Patent Application
20210330611
In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Neurodegeneration is the progressive loss of structure and/or function of neurons, which may lead to the death of the affected neurons. Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and multiple sclerosis. Although these diseases have different etiologies and symptoms, they all result in progressive degeneration and/or death of neuron cells. Despite their differences, these diseases also display similarities that can relate these diseases on a cellular or molecular level. Such similarities offer therapeutic advances using modalities common to each of these diseases.
Clinical management of neurodegenerative remains a significant challenge in medicine, however, as they do not address the cellular or molecular basis of the disease. Although some degree of axonal remyelination by oligodendrocytes takes place early during the course of MS, the ability to repair the CNS eventually fails, leading to irreversible tissue injury and an increase in disease-related disabilities.
Currently approved therapies for CNS demyelinating diseases, such as multiple sclerosis (MS), are primarily immunomodulatory, and typically do not have direct effects on CNS repair. Similarly, drugs for other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease do not address the neuronal death and loss of function, but rather ameliorate associated symptoms.
Thus, there is a need for additional therapies that prevent and/or ameliorate neurodegeneration. The present invention meets such need.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present invention provides methods and compositions for treating a CNS disease, disorder or injury. The present invention also provides methods and compositions for preserving or protecting neural structure and/or function in a subject in need thereof, such as in a mammalian subject by administering one or more agents and/or compositions described herein to the subject.
One embodiment provides a method of treating or preventing neurodegeneration in a mammal, such as a human, comprising administering to the mammal in need thereof an effective amount of a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
In a related aspect, the invention features a method of preventing progression of a CNS disorder in a subject in need of treatment. The method comprises administering to the subject a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof in an amount sufficient to thereby arrest the CNS disorder and prevent further neuronal injury and/or death. In certain embodiments, said treatment may result in reduction of one or more symptoms associated with the disease. In some embodiments, the treatment results in reducing, retarding or preventing a relapse, or the worsening of progression of the disease in the subject.
Some embodiments provide for methods and compositions for preventing or ameliorating demyelination in a subject, such as mammalian subject, by administering one or more compositions that comprise a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
Other embodiments provide methods and compositions for enhancing myelination and/or re-myelination in a mammalian subject, such as a human subject, by administering one or more compositions that comprise a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
Further embodiments provide methods and compositions for decreasing neurodegeneration associated with plaque formation (e.g., amyloid plaque formation) in a mammalian subject, such as a human subject.
In some embodiments, the methods and compositions of the invention are used for decreasing neurodegeneration in a patient with multiple sclerosis.
In some embodiments, the methods and compositions of the invention are used for decreasing neurodegeneration in a patient with Alzheimer's disease.
Other embodiments provide methods and compositions for decreasing neurodegeneration in a mammalian subject with a genetic predisposition for Alzheimer's disease. Examples of such genetic predisposition include mutations m the amyloid precursor protein (APP) gene, Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) genes, and ApoE4.
One embodiment provides a method of treating or preventing or decreasing oxidative stress in a mammal comprising administering to the mammal in need thereof an effective amount of a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
Another embodiment provides a method for inhibiting microglial activation in the CNS of a mammal with a disease, disorder, or injury involving demyelination, dysmyelination, or neurodegeneration, comprising administering to the mammal an effective amount of a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
One embodiment provides a method of preventing demyelination and neuronal injury in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof so as to prevent an increase in demyelination and injury of CNS neurons in said mammal.
Another embodiment provides a method for promoting survival of CNS neurons in a mammal, comprising administering to a mammal in need thereof an effective amount of a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof so as to decrease neuronal injury/promote survival of neurons in the CNS of said mammal.
One embodiment provides a method of inhibiting microglial activation in CNS neurons comprising contacting CNS neurons with a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
Another embodiment provides a method for promoting remyelination in a mammal comprising administering to the mammal an effective amount of a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
One embodiment provides a method for reducing the rate of demyelination and neuronal injury in a mammal comprising administering to the mammal an effective amount of a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of calcium signaling, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
One embodiment provides a method to treat or prevent neurodegeneration in a mammal comparing administering to said mammal an effect amount of one or more of the small molecules provided in Table 1 and/or Table 2, provided that the small molecule is not acivicin.
Another embodiment provides a pharmaceutical composition comprising at least one modulator of calcium signaling, at least one modulator of microtubule dynamics, at least one modulator of calcium signaling, at least one modulator of chemokine signaling, at least one modulator of DNA replication, at least one modulator of dopamine receptor signaling, at least one modulator of cAMP signaling, at least one modulator of glucocorticoid-receptor signaling, at least one modulator of purine nucleotide biosynthesis, at least one modulator of neurotransmitter transport or a combination thereof, in an amount effective to treat or prevent neurodegeneration, and a pharmaceutically acceptable carrier, diluent, or excipient.
One embodiment provides a pharmaceutical composition with selectivity to inhibit fibrin-induced microglia activation, but not LPS-induced activation comprising at least one modulator of microtubules, at least one modulator of glucocorticoids, at least one modulator of progesterone, at least one modulator of bacterial growth, at least one modulator of β2-adrenoceptor signaling, at least one modulator of parasite growth, at least one modulator of alpha 2-adrenergic receptors, at least one modulator of alpha 1-adrenergic receptor, at least one modulator of 5-HT4 receptors, at least one modulator of HMG-CoA reductase, at least one modulator that decreases free radicals, at least one modulator that decreases prostaglandins, at least one modulator that regulates mineralocorticoid receptor signaling or a combination thereof, in an amount effective to treat or prevent neurodegeneration, and a pharmaceutically acceptable carrier, diluent, or excipient.
In one embodiment, the modulator of calcium signaling is a calcium channel blocker, a vasodilator or an adrenoreceptor agonist. In another embodiment, calcium channel blocker comprises fendiline. In one embodiment, the vasodilator comprises nylidrin. In one embodiment, the modulator of microtubule dynamics is an inhibitor of microtubule assembly. In one embodiment, the inhibitor of microtubule assembly comprises vinblastine (vinblastine sulfate), colchicine and/or podofilox. In another embodiment, the modulator of chemokine signaling comprises tannic acid. In one embodiment, the modulator of DNA replication is an inhibitor of DNA topoisomerase II, such as teniposide. In one embodiment, the modulator of dopamine receptor signaling is blocker of dopamine receptors, such as prochlorperazine or thioridazine. In another embodiment, the modulator of cAMP signaling comprises nylidrin, prochlorperazine or thioridazine. In one embodiment, the modulator of glucocorticoid-receptor signaling comprises betamethasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone hemisuccinate, hydrocortisone sodium phosphate, methylprednisolone, prednisolone or triamcinolone diacetate. In another embodiment, the modulator of purine nucleotide biosynthesis is an inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH), such as mycophenolic acid (MPA). In one embodiment, the modulator of neurotransmitter transport is an inhibitor of norepinephrine reuptake, such as maprotiline.
