Patent Document

CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Ser. No. 62/103,629 filed Jan. 15, 2015. This disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application. 
     
    
     TECHNICAL FIELD 
       [0002]    This disclosure generally relates to methods of treating neurological inflammatory diseases. 
       BACKGROUND 
       [0003]    Molybdenum cofactor (MoCo) is an evolutionarily conserved molybedenum (Mo) coordinated pterin-compound and is necessary for the activity of all Mo-enzymes, with the exception of nitrogenase. MoCo is synthesized by a unique and evolutionarily conserved multi-step pathway, from which only two intermediates have been identified: the sulphur- and metal-free pterin derivative, precursor Z, also known as cPMP, and molybdopterin (MPT), a pterin with an ene-dithiol function, which is essential for the Mo-linkage. 
       SUMMARY 
       [0004]    Methods of treating neurological inflammatory disease or seizures caused by neuroinflammation by administering cPMP are described. Treating neuroinflammatory and neurometabolic diseases with cPMP is described so as to override dyshomeostasis in the MoCo synthesis pathway and control synaptic inhibition in the gephyrin-GABAR pathway. This is a novel strategy for preventing neural circuit dyshomeostasis by stabilizing inhibitory synapses. 
         [0005]    In one aspect, a method of treating a neurological inflammatory disease in an individual is provided. Such a method typically includes administering an effective amount of cPMP to the individual, thereby treating the individual. Representative neurological inflammatory diseases include, without limitation, central nervous system (CNS) autoimmune disorders such as multiple sclerosis (MS), neuromyelitis optica (NMO), anti-NMDA receptor encephalitis, and autoimmune epilepsies; Alzheimer&#39;s disease; amyotrophic lateral sclerosis (ALS); schizophrenia; autism; epilepsy and other seizure disorders (e.g., febrile seizures without underlying infection); CNS infectious diseases (e.g., viral, bacterial, parasitic); MoCo deficiencies; and other neurodegenerative diseases involving microglial and astrocytic inflammatory responses. 
         [0006]    In some embodiments, the administering step is selected from the group consisting of orally, topically, and parenterally. In some embodiments, such a method further includes identifying an individual having a neurological inflammatory disease (e.g., identifying an individual having ALS, epilepsy or another seizure disorder, or autism or schizophrenia). In some embodiments, such a method further includes identifying an individual having a mutation in a gene selected from the group consisting of gephyrin, MOCS1, and MOCS2. In some embodiments, such a method further includes monitoring the individual for the amount of MPT, MoCo, MoCo—S or another intermediate or by-product of the MoCo biosynthesis pathway. 
         [0007]    In another aspect, a method of treating ALS, epilepsy or another seizure disorder in an individual is provided. Such a method generally includes administering an effective amount of cPMP to the individual, thereby treating the individual. In some embodiments, the administering step is selected from the group consisting of orally, topically, and parenterally. In some embodiments, such a method further includes identifying an individual having ALS, epilepsy or another seizure disorder. In some embodiments, such a method further includes monitoring the individual for the amount of MPT, MoCo, MoCo—S or another intermediate or by-product of the MoCo biosynthesis pathway. 
         [0008]    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0009]      FIG. 1A  is a schematic showing the synthesis of molybdenum co-factor under homeostatic conditions. 
           [0010]      FIG. 1B  are the chemical structures of the first three compounds shown in  FIG. 1A : guanosine triphosphate (GTP), cyclic pyranopterin monophosphate (cPMP), and molybdopterin (MPT). 
           [0011]      FIG. 2  is a schematic showing the synthesis of molybdenum co-factor during inflammatory conditions. 
           [0012]      FIG. 3  is a schematic showing a summary of the impact of inflammatory cytokines on elements of the molybdenum biosynthesis pathway and the concomitant dysregulation of inhibitory synaptic function that results in hyperexcitability and seizure. 
           [0013]      FIG. 4  demonstrates the IFNgamma-induced dysregulation of the MoCo pathway and down-regulation of inhibitory synaptic proteins. Mouse cortical neurons were cultured in a two-chamber device that separates cell bodies from axons. Panel A is a schematic of the chambered device constructed in PDMS polymer. Panel B is a low-magnification image of the corresponding regions shown in Panel A stained with an antibody against neurofilament (an axon-specific protein). Higher magnification images are shown of the cell body chamber (Panel C), the axon grooves (Panel D), and the axon chamber (Panel E). DAPI staining indicates the complete absence of any cells in the axon chamber. Panel F is a schematic showing the experimental design: IFNgamma was added to the pure axons in the axon chamber; RNA was collected from the cell body chamber 72 hours later and analyzed by microarray to identify changes in gene expression. 
           [0014]      FIG. 5  are graphs showing that IFNgamma stimulation of the distal axons stimulated a transcriptional program in the neuron cell bodies that is marked by simultaneous down-regulation of numerous components of inhibitory synapses (gephyrin (Panel A), glycine receptor beta subunit (Panel B), numerous GABA receptor elements (not shown), and multiple gephyrin-binding scaffolds (not shown)) and robust up-regulation of MOCOS (Panel C). At the same time, GTP cyclohydrolase I (Panel D), xanthine dehydrogenase (Panel E), and aldehyde dehydrogenase (Panel F) were significantly up-regulated, indicating substantial changes in the MoCo pathway. 
           [0015]      FIG. 6  shows spontaneous calcium levels in neurons stimulated for 24 hr with TNF-alpha or unstimulated (PBS, vehicle control). The PBS control cultures show low amplitude calcium transients that are non-synchronous. In contrast, stimulation with TNFalpha drove the cells to exhibit large amplitude calcium signals that were highly synchronized, indicating a general reduction in synaptic inhibition in the neural network. Calcium levels were monitored using a fluorescent reporter transduced into the neurons with adenovirus. 
           [0016]      FIG. 7  demonstrates that spontaneous activity in neurons stimulated with TNFalpha or IFNgamma is highly correlated (hence, synchronous), in contrast to vehicle control-stimulated cultures. The Pearson correlation matrix was calculated for all of the neurons showing spontaneous activity in the culture and heat-mapped (red=highly correlated activity; blue=uncorrelated activity). In the vehicle control neurons, there is a small cluster of synchronous activity but the overall network is uncorrelated. TNFalpha or IFNgamma stimulation resulted in nearly complete correlation across the entire network. Moreover, the absolute number of spontaneously active cells is clearly increased in the IFNgamma and TNFalpha stimulated networks (137 neurons and 151 neurons, respectively, versus only 42 neurons in the vehicle control). These findings indicate that inhibition in the network is highly suppressed by TNFalpha or IFNgamma stimulation. 
           [0017]      FIG. 8  are graphs showing the averaged calcium responses in cytokine-stimulated neurons.  FIG. 8A  shows the basal levels of activity in the neuron cultures. Non-synchronized calcium responses occur in the control cultures, resulting in an overall low level of synaptic activity in the network.  FIGS. 8B and 8C  show that stimulation with IFN gamma or TNF alpha results in network bursting and highly synchronized synaptic activity in which many cells in the culture flux calcium at the same time.  FIG. 8D  shows that treatment of control cultures with picrotoxin (2.4 μM), a small molecule inhibitor of inhibitory GABAergic channels, induces network synchrony and bursting that phenocopies the response observed in cytokine-stimulated cultures.  FIG. 8E  shows that the addition of GABA (27 μM) to control cultures completely suppresses synaptic activity, consistent with enhanced inhibition. 
       
