Kalipada Pahan - US grants

DESCRIPTION (adapted from applicant's abstract): Multiple sclerosis (MS) is a chronic human demyelinating disorder of the central nervous system (CNS). The identification of a wide range of inflammatory molecules (pro-inflammatory cytokines, cell adhesion molecules, inducible nitric oxide synthase and peroxynitrite) in brain lesions of patients with MS and rats/mice with experimental allergic encephalomyelitis (EAE), an animal model of MS, suggests that a broad spectrum of inflammatory processes plays the crucial role in the pathogenesis of MS/EAE. Therefore, analysis of molecular mechanisms for the regulation of these inflammatory molecules in activated glial cells and in the CNS of animals with EAE should decipher the said mechanisms of the disease process in MS/EAE and further the possibility of developing effective therapies for MS patients with drugs that block the activation of inflammatory molecules. Activation of NF-kB, a pro-inflammatory transcription factor, is important for the induction of inflammatory molecules. The investigators have found that NF-kB is induced in the neural tissues of rats and mice with EAE. However, the role of this induced activation of NF-kB is not known. Therefore, Specific Aim I has been designed to delineate the role of NF-kB activation in the disease process of EAE. The investigators have found that activation of p21ras by the expression of a dominant-negative mutant of p21ras inhibits the activation of NF-kB in activated glial cells suggesting that p21ras may regulate the in vivo activation of NF-kB in the CNS of EAE animals and the disease process of EAE. Therefore, Specific Aim II will examine the effects of inhibitors of p21ras on the disease process of EAE. Since cAMP-dependent proteinase kinase (PKA) differentially regulates the activation of NF-kB in astrocytes and macrophages, Specific Aim III has been devoted to investigate the molecular basis for the differential regulation of NF-kB activation in two different cell types (astrocytes and macrophages) by PKA. The proposed studies are significant and will utilize the state of the art methodologies in cellular and molecular biology to enhance our understanding of the signaling pathways for the activation of NF-kB in vitro in glial cells and in vivo in the spinal cord of EAE and to provide promising therapeutic strategies for MS patients.

One mechanism leading to neurodegeneration in Alzheimer's disease (AD) is amyloid-beta (Abeta) peptide neurotoxicity. The presence of apoptotic bodies in the brain of AD patients and the ability of Abeta to elicit apoptosis and cell death in cultured CNS neurons suggest that the aberrant induction of apoptosis by Abeta peptides may be an important cause in removing the irreplaceable neurons in AD. However, the mechanism by which Abeta causes apoptosis and cell death neurons remains largely unknown. Since ceramide, a lipid second messenger, produced by the degradation of sphingomyelin is an important inducer of apoptosis and cell death in various cell types including neuronal cells, we decided to investigate whether Abeta induces the production of ceramide in neuronal cells. Interestingly, we have found that Abeta induces the production of ceramide by more than 10 fold within 24 h in rat pheochromocytoma (PC 12) cells. Furthermore, PP2, a specific inhibitor of Src tyrosine kinases, but not PP3, a negative control of PP2, inhibits Abeta-mediated induction of ceramide production. Taken together, these observations suggest that src tyrosine kinase(s) may play an important role in Abeta-induced production of ceramide in neuronal cells and that ceramide may participate in the pathogenesis of AD. To establish this hypothesis, we have proposed three simple specific aims. Since ceramide produced from the degradation of sphingomyelin by sphingomyelinases (neutral, NSMase; acidic, ASMase), Specific aim I will investigate whether Abeta induces the activation of sphingomyelinases (N-SMase and/or A-SMase) neuronal cells. Specific aim II has been designed to investigate whether Src is involved in Abeta-mediated degradation of sphingomyelin to ceramide in neuronal cells. Specific aim III has been devoted to analyze the levels of ceramide and sphingomyelin in postmortem brains of AD and non-demented controls. Results obtained from this pilot proposal will significantly enhance our understanding about the cause of neuronal loss in AD and will provide promising therapeutic strategies for AD patients.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders in humans. Despite intense investigations, no effective therapy is available. Although its etiology remains unknown, it is believed that complex interaction between environmental and genetic factors leads to activation of glia and induction of broad-spectrum inflammatory reactions followed by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Consistently, enhanced expression of inducible nitric oxide synthase (iNOS) and pro inflammatory cytokines (IL-1a, IL-6 and TNF-a) has been found in association with glial cells in the SNpc of patients with PD. We have found that lovastatin, a drug approved for hypercholesterolemia, and sodium phenylacetate (NaPA), a drug approved for urea cycle disorders in children, inhibit the expression of iNOS and pro inflammatory cytokines in glial cells by inhibiting Ras - NF-kB. Our recent studies have shown that drinking water containing NaPA markedly inhibits the disease process of experimental allergic encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS), as well as the expression of proinflammatory molecules in the CNS of EAE mice. Similarly sodium phenylbutyrate (NaPB), a synthetic precursor of NaPA and a FDA-approved drug, also inhibited the inflammatory disease process of EAE. Consistently, it has been also shown that lovastatin inhibits the disease process of EAE in different animal models. These results raise the possibility that lovastatin, NaPA and NaPB may turn out to be antineuroinflammatory drugs. Unlike MS, PD is not an autoimmune disorder. However, similar to MS, inflammation within the CNS plays a major role in the loss of dopaminergic neurons in SNpc of PD patients. Therefore, it is of worth trying if oral administration of lovastatin, NaPA and NaPB attenuates the expression of proinflammatory molecules (iNOS and cytokines) and the loss of dopaminergic neurons in an animal model of PD. A positive outcome of this grant proposal will further attest the anti neuroinflammatory role of lovastatin, NaPA and NaPB, and enhance the possibility of treating PD patients with these FDA approved drugs as primary or adjunct therapy.