In another embodiment, said mammal has been diagnosed with a disease, disorder, or injury involving demyelination, dysmyelination, or neurodegeneration. In one embodiment, said disease, disorder, or injury is selected from the group consisting of multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barre syndrome, Marie-Charcot-Tooth disease and Bell's palsy.
One embodiment also includes pharmaceutical compositions and kits that contain one or more agent that can be used to inhibit degeneration of a neuron or a portion thereof, as described herein. The pharmaceutical compositions and kits can optionally include one or more pharmaceutically acceptable excipients.
Another embodiment features a packaged composition (e.g., a packaged pharmaceutical composition) that includes at least one agent disclosed herein that is labeled and/or contains instructions for use of said agent for treating a CNS disorder and/or oxidative stress. The agent can be in a form suitable for any route of administration, e.g., oral administration, peripheral administration, intrathecal administration, etc. One or more active agents can be included in the packaged pharmaceutical composition.
These aspects and other features and advantages of the invention are described below in more detail. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The practice of the methods and compositions described herein may employ, unless otherwise indicated, conventional techniques of pharmaceutical chemistry, drug formulation techniques, dosage regimes, and biochemistry, all of which are within the skill of those who practice in the art. Such conventional techniques include the use of combinations of therapeutic regimes including but not limited to the methods described herein; technologies for formulations of adjunct therapies used in combination with known, conventional therapies and/or new therapies for the treatment of neurodegeneration, delivery methods that are useful for the compositions of the invention, and the like. Specific illustrations of suitable techniques can be had by reference to the examples herein.
Oxidative stress is a central part of innate-immune induced neurodegeneration. However, the transcriptomic landscape of the central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available.
Provided herein is the oxidative stress innate immune cell atlas in neuroinflammatory disease and disclosure of the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells (Tox-seq) identified a core oxidative stress gene signature coupled to coagulation and glutathione pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen (HTS) and oxidative stress gene network analysis, identified several candidates including the glutathione regulator acivicin with potent therapeutic effects decreasing oxidative stress and axonal damage in chronic and relapsing models of multiple sclerosis (MS). Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and can enable the discovery of selective neuroprotective strategies.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known/available to those skilled in the art have not been described in order to avoid obscuring the invention.
For the purposes of clarity and a concise description, features can be described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are intended to aid the reader in understanding the present invention but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.
As used herein, the indefinite articles “a”, “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise. Thus, for example, reference to “an inhibitor” refers to one or more agents with the ability to inhibit a target molecule, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
The phrase “and/or,” as used herein, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases.
As used herein, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating a listing of items, “and/or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
As used herein, the term “about” means plus or minus 10% of the indicated value. For example, about 100 means from 90 to 110.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
A “CNS disorder” can be any disease, disorder or injury associated with the toxicity of a population of cells within the CNS. In one example, the CNS disorder is associated with a pathological process such as neurodegeneration, demyelination, dysmyelination, axonal injury, and/or dysfunction or death of an oligodendrocyte or a neuronal cell, or loss of neuronal synapsis/connectivity. In other examples, the CNS disorder is a disease associated with plaque formation, e.g., amyloid plaque formation. CNS disorders include neurodegenerative disorders that affect the brain or spinal cord of a mammal. In certain embodiments, the CNS disorder has one or more inflammatory components.
The term “neurodegenerative diseases” includes any disease or condition characterized by problems with movements, such as ataxia, and conditions affecting cognitive abilities (e.g., memory) as well as conditions generally related to all types of dementia. “Neurodegenerative diseases” may be associated with impairment or loss of cognitive abilities, potential loss of cognitive abilities and/or impairment or loss of brain cells. Exemplary “neurodegenerative diseases” include Alzheimer's disease (AD), diffuse Lewy body type of Alzheimer's disease, Parkinson's disease, Down syndrome, progressive multiple sclerosis (MS), dementia, mild cognitive impairment (MCI), amyotrophic lateral sclerosis (ALS), traumatic brain injuries, ischemia, stroke, cerebral ischemic brain damage, ischemic or hemorrhaging stroke, multi-infarct dementia, hereditary cerebral hemorrhage with amyloidosis of the Dutch-type, cerebral amyloid angiopathy (including single and recurrent lobar hemorrhages), neurodegeneration induced by viral infection (e.g. AIDS, encephalopathies) and other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy and dementia associated with cortical basal degeneration, epilepsy, seizures, and Huntington's disease.
As used herein, a disease, disorder or condition is “treated” if at least one pathophysiological measurement associated with the disease is decreased and/or progression of a pathophysiological process is reversed, halted or reduced. For example, a disease, disorder or condition can be “treated” if the number of plaques present in the CNS of a patient with a neurodegenerative disease is reduced, remains constant, or the creation of new plaques is slowed by the administration of an agent. In another example, a disease, disorder or condition can be “treated” if one or more symptoms of the disease or disorder is reduced, alleviated, terminated, slowed, or prevented. Measurement of one or more exemplary clinical hallmarks and/or symptoms of a disease can be used to aid in determining the disease status in an individual and the treatment of one or more symptoms associated therewith.
The term “administering” as used herein refers to administering to a subject and/or contacting a neuron or portion thereof with an inhibitor as described herein. This includes administration of the inhibitor to a subject in which the neuron is present, as well as introducing the inhibitor into a medium in which a neuron is cultured. Administration “in combination with” one or more further agents includes concurrent and consecutive administration, in any order.
The term “neuron” as used herein denotes nervous system cells that include a central cell body or soma, and two types of extensions or projections: dendrites, by which, in general, the majority of neuronal signals are conveyed to the cell body, and axons, by which, in general, the majority of neuronal signals are conveyed from the cell body to effector cells, such as target neurons or muscle. Neurons can convey information from tissues and organs into the central nervous system (afferent or sensory neurons) and transmit signals from the central nervous systems to effector cells (efferent or motor neurons). Other neurons, designated interneurons, connect neurons within the central nervous system (the brain and spinal column). Certain specific examples of neuron types that may be subject to treatment according to the invention include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons.