    
    
     DETAILED DESCRIPTION 
       [0018]    As described herein, modulation of the molybdenum cofactor biosynthetic pathway returns cells that are under inflammatory stress to homeostasis by reducing shunting of gephyrin away from stabilization of inhibitory synapses and renormalizing inhibitory control of neural networks. Therefore, as described herein, cPMP can be administered to an individual to treat a number of different neurological inflammatory diseases or relieve the symptoms that are a result of a number of different neurological disorders. 
       Molybdenum Cofactor (MoCo) and the Genetics Associated Therewith 
       [0019]    All of the molybdenum (Mo) containing enzymes of humans, animals, plants, arachaea and bacteria, with the exception of nitrogenase from prokaryotes, require a co-factor that includes an organic moiety, molybdopterin (MPT), and molybdenum. This molybdenum cofactor (MoCo) possesses, across all phylogenetic groups, the same base structure that is very unstable in its free form, in particular under aerobic conditions when it is not bound to an apoprotein. The biosynthetic pathway, discussed in more detail below, is evolutionarily conserved and the corresponding proteins from various organisms are extremely homologous. 
         [0020]    A mutational defect in MoCo-biosynthesis leads to simultaneous loss of the activities of all Mo enzymes, inclusive the sulphite oxidase. Human MoCo deficiency is a severe, autosomal-recessive genetic disorder, which clinically cannot be differentiated from the less frequently occurring sulphite-oxidase deficiency. Most afflicted patients exhibit neurological abnormalities such as non-treatable seizures and lack of development of the brain, which can be traced back to the toxicity of sulphite, a lack of sulphate or both. Most afflicted patients usually die in early childhood. 
         [0021]    A eukaryotic gene encoding a protein involved in MoCo-biosynthesis was obtained from  Arabidopsis thaliana . Subsequently, a human gene encoding a protein involved in MoCo-biosynthesis, was obtained and designated MOCS1. Due to alternate splicing of the MOCS1 gene, the MOCS1A and MOCS1B proteins are produced and convert a guanosine derivative into the sulphur-free precursor Z (i.e., cPMP). Patients having a mutation in the MOCS1 gene are referred to as having MoCo-deficiency type A. In a subsequent step, precursor Z (i.e., cPMP) is converted to MPT by an enzyme, which is encoded by a gene designated MOCS2 and activated by the protein encoded by the MOCS3 gene. Patients having a mutation in the MOCS2 gene are referred to as having MoCo-deficiency type B. Finally, Mo is inserted into MPT by a protein referred to as gephyrin. Patients having a mutation in the gene encoding gephyrin, GEPHN, are referred to as having MoCo-deficiency type C. 
       Inflammation-Induced Injury and the MoCo Biosynthesis Pathway 
       [0022]    Inflammation triggers neuronal and axonal injury via multiple mechanisms. However, perhaps the most physiologically relevant mechanism of neuronal injury is inflammation-induced synaptic dysfunction and derailment of homeostatic electrophysiological activity in neural circuits. For example, TNFalpha and IFNgamma are known to induce hippocampal injury by triggering excitotoxicity. There are multiple mechanism by which these inflammatory cytokines alter synaptic function—for example, by altering excitatory receptor function and increasing synaptic calcium levels. However, an equally important mechanism of cytokine-mediated synaptic dysregulation may be down-regulation of inhibitory receptors. Reduced inhibition will raise the overall level of synaptic activity and create a feedback loop in which excitatory synaptic activity builds, calcium accumulates in the synapse, and calcium-dependent proteases degrade synaptic connections. This feedback loop likely exacerbates the loss of inhibition, creating spreading synaptic dysregulation, neural injury, and neural circuit hyperactivity and/or failure. 
         [0023]    Gephyrin is a critical scaffolding protein that controls the localization, clustering, and inhibitory function of glycine and GABA receptors at synaptic sites. Gephyrin function is directly tied to inhibitory control of neural circuitry, and down-regulation of gephyrin is linked to seizures and hyperexcitability of neurons. Genetic defects in gephyrin are associated with autism, epilepsy, and schizophrenia. 
         [0024]    The inhibitory receptor scaffolding function of gephyrin is mediated by a C domain that links evolutionarily conserved G and E domains. Crucially, the G and E domains of gephyrin are necessary for the synthesis of molybdenum cofactor (MoCo), a molecule that is required for activation of molybdenum-dependent enzymes necessary for survival. Humans with mutations in the non-scaffolding domains of gephyrin exhibit MoCo deficiencies and severe neurological and developmental abnormalities. 
         [0025]    Under homeostatic conditions, guanosine triphosphate (GTP) is converted to a coordination complex of molybdopterin and a molybdenum oxide (MoCo) by the action of several catalytic enzymes including gephyrin. MoCo must be sulfurated by the molybdenum cofactor sulfurase (MOCOS) in order to function as a co-factor for xanthine dehydrogenase and other molybdenum-dependent enzymes. Xanthine dehydrogenase catalyzes the conversion of xanthine and NAD+ to urate and NADH, providing a fundamental reducing agent necessary for redox metabolism and the production of cellular energy stores in the form of ATP. 
         [0026]    Increases in cellular calcium lead to the activation of calcium-dependent proteases such as calpain. Calpain targets two components of the MoCo biosynthesis pathway, resulting in disruption of cellular homeostasis. Calpain irreversibly converts xanthine dehydrogenase to xanthine oxidase, creating a powerful source of reactive oxygen species that directly damage the cell. Moreover, the conversion of xanthine dehydrogenase to xanthine oxidase shunts cellular metabolism away from the production of NADH and ATP, compromising cellular energy balance. Calpain also cleaves gephyrin, resulting in loss of scaffolding function and down-regulation of inhibitory synaptic function. Calpain-cleaved gephyrin also exhibits altered MoCo synthesis function caused by the physical separation of the G and E domains. 
         [0027]    Calpain-mediated cleavage of gephyrin at synapses creates a feedback loop in which reduced inhibitory receptor function results in increased excitatory receptor activity, increased calcium influx, and further activation of calpain. 
         [0028]    Inflammatory cytokines such as IFNgamma and TNFalpha directly alter calcium flux in target cells and increase expression and activation of calpain. Inflammatory cytokine exposure will therefore reduce inhibitory synaptic function, increase excitatory load, alter MoCo synthesis, and drive the target cell toward reactive oxygen species production. 
         [0029]    Inflammatory cytokines also increase the expression of GTP cyclohydrolase I, the rate limiting step in the de novo synthesis of 5,6,7,8-tetrahydrobiopterin from GTP. Inflammatory cytokine exposure will therefore shunt GTP away from MOCS1A/MOCS1AB-mediated production of cyclic pyranopterin monophosphate (cPMP), resulting in decreased MoCo synthesis. 
       Seizures and the MoCo Biosynthesis Pathway 
       [0030]    Febrile seizures are the most common type of neurologic complication in infants and preschool children. Febrile seizures occur at body temperatures over 38° C. in the absence of acute electrolyte imbalance or dehydration, in the absence of direct CNS infection, and without previous evidence of unprovoked seizures (Commission on Epidemiology and Prognosis, 1993, Epilepsia, 34:592-6). It is estimated that 1 in 25 children will experience at least one febrile seizure, and the occurrence of febrile seizure is associated with heightened susceptibility to future seizures—1 in 3 individuals with childhood febrile seizure will experience another seizure of some type within 20 years. Moreover, the risk of epilepsy among individuals experiencing a childhood febrile seizure is higher than the general population, with incidence reports ranging from 6% to 13%, rates that are more than 10 times higher than in the general population. Of note, an increased frequency of febrile seizure is associated with some vaccines in children, including measles-containing and pertussis vaccines. For example, the diptheria-tetanus-pertussis vaccine is associated with an increase of 6-9 cases of febrile seizure per 100,000 vaccinations and fever is observed in 50% of vaccinated infants. The measles-mumps-rubella (MMR) vaccine is associated with an increase of up to 16 cases of febrile seizure per 100,000 vaccinations, and the addition of varicella to the same vaccine increases the risk even further. Finally, acute seizures associated with viral, bacterial, and parasitic infections in children, whether systemic or localized to the CNS, are a primary factor in the development of epilepsy. For example, during the 2009-2010 influenza A (H1N1) pandemic, in which more than 70% of infected individuals were younger than 24 years of age, up to 6% of infections resulted in neurological complications, with over 10% of children less than 15 years of age presenting with neurological symptoms. Of these complications, seizure and abnormal EEG were the most common. Likewise, infection with enterovirus 71, a picornavirus with widespread epidemic infectivity throughout the Asia-Pacific region, is associated with neurologic complications in almost 20% of infected children. A high incidence of seizure also occurs in children infected with  Plasmodium, Taenia solium  and other parasites. The common factor across all of these seizure events, whether febrile or afebrile, is the production of inflammatory cytokines in the CNS. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) are powerful pyrogens that are elevated in the brain during febrile seizures, and experimental evidence directly supports a role for these factors in the initiation of seizures. Likewise, TNFalpha, interleukin-6 (IL-6), and interferon gamma (IFNgamma) are produced and/or released in the CNS during acute infection. 
       Methods of Treating Neurological Inflammatory Diseases 
       [0031]    As described herein, bypassing GTP-to-cPMP conversion by providing exogenous cPMP can stabilize MoCo synthesis and provide regulatory feedback control to drive a dysregulated system back toward homeostasis. Similarly, blocking GTP cyclohydrolase I to push GTP back into the MoCo pathway; increasing expression or activity of MOCS1A and/or MOCS1AB activity to push GTP to cPMP; or increasing expression or activity of MOCS2A, MOCS2B, and/or MOCS3 activity to push cPMP to MPT also can stabilize MoCo synthesis and provide regulatory feedback control to drive a dysregulated system back toward homeostasis. Likewise, increasing expression or activity of gephyrin to increase MoCo synthesis and to stabilize inhibitory synapses; or blocking calpain to prevent the conversion of xanthine dehydrogenase to xanthine oxidase can stabilize MoCo synthesis and provide regulatory feedback control to drive a dysregulated system back toward homeostasis. Supplementation with cPMP may be enhanced by simultaneously blocking calpain to prevent aberrant xanthine oxidase-dependent production of reactive oxygen species during an inflammatory drive and to maintain gephyrin-dependent synaptic stabilization. In addition, given the link between inflammation-induced seizures and gephyrin/GABAR, treatment with glycogen synthase kinase 3beta (GSK3beta) inhibitors may increase gephyrin activity. For example, GSK-3 Inhibitor IX (CAS 667463-62-9) or lithium chloride may suppress seizures by enhancing gephyrin function and overcome inflammation-induced shunting of gephyrin away from inhibitory synapse stabilization. Thus, methods of treating a neurological inflammatory disease are described herein. 
