Manisha Patel - US grants

DESCRIPTION (adapted from applicant's abstract): Neuronal death is a major devastating consequence of status epilepticus. Seizure-induced neuronal death itself likely underlies status epilepticus-induced neurological deficits such as memory loss and may also contribute to a worsening of the epileptic condition. The mechanisms of seizure-induced neuronal death remain obscure. Understanding these mechanisms in molecular terms may provide novel therapeutic approaches aimed at preventing seizure-induced neuronal death. It is hypothesized that prolonged seizure activity kills vulnerable neurons by an oxidative mechanism involving the formation of superoxide (O2-) radicals. The hypothesis predicts that overproduction of O2- and related reactive species oxidizes critical cellular targets (i.e. proteins, lipids and DNA), ultimately resulting in neuronal death. Seizure-induced O2- production is hypothesized to arise from the mitochondria via the electron transport chain and the extracellular space via activation of the NADPH oxidase complex. Using the kainic acid model of epilepsy and a diversity of biochemical, molecular biological and analytical tools and techniques, the goal of this proposal is to define the role of O2- radicals in seizure induced neuronal death. Specific Aim 1 will determine if seizures produce oxidative damage and if this correlates with subsequent neuronal death. Aconitase inactivation (an index of O2- production), F2-isoprostanes (a non-enzymatic product of lipid peroxidation) and oxidized bases of DNA will be used as markers of oxidant damage to proteins, lipids and DNA respectively. Metalloporphyrin superoxide dismutase (SOD) mimetics that eliminate O2- and related reactive species will be tested for their ability to protect against oxidative damage and neuronal death. Specific Aim 2 will determine if compartmentalized O2- production modulates seizure-induced neuronal death. Genetically modified mice that over express or lack endogenous SOD1, SOD2 and SOD3 will be used to define the roles of cytosolic, mitochondrial and extracellular O2-, respectively. Specific Aim 3 will determine the roles of the mitochondrial electron transport chain and the plasma membrane NADPH oxidase as two potential cellular sources of seizure-induced O2- production. Polarographic studies in intact and submitochondrial particles as well as oxidative phosphorylation enzymology will be employed to determine the site(s) of mitochondrial O2- generation. These studies can advance our understanding of seizure-induced neuronal death and suggest novel therapeutic strategies for rescuing neurons in the context of status epilepticus in humans

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to elucidate the mechanism by which mitochondrial oxidative stress produces dopaminergic neuronal death in Parkinson's Disease (PD). The precise mechanism by which mitochondrial oxidative stress, bioenergetic decline and iron overload arise and collaborate to produce age-related neuronal death in Parkinson's disease remains unclear. It is hypothesized that neuronal damage in Parkinson's disease results, in part from direct superoxide radical toxicity due to oxidative inactivation of mitochondrial aconitase. The hypothesis predicts that superoxide production, arising from Complex I inhibition or abnormal dopamine metabolism, inactivates [4Fe-4S]Z+-containing mitochondrial aconitase, resulting in loss of aconitase activity and release Fe z+ and H202. Posttranslational modification of this key TCA cycle enzyme can therefore result in an increased iron load, oxidant burden and bioenergetic decline. The presence of an iron responsive element (IRE) in the 5' untranslated region of the mitochondrial aconitase Mrna provides an additional mechanism for iron dysregulation in Parkinson's disease. The proposal will utilize human PD samples, animal models of PD (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and 6-hydroxydopamine) and dopaminergic cell culture models in conjunction with a diversity of tools and techniques that include biochemical analyses, confocal microscopy, molecular biology and transgenic/knockout/aging mice. Specific Aim 1 will determine whether mitochondrial aconitase is inactivated in human and experimental Parkinson's disease. The influence of aging and chronic mitochondrial oxidative stress will be determined using mice deficient in MnSOD, a critical mitochondrial antioxidant. Specific Aim 2 will determine whether mitochondrial aconitase inactivation contributes to impaired iron homeostasis. Specific Aim 3 will determine whether scavenging mitochondrial superoxide using native or synthetic antioxidants (e.g. MnSOD transgenic mice or metalloporphyrins) protect against mitochondrial aconitase inactivation in a manner that correlates with decreased iron overload and neuronal death in experimental Parkinson's disease. Specific Aim 4 will determine the downstream consequences of mitochondrial aconitase inactivation in experimental Parkinson's disease. Specifically, regulation of brain mitochondrial aconitase synthesis by the 5' IRE in its mRNA, impact on the TCA cycle capacity and direct neurotoxicity of aconitase gene silencing will be examined. These studies can advance our understanding of the oxidative mechanisms of neuronal death in Parkinson's disease and suggest novel therapeutic strategies for rescuing neurons from age-related neurodegeneration. Additionally, this line of investigation may explain Parkinson's disease arising from genetic factors uncovered by aging as well as environmental factors. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The long-term goal of this translational research proposal is to develop novel therapeutic agents for the treatment of epilepsies associated with mitochondrial dysfunction. This proposal is intended to address an important NINDS intiative aimed at developing screening models and identifying candidate therapeutics for devastating neurological disorders. Epileptic seizures are the most common feature observed in children with inherited mitochondrial diseases. Work in this laboratory suggests an emerging role of mitochondrial dysfunction in seizure-induced brain injury as well as seizure susceptibility. Based on these studies, it is hypothesized that mitochondrial dysfunction is an attractive target for therapeutic intervention. The goal of this proposal is to develop and validate a two-step screening model to identify therapeutic agents that preferentially ameliorate mitochondrial dysfunction and would therefore benefit catastrophic childhood mitochondrial epilepsies. The model is based on a strain of mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or Sod2), that develop frequent spontaneous seizures in postnatal life. The first screen (Specific Aim 1) will utilize rat brain mitochondria to select compounds that decrease mitochondrial oxidative stress. Selected compounds from this screen will be tested in a second in vivo model of Sod2-/- mice. The goal of the second therapeutic screen (Specific Aim 2) is to develop an in vivo model of mitochondrial dysfunction in B6D2F1 Sod2-/- mice. Model development will involve video monitoring, EEG recordings, mitochondrial enzymology, oxidative stress and survival analysis. The model will be validated with a series of lipophilic metalloporpyrin catalytic antioxidants designed to cross the blood-brain barrier. Together, the in vitro and in vivo approaches can be utilized sequentially to identify candidate therapeutic agents. This screening procedure will allow us to participate the the NINDS cooperative program designed to specifically develop candidate therapies for clinical development. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to determine the mechanisms by which environmental agents produce neurodegeneration. Environmental neurotoxicants are strongly implicated in the etiology of neurodegenerative diseases such as Parkinson's disease (PD). Redox cycling agents such as the herbicide paraquat (PQ2+) are found in the environment and several compounds in this class have come under investigation as neurotoxic agents based on the ability to produce reactive oxygen species (ROS) in an aerobic environment and epidemiological reports linking their exposure with increased risk of PD. However, the cellular and molecular mechanisms by which environmental redox cycling agents produce ROS and resultant neurotoxicity remain incompletely understood. It is hypothesized that the mitochondria play a key role in ROS production by redox cycling agents and consequent neurotoxicity. The hypothesis predicts that redox cycling agents such as PQ2+ increase mitochondrial ROS production by a mechanism involving its partial reduction by electrons of the electron transport chain to form the PQ+. radical via complex III as the redox enzyme. The hypothesis further predicts that mitochondria are a target of redox cycling agents and scavenging mitochondrial oxidants will ameliorate neurotoxicity. To address this, the following specific aims are proposed. Specific Aim 1: Determine the mitochondrial mechanism of ROS generation and neurotoxicity by redox cycling agents. Specific Aim 2: Determine if mitochondria are a source and a target of oxidative stress produced by redox cycling agents in dopaminergic cells in vivo. Specific Aim 3: Determine if mitochondrially targeted therapies ameliorate mitochondrial oxidative stress and neurotoxicity produced by in vivo administration of redox cycling agents. These studies can elucidate the mechanism by which exposure to environmental redox cycling agents can injure dopaminergic neurons and provide a rational therapy to treat neurotoxicant-induced Parkinson's disease. PUBLIC HEALTH RELEVANCE A steadily growing body of literature suggests that environmental agents alone, or in combination with genetic factors or other toxicants may predispose individuals to neurodegenerative diseases such as Parkinson's disease (PD). Paraquat and diquat are widely used prototypical redox cycling environmental agents with the potential of causing parkinsonism. The extensive use of these agents as a landscape and aquatic herbicides underscores the importance of their environmental and occupational risk. Therefore, elucidating the molecular mechanisms of such agents and development of rational therapeutic strategies that penetrate the blood brain barrier is critical. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a prevalent, often drug-resistant form of epilepsy usually preceded by injury which progressively leads to the development of recurrent unprovoked seizures by a process known as epileptogenesis. The role of mitochondria in TLE is recently emerging; however whether and how mitochondrial functions contribute to TLE remains unknown. It is hypothesized that mitochondrial oxidative stress plays a key role in epileptogenesis. We seek to determine whether and how mitochondrial reactive oxygen species (ROS) contribute to epileptogenesis. Preliminary data suggests that mitochondrial oxidative stress occurs to varying extent throughout epileptogenesis in two chemoconvulsant models of epilepsy. Using a diversity of approaches including state-of-the-art mitochondrial redox techniques, mass spectrometry and continuous video-EEG monitoring in two chemoconvulsant animal models, the following specific aims will be examined. Specific Aim 1 will determine the occurrence of mitochondrial oxidative stress during epileptogenesis and establish a cause-effect relationship between oxidative stress and epilepsy development. Specific Aim 2 will determine how mitochondrial ROS contributes to epileptogenesis. Specifically the role of posttranslational oxidative modification of complex I and mitochondrial DNA damage and repair will be examined. Specific Aim 3 will determine if pharmacological inhibition of mitochondrial oxidative stress can prevent epileptogenesis. These studies will establish a potential role of mitochondrial oxidative stress in epileptogenesis and suggest novel therapeutic approaches for modifying the progression of TLE.

DESCRIPTION (provided by applicant): Chemical warfare agents are a threat to both the civilian population and military personnel and there is an urgent need for rapid development of medical countermeasures. In particular, there is a need for neuroprotective therapies to counteract the central nervous system effects of chemical warfare agents. Recent efforts by the NIH CounterAct program to develop medical countermeasures have identified AEOL10150 as a lead compound that rescues lung injury caused by mustard and chlorine. The goal of this project is to determine if AEOL10150 is a neuroprotective medical countermeasure against surrogate and primary nerve agents. Based on our preliminary results and prior work, catalytic removal of reactive species by AEOL10150 is predicted to blunt oxidative stress and thereby prevent downstream changes such as metabolic dysfunction, gliosis and neuronal loss. Specific goals of the project are to determine the pharmacokinetic profile of AEOL10150 in rats (Specific Aim 1) and evaluate the neuroprotective efficacy of AEOL10150 against pilocarpine-induced neurotoxicity in rats (Specific Aim 2) using a variety of biochemical, pharmacological and analytical tools and techniques. These studies can help identify AEOL10150 as a versatile medical countermeasure against chemical warfare agents.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a prevalent, often drug resistant form of acquired epilepsy that frequently presents with co-morbidities such as cognitive dysfunction. Oxidative stress has been implicated in various neurological diseases including experimental models of TLE. However, whether oxidative stress contributes to chronic seizures and/or cognitive decline in TLE is unknown. Isoketals (IsoKs) and neuroketals (NeuroKs) are highly reactive gamma-ketoaldehydes (?KAs) formed via the non-enzymatic, free radical catalyzed, peroxidation of arachidonic acid and docosahexaenoic acid, respectively which are highly enriched in brain. ?KAs rapidly and irreversibly adduct to lysine residues and readily crosslink proteins which can lead to cell dysfunction. Elevated IsoKs in plasma and tissues occur in pathological conditions including Alzheimer's disease, atherosclerosis, and inflammation. Pharmacological scavenging of ?KAs has been shown to markedly inhibit cognitive impairment in humanized apoE4 mice, an animal model of Alzheimer's disease. The goals of this project are to 1) determine whether IsoK and/or NeuroK adduct formation occurs during epileptogenesis, 2) Identify candidate hippocampal proteins adducted by IsoKs/NeuroKs using mass spectrometry during epileptogenesis, 3) determine if a pharmacological scavenger of ?KAs, salicylamine (SA) can inhibit cognitive decline and/or chronic seizures associated with epileptogenesis and 4) determine if SA can inhibit neuronal death and/or reactive gliosis associated with epileptogenesis. Collectively, this project can identify a novel role of ?KAs as mediators of oxidative stress in chronic epilepsy and/or cognitive impairment associated with TLE and provides a therapeutic approach for its treatment.