The terms “mammal” and “mammalian subject” as used herein refers to any animal classified as a mammal, including humans, higher non-human primates, rodents, and domestic and farm animals, such as cows, horses, dogs, and cats. In some embodiments of the invention, the mammal is a human.
The term “pharmaceutical composition” refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a tablet, capsule, or a vial. The quantity of active ingredient in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
The phrase “therapeutically effective amount” or “effective amount” used in reference to an agent of the invention is an art-recognized term. In certain embodiments, the term refers to an amount of an agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.
“Inhibitors,” “activators,” and “modulators” are used to refer to activating, inhibitory, or modulating (increase, inhibit, decrease or activate expression or activity as compared to control (an untreated or healthy subject/mammal) molecules. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate activity, e.g., agonists.
In certain embodiments, a therapeutically effective amount of an agent for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent. In certain embodiments, a therapeutically effective amount is the amount effective to induce endogenous oligodendrocyte precursor cell differentiation and/or maturation, thereby promoting myelination in the subject's central nervous system.
As used herein, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are intended to be inclusive similar to the term “comprising.”
As used herein, said “contain”, “have” or “including” include “comprising”, “mainly consist of”, “basically consist of” and “formed of”; “primarily consist of”, “generally consist of” and “comprising of” belong to generic concept of “have” “include” or “contain”.
Discovery of small molecule compounds that inhibit innate immune activation: To identify druggable pathways shared by both microglia and macrophages, function-selective transcriptomics was combined with drug network analysis of pathways identified through a small molecule screen in primary microglia. A high-content, high-throughput screen (HTS) was developed to discover new small molecule inhibitors that suppress microglia activation. Primary microglia were stimulated by the innate immune activators fibrin or lipopolysaccharide (LPS). 1,907 clinical drugs and bioactive compounds were screened using increased cell size (≥800 μm2) as a marker of activation and decreased size (<150 μm2) as a marker of toxicity. 128 compounds were identified that inhibited fibrin-induced microglia activation by ≥50% without toxicity (≤3% cell death) (Table 1). A total of 31 compounds, of which 27 of them inhibited fibrin- and/or LPS-induced microglia activation, with known molecular targets and potential clinical relevance for neurological diseases (Table 2) were selected to generate a “microglia drug-target network” containing ten subnetworks targeting different pathways: chemokine signaling, GGT, calcium signaling, purine nucleotide biosynthesis, cAMP signaling, neurotransmitter transport, DNA replication, dopamine-receptor signaling, microtubule dynamics, multidrug resistance, and glucocorticoid-receptor signaling.
Agents/Compounds
Agents of the invention include, but are not limited to: Modulators of calcium signaling include, but are not limited to, a calcium channel blocker, a vasodilator, β2 adrenoreceptor agonist, fendiline (fendiline hydrochloride) and/or nylidrin (buphenine, nylidrin hydrochloride).
Modulators of microtubule dynamics include, but are not limited to, inhibitors of microtubule assembly, vinblastine (vinblastine sulfate), colchicine and/or podofilox.
Modulators of chemokine signaling include, but are not limited to, tannic acid.
Modulators of DNA replication include, but are not limited to, teniposide (an inhibitor or DNA topoisomerase II).
Modulators of beta2-adrenoreceptor signaling, alpha 2-adrenergic receptor signaling, and alpha 1 adrenergic receptor signaling include, but are not limited to, Ritodrine Hydrochloride, Levonordefrin, Salmeterol, Xylazine Hydrochloride, Idazoxan Hydrochloride, Naftopidil Dihydrochloride
Modulators of progesterone include, but are not limited to Medroxyprogesterone Acetate, Melengestrol Acetate.
Modulators of dopamine receptor signaling include, but are not limited to, prochlorperazine (prochlorperazine dimaleate) (blocker of dopamine receptors) and/or thioridazine (thioridazine hydrochloride)(antagonist of dopamine; blocks dopamine receptors).
Modulators of cAMP signaling include, but are not limited to, nylidrin (buphenine, nylidrin hydrochloride), prochlorperazine (prochlorperazine dimaleate) and/or thioridazine (thioridazine hydrochloride).
Modulators of glucocorticoid-receptor signaling (including glucocorticoid receptor (NR3C1)) include, but are not limited to, betamethasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone hemisuccinate, hydrocortisone sodium phosphate, methylprednisolone, prednisolone, and/or triamcinolone diacetate.
Modulators of purine nucleotide biosynthesis include, but are not limited to, inhibitors of inosine-5′-monophosphate dehydrogenase (IMPDH) and/or mycophenolic acid (MPA; mycophenolate).
Modulators of neurotransmitter transport include, but are not limited to, an inhibitor of norepinephrine reuptake and/or maprotiline (maprotiline hydrochloride).
Administration
Pharmaceutical formulations of the agents described herein are prepared by combining the agent having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, BHA, and BHT; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counter-ions such as sodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG.
Agents to be used for in vivo administration can be sterile, which can be achieved by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. Therapeutic compositions may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial.
Agents described herein can be optionally combined with or administered in concert with each other or other agents known to be useful in the treatment of the relevant disease or condition.
Thus, in the treatment of demyelinating diseases, the agents can be administered in combination with injectable compositions including interferon beta 1a inhibitors or interferon beta 1b inhibitors, glatiramer acetate, and daclizumab; oral medications such as teriflunomide, fingolimod, and dimethyl fumarate; or infused medications such as alemtuzumab, mitoxantrone, or natalizumab.
In the treatment of Alzheimer's disease, agents can be administered with acetylcholinesterase inhibitors (e.g., donepezil, galantamine, and rivastigmine) and/or NMDA receptor antagonists (e.g., memantine).
In the treatment of ALS, for example, agents can be administered in combination with Riluzole (Rilutek), minocycline, insulin-like growth factor 1 (IGF-1), and/or methylcobalamin.
In another example, in the treatment of Parkinson's disease, agents can be administered with L-dopa, dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, cabergoline, apomorphine, and lisuride), dopa decarboxylase inhibitors (e.g., levodopa, benserazide, and carbidopa), and/or MAO-B inhibitors (e.g., selegiline and rasagiline).
The combination therapies can involve concurrent or sequential administration, by the same or different routes, as determined to be appropriate by those of skill in the art. The invention also includes pharmaceutical compositions and kits.
The route of administration of the agents is selected in accordance with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems as described below.