         [0032]    As used herein, neurological inflammatory diseases include, without limitation, central nervous system (CNS) autoimmune disorders such as multiple sclerosis (MS), neuromyelitis optica (NMO), anti-NMDA receptor encephalitis, and autoimmune epilepsies; Alzheimer&#39;s disease; amyotrophic lateral sclerosis (ALS); schizophrenia; autism; epilepsy and other seizure disorders (e.g., febrile seizures without underlying infection); CNS infectious diseases (e.g., viral, bacterial, parasitic); MoCo deficiencies (e.g., due to genetic mutations); and other neurodegenerative diseases involving microglial and astrocytic inflammatory responses. A neurological inflammatory disease for which the methods described herein are particularly useful is neuroinflammation-induced seizures. “Treating” as used herein refers to relieving, reducing or ameliorating the symptoms of any of such neurological inflammatory diseases. 
         [0033]    As described herein, methods of treating a neurological inflammatory disease can include administering an effective amount of cPMP to an individual. In some instances, an individual may be identified as having a neurological inflammatory disease (e.g., central nervous system (CNS) autoimmune disorders such as multiple sclerosis (MS), neuromyelitis optica (NMO), anti-NMDA receptor encephalitis, and autoimmune epilepsies; Alzheimer&#39;s disease; amyotrophic lateral sclerosis (ALS); schizophrenia; autism; epilepsy and other seizure disorders (e.g., febrile seizures without underlying infection); CNS infectious diseases (e.g., viral, bacterial, parasitic); MoCo deficiencies (e.g., due to genetic mutations); and other neurodegenerative diseases involving microglial and astrocytic inflammatory responses prior to being administered an effective amount of cPMP. In some instances, an individual may be identified as having a mutation in the MOCS1 or MOCS2 gene or the gene encoding gephyrin prior to being administered an effective amount of cPMP. cPMP can be administered on a long-term basis (e.g., when genetic mutations are present) or cPMP can be administered as an acute intervention to renormalize inhibitory synapses. 
         [0034]    Also as described herein, methods of treating a neurological inflammatory disease can further include monitoring the individual. Simply by way of example, the amount of MPT, MoCo, MoCo—S or another intermediate or by-product of the MoCo biosynthesis pathway (e.g., levels of xanthine, hypoxanthine, uric acid, sulfite, and S-sulfocysteine) can be monitored in an individual (e.g., in urine) and can be used as biomarkers for effective cPMP dosing. In some instances, the individual&#39;s symptoms can be monitored (e.g., for improvement) or feedback from EEG can be used to monitor treatment and/or establish dosing. Depending upon the results of the monitoring step, the effective amount of cPMP can be adjusted as desired. 
         [0035]    Typically, an effective amount of cPMP is an amount that treats (e.g., ameliorates, relieves or reduces the symptoms of) a neurological inflammatory disease without inducing any adverse effects. An effective amount of cPMP can be formulated, along with a pharmaceutically acceptable carrier, for administration to an individual. The particular formulation, will be dependent upon a variety of factors, including route of administration, dosage and dosage interval of a compound the sex, age, and weight of the individual being treated, the severity of the affliction, and the judgment of the individual&#39;s physician. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all excipients, solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with administration. The use of such media and agents for pharmaceutically acceptable carriers is well known in the art. Except insofar as any conventional media or agent is incompatible with a compound, use thereof is contemplated. 
         [0036]    Pharmaceutically acceptable carriers are well known in the art. See, for example Remington:  The Science and Practice of Pharmacy , University of the Sciences in Philadelphia, Ed., 21st Edition, 2005, Lippincott Williams &amp; Wilkins; and  The Pharmacological Basis of Therapeutics , Goodman and Gilman, Eds., 12th Ed., 2001, McGraw-Hill Co. Pharmaceutically acceptable carriers are available in the art, and include those listed in various pharmacopoeias. See, for example, the U.S. Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British pharmacopeia (BP); the U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) publications (e.g., Inactive Ingredient Guide (1996)); and Ash and Ash, Eds. (2002) Handbook of Pharmaceutical Additives, Synapse Information Resources, Inc., Endicott, N.Y. The type of pharmaceutically acceptable carrier used in a particular formulation can depend on various factors, such as, for example, the physical and chemical properties of cPMP, the route of administration, and the manufacturing procedure. 
         [0037]    A pharmaceutical composition that includes cPMP as described herein typically is formulated to be compatible with its intended route of administration. Suitable routes of administration include, for example, oral, rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteral administration. Routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration, as well as intraperitoneal, intra-arterial, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration. 
         [0038]    For intravenous injection, for example, the composition may be formulated as an aqueous solution using physiologically compatible buffers, including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH, and a tonicity agent, such as, for example, sodium chloride or dextrose. For oral administration, a compound can be formulated in liquid or solid dosage forms, and also formulation as an instant release or controlled/sustained release formulations. Suitable dosage forms for oral ingestion by an individual include tablets, pills, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, and emulsions. 
         [0039]    Oral dosage forms can include excipients; excipients include, for example, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, anti-adherants, cationic exchange resins, wetting agents, antioxidants, preservatives, coloring, and flavoring agents. Specific examples of excipients include, without limitation, cellulose derivatives, citric acid, dicalcium phosphate, gelatine, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate, sorbitol, starches, stearic acid or a salt thereof, sugars (e.g., dextrose, sucrose, lactose), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes. 
         [0040]    cPMP as described herein also can be formulated for parenteral administration (e.g., by injection). Such formulations are usually sterile and, can be provided in unit dosage forms, e.g., in ampoules, syringes, injection pens, or in multi-dose containers, the latter usually containing a preservative. The formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain other agents, such as buffers, tonicity agents, viscosity enhancing agents, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives. Depending on the injection site, the vehicle may contain water, a synthetic or vegetable oil, and/or organic co-solvents. In certain instances, such as with a lyophilized product or a concentrate, the parenteral formulation would be reconstituted or diluted prior to administration. Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof, can serve as controlled or sustained release matrices, in addition to others well known in the art. 
         [0041]    In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims. 
       EXAMPLES 
     Example 1—Effects of IFN Gamma on Neurons 
       [0042]    Mouse cortical neurons were cultured in a two-chamber device that separates cell bodies from axons. A schematic of the chambered device constructed in PDMS polymer is shown in  FIG. 4A . 
         [0043]    The experimental design is shown in  FIG. 4F . IFN gamma was added to the pure axons in the axon chamber, and RNA was collected from the cell body chamber 72 hours later. The RNA was analyzed by microarray to identify changes in gene expression. 
         [0044]    A low-magnification image of the regions shown in Panel A designated “C,” “D” and “E” are shown in  FIG. 4B , and were stained with an antibody against neurofilament, an axon-specific protein. Higher magnification images were obtained of the cell body chamber ( FIG. 4C ), of the axon grooves ( FIG. 4D ), and of the axon chamber ( FIG. 4E ). DAPI staining indicated the complete absence of any cells in the axon chamber. 
         [0045]    The results of these experiments demonstrated IFN gamma-induced dysregulation of the MoCo pathway and down-regulation of inhibitory synaptic proteins. 
       Example 2—Genes Regulated by IFN Gamma 
       [0046]    IFN gamma stimulation of the distal axons stimulated a transcriptional program in the neuron cell bodies that is marked by simultaneous down-regulation of numerous components of inhibitory synapses including, for example, gephyrin ( FIG. 5A ), glycine receptor beta subunit ( FIG. 5B ), numerous GABA receptor elements (not shown), and multiple gephyrin-binding scaffolds (not shown) as well as robust up-regulation of MOCOS ( FIG. 5C ). At the same time, GTP cyclohydrolase I ( FIG. 5D ), xanthine dehydrogenase ( FIG. 5E ), and aldehyde dehydrogenase ( FIG. 5F ) are significantly up-regulated. Taken together, these results indicate substantial changes in the MoCo pathway. 
       Example 3—Pathogenic Model 
       [0047]    While it is known that inflammatory cytokines alter neuron excitability, the underlying mechanism by which this occurs is poorly understood. Some evidence indicates that cytokines such as TNF alpha induce changes in the distribution of excitatory and inhibitory receptors on the plasma membrane of synapses, resulting in an overall alteration in excitability (Stellwagen et al., 2005, J. Neurosci., 25:3219-28). And while the impact of inflammatory cytokines on synaptic function has been widely reviewed (see, e.g., Fouregeaud and Boulanger, 2010, Eur. J. Neurosci., 32:207-17; Koller et al., 1997, Prog. Neurobiol., 52:1-26; Schafers and Sorkin, 2008, Neurosci. Lett., 437:188-93), the field stills lacks a therapeutically tractable pathogenic model for the described phenomenon. 
         [0048]    It is proposed herein that a key mechanism of hyperexcitability and seizure induction by inflammatory cytokines is the destabilization of the homeostatic molybdenum cofactor biosynthesis pathway via a reduction in gephyrin-mediated transition from MPT to MoCo, a disruption of gephyrin-mediated inhibitory neurotransmitter receptor synaptic clustering, a metabolic switch from energy production via xanthine dehydrogenase to energy failure via xanthine oxidase activity, the reversal of cPMP production from GTP to the production of 7,8-DHNP-3′-TP with concomitant amplification of nitric oxide production, the stress-dependent down-regulation of multiple components of the inhibitory neurotransmitter receptor machine, and the compensatory up-regulation of elements of the molybdenum biosynthetic apparatus. This is a novel hypothesis that places molybdenum cofactor synthesis, and particularly cPMP, at the center of a pathogenic cascade that results in severe clinical sequelae for many children.  FIGS. 2 and 3  outline variations of this proposed model. 
       Example 4—Expression Data 
       [0049]    Cortical neurons were prepared from embryonic day 15 C57BL/6 mouse fetuses, following published protocols (Sauer et al., 2013, Neurobiol. Dis., 59:194-205). In preliminary experiments, after one week in vitro, the neurons were stimulated for 24 hr with IFN gamma (500 U/mL). Quadruplicate RNA samples were collected under treated and untreated conditions, and changes in gene expression were assessed using the Illumina BeadArray system. Only genes that were detected at P&lt;0.05 on the array were considered for further analysis. Expression levels were un-normalized and the relative level of expression following IFN gamma stimulation was compared to untreated controls. Table 1 provides mean±95% confidence intervals; the appropriate statistical test was chosen based on normality and equal variance tests. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 IFN gamma-induced Transcriptional Changes in Mouse Cortical Neurons 
               