DESCRIPTION (provided by applicant): Despite extensive research on the pathogenesis of PD, no effective therapy is available to halt this progressive neurodegenerative disorder. One of the major roadblocks for discovering drugs against PD is the unavailability of a true chronic persistent animal model for PD. Therefore, developing a chronic persistent animal model to study the pathogenesis of PD is of paramount importance. Based on our exciting preliminary results, here studies have been proposed from various angles to delineate if simple castration of male mice leads to PD-related nigrostriatal pathologies. First, activation of microglia and astroglia and production of proinflammatory molecules in the nigra play an important role in the pathogenesis of PD. Our studies will indicate if castration leads to the activation of glial cells and the expression of proinflammatory molecules in the nigra of male mice. Second, GDNF supports normal health and viability of dopaminergic neurons and the level of GDNF goes down in the nigra of PD patients. Therefore, we will investigate if castration decreases nigral level of GDNF. Third, it has been shown that in PD patients, nigrostriatal dopaminergic neurons and neurotransmitters disappear. Therefore, we will investigate these nigrostriatal pathologies in castrated mice. Fourth, we will test if castration leads to an impairment of locomotor activities in male mice. In summary, if this project becomes successful, castrated male C57/BL6 mice may be used as a simple toxin-free chronic persistent animal model to study PD-related nigrostriatal pathologies paving the way for easy drug screening against PD. PUBLIC HEALTH RELEVANCE: One of the major roadblocks for discovering drugs against PD is the unavailability of a true chronic persistent animal model for PD. Here we will try to describe a simple toxin-free non-transgenic chronic persistent mouse model to study PD-related pathologies paving the way for easy drug screening against PD. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders in human. In spite of extensive research on the pathogenesis of PD, no effective therapy is available to halt this devastating neurodegenerative process. Recently we have demonstrated that Parkinsonian neurotoxin MPP+ induces the activation of NF-?B in human and mouse glial cells, that NF-?B activation is induced in vivo in the SNpc of PD patients and MPTP-intoxicated mice and that selective inhibition of NF-?B by NBD peptide results in significant protection of nigrostriatal neurons against MPTP-induced neurodegeneration in mice. These mice results suggest that this NF-?B-based therapeutic approach may be useful in PD. As we know mice results are not always translated to humans. Had all mice results been replicated in PD in human, the PD would have been cured long ago. Therefore, we would like to examine the efficacy of NEMO-binding domain peptides in inhibiting the disease process in a monkey model of PD, the best available model for human PD. First, we will investigate if this peptide enters into monkey brain. Second, activation of microglia and astroglia and production of proinflammatory molecules play an important role in the pathogenesis of several neurodegenerative disorders including PD. Our studies will indicate if NBD peptide treatment is capable of inhibiting the activation of microglia and astroglia and attenuating the expression of proinflammatory molecules in MPTP-intoxicated hemiparkinsonian monkeys. Third, it has been shown that nigrostriatal dopaminergic neurons in PD patients as well as in MPTP-intoxicated monkeys disappear. We will investigate if NBD peptide can protect these neurons in MPTP-intoxicated hemiparkinsonian monkeys. Fourth, this proposal will examine if NBD peptide can restore the level of dopamine in the striatum of MPTP-intoxicated monkeys. Fifth, similar to PD, MPTP intoxicated monkeys also display locomotor abnormalities. We will test if NBD peptide can improve locomotor activities in MPTP-intoxicated monkeys. If our mice results are replicated in monkeys, it could be a remarkable advancement in the treatment of PD. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: In spite of extensive research on the pathogenesis of Parkinson's disease (PD), no effective therapy is available to halt this devastating neurodegenerative process. Here we would like to test the efficacy of NBD peptide, a specific inhibitor of induced NF-?B activation, in inhibiting the disease process in a monkey model of PD, the best available model for human PD. Results obtained from this proposal will increase the possibility of treating PD patients with NBD peptides as primary or adjunct therapy. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