For intracerebral use, the agents can be administered continuously by infusion into the fluid reservoirs of the CNS, although bolus injection may be acceptable. The agents can be administered into the ventricles of the brain or otherwise introduced into the CNS or spinal fluid. Administration can be performed by use of an indwelling catheter and a continuous administration means such as a pump, or it can be administered by implantation, e.g., intracerebral implantation of a sustained-release vehicle. More specifically, the agents can be injected through chronically implanted cannulas or chronically infused with the help of osmotic minipumps. Subcutaneous pumps are available that deliver proteins through a small tubing to the cerebral ventricles. Highly sophisticated pumps can be refilled through the skin and their delivery rate can be set without surgical intervention. Examples of suitable administration protocols and delivery systems involving a subcutaneous pump device or continuous intracerebroventricular infusion through a totally implanted drug delivery system are those used for the administration of dopamine, dopamine agonists, and cholinergic agonists to Alzheimer's disease patients and animal models for Parkinson's disease, as described by Harbaugh, J. Neural Transm. Suppl. 24:271, 1987; and DeYebenes et al., Mov. Disord. 2:143, 1987.
Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167, 1981; Langer, Chem. Tech. 12:98, 1982), ethylene vinyl acetate (Langer et al., Id), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include liposomally entrapped compounds, which can be prepared by methods known per se (Epstein et al., Proc. Natl. Acad. Sci. U.S.A. 82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A. 77:4030, 1980; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy.
A therapeutically effective amount of an agent will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from, for example, about 1 μg/kg to up to 100 mg/kg or more (e.g., about 1 μg/kg to 1 mg/kg, about 1 μg/kg to about 5 mg/kg, about 1 mg/kg to 10 mg/kg, about 5 mg/kg to about 200 mg/kg, about 50 mg/kg to about 150 mg/mg, about 100 mg/kg to about 500 mg/kg, about 100 mg/kg to about 400 mg/kg, and about 200 mg/kg to about 400 mg/kg), depending on the factors mentioned above. Typically, the clinician will administer an active inhibitor until a dosage is reached that results in improvement in or, optimally, elimination of, one or more symptoms of the treated disease or condition. The progress of this therapy is easily monitored by conventional assays. One or more agent provided herein may be administered together or at different times (e.g., one agent is administered prior to the administration of a second agent). One or more agent may be administered to a subject using different techniques (e.g., one agent may be administered orally, while a second agent is administered via intramuscular injection or intranasally). One or more agent may be administered such that the one or more agent has a pharmacologic effect in a subject at the same time. Alternatively, one or more agent may be administered, such that the pharmacological activity of the first administered agent is expired prior the administration of one or more secondarily administered agents.
One skilled in the art, upon reading the present specification, will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
The present invention also provides a therapeutic kit containing materials useful for the treatment or prevention of the disorders and conditions described above is provided. The therapeutic kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a pharmaceutical composition that is by itself or when combined with another agent effective for treating or preventing the condition and may have a sterile access port (e.g., an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the pharmaceutical composition is one of the agents described herein above. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the kit may include (a) a first container with a pharmaceutical composition contained therein, wherein the composition includes an agent described herein; and (b) a second container with a pharmaceutical composition contained therein, wherein the composition includes a different agent. The therapeutic kit in this embodiment of the invention may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the therapeutic kit may further include a second (or third) container including a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Assessment of Treatment
In some embodiments, the successful treatment of a subject with an agent described herein is determined by at least about a 10%-100% decrease in one or more symptoms of a CNS disorder. Examples of such symptoms include, but are not limited to, slowness of movement, loss of balance, depression, decreased cognitive function, short-term memory loss, long-term memory loss, confusion, changes in personality, language difficulties, loss of sensory perception, sensitivity to touch, numbness in extremities, tremors, ataxia, muscle weakness, muscle paralysis, muscle cramps, muscle spasms, significant changes in eating habits, excessive fear or worry, insomnia, delusions, hallucinations, fatigue, back pain, chest pain, digestive problems, headache, rapid heart rate, dizziness, and visual changes.
For example, clinical signs of MS are routinely classified and standardized, e.g., using an EDSS rating system based on neurological examination and long-distance ambulation. As used herein, the “Expanded Disability Status Scale” or “EDSS” is intended to have its customary meaning in the medical practice. EDSS is a rating system that is frequently used for classifying and standardizing MS. The accepted scores range from 0 (normal) to 10 (death due to MS). Typically, patients having an EDSS score of about 4-6 will have moderate disability (e.g., limited ability to walk), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair). More specifically, EDSS scores in the range of 1-3 refer to an MS patient who is fully ambulatory, but has some signs in one or more functional systems; EDSS scores in the range higher than 3 to 4.5 show moderate to relatively severe disability; an EDSS score of 5 to 5.5 refers to a disability impairing or precluding full daily activities; EDSS scores of 6 to 6.5 refer to an MS patient requiring intermittent to constant, or unilateral to bilateral constant assistance (cane, crutch or brace) to walk; EDSS scores of 7 to 7.5 means that the MS patient is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair; EDSS scores of 8 to 8.5 refer to patients that are restricted to bed; and EDSS scores of 9 to 10 mean that the MS patient is confined to bed, and progressively is unable to communicate effectively or eat and swallow, until death due to MS.
In certain embodiments, the evaluation of disease progression includes a measure of upper extremity function (e.g., a 9HP assessment). Alternatively, or in combination, disease progression includes a measure of lower extremity function. Alternatively, or in combination, disease progression includes a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW). Alternatively, or in combination, disease progression includes a measure of ambulatory function, e.g., longer distance ambulatory function (e.g., a 6-minute walk test). In one embodiment, the disease progression includes a measure of ambulatory function other than EDSS ambulatory function. In one embodiment, disease progression includes a measure of upper extremity function e.g., a 9HP assessment, and a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW). In one embodiment, disease progression includes a measure of upper extremity function (e.g., a 9HP assessment) and a measure of lower extremity function. In one embodiment, disease progression includes a measure of upper extremity function (e.g., a 9HP assessment), a measure of lower extremity function, and a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW) and/or longer distance ambulatory function (e.g., a 6-minute timed walk test (e.g., 6MWT)). In one embodiment, one, two or the combination of the T25FW, 6MWT and 9HP assessments can be used to acquire a disease progression value. The measure of ambulatory function (e.g., short distance ambulatory function (e.g., T25FW) or longer distance ambulatory function (e.g., a timed (e.g., 6-minute) walk test (e.g., 6MWT)) and/or measure of upper extremity function (e.g., a 9HP assessment) can further be used in combination with the EDSS to evaluate MS, e.g., progressive forms of MS.