DESCRIPTION (provided by applicant): Chemical warfare agents are a threat to both the civilian population and military personnel and there is an urgent need for rapid development of medical countermeasures. Controlling seizure activity and downstream consequences is critical for neuroprotection and survival after organophosphate (OP) exposure. The goal of this research project is to develop a novel and efficacious neuroprotective countermeasure against OP nerve agents. Recent efforts by the NIH CounterACT program to develop medical countermeasures have identified AEOL10150 as a lead compound that rescues lung injury caused by mustard and chlorine. The goal of this project is to determine if AEOL10150 is a neuroprotective medical countermeasure against a model OP and a primary nerve agent. Based on our preliminary results and prior work using a surrogate nerve agent, catalytic removal of reactive species by AEOL10150 is predicted to blunt oxidative stress and thereby prevent downstream changes such as neuronal loss and cognitive dysfunction. Specific goals of the project are to characterize oxidative stress as a target in two OP models, determine pharmacokinetics, bioavailability, and neuroprotective efficacy of AEOL10150 in these models using a variety of biochemical, pharmacological and analytical tools and techniques. These studies can help identify AEOL10150 as a versatile medical countermeasure against chemical warfare agents.

? DESCRIPTION (provided by applicant): Mitochondria control critical cell functions that can impact epilepsy and its comorbidities. Key features of temporal lobe epilepsy (TLE) such as its progressive nature, rising incidence with aging and bioenergetic demands suggest mitochondrial involvement. Mitochondrial dysfunction has been implicated in various neurological diseases including experimental models of TLE. However, the precise mechanisms underlying mitochondrial dysfunction in TLE remain unclear. The proposal builds upon our previous discoveries of impaired mitochondrial redox status and bioenergetics in experimental models of TLE. A central mediator that links mitochondrial oxidative stress with bionenergetic dysfunction is sirtuin-3 (SIRT3), a mitochondrial nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase known to maintain metabolic homeostasis. SIRT3 is exquisitely sensitive to aging and serves as a major switch to regulate mitochondrial protein acetylation to control metabolic flux and energy. It is hypothesized that decreased SIRT3 activity and consequent increases in mitochondrial protein acetylation contribute to the impaired bioenergetics in TLE. Using novel mass spectrometry-based quantitative mitochondrial acetylomics, brain-specific SIRT3 conditional knockout mice and restorative therapies, we plan to explore the role of SIRT3 in TLE. Aim 1 will determine if SIRT3 dysfunction occurs following chemoconvulsant-induced epilepsy. Aim 2 will determine if conditional deletion of SIRT3 or its target, Sod2 is sufficient to cause age-related epilepsy and cognitive dysfunction and Aim 3 will determine if restoration of SIRT3 activity by supplementation with NAD+ precursor attenuates deficits observed in chemoconvulsant-induced TLE. Collectively, this project can identify a novel role of SIRT3 as a mediator of mitochondrial dysfunction in chronic seizures and/or cognitive impairment associated with TLE and provide a therapeutic approach for its treatment.