DESCRIPTION (provided by applicant): In spite of extensive research on the pathogenesis of MS, no effective therapy is available to halt this demyelinating disease process. In MS, myelin repair is generally insufficient despite the relative survival of oligodendrocytes within the plaques and the recruitment of oligodendrocyte precursors. Promoting remyelination, therefore, appears to be a crucial therapeutic challenge. While neurotrophins (NGF, NT-3, NT-4/5, and BDNF) and glial cell line-derived neurotrophic factor (GDNF)-related factors (GDNF, neurturin) do not increase myelinogenesis, ciliary neurotrophic factor (CNTF) induces a strong promyelinating effect. Therefore, increasing the level of CNTF in the CNS is an important step in repairing axonal damage in MS. Although gene manipulation and stereotaxic injection of CNTF into the brain are available options, it seems from the therapeutic angle, the best option is to stimulate/induce the production of CNTF within the CNS of patients with MS. Is it really possible? Our exciting preliminary results demonstrate that it is possible by a natural compound. Cinnamon is a natural spice and flavoring material used for centuries throughout the world. We have found that oral feeding of ground cinnamon increases the level of CNTF in the CNS of normal mice and mice with experimental allergic encephalomyelitis (EAE); an animal model of MS. Consistently, cinnamon metabolite sodium benzoate (NaB) also increases the expression of CNTF in astrocytes. Therefore, from the academic angle, we have planned experiments in Specific aim I to investigate molecular mechanisms by which cinnamon metabolite NaB increases CNTF in astrocytes. Specific aim II has been devoted for therapeutic purposes. Here we would like to delineate the efficacy of orally administered cinnamon in inhibiting the disease process of EAE. Specific aim III has been enshrined to delineate if cinnamon requires CNTF to inhibit the disease process of EAE. If our study becomes successful, it will describe a novel myelinotrophic activity of cinnamon and ~ 1 teaspoonful of ground cinnamon per day may help MS patients to manage the disease process bringing down the drug cost to <$10 per month for each patient.