Alzheimer's disease (AD) is a neurodegenerative disorder that results in the loss of cortical neurons, especially in the associative neocortex and hippocampus which in aim leads to slow and progressive loss of cognitive functions, ultimately leading to dementia and death. Major hallmarks of the disease are aggregation and deposition of misfolded proteins such as aggregated beta-amyloid peptide as extracellular senile or neuritic ‘plaques’, and hyperphosphorylated tau protein as intracellular neurofibrillary tangles.
Genetic predispositions for AD are divided into two forms: early-onset familial AD (<60 years), and late-onset sporadic AD (>60 years). Rare, disease causing mutations in Amyloid precursor protein (APP), Presenilin 1 (PSEN1), and Presenilin 2 (PSEN2) genes are known to result in early-onset familial AD while, APOE (allele 4) is the single most important risk factor for late-onset AD. In specific embodiments, the methods of the invention are used to treat subjects with a genetic predisposition for wither early onset familial AD or late-onset sporadic AD.
Although Alzheimer's disease develops differently for every individual, there are many common symptoms. In the early stages, the most common symptom is difficulty in remembering recent events. As the disease advances, symptoms can include confusion, irritability, aggression, mood swings, trouble with language, and long-term memory loss.
Clinical Decision Support Systems (CDSS) comprising computer hardware, software, and/or systems can be used to determine a diagnosis for a patient who has certain symptoms associated with Alzheimer's disease. CDSS often include at least three component parts: a knowledge basis, an inference engine, and a communication mechanism. The knowledge base may comprise compiled information about symptoms, pharmaceuticals, and other medical information. The inference engine may comprise formulas, algorithms, etc. for combining information in the knowledge base with actual patient data. The communication mechanism may be ways to input patient data and to output helpful information based on the knowledge base and inference engine. For example, information may be inputted by a physician using a computer keyboard or tablet and displayed to the physician on a computer monitor or portable device.
In certain aspects, the assessment of treatment includes radiological assessment, e.g., single photon emission computed tomography (SPECT), Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) and scintigraphy. For example, multiple sclerosis can be assessed using radiologic assessment of CNS plaques, e.g. by MRI. In another example, AD plaque load can be assessed, e.g., using Aβ-PET.
The assessment of treatment according to the present invention may also be performed using scanning database systems and methods such as those described in US Appln. No. 20150039346.
The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is the example intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.
Transcriptional Profiling and Therapeutic Targeting of Oxidative Stress in Neuroinflammation
Oxidative injury is a pathologic feature linked to neurodegeneration, myelin damage and disease progression in MS and other neurodegenerative diseases (1-6). Oxidative stress mediated by reactive oxygen species (ROS) release from CNS innate immune cells promotes neurodegeneration and demyelination (1,3,7-10). Innate immune-mediated oxidative injury has been proposed as a critical process underlying the progression of MS from the relapsing phenotype to relentless neurodegeneration (11,12). In progressive MS, neurodegeneration is associated with robust microglia activation and oxidative stress (12,13). However, the mechanisms in innate immune cells that trigger oxidative injury in neuroinflammation remain poorly understood. Single-cell technology has led to an appreciation of the heterogeneity of CNS innate immune responses with distinct gene profiles between microglia and CNS infiltrating macrophages in MS, Alzheimer's disease (AD), and related animal models (14-21). However, the functional transcriptomic landscape of oxidative stress inducing innate immune cells is unknown. Furthermore, the discovery of drugs capable of selectively suppressing innate immune-driven neurodegeneration has been hindered by the lack of molecular understanding of the neurotoxic functions of CNS innate immune cells.
Here, the innate immune cell atlas of oxidative stress in neuroinflammatory disease and the discovery of new therapeutic targets is reported. To functionally dissect the oxidative stress signature of CNS innate immunity at the single-cell level, a Toxic-RNA-seq (Toxseq) was developed to transcriptionally profile ROS+innate immune cells. A core oxidative stress signature shared among a microglia cluster and subsets of infiltrating myeloid cells in mice were identified, as well as microglia from MS lesions. Tox-seq followed by microglia HTS of a library of 1,907 clinical drugs and bioactive compounds and oxidative stress gene network analysis identified glutathione transferase activity and the compound acivicin, which inhibits the degradation of the antioxidant glutathione by targeting γ-glutamyl transferase (GGT). Therapeutic administration of acivicin reversed clinical signs, decreased oxidative stress, and protected from neurodegeneration in chronic EAE, even when administered eighty days after disease onset. Thus, these studies determine the transcriptomic landscape of oxidative stress in CNS innate immunity and provide druggable pathways for therapeutic targeting of neurotoxic innate immune populations.
Materials and Methods
Animals SJL/J, NOD, C57BL/6, and Ggt1dwg/dwg 38 mice were purchased from The Jackson Laboratory, and Sprague-Dawley rat Po litters were purchased from Charles River Laboratories. Ccr2RFP/RFP mice (56) on C57BL/6 background (provided by I. F. Charo, Gladstone Institutes) were crossed with Cx3cr1GFP/GFP mice (57) to generate Cx3cr1GFP/+ Ccr2RFP/+ mice. Mice were housed under IACUC guidelines in a temperature and humidity-controlled facility with 12 h light-12 h dark cycle and ad libitum feeding. All animal protocols were approved by the Committee of Animal Research at the University of California, San Francisco, and were in accordance with the National Institutes of Health guidelines.
EAE. Active EAE was induced in 8- to 10-week-old female SJL/J mice, C57BL/6 mice, and Cx3cr1GFP/+ Ccr2RFP/+ mice by subcutaneous immunization with 50 μg MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) or 100 μg PLP139-151 peptide (HSLGKWLGHPDKF; Auspep) in complete Freund's Adjuvant (Sigma-Aldrich) supplemented with 400 μg of heat-inactivated Mycobacterium tuberculosis H37Ra (Difco Laboratories). At day 0 and 2 after immunization, mice were given intraperitoneal injection of 200 ng (C57BL/6) or 75 ng (SJL/J) pertussis toxin (Sigma-Aldrich). For chronic NOD EAE model, 10- to 12-week-old NOD mice were immunized with 150 μg MOG35-55 peptide, followed by administration of 200 ng pertussis toxin on days 0 and 2 as described 43. For adoptive transfer of EAE in SJL/J mice, donor SJL/J mice were immunized as described above, and on day 10 post immunization, cells were isolated from the draining lymph nodes and spleen. Lymphocytes were re-stimulated with 20 μg/ml PLP139-151 and 10 ng/ml IL-12 (eBioscience) for 4 d, 3×107 restimulated cells were transferred to healthy SJL/J recipient mice as described (9). For prophylactic treatment, mice were each administered with 5 mg/kg acivicin (Santa Cruz Biotechnology, dissolved at 1 mg/ml in saline), 5 mg/kg GGsTop (Tocris) or saline daily from day 0. For therapeutic treatment, acivicin or saline was injected daily starting at the peak of the initial paralytic episode or at day 80 during the chronic phase of EAE in NOD mice. Mice were randomly assigned to treatment groups, scored and drug treated in a blinded manner Mice that did not develop symptoms of EAE were not excluded from the analysis. Experimental groups were unblinded to treatment assignment at the end of the experiments. Mice were observed daily, and clinical scores were assigned as follows by observers blinded to treatment: 0, no symptoms; 1, loss of tail tone; 2, ataxia; 3, hindlimb paralysis; 4 hindlimb and forelimb paralysis; 5, moribund. EAE onset was defined by weight loss (>1 gram) and first day of symptoms (score >0).