Inflammation and oxidative stress are two important processes known to occur concurrently in epilepsy. Neuroinflammation is a well-recognized mediator of acquired epilepsy and a highly investigated target for therapeutic intervention. Glutathione depletion occurs following insults that cause epilepsy and its repletion can modify seizures and cognitive impairment. Recent work in our laboratory has shown that elevation of glutathione can inhibit neuroflammation. The long term goal of this proposal is to elevate brain glutathione levels in models of acquired epilepsy by a novel mechanism i.e. direct posttranslational activation of its rate-limiting synthetic enzyme, ?- glutamyl cysteine ligase. The goal of this proposal is to test the hypothesis that increased de novo synthesis of glutathione decreases neuroinflammation, seizures and/or cognitive dysfunction. Aim. 1 will test select compounds for their ability to increase glutathione, inhibit neuroinflammation and neuronal excitability in vitro. Aim 2 will determine if compounds capable of elevating glutathione inhibit seizures, epilepsy and/or cognitive dysfunction. These studies can identify a novel mechanism of reducing neuroinflammation in acquired epilepsy and suggest redox-based compounds for the treatment of epilepsy and associated comorbidities.

Although altered metabolism is rapidly emerging as a key feature of epilepsies, it has not been systematically investigated in any genetic form of pediatric epilepsy. Dravet syndrome (DS), a catastrophic childhood epilepsy associated with de novo mutations in a voltage-activated sodium channel, Nav1.1 is one of the most common genetic epilepsies. DS patients suffer with intractable early-life seizures, and debilitating comorbidities. Energy metabolism in comorbidities associated with DS remain virtually unexplored. To address this unmet need, recent collaborative research in our two laboratories revealed decreased glycolytic and oxygen consumption rates in a validated zebrafish model of DS i.e., scn1Lab mutants. This was accompanied by downregulation of key enzymes, pck1 and pck2, in the gluconeogenesis pathway. Here, we hypothesize that energy disruption occurs in DS due to glucose dysregulation resulting in seizures and/or comorbidities. The following aims are proposed to test this hypothesis. Aim 1 will determine if pharmacological inhibition of pck1 and/or pck2 phenocopies metabolic and behavioral deficits in wildtype zebrafish. Aim 2 will determine if pharmacological manipulation of pck1 and/or pck2 is therapeutic in scn1Lab mutant zebrafish. These studies promise to provide a mechanistic explanation of the metabolic defects observed in DS and could suggest novel avenues for therapeutic intervention.

Mitochondrial Reactive Oxygen Species in Epilepsy-Associated Astrogliosis The parent grant focuses on the interplay between neuroinflammation and redox status. The supplemental research addresses a related but distinct goal to investigate the role of mitochondria in seizure-induced neuroinflammation. Mitochondria integrate the energy requirements of neuronal cells and circuits with nutrients, ions, inflammatory mediators and redox status. Mitochondrial dysfunction, oxidative stress and neuroinflammation have been linked with pathophysiological hyperexcitability associated with epilepsy. Neuroinflammation specifically has been identified as a therapeutic target for epilepsy, however the detailed mechanisms underlying seizure-induced neuroinflammation, including upregulation of astrocyte- specific glial fibrillary acidic protein (GFAP), remain at large. One clue regarding upregulation of GFAP arises from recent studies in our laboratory showing a robust transcriptional upregulation of GFAP in mice lacking the anti-oxidant mitochondrial manganese superoxide dismutase-2 (Sod2) in forebrain neurons, a finding replicated in primary neuronal-glial culture. In contrast, mixed cultures in which neurons were selectively depleted displayed no such upregulation. The goal of this project is to determine if mitochondrial reactive oxygen species (mtROS) generated within neurons can activate neuroinflammation via upregulation of GFAP expression as a result of posttranslational redox modification of a conserved cysteine (Cys294) in GFAP, resulting in long-lasting neuroinflammation. Aim 1 will determine if neuronal mtROS is sufficient to induce GFAP upregulation and astrogliosis in vitro using mixed rat neuronal culture and in vivo using Nex-Cre/Sod2f/f mice microinjected with viral vectors driving GCaMP6f within astrocytes. Aim 2 will determine if this astrogliosis involves redox modification of GFAP protein by mutating Cys294. These studies will reveal novel redox-based therapeutic targets to treat epilepsy.