Parkinson's disease (PD) is the second most common and debilitating age-associated human neurodegenerative disorder. Clinically, PD is characterized by tremor, slowness of movement, stiffness, and postural instability. Pathologically, it is indicated by activation of glial cells and progressive degeneration of the nigrostriatal dopaminergic neurons associated with the presence of intracytoplasmic inclusions (Lewy bodies). This application addresses an important aspect of PD. Although the rate of disease progression varies from patient to patient, PD is a progressive neurodegenerative disorder. However, the mechanism behind disease progression is poorly understood. We hypothesize that RANTES and eotaxin could hold the key for driving disease progression and that targeting these two chemokines may be an important strategy to control T cell infiltration and hence the disease progression in PD. Here this hypothesis will be tested from several experiments on mice, monkeys and humans. It is known that nigrostriatal pathology does not persist in acute MPTP mouse model. Under Specific aim I, we will investigate if supplementation of RANTES and eotaxin induces persistent and progressive disease in acute MPTP-intoxicated mice. Specific aim II has been planned to determine whether PD patients have higher levels of RANTES and eotaxin by monitoring the level of these two chemokines in serum of PD patients and age-matched controls. Finally, we have devoted the Specific aim III to delineate if blocking the functions of RANTES and eotaxin by maraviroc, an inhibitor of CCR5 and a FDA- approved drug, halt the disease progression in hemiparkinsonian monkeys. A positive outcome of this study will establish RANTES and eotaxin as targets for PD, translate CCR5-based treatment (maraviroc) to PD clinic, uncover the clue for the progression of PD, and find a drug to stop the progression of PD.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common human neurodegenerative disorder of the CNS and one of the most common signs of AD is memory loss. Despite intense investigations, no effective therapy is available to improve memory in AD. Peroxisome proliferator-activated receptor (PPAR) ? is a transcription factor that regulates genes involved in fatty acid catabolism. Although hippocampus does not metabolize fat, recently we have demonstrated that PPAR? is constitutively expressed in nuclei of hippocampal neurons and surprisingly controls calcium influx and the expression of various plasticity-related genes via direct transcriptional regulation of CREB. Being a nuclear hormone receptor, PPAR? needs ligand(s) for translocation into the nucleus. Because PPAR? is constitutively present in nuclei of hippocampal neurons, ligands must be constitutively present in the hippocampal neurons as well. Interestingly, we have identified three novel ligands (Hexadecanamide, Octadecenamide and 3-hydroxy, 2, 2-dimethyl butyrate) from hippocampal extracts of normal mice. Here, we would like to examine functions of these novel ligands in the hippocampus, compare levels of these ligands and their receptor PPAR? in the hippocampus of patients with AD, mild cognitive impairment (MCI) and age-matched controls with no cognitive impairment, and delineate whether these ligands improve memory and learning in an animal model of AD via PPAR?. A positive outcome of this grant proposal will highlight the discovery of novel hippocampal ligands of PPAR?, allowing us to develop hippocampus-based drugs to enhance synaptic plasticity and protect memory and learning in cognitive disorders including AD.

Although the classical role of peroxisome proliferator-activated receptor ? or ? (PPAR?) is to regulate genes involved in fatty acid synthesis, PPAR? is highly expressed in the CNS and it participates in many brain functions including myelination. Being a nuclear hormone receptor, PPAR? needs ligand(s) for its activation and nuclear translocation. Although there are many synthetic ligands of PPAR?, nothing is known about the physiological activation of PPAR? by endogenous ligands in the brain. Therefore, we have identified three possible physiological ligands [1,3-di-tert-butylbenzene (DBB); 2,4-di-tert-butylphenol (DBP); hexadecanoic acid, methyl ester (HAM)] of PPAR? from nuclear extracts of mouse cerebellum. Here, by using in silico interaction studies, time-resolved FRET analyses, thermal shift assay, site-directed mutagenesis, and mass spectrometry, we will characterize whether these physiological molecules are true ligands with PPAR?. Furthermore, we would like to examine whether these cerebellar ligands exhibit promyelinating effect via PPAR?. A positive outcome of this grant proposal will highlight the discovery of novel cerebellar ligands of PPAR?, allowing us to develop cerebellum-based drugs in the future to promote remyelination in demyelinating disorders.

Although the classical role of peroxisome proliferator-activated receptor ? or ? (PPAR?) is to regulate genes involved in fatty acid synthesis, PPAR? is highly expressed in the CNS and it participates in many brain functions including myelination. Being a nuclear hormone receptor, PPAR? needs ligand(s) for its activation and nuclear translocation. Although there are many synthetic ligands of PPAR?, nothing is known about the physiological activation of PPAR? by endogenous ligands in the brain. Therefore, we have identified three possible physiological ligands [1,3-di-tert-butylbenzene (DBB); 2,4-di-tert-butylphenol (DBP); hexadecanoic acid, methyl ester (HAM)] of PPAR? from nuclear extracts of mouse cerebellum. Here, by using in silico interaction studies, time-resolved FRET analyses, thermal shift assay, site-directed mutagenesis, and mass spectrometry, we will characterize whether these physiological molecules are true ligands with PPAR?. Furthermore, we would like to examine whether these cerebellar ligands exhibit promyelinating effect via PPAR?. A positive outcome of this grant proposal will highlight the discovery of novel cerebellar ligands of PPAR?, allowing us to develop cerebellum-based drugs in the future to promote remyelination in demyelinating disorders.