Cell isolation from spinal cord. Mice were perfused with ice-cold phosphate buffered saline (PBS) and spinal cord tissue was flushed out from the spinal column with PBS. Spinal cords were incubated in HBSS with 0.2% collagenase, type 3 (Worthington) gently shaking for 30 min at 37° C. under trypsin-free, mild dissociation conditions. Tissue were mechanically dissociated and passed through a 40 μm cell strainer (Falcon). Single-cell suspension was collected in 5 ml RPMI-1640 medium without phenol red supplemented with 25 mM HEPES, 1% Penicillin/Streptavidin and 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and myelin was depleted following the myelin removal beads II manufactures guidelines (Miltenyi Biotec). Myelin-depleted cell suspensions were processed for flow cytometry, bulk RNA-seq or scRNA-seq as described below. For bulk RNA-seq, myelin depleted (myelin removal beads II, Miltenyi Biotec) cell suspensions from spinal cords of five mice per experiment were pooled. For scRNAseq, single cell suspensions from individual animals were processed independently.
ROS labeling for Tox-seq. Following surface antigen flow cytometry staining, live cells isolated from spinal cord were stained for intracellular ROS in vitro using membrane permeable, fluorescent reagent 2′,7′-dichlorofluorescein diacetate (DCFDA; Abcam) as described (22). DCFDA recognizes ROS and is also a redox indicator probe that responds to cellular oxidant stress including reactive nitrogen species and elevated iron. For Bulk RNA-seq, cells were incubated with 10 μM DCFDA in PBS supplemented with 2% FBS, 2 mM EDTA (Gibco) for 30 min at 37° C., and directly analyzed by flow cytometry. For scRNA-seq, to minimize cell activation and cell death cells were incubated with 10 μM DCFDA in PBS supplemented with 2% FBS without EDTA at 4° C. for 30 min prior to cell sorting.
Fluorescence-activated cell sorting analysis for Tox-seq. For the scRNA-seq experiment, myelin-depleted cell suspensions were treated for 5 min at 4° C. with Fc-block in BSA staining buffer (BD) and then incubated for 30 min at 4° C. with CD11 b APC-Cy7 (M1/70) antibodies. Cells were washed with stain buffer once and then in vitro ROS labeling (DCFDA) was performed as described above. Cells were incubated for 5 min at 4° C. with 1 μM sytox blue live/dead stain and then cells were sorted using FACSAria II. After centrifugation at 300 g and 4° C. for 10 min, cell pellets were resuspended in cold PBS supplemented with 2% FBS at 333 cells/μl and immediately processed for scRNA-seq as described below.
Droplet-based scRNA-seq. For Tox-seq, 30 μL of each live sytox blue −CD11b+ROS− and live sytox blue −CD11b+ROS+ sorted cell population (233 cells/μl) from healthy and EAE spinal cords (disease score 1.0) were run on the 10× Genomics Chromium platform, and libraries were prepared following manufactures instructions for the Chromium Single Cell 3′ v2 Reagent Kit (
Unbiased graph-based clustering analysis of scRNA-seq data. The R toolkit Seurat (23) was used for quality control processing, graph-based clustering, visualizations, and differential gene expression analyses of scRNA-seq data and performed in R version 3.4.2. Cellranger Aggr aggregated dataset of 9,079 cells were filtered through quality control parameters that included parameters to keep cells with 200-5,000 nFeature_RNA per cell, and eliminate unhealthy cells with >5% and >25% mitochondrial and ribosomal genes, respectively. The percent of mitochondrial (percent.mito) and ribosomal (percent.ribo) genes were regressed out. All remaining variable genes were used for downstream analyses, including immediate early response genes induced by cell isolation procedure 24. Following QC, 17,814 genes across 8,701 single cells were subjected to downstream analyses following Seurat version 3 default parameters unless otherwise stated (Supp
Bulk RNA-seq and data analysis. For bulk RNA-seq experiment, surface stained cells were resuspended in 100 μl PBS with 1% FBS for cell sorting using FACSAria II (BD Bioscience). CompBeads Compensation Particles (BD Biosciences) individually stained with each of the fluorescently labeled antibodies were used for color compensation. In each experiment, the maximal cell number of sorted microglia and macrophages was assessed based on CD11b (M1/70) and CD45 (30-F11) signal intensities. The sum of sorted cells from three independent experiments (n=15 mice total) was 240,606 microglia and 183,530 macrophages. ROS+ (DCFDA) cells were gated based on FITC signals (cut-off 2×103), and MHC II+ cells gated based on PE-Cy7 signal (cut-off 3×103) compared to the unstained cells. Microglia and macrophage subpopulations were classified into MHC II+ ROS+, MHC II− ROS−(baseline), MHC II− ROS+ and MHC II+ROS−. In each sorting experiment, eight distinct cell populations were collected separately on ice. After centrifugation at 300 g and 4° C. for 6 min, cell pellets were stored at −80° C. in 150 μl RLT buffer (Qiagen) adding 1% 2-mercaptoethanol (Gibco) prior to RNA isolation for bulk RNA-seq.