             
          
           
               
                 Gene 
                 Role 
                 Fold Change 
                 Statistics 
               
               
                   
               
               
                 GABA A R α3 
                 inhibitory neurotransmitter receptor 
                 0.66 ± 0.10 
                 P = 0.0009 
               
               
                   
                 subunit 
               
               
                 GABA A R β1 
                 inhibitory neurotransmitter receptor 
                 0.56 ± 0.09 
                 P = 0.0001 
               
               
                   
                 subunit 
               
               
                 GABA A R β2 
                 inhibitory neurotransmitter receptor 
                 0.80 ± 0.09 
                 P = 0.03 
               
               
                   
                 subunit 
               
               
                 GABA A R β3 
                 inhibitory neurotransmitter receptor 
                 0.75 ± 0.07 
                 P = 0.0009 
               
               
                   
                 subunit 
               
               
                 GABA A R γ2 
                 inhibitory neurotransmitter receptor 
                 0.64 ± 0.05 
                 P = 0.007 
               
               
                   
                 subunit 
               
               
                 GABA B R2 
                 inhibitory neurotransmitter receptor 
                 0.71 ± 0.10 
                 P = 0.004 
               
               
                   
                 subunit 
               
               
                 Glycine R β1 
                 inhibitory neurotransmitter receptor 
                 0.59 ± 0.07 
                 P = 0.0001 
               
               
                   
                 subunit 
               
               
                 Gephyrin 
                 GABA and glycine receptor scaffold 
                 0.75 ± 0.05 
                 P &lt; 0.0001 
               
               
                   
                 molybdenum cofactor synthesis 
               
               
                   
                 catalyst 
               
               
                 Collybistin 
                 gephyrin clustering regulator 
                 0.71 ± 0.06 
                 P = 0.0006 
               
               
                 MOCOS 
                 molybdenum cofactor sulfurase 
                 2.97 ± 0.21 
                 P &lt; 0.0001 
               
               
                 Xdh 
                 xanthine dehydrogenase; energy 
                 1.47 ± 0.14 
                 P = 0.0001 
               
               
                   
                 metabolism 
               
               
                 Aldh1a1 
                 aldehyde dehydrogenase; metabolism 
                 2.46 ± 0.08 
                 P &lt; 0.0001 
               
               
                 Gch1 
                 GTP cyclohydrolase I; biopterin 
                 1.59 ± 0.04 
                 P = 0.003 
               
               
                   
                 synthesis 
               
               
                 Actb 
                 actin; housekeeping 
                 1.04 ± 0.06 
                 P = 0.407 
               
               
                 Gapdh 
                 glyceraldehyde 3-phosphate 
                 1.03 ± 0.06 
                 P = 0.850 
               
               
                   
                 dehydrogenase 
               
               
                   
               
             
          
         
       
     