Effective clearance of aggregated ?-syn from the brain parenchyma is expected to reduce the development and progression of both sporadic and familial PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been shown that ?-syn induces microglial activation via TLR2. However, until now, there is no specific inhibitor of TLR2. Knockdown of TLR2 may not be a feasible approach as it would wipe out both basal as well as induced TLR2 signaling pathways. Since TLR2 is known to function via Myd88, to target this induced TLR2 signaling from the therapeutic angle, we have designed a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that selectively inhibits TLR2 signaling pathway including fibrillar ?-syn-mediated microglial activation. Therefore, here, we want to test a novel hypothesis that intranasal administration of wtTIDM peptide suppresses neuroinflammation (Specific aim I) and decreases ?-synucleinopathy (Specific aim II) in A53T transgenic mice via TLR2. A positive outcome of this cutting- edge R21 grant proposal will delineate if selective targeting of activated status of one component of the innate immune system (TLR2) by wtTIDM peptide reduces Lewy body pathology, highlighting the discovery of a prospective intranasal agent to reduce ?-synucleinopathy in PD, DLB and MSA. 1

Pathologically, multiple sclerosis (MS) can be identified by the presence of diffuse, discrete demyelinated areas, called plaques. Demyelination is a major feature of MS and therefore, an approach to the management of MS involves an increase in remyelination of axons, resulting in clinical improvement. Peroxisome proliferator-activated receptor ? or ? (PPAR?) being highly expressed in the CNS participates in many brain functions including myelination. Being a nuclear hormone receptor, PPAR? needs ligand(s) for its activation and nuclear translocation. Therefore, identification of new nontoxic ligand of PPAR? would be very important for promoting remyelination. The ?-hydroxy ?-methylbutyrate (HMB) is available in local GNC stores as a muscle-building supplement in human. It is a physiological molecule that is produced in human through the metabolism of L-leucine. HMB is known to increase exercise-induced gains in muscle size and muscle strength and improve exercise performance. Here, we will test an exciting hypothesis that HMB binds to the ligand-binding domain of PPAR? (Specific aim I) and that HMB and its precursor L-leucine promote maturation of OPCs (Specific aim II) and stimulate remyelination in animal models (cuprizone and experimental autoimmune encephalomyelitis or EAE) of CNS demyelination (Specific aim III) via OPC-specific and/or microglia-specific PPAR?. To investigate whether the muscle building effects of L-leucine and HMB could contribute to improved motor function in cuprizone and EAE models, Specific aim III will also examine the role of skeletal muscle-specific PPAR?. A positive outcome of this cutting-edge R01 proposal will delineate easily available muscle-building supplement HMB as a physiological ligand of PPAR? and enhance the possibility of promoting remyelination and treating patients with MS and other demyelinating disorders with HMB and its precursor L-leucine as primary or adjunct therapy.

Effective reduction of aggregated tau from the brain parenchyma is expected to reduce the development and progression of both sporadic and familial Alzheimer?s disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), and other tauopathies. However, pathways for lowering aggregated tau from the brain are poorly understood. Neuroinflammation is another hallmark of neurodegenerative disorders and recently it has been shown that the progression of phospho-tau pathology is driven by microglia and that microglial activation forms the crucial link between tau aggregation and brain damage. Here, we want to test a novel hypothesis that intranasal administration of wild type TLR2-interacting domain of MyD88 (wtTIDM) peptide suppresses microglial inflammation and decreases tauopathy in P301S transgenic mice via TLR2. A positive outcome of this grant proposal will delineate a new crosstalk between TLR2 and tauopathy and describe if selective targeting of activated status of TLR2 by wtTIDM peptide reduces tangle pathology, highlighting the discovery of a prospective intranasal agent to reduce tau pathology in AD, PSP, FTD, and other tauopathies. Administrative supplement: TLR2 is present in different cell types in the brain. For example, in the brain, in addition to microglia, TLR2 is also expressed in neurons. Therefore, here, we will investigate the role of neuronal and microglial TLR2 in tauopathy and wtTIDM peptide-mediated clearance of tauopathy and improvement in cognitive behaviors in P301S transgenic mice.