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions. cDNA was generated from full-length RNA using the NuGEN Ovation RNA-seq V2 kit, and then sheared by the Covaris S2 Sonicator to yield uniform size fragments. The NuGen Ovation Ultralow V2 kit was used to add adapters, barcoding and amplification. Libraries were purified using Agencourt XP magnetic beads and quantified by KAPA qPCR (Illumina) Four libraries per lane were pooled for a single end (SE 50 bp) run on the Illumnia HiSeq 4000 platform. Input sequences were provided in FASTQ format. Trimming of known adapters and low-quality regions of reads was performed using Fastq-mcf59. Sequence quality control was assessed using the program FastQC and RSeQC. Reads were aligned to the mm9 mouse genome assembly using Tophat 2.0.13, and the number of reads mapping to each gene were counted using “featureCounts”, part of the Subread suite. Differential analysis was performed using edgeR60 Bioconductor package. The data set was filtered by including all genes which had at least in two replicates a CPM (counts per million) between 0.5 and 5000. The remaining genes were normalized using calcNormFactors (TMM) (“weighted trimmed mean of M-values”) in edgeR 61. Calculation of P-values was performed in edgeR for the differential expression between samples. The built-in R function “p.adjust” was used to calculate the FDR (false discovery rate) for each P value using the Benjamini-Hochberg method. To identify differentially expressed genes that were overrepresented in existing annotated genes, the data was analyzed with GO Elite (62). Clustering was performed first by doing k-means clustering with 100 clusters on log transform expression levels then by Hierarchical Ordered Partitioning and Collapsing Hybrid (HOPACH) (63) on the k-means clusters to prune highly similar clusters.
Co-expression clusters from enriched GO terms for bulk RNA dataset. Functional enrichment analysis of a data set with 2,145 differentially expressed genes in ROS+ versus MHC II+ microglia was performed in Cytoscape (32) using BiNGO plugin and GO annotations downloaded on 8 Dec. 2016. Filtering for GO term results with <2000 genes, 592 Biological Process terms with a corrected p-value <0.05 were found. GO term enrichment was also performed on the exclusive subsets of ROS-expressed genes (1,613) and MHC II-expressed genes (924), resulting in 58 and 783 filtered Biological Process terms, respectively. From these GO term results, 6 were selected that represented processes of interest, maximum specificity (i.e., low total gene membership) and exclusive significance (i.e., non-overlapping terms). Interaction networks were constructed for each set of differentially expressed genes associated with these 6 terms using GeneMANIA (64). Default co-expression and physical interaction sources were selected from the network construction; the option to add a number of related genes was set to zero; all other settings were default values. The interaction data from GeneMANIA was imported into Cytoscape for visualization and data overlay. Co-expression clusters were defined as the largest connected set of either up- or down-regulated genes and extracted as subnetworks.
Preparation of fibrin plates for HTS. Fibrin-coated 384-well plates were prepared as follows: Using a EL406 liquid handler (BioTek), columns 1-22 received 30 μL of 2 U/mL Thrombin (Sigma-Aldrich) in a buffer of 20 mM HEPES, pH 7.4, and 14 mM CaCl2, followed by 30 μL of 12 μg/mL human plasminogen-free fibrinogen (EMD Milipore) in 20 mM HEPES, pH 7.4 for a final concentration of 6 μg/mL of fibrinogen. After incubation for 90 min in 37° C. to allow fibrin formation, plates were dried overnight in a 37° C. incubator equipped with fans to circulate the air.
Image-based HTS of small molecule inhibitors of microglia activation. Primary rat microglia were isolated and cultured in the presence of heat-inactivated Performance Plus FBS (Thermo Fisher Scientific) as described (18,65). FBS in microglia culture was used to obtain a sufficiently high yield of microglia that was required for the HTS of 2,000 compounds. Since microglia activation can be influenced by the culture conditions (18,65), FBS was batch-tested with three quality control criteria: high cell yield, no effect on morphologic activation at baseline, and response to LPS induced morphologic activation by at least 50%. Microglia HTS was performed in 384-well PDL coated plates (Greiner). To screen for compounds that inhibited fibrin-activated microglia, 50 μL of DMEM containing 10% FBS was added to the fibrin-coated plate using an EL406 liquid dispenser-aspirator (Biotek). A library of 1,907 clinical drugs and bioactive compounds, compiled by the Small Molecule Discovery Center at the University of California, San Francisco, was screened at 10 μM. 100 nL of each compound (10 mM stock solution in DMSO) was added using a Biomek FXP automated laboratory workstation (Beckman Coulter) outfitted with a 50 nL pintool (V&P Scientific). Columns 1-2 contained DMSO-treated control cells (defining maximal activation/0% inhibition); columns 3-22 contained test compounds, and columns 23-24 contained unstimulated control cells (defining minimal activation/100% inhibition). 3,000 microglia cells were added to each well in 50 μL of DMEM containing 10% FBS, giving a final compound concentration of 10 μM in 0.1% DMSO. Assay plates were incubated at 37° C., 5% CO2 for 48 h. Using the EL406 liquid dispenser-aspirator, cells were then fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton-X100, and stained with 0.5 μg/mL CellMask Red (Thermo Fisher Scientific) and 2 μg/mL Hoechst nuclear dye (Thermo Fisher Scientific) with PBS washes between steps. The plates were stored in PBS for readout by imaging.
To screen for inhibitors of LPS (Sigma-Aldrich)-activated microglia, PDL-precoated 384-well plates (Greiner) were used. 50 μL of microglia cell suspension (3,000 cells per well) were added to each well with a WellMate multi-channel liquid dispenser (Thermo Fisher Scientific). 100 nL of each test compound (10 mM stock solutions in DMSO) was then added to columns 3-22 using the Biomek FXP and 50 nL pintool. One hour after the addition of compounds, 50 μL of a 1 ng/mL LPS solution was added to columns 1-22 using the EL406 liquid dispenser-aspirator, yielding an assay concentration of 0.5 ng/mL LPS in 100 μL assay volume. Columns 1-2 contained stimulated cells treated with DMSO (defining maximal activation/0% inhibition) and columns 22-24 contained unstimulated cells (defining minimal activation/100% inhibition). Assay plates were incubated at 37° C., 5% CO2 for 48 h. Cells were then fixed, permeabilized, stained and washed as described for the fibrin assay (9).