         [0050]    In order to determine the relevance of these genes to acute inflammatory events in vivo, C57BL/6 mice were infected with the Theiler&#39;s murine encephalomyelitis virus, as per standard protocols (Howe et al., 2012a, J. Neuroinflamm., 9:50; Howe et al., 2012b, Sci. Rep., 2:545; Lafrance-Corey and Howe, 2011, J. Vis. Exp., 52:2747). The hippocampus was excised at 24 hr after infection, RNA was collected, and Illumina BeadArray analysis was performed to compare gene expression levels to sham infected mice. This time point coincided with a robust inflammatory infiltrate present in the hippocampus, and it was shown that this infiltrate triggers hippocampal neural circuitry changes that result in seizures between 3 and 7 days after infection, followed by the development of epilepsy after 60 days post-infection. It was found that GTP cyclohydrolase I was up-regulated more than 4-fold in the TMEV infected mice; likewise, MOCOS was up-regulated over 3-fold, Xdh was increased by 5-fold, several aldehyde dehydrogenase isoforms were up-regulated, as was collybistin. In contrast, the delta subunit of the GABA A receptor was down-regulated by 30%, and numerous GABA receptor subunits and binding proteins were down-regulated by 10%. 
       Example 5—The Impact of Inflammatory Cytokines on Molybdenum Co-Factor Biosynthesis Gene Expression and Inhibitory Neurotransmitter Receptor Function Gene Expression 
       [0051]    Cortical and hippocampal neurons are prepared from C57BL/6 mice and are cultured under conditions that promote formation of mature synaptic networks. Cultures are exposed to TNFalpha, IL-1β, IL-6, and IFNgamma at several doses (0, 1, 3, 10, 30, 100, 300 ng/mL) and for different times (6, 12, 24, 48, 72, and 96 hr). In parallel cultures, the amount of cell death is assessed using the MTT assay, and doses that kill greater than 10% of the culture are excluded from analysis. RNA is collected using Qiagen RNeasy kits and cDNAs are generated using the Roche Transcriptor first strand cDNA synthesis kit and random hexamer primers. Probe-based real-time PCR is performed on the samples using the Roche LightCycler 480 Probes Master system, and the primer pairs and Roche Universal Probe Library hydrolysis probes defined in Table 2. Expression is normalized to Aco2 and UROD, genes that previously have been defined as suitable housekeeping factors. A multi-factor normalization scheme is used to quantify relative differences in gene expression between controls and cytokine treated samples (Anderson et al., 2004, Cancer Res., 64:5245-50). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 RT-PCR Analysis of Inflammatory Cytokine-Induced Changes 
               
               
                 in MOCO Biosynthesis Pathway and Inhibitory/Excitatory Receptor Expression. 
               
             
          
           
               
                   
                   
                 SEQ 
                   
                 SEQ 
                 UPL 
               
               
                 Gene Target 
                 FORWARD 
                 ID NO 
                 REVERSE 
                 ID NO 
                 PROBE 
               
               
                   
               
               
                 Gephyrin var 1 (NM_145965) 
                 tggtctcatcagttattcccatc 
                   1 
                 cgagaaatgatggagtctgga 
                   2 
                  96 
               
               
                   
               
               
                 Gephyrin var 2 (NM_172952) 
                 tgatcttcatgctcagatcca 
                   3 
                 gcaaatgttgttggcaagc 
                   4 
                  53 
               
               
                   
               
               
                 Collybistin var 1 
                 tgaataaaaaggcaacctaccg 
                   5 
                 tgggagatgtcaatctctgttc 
                   6 
                  83 
               
               
                 (NM_001033329) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Collybistin var 2 
                 tgagaaaagcttctaaacagaaagg 
                   7 
                 gtactggccctggtttaacg 
                   8 
                  77 
               
               
                 (NM_001290384) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Mocos (NM_026779) 
                 aagcaaagtccacacttccag 
                   9 
                 gtagtaccgggaggctgacc 
                  10 
                  72 
               
               
                   
               
               
                 Mocs1 (NM_020042) 
                 ggatgtggtggacatcgtg 
                  11 
                 gaggccgatggttctcag 
                  12 
                  11 
               
               
                   
               
               
                 Mocs2 (NM_013826) 
                   
                  13 
                 tgtgagctgaagacacagca 
                  14 
                  47 
               
               
                   
               
               
                 Mocs3 (NM_001160330) 
                 ggccagatgaccgtctacc 
                  15 
                 gccgtctgcacagttggt 
                  16 
                  94 
               
               
                 [non-intron spanning] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Suox (NM_173733) 
                 tctaccatgagcatcggtgt 
                  17 
                 catcgaagacctcagagccta 
                  18 
                 109 
               
               
                   
               
               
                 Xdh (NM_011723) 
                 agggattccggacctttg 
                  19 
                 gcagcagtttgggttgtttc 
                  20 
                  69 
               
               
                   
               
               
                 Aldh1a1 (NM_013467) 
                 gccatcactgtgtcatctgc 
                  21 
                 catcttgaatccaccgaagg 
                  22 
                  26 
               
               
                   
               
               
                 Aldh1a2 (NM_009022) 
                 catggtatcctccgcaatg 
                  23 
                 gcgcatttaaggcattgtaac 
                  24 
                  33 
               
               
                   
               
               
                 Aldh1a3 (NM_053080) 
                 aacctggacaaagcactgaag 
                  25 
                 aatgcattgtagcagttgatcc 
                  26 
                  66 
               
               
                   
               
               
                 Aldh1a7 (NM_011921) 
                 caagctggctgacttaatgga 
                  27 
                 gactttcccagcattcatcg 
                  28 
                  84 
               
               
                   
               
               
                 Aldh1b1 (NM_028270) 
                 gaccggagaacgctgatacta 
                  29 
                 agtcgggcagtcagcatc 
                  30 
                  45 
               
               
                   
               
               
                 Aldh111 (NM_027406) 
                 tccctacttcccgtctttga 
                  31 
                 acaggctctgcccgattac 
                  32 
                  97 
               
               
                   
               
               
                 Aldh112 (NM_153543) 
                 cgctcgctcctctacatcat 
                  33 
                 gccaacttcagcttgtttttg 
                  34 
                  22 
               
               
                   
               
               
                 Aldh2 (NM_009656) 
                 tgttcggggacgtaaaagac 
                  35 
                 tgaggatttgcatcactggt 
                  36 
                  63 
               
               
                   
               
               
                 Aldh3a1 (NM_007436) 
                 tgtggagaagctcaaaaagtcac 
                  37 
                 cccatagtcatgggactgct 
                  38 
                  60 
               
               
                   
               
               
                 Aldh3a2 (NM_007437) 
                 gctgaagcagttcaacaaagg 
                  39 
                 aggagaggcaacaaggaagtc 
                  40 
                  89 
               
               
                   
               
               
                 Aldh3b1 (NM_026316) 
                 ctggatgaagccatcgagtt 
                  41 
                 ccaacacctgtttgataacctg 
                  42 
                   7 
               
               
                   
               
               
                 Aldh3b2 (NM_001177438) 
                 aagagttgctgcacatctgg 
                  43 
                 gtgcagtgtggcctcaga 
                  44 
                  84 
               
               
                   
               
               
                 Aldh4a1 (NM_175438) 
                 accggttccgtaccttcc 
                  45 
                 agctgtgcacaaaatggaaat 
                  46 
                  19 
               
               
                   
               
               
                 Aldh5a1 (NM_172532) 
                 aacagctggaaaggggtctc 
                  47 
                 cattaagtcgtaccatttacggagt 
                  55 
                   
               
               
                   
               
               
                 Aldh6a1 (NM_134042) 
                 ggtaaatgccacatggtatcc 
                  49 
                 tgattcaacaaattttccatcaat 
                  88 
                   
               
               
                   
               
               
                 Aldh7a1 (NM_138600) 
                 tgtccttcactgggagcact 
                  51 
                 gtttcctccaagctccaaca 
                  52 
                  10 
               
               
                   
               
               
                 Aldh8a1 (NM_178713) 
                 ggcaagaatcctgctatcatct 
                  53 
                 actggtacaaaggcaaatctcc 
                  54 
                  25 
               
               
                   
               
               
                 Aldh9a1 (NM_019993) 
                 ctggcagtgcctggaatact 
                  55 
                 cctcctgggagctgaatgt 
                  56 
                  71 
               
               
                   
               
               
                 Aldh16a1 (NM_145954) 
                 ctcttgggccaccatgtc 
                  57 
                 ctggccaggttttctcctg 
                  58 
                   5 
               
               
                   
               
               
