes and retinal ganglion cells (RGCs). Understanding the pathophysiology of optic nerve degeneration in EAE has potential therapeutic implications to develop agents that restore visual function. Demonstration of increased activity and expression of calpain, a calcium (Ca2+)-dependent protease, in optic nerve using an animal model of MS, experimental allergic encephalomyelitis (EAE), in Lewis rats has implied a role for calpain in optic nerve damage in EAE. Since the precise timing of inflammation and mechanisms of cell and axon damage in optic nerve are not fully understood, the potential to prevent inflammation and molecular events that lead to axonal and cell damage, may improve and restore function. We hypothesize that calpain-mediated activation of auto-reactive T cells and immune cell infiltration into the CNS will result in loss of visual function due to inflammation leading to axon and oligodendrocyte damage in EAE optic nerve with subsequent loss of RGCs which may occur prior to clinical symptoms of disease. A corollary hypothesis is that inhibiting calpain at different time-points following challenge will restore visual function by preventing inflammation in the periphery and in optic nerve, and ameliorate neurodegeneration after damage to the optic nerve have begun. Data indicate that Ca2+ influx, calpain expression, axonal damage, cell death, and retinal damage are increased in EAE optic nerve with several parameters affected before disease onset. Treatment with calpain inhibitors reduced immune cell infiltration, calpain expression, cells death, and retinal damage, resulting in improved visual responses compared to untreated EAE animals. In vitro interferon (IFN?) induced calpain activation in glial cells and MBP-specific T cell supernatant degraded MBP. The following specific aims have been designed to test these hypotheses: (1) determine the timing of inflammatory responses, Ca2+ influx, Ca2+-dependent events, cell death, and axonal/myelin degeneration in EAE optic nerve and examine the status of RGCs in correlation with visual dysfunction in EAE animals following challenge;(2) investigate whether treatment with calpain inhibitors will restore visual function by altering the immune arm (T cell activation, immune cell infiltration into optic nerve) and/or the neurodegenerative arm (cell death, axonal damage) in acute EAE, as compared to vehicle treated animals;and (3) examine the effects of calpain inhibitors in preserving and protecting function of RGCs in vitro when subjected to pro-inflammatory cytokines or incubated with supernatant from activated MBP- specific T cells. Understanding the process and timing of optic nerve damage will further the development of treatment of strategies to best restore the impairment caused by optic nerve degeneration in MS. PUBLIC HEALTH RELEVANCE: Damage to the optic nerve resulting in impaired vision is one of the most common symptoms of patients with multiple sclerosis (MS). MS-associated inflammation of the optic nerve due to an influx of immune cells is thought to contribute to deficits leading to impaired vision, including demyelination, axonal degeneration, and loss of retinal ganglion cells (RGCs) and oligodendrocytes. Understanding the mechanisms by which inflammatory and neurodegenerative events contribute to optic nerve damage may have important and potentially therapeutic implications for developing agents that protect cells, preserve axons and myelin, and thus, improve vision and ultimately retard the development of MS.

Summary Parkinson's disease (PD) is a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN) along with the accumulation of ?-synuclein (?-syn) in the brain, activation of microglia, production of inflammatory cytokines/chemokines, infiltration of CD4+ T-cells, and neurodegeneration. The most potent therapy, L-dopa, does not block disease progression, and the mechanism of the progressive nature is unclear. Calpain, a cysteine protease regulated by calcium, plays a pivotal role in SN and SC (spinal cord) degeneration in PD, and its role in ?-syn aggregation, activation of microglia, T cells and their migration indicate calpain to be crucial in promoting the inflammatory process and disease progression. While calpain-1 cleavage of ?-syn promotes synuclein aggregation in PD-like diseases, the precise involvement of the two major calpain isoforms, calpain-1 and calpain-2, in ?-syn presentation to CD4+ T-cells remains unknown. Preliminary studies here identified a subtype of CD4+ T cells in MPTP mice, which was abolished by calpain inhibitor, suggesting that activation of calpain and CD4+ T cells may play critical roles in the inflammatory process and disease progression in PD. Preliminary data also suggest that siRNA-mediated knockdown of calpain-2 diminishes antigen presentation by human B-cells and inhibits activation of CD4+ T cells. Thus, we hypothesize that activation of distinct calpain isoforms may favor expansion of a subtype of ?-syn-reactive CD4+ T cells in PD-like disease. We also hypothesize that calpain inhibition may attenuate ?-syn aggregation and expansion of inflammatory T cells, reduce inflammation and support neuronal survival and improved outcome in PD patients. Two specific aims are proposed to test the hypothesis: (Aim 1) To investigate whether activation of calpain-1 or calpain-2 is linked with microglial presentation of ?-syn to CD4+ T cells resulting in disease progression and neurodegeneration in PD-like disease in mice. (Aim 2) To determine whether inhibition of calpain-2 reduces microglial presentation of ?-syn and generation of pathogenic CD4+ T cells, attenuating disease progression in mouse models of PD. The goal of this study is to investigate the role of calpain-1 and calpain-2 in generating ?-syn-reactive pathogenic CD4+ T cells, and whether a subpopulation of CD4+ T cells from MPTP mice can induce PD-like disease in immunodeficient mice. Studies are planned to determine whether deletion of calpain-1 attenuates ?-syn aggregation and expansion of CD4+ T cells using calpain-1 knockout (KO) mice. In addition, the role of calpain-2 inhibitor will be assessed in the study of alteration of inflammatory CD4+ T cell response and production of cytokines/chemokines in calpain-1 KO mice. Testing the effects of distinct calpain isoforms in the generation of ?-syn-reactive pathogenic T cells and induction neuronal death and degeneration may lead to development of a novel approach for treating PD as well as other neurodegenerative disorders.