                 Aldh18a1 (NM_019698) 
                 gatgtcatcgtcacagagaacg 
                  59 
                 aagacacaggcgctgtcc 
                  60 
                  67 
               
               
                   
               
               
                 GTP Cyclohydrolase 1 Gch1 
                 gcctcaccaaacagattgc 
                  61 
                 cacgcctcgcattaccat 
                  62 
                  40 
               
               
                 (NM_008102) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Nitric Oxide Synthase 1 
                 ggcgttcgtgattactgtga 
                  63 
                 tcttcctcatgtccaaatcca 
                  64 
                  69 
               
               
                 (neuronal) Nos1 
                   
                   
                   
                   
                   
               
               
                 (NM_008712) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Nitric Oxide Synthase 2 
                 ctttgccacggacgagac 
                  65 
                 tcattgtactctgagggctgac 
                  66 
                  13 
               
               
                 (inducible) Nos2 
                   
                   
                   
                   
                   
               
               
                 (NM_010927) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycine Receptor Glra1 
                 caacacaaggaactgcttcg 
                  67 
                 gatttagcatggggctcttg 
                  68 
                  69 
               
               
                 (NM_001290821) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycine Receptor Glra2 
                 cataaggagttccttcgtctc 
                  69 
                 cgactttcacgagtaacatcttctt 
                  70 
                  34 
               
               
                 (NM_183427) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycine Receptor Glra3 
                 gggaagccgcactgttact 
                  71 
                 gagatcgcgcactgtttgt 
                  72 
                  49 
               
               
                 (NM_080438) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycine Receptor Glrb1 
                 ctgatgctagtgctgccaga 
                  73 
                 gtgcactctgaggccaaact 
                  74 
                 105 
               
               
                 (NM_010298) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor Gabra1 
                 gcccactaaaattcggaagc 
                  75 
                 cttctgctacaaccactgaacg 
                  76 
                  93 
               
               
                 (NM_010250) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor alpha 
                 acaaaaagaggatgggcttg 
                  77 
                 tcatgacggagcctttctct 
                  78 
                 103 
               
               
                 Gabra2 (NM_008066) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor alpha 
                 cttgggaaggcaagaaggta 
                  79 
                 tggagctgctggtgttttct 
                  80 
                  80 
               
               
                 Gabra3 (NM_008067) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor alpha 
                 aaagcctcccccagaagtt 
                  81 
                 catgttcaaattggcatgtgt 
                  82 
                  68 
               
               
                 Gabra4 (NM_010251) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor alpha 
                 caagtgtctttctggattaataaggag 
                  83 
                 gtcatggttaaaacagtggtgatt 
                  84 
                  95 
               
               
                 Gabra6 (NM_001099641) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor beta 
                 ccctctggatgagcaaaact 
                  85 
                 aattcgatgtcatccgtggta 
                  86 
                  80 
               
               
                 Gabrb1 (NM_008069) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor beta 
                 gggtctccttttggattaactatga 
                  87 
                 ggtcattgttaggacagttgtaattc 
                  88 
                  84 
               
               
                 Gabrb2 (NM_008070) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor beta 
                 ctccattgtagagcaccgtct 
                  89 
                 tcaatgaaagtcgaggataggc 
                  90 
                  80 
               
               
                 Gabrb3 (NM_008071) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor delta 
                 cggagctgatgaacttcaaat 
                  91 
                 atgtagacgccccggttc 
                  92 
                  11 
               
               
                 Gabrd (NM_008072) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor gamma 2 
                 acagaaaatgacgctgtgga 
                  93 
                 catctgacttttggcttgtgaa 
                  94 
                  68 
               
               
                 Gabrg2 (NM_008073) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor gamma 3 
                 atgcgacaccagcaagaac 
                  95 
                 caatggtgctgagtgtggtc 
                  96 
                   9 
               
               
                 Gabrg3 (NM_008074) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor pi Gabrp 
                 atttcaccctggtcaccgta 
                  97 
                 gctcaaattgcaaaaccaatc 
                  98 
                  18 
               
               
                 (NM_146017) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA A Receptor rho Gabrr1 
                 tgcctgctagagtcccctta 
                  99 
                 ccgtgatgatggtggacat 
                 100 
                   9 
               
               
                 (NM_008075) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA B Receptor 1 Gabbr1 
                 gacattgatgtctccattctgc 
                 101 
                 gcagccctttgtaaccataga 
                 102 
                  78 
               
               
                 (NM_019439) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GABA B Receptor 2 Gabbr2 
                 gaacatggcagcgaaagtct 
                 103 
                 ctggtacttgctgccaaaca 
                 104 
                  91 
               
               
                 (NM_001081141) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor AMPA 1 
                 agggatcgacatccagagag 
                 105 
                 tgcacatttcctgtcaaacc 
                 106 
                  62 
               
               
                 Gria1 (NM_008165) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor AMPA 2 
                 ggggaggtgattccaagg 
                 107 
                 cccccgacaaggatgtaga 
                 108 
                  67 
               
               
                 Gria2 (NM_013540) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor AMPA 3 
                 agccgtgtgatacgatgaaa 
                 109 
                 caaggtttacaggcgttcct 
                 110 
                  31 
               
               
                 Gria3 (NM_016886) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor AMPA 4 
                 ctgccaacagttttgctgtg 
                 111 
                 aaatggcaaacacccctcta 
                 112 
                  48 
               
               
                 Gria4 (NM_019691) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Delta 1 
                 agacttttggcaaagacatgc 
                 113 
                 actgccattcaagcccttc 
                 114 
                  95 
               
               
                 Grid1 (NM_008166) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Delta 2 
                 ccctaccgtgatgtcttttca 
                 115 
                 agaatgtccatgtcgccact 
                 116 
                   1 
               
               
                 Grid2 (NM_008167) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Kainate 
                 tctggtttggcgttggag 
                 117 
                 tcctccaactattctggtcgat 
                 118 
                 105 
               
               
                 1 Grik1 (NM_146072) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Kainate 
                 agtgccaccataccatccag 
                 119 
                 gctggcacttcagagacattc 
                 120 
                  31 
               
               
                 2 Grik2 (NM_010349) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Kainate 
                 cacttcatcttcaccactctgg 
                 121 
                 actcccgagtagcggtagg 
                 122 
                  85 
               
               
                 3 Grik3 (NM_001081097) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Kainate 
                 gccattgagtatggcacgat 
                 123 
                 tggtaacgggaattttggaa 
                 124 
                  67 
               
               
                 4 Grik4 (NM_175481) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor Kainate 
                 cccctcagctagcctcatct 
                 125 
                 gcctcgcaccagttcttcta 
                 126 
                  40 
               
               
                 5 Grik5 (NM_008168) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 1 
                 catttagggctatcacctcca 
                 127 
                 cactgtgtctttttggttttgc 
                 128 
                  78 
               
               
                 Grin1 (NM_008169) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 2A 
                 attcaaccagaggggcgta 
                 129 
                 ttcaagacagctgcgtcatag 
                 130 
                  48 
               
               
                 Grin2a (NM_008170) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 2B 
                 gggttacaaccggtgccta 
                 131 
                 ctttgccgatggtgaaagat 
                 132 
                  53 
               
               
                 Grin2b (NM_008171) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 2C 
                 gaagcgggccatagacct 
                 133 
                 tggcagatccctgagagc 
                 134 
                  94 
               
               
                 Grin2c (NM_010350) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 2D 
                 tgcgatacaaccagccaag 
                 135 
                 agatgaaggcgtccagtttc 
                 136 
                  25 
               
               
                 Grin2d (NM_008172) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 3A 
                 cacgaatcaaaaacaaatccaa 
                 137 
                 tgtgtttaatgctctgtgaaacc 
                 138 
                  71 
               
               
                 Grin3A (NM_001276355) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 3B 
                 cgtcctacggagggaggt 
                 139 
                 ccagggacaccagcacat 
                 140 
                  80 
               
               
                 Grin3b (NM_130455) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glutamate Receptor NMDA 
                 ctcaaggaggctacccacag 
                 141 
                 tagttcccatgctgaggtga 
                 142 
                  77 
               
               
                 AP1 Grina (NM_023168) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 PSD-95 D1g4 (NM_007864) 
                 tctgtgcgagaggtagcaga 
                 143 
                 cggatgaagatggcgatag 
                 144 
                 102 
               
               
                   
               
             
          
         
       
     
       Example 6—The Impact of Inflammatory Cytokines on Molybdenum Co-Factor Biosynthesis and Inhibitory Neurotransmitter Receptor Function Protein Expression 
       [0052]    Similar cultures and treatment conditions as described in Example 5 are used to generate protein lysates for analysis of expression of GABA receptor subunits, glycine receptor subunits, gephyrin, GTP cyclohydrolase I, and MoCoS. Neurons grown in glass multi-well chambered slides are used for the analysis of expression of these targets by immunofluorescence microscopy. For IF, cells are stimulated for 24, 48, 72, or 96 hrs at 100 ng/mL (or at a dose defined in Example 5 as optimal for gene induction) prior to fixation and immunostaining. Table 3 lists the relevant antibodies that are employed. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Antibodies 
               
             
          
           
               
                 Target 
                 Source 
               
               
                   
               
               
                 GABA A R α1 
                 Synaptic Sys #224203, 1:500 (WB), rabbit 
               
               
                   
                 Alomone #AGA-001, 1:400 (IF), rabbit 
               
               
                 GABA A R α2 
                 Synaptic Sys #224102, 1:500 (WB), rabbit 
               
               
                   
                 Alomone #AGA-002, 1:200 (IF), rabbit 
               
               
                 GABA A R α3 
                 Synaptic Sys #224302, 1:500 (WB), rabbit 
               
               
                   
                 Alomone #AGA-003, 1:200 (IF), rabbit 
               
               
                 GABA A R β2/3 
                 Millipore #MAB341, 1:50 (WB), 1:25 (IF), mouse 
               
               
                 GABA A R γ2 
                 Alomone #AGA-005, 1:200 (WB), 1:100 (IF), rabbit 
               
               
                 GABA B R2 
                 Synaptic Sys #322205, 1:1000 (WB), 1:500 (IF), 
               
               
                   
                 guinea pig 
               
               
                 Glycine R β 
                 Alomone #AGR-014, 1:200 (WB), rabbit 
               
               
                   
                 Synaptic Sys #146211, 1:500 (IF), mouse 
               
               
                 Gephyrin 
                 Synaptic Sys #147004, 1:1000 (WB), guinea pig 
               
               
                   
                 Synaptic Sys #147021, 1:250 (IF), mouse 
               
               
                 Collybistin 
                 Synaptic Sys #261003, 1:1000 (WB), 1:500 (IF), 
               
               
                   
                 rabbit 
               
               
                 GTP 
                 Sigma #SAB4200046-200UL, 1:1000 (WB) 
               
               
                 Cyclohydrolase I 
               
               
                 MoCo-S 
                 Santa Cruz #SC-85066, 1:200 (WB), rabbit 
               
               
                   
                 Pierce #PA5-30662, 1:500 (WB), rabbit 
               
               
                   
               
             
          
         
       
     
       Example 7—The Impact of Inflammatory Cytokines on Spontaneous and Evoked Synaptic Activity 
       [0053]    Neurons are cultured in glass imaging chambers under conditions that promote formation of mature synaptic networks. Cells are infected with an AAV1.Syn.GCaMP6f calcium reporter that provides fast optical tracking of intracellular calcium levels (Akerboom et al., 2012, J. Neurosci., 32:13819040; Chen et al., 2013, Nature, 499:295-300). Calcium levels are monitored in real-time using a Zeiss 5-Live confocal microscope equipped with environmental chamber. Following collection of baseline spontaneous activity levels at low magnification, inflammatory cytokine is added at the optimal cytokine concentration determined above, and cells are followed for up to 60 minutes. Images are post-processed in Image J to measure calcium transient amplitudes and frequencies within defined cells. In some experiments, an Olympus multi-photon microscope is used at high magnification to track activity in individual synapses. In addition to spontaneous activity, calcium flux elicited by addition of glutamate to cultures that have been pretreated with inflammatory cytokines for different times (0, 1, 3, 6, 12, 24, 48, 72, or 96 hr) prior to stimulation also is measured. See  FIGS. 6 and 7 . 
       Example 8—The Effect of cPMP Supplementation on Synaptic Changes Induced by Inflammatory Cytokines 
       [0054]    Hippocampal and cortical neurons are treated with inflammatory cytokines at the optimized dose and time identified above in the presence of different concentrations of cPMP. Extrapolating from the field of purinergic signaling, concentrations ranging from nanomolar to millimolar (1, 3, 10, 30, 100, 300 nM; 1, 3, 10, 30, 100, 300 μM; 1, 3 mM) are tested. In preliminary experiments, the survival of naive neurons treated with cPMP for different times (1, 3, 6, 12, 24, 48, 72, or 96 hr) is assessed by MTT or LDH assay, and doses that kill more than 10% of cells are excluded from further analysis. In some instances, the cPMP is encapsulated in liposomes (for example, lipofectin or lipofectamine) (Hughes et al., 2010, Methods Mol. Biol., 605:445-59). After optimizing cPMP delivery, neurons are stimulated with inflammatory cytokines in the presence or absence of cPMP under conditions that alter spontaneous and/or evoked calcium flux. If cPMP treatment reverses the effect of inflammatory cytokines on dynamic synaptic activity, the effect of cPMP on expression and localization of the protein targets explored in Example 6 also is examined, and the effect of cPMP on the expression of genes measured in Example 5 is tested. 
       Example 9—MoCo Pathway and Neurotransmitter Receptor Changes Induced by Acute Virus Infection of the Brain 
       [0055]    Young (4 week old) mice were infected with the Theiler&#39;s murine encephalomyelitis virus for 24 hr to model acute childhood brain infection. Illumina microarray was employed to assess transcriptional changes. Table 4 shows maximal up-regulation or down-regulation of relevant genes during the first 24 hr of infection. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Maximal Up-regulation or Down-regulation of Relevant Genes 
               
             
          
           
               
                   
                 Fold- 
                   
                   
                 Fold- 
                   
               
               
                 Gene 
                 change 
                 Function 
                 Gene 
                 change 
                 Function 
               
               
                   
               
               
                 MoCos 
                 3.0 
                 molybdenum cofactor 
                 Gphn 
                 −1.4 
                 gephyrin (Mo 
               
               
                   
                   
                 biosynthesis 
                   
                   
                 pathway and 
               
               
                   
                   
                   
                   
                   
                 inhibitory synapse) 
               
               
                 Xdh 
                 4.9 
                 xanthine 
                 Glra2 
                 −1.4 
                 glycine receptor 
               
               
                   
                   
                 dehydrogenase/oxidase 
                   
                   
                 (inhibitory) 
               
               
                 Gch1 
                 4.3 
                 GTP cyclohydrolase 1 
                 Gabrd 
                 −1.6 
                 GABA receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Aldh1b1 
                 2.5 
                 aldehyde dehydrogenase 1 
                 Gabrb1 
                 −1.4 
                 GABA receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Nos3 
                 1.3 
                 nitric oxide synthase 3 
                 Gabrg1 
                 −1.3 
                 GABA receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Gria2 
                 1.3 
                 AMPA receptor 
                 Gabbr2 
                 −1.3 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grin2c 
                 1.2 
                 NMDA receptor 
                 Gabbr3 
                 −1.3 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Dlg5 
                 1.3 
                 disks large homolog 
                 Gabbr1 
                 −1.3 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Capn2 
                 1.5 
                 large calpain subunit 
                 Gabra1 
                 −1.3 
                 GABA receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Capn5 
                 1.3 
                 large calpain subunit 
                 Gad1 
                 −1.3 
                 glutamate 
               
               
                   
                   
                   
                   
                   
                 decarboxylase 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Capns1 
                 1.3 
                 small calpain subunit 
               
               
                 Gpx1 
                 1.5 
                 glutathione peroxidase 1 
               
               
                 Akr1b8 
                 1.8 
                 aldo/keto reductase 1 
               
               
                 G6pdx 
                 1.3 
                 glucose-6-P 
               
               
                   
                   
                 dehydrogenase 
               
               
                   
               
             
          
         
       
     
         [0056]    These measurements indicate that acute infection of the brain, consistent with elevated TNF alpha (6-fold increase at 24 hr) and IL1 beta (10-fold increase at 24 hr) in this model system, induces increased synthesis of MoCo pathway-related factors, increased production of oxidative stress factors, up-regulated calpain production, and increased expression of excitatory neurotransmitter receptors. Simultaneously, acute infection triggers down-regulated expression of gephyrin and a host of GABAergic receptors, resulting in suppression of synaptic inhibition. 
       Example 10—MoCo Pathway and Neurotransmitter Receptor Changes in Human Neurons Induced by Inflammatory Cytokines 
       [0057]    Neurons were induced from human neural stem cells and grown under conditions that foster mixed excitatory and inhibitory neuron phenotypes. These cells were then stimulated with TNF alpha, IL1 beta, or IFN gamma for 24 hrs, and transcriptional changes were assessed by microarray. Responses were variable between cytokines but, in general, the inflammatory stimuli induced changes that are summarized in Table 5. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Inflammatory Stimuli Induced Genes 
               
             
          
           
               
                   
                 Fold- 
                   
                   
                 Fold- 
                   
               
               
                 Gene 
                 change 
                 Function 
                 Gene 
                 change 
                 Function 
               
               
                   
               
               
                 MoCos 
                 2.0 
                 molybdenum cofactor 
                 Gphn 
                 −1.2 
                 gephyrin (Mo 
               
               
                   
                   
                 biosynthesis 
                   
                   
                 pathway and 
               
               
                   
                   
                   
                   
                   
                 inhibitory synapse) 
               
               
                 Gch1 
                 3.7 
                 GTP cyclohydrolase 1 
                 Glra2 
                 −1.7 
                 glycine receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Aldh1 
                 1.5 
                 aldehyde 
                 Glrb 
                 −1.7 
                 glycine receptor 
               
               
                   
                   
                 dehydrogenase 1 
                   
                   
                 (inhibitory) 
               
               
                 Aldh2 
                 1.4 
                 aldehyde 
                 Gabrd 
                 −1.6 
                 GABA receptor 
               
               
                   
                   
                 dehydrogenase 2 
                   
                   
                 (inhibitory) 
               
               
                 Nos3 
                 1.6 
                 nitric oxide synthase 3 
                 Gabbr2 
                 −5.0 
                 GABA receptor 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Gria3 
                 1.4 
                 AMPA receptor 
                 Gabbr3 
                 −2.5 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grina 
                 1.4 
                 NMDA receptor 
                 Gad1 
                 −1.8 
                 glutamate 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 decarboxylase 
               
               
                   
                   
                   
                   
                   
                 (inhibitory) 
               
               
                 Grin3a 
                 2.3 
                 NMDA receptor 
               
               
                   
                   
                 (excitatory) 
               
               
                 Grik2 
                 1.5 
               
               
                 Grm5 
                 3.9 
                 metabotropic 
               
               
                   
                   
                 glutamate receptor 
               
               
                   
                   
                 (excitatory) 
               
               
                 Dlg3 
                 1.2 
                 disks large homolog 
               
               
                   
                   
                 (excitatory) 
               
               
                 Capn2 
                 1.5 
                 large calpain subunit 
               
               
                 Capn5 
                 5.3 
                 large calpain subunit 
               
               
                 Capns1 
                 1.2 
                 small calpain subunit 
               
               
                   
               
             
          
         
       
     
       Example 11—MoCo Pathway and Neurotransmitter Receptor Changes in Human Neurons Induced by Inflammatory Cytokines 
       [0058]    Neurons were cultured from neonatal mice and stimulated with TNF alpha or IFN gamma for 24 hr. Transcriptional changes were assessed by quantitative RT-PCR. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Transcriptional Changes 
               
             
          
           
               
                   
                 Fold- 
                   
                   
                 Fold- 
                   
               
               
                 Gene 
                 change 
                 Function 
                 Gene 
                 change 
                 Function 
               
               
                   
               
               
                 Gria1 
                 1.2 
                 AMPA receptor 
                 Gphn 
                 −2.1 
                 gephyrin (Mo 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 pathway and 
               
               
                   
                   
                   
                   
                   
                 inhibitory synapse) 
               
               
                 Gria2 
                 1.7 
                 AMPA receptor 
                 Glra2 
                 −1.6 
                 glycine receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Gria3 
                 4.1 
                 AMPA receptor 
                 Glra3 
                 −2.3 
                 glycine receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grid1 
                 1.3 
                 glutamate receptor 
                 Glrb1 
                 −4.5 
                 glycine receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grid2 
                 2.4 
                 glutamate receptor 
                 Gabra1 
                 −1.2 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grik3 
                 6.2 
                 kainate receptor 
                 Gabra2 
                 −2.3 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grik4 
                 3.0 
                 kainate receptor 
                 Gabra4 
                 −2.9 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grik5 
                 1.8 
                 kainate receptor 
                 Gabrb1 
                 −1.4 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grin2b 
                 2.6 
                 NMDA receptor 
                 Gabrd 
                 −1.6 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grin2d 
                 1.9 
                 NMDA receptor 
                 Gabrg2 
                 −4.8 
                 GABA receptor 
               
               
                   
                   
                 (excitatory) 
                   
                   
                 (inhibitory) 
               
               
                 Grin3a 
                 2.7 
                 NMDA receptor 
               
               
                   
                   
                 (excitatory) 
               
               
                 Grina 
                 2.6 
                 NMDA receptor 
               
               
                   
                   
                 (excitatory) 
               
               
                   
               
             
          
         
       
     
       Example 12—Inflammatory Cytokines Induce Network Synchronization that Phenocopies Loss of Inhibition 
       [0059]    Neurons were cultured from neonatal mice and stimulated with TNF alpha (100 ng/mL) or IFN gamma (500 U/mL) for 24 hr. Following transduction with an AAV-encoded GCaMPf reporter, fast calcium transients were imaged in the cells. Regions of interest outlining individual neurons were defined in each frame of movies collected over several minutes, and the fluorescence intensity of each cell was graphed through time to reveal patterns in the population response.  FIG. 8  shows the averaged calcium response traces calculated for dozens of cytokine-stimulated neurons in each experiment and are representative of more than 4 separate experiments and more than 4 separate cell preps within each experiment. 
         [0060]      FIG. 8A  shows the basal level of calcium activity in the neuron cultures. Non-synchronized calcium responses occur in the control cultures, resulting in an overall low level of synaptic activity in the network.  FIGS. 8B and 8C  show the stimulation of calcium activity in neurons following treatment with IFN gamma or TNF alpha, respectively.  FIGS. 8B and 8C  show that treatment with IFN gamma or TNF alpha results in network bursting and highly synchronized synaptic activity in which many cells in the culture flux calcium at the same time. 
         [0061]      FIG. 8D  shows that treatment of control cultures with 2.4 μM picrotoxin, a small molecule inhibitor of inhibitory GABAergic channels, induces network synchrony and bursting that phenocopies the response observed in cytokine-stimulated cultures (compare with  FIGS. 8B and 8C ).  FIG. 8E  shows that addition of 27 μM GABA to control cultures completely suppresses synaptic activity, consistent with enhanced inhibition. 
         [0062]    These findings indicate that TNF alpha and IFN gamma induce the suppression of inhibitory neurotransmission in the neuronal network, resulting in synchronous bursting behavior. Given the transcriptional profiles measured in cytokine-stimulated neurons, this network behavior is consistent with a reduction in inhibitory neurotransmitter receptors linked to reduced gephyrin expression and alteration of the MoCo synthesis pathway. 
         [0063]    It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims. 
         [0064]    Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

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