To measure microglial activation, assay plates were imaged using an INCell Analyzer 2000 automated fluorescent microscope (GE Healthcare) equipped with a 10× objective and excitation/emission filter pairs of 350 nm/455 nm (Hoechst stain) and 579 nm/624 nm (CellMask Red). Images were analyzed with the INCell Developer Toolbox feature extraction software (GE Healthcare). Cell nuclei stained with Hoechst dye were segmented using a “nuclear” segmentation method, with a minimum target area of 30 μm2 and sensitivity of 75%. Exclusion criteria for cell segmentation was set to intensity <120 units, or area >1000 μm2 The CellMask Red-stained cell bodies were segmented using an “intensity” segmentation method with a set threshold between 200-4095 intensity units. The borders of adjacent contacting cells were resolved using the “clump breaking” post-processing segmentation method that utilized discrete nuclei as seeds. Only cells containing a nucleus within the cell body area were analyzed. Activated microglia were defined as cells with a size ≥800 μm2, whereas cell size <150 μm2 was classified as dead cells. The number of activated microglia was divided by the total number of cells in the well to yield a fraction of activation. This activated fraction was normalized to the stimulated/untreated wells (columns 1-2) and unstimulated wells (columns 23-24) to determine the percentage of inhibition of microglial activation. Similarly, the percentage of dead cells was calculated by comparing the fraction of dead cells in a well to the stimulated and unstimulated controls. For each assay plate, Z-prime values were calculated as described (9). The average Z′ value was 0.5 for the six fibrin screening plates and 0.63 for the six LPS screening plates.
Compounds that were toxic to fewer than 3% of microglial cells and inhibited activation by >50% in either LPS or fibrin-treated plates were considered active. The 50% inhibition cutoff value was set as 3 standard deviation from the mean value of untreated control. Active compounds were reconfirmed in dose-response assays conducted in triplicates of 10 concentrations, with 3-fold serial dilutions ranging from 0.001 μM-20 μM. IC50 values were estimated from normalized % inhibition values, using the four-parameter non-linear regression analysis (Graphpad Prism).
Oxidative stress pathway modeling. A novel oxidative stress pathway was constructed based on compiling information from the literature and existing pathway diagrams, that included glutathione metabolism, redox, biosynthesis, uptake, breakdown, glutathionylation and acivicin inhibition. The gamma-glutamyl cycle was simplified for clarity. PathVisio (33) was used to construct the pathway model, which was deposited at WikiPathways (WP4466) (66) and then imported into Cytoscape for RNA-seq data overlay.
Immunohistochemistry. For immunohistochemical analysis, spinal cords were processed as Described (9,29,30,67,68). Antibodies used were as follows: mouse anti-gp91 (CYBB, 1:200; 53, BD Biosciences), mouse anti-iNOS (1:500, 610329, BD Biosciences), rabbit anti-Iba-1 (1:1000; 019-19741, Wako), rat anti-MHC II (1:300; M5/114.15.2, Thermo Fisher Scientific), rabbit coagulation factor X (F10; NBP1-33320, NOVUS), mouse anti-CLEC4E (1:700; AT16E3, abcam), mouse anti-neurofilament H non-phosphorylated (1:100; SMI-32, BioLegend), mouse anti-myelin basic protein (1:100; SMI-99, BioLegend), rabbit polyclonal anti-GGT1 (1:100; SAB2701966, Sigma), or goat polyclonal anti-4HNE (1:200; ab46544, Abcam) and Alexa 647, 488, 405 (1:500; Jackson ImmunoResearch) the Vector-Red and Vector-Blue alkaline phosphatase substrate kit (Vector Labs) for detection. Sections were stained with DAPI (1:1000, Thermo Fisher Scientific) for 3 min at room temperature. For mouse primary antibodies, the Mouse on Mouse (M.O.M.) kit (Vector Labs) was used according to the manufacturer's protocol. Analysis was based on an established method of neuropathology for sampling multiple spinal cord sections and comparing similar anatomical regions as described (68-70). Images were acquired with an Axioplan II epifluorescence microscope (Zeiss) equipped with Plan-Neofluar objectives (10×0.3 NA, 20×0.5 NA, or 40×0.75 NA) or all-in-one BZ-X700 fluorescence microscope (Keyence), Fluoview FV 1000 (Olympus) confocal microscope and Fluoview software v3.1b with Olymus 40× and 0.8 NA water-immersion lens as described (9), or Aperio Versa scanner (Leica) with Aperio Imagescope 12.4 and 1.25×, 10×, and 20× lenses. Images of similar anatomical locations were quantified using NIH ImageJ (version 1.50) by observers blinded to experimental conditions.
Flow cytometry. Cells were incubated with anti-mouse CD16/CD32 antibody (2.4G2) diluted in PBS with 2% FBS, and 2 mM EDTA at 4° C. for 15 min to block Fc receptor binding. Cells were stained with fluorescent-conjugated antibodies: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD44 (1M7), CD25 (3C7), CD11b (M1/70), CD45 (30-F11), and MHC II (M5/114.15.2). For surface labeling of GGT1, unconjugated mouse monoclonal anti-GGT1 (1:100; ab55138, Abcam) was incubated with surface markers for 1 hr at 4° C. Then FITC conjugated species-specific secondary antibody (1:100) was added for 1 hr at 4° C. For intracellular staining of IFN-γ and IL-17A, T cells were stimulated for 4 hr with Cell Activation Cocktail (BioLegend). Cells were then fixed with Cytofix/Cytoperm solution (BD Bioscience) and stained with antibodies to IL-17A (TC11-18H10.1), or IFN-γ (XMG1.2). Foxp3 (FJK-16S) staining was performed according to the manufacturer's protocol (eBioscience). Cells were analyzed by flow cytometry on an LSR II (BD Biosciences) with FlowJo software (Tree Star Inc.). Antibodies were purchased from BioLegend, BD Biosciences, or eBioscience.
Bone marrow—derived macrophage cultures. Bone marrow—derived macrophages (BMDMs) were prepared as described (67). In brief, bone-marrow cells were isolated from tibia and femur of C57BL/6 mice, Ggt1+/+ mice or Ggt1dwg/dwg mice and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin (Corning), and 10 ng/ml murine M-CSF (14-8983-80, Thermo Fisher Scientific). On days 6-7, adherent BMDMs were harvested by adding PBS with 5 mM EDTA to the plates and used for assays.
Human macrophage cultures. Human peripheral blood mononuclear cells (PBMCs) were purchased from AllCells, LLC (Alameda, Calif.). To differentiate PBMC into monocyte-derived macrophages, 2×106 PBMC/mL were plated in RPMI-1640 media supplemented with 10% FBS, 1% penicillin-streptomycin (Corning), and 50 ng/ml human M-CSF (300-25, Peprotech) in tissue-culture treated dishes (Corning). After 24 h, non-adherent cells were removed, and adherent cells were cultivated for 7-8 additional days at 37° C. in 5% CO2 to promote their full differentiation into macrophages. Oxidant detection with DHE. Intracellular concentrations of ROS were measured using dihydroethidium (DHE) as described (9). Cultured BMDMs or human macrophages were incubated in medium containing 5 μM DHE (Invitrogen) for 30 min Cells were plated on 96-well, black μ-clear-bottom microtiter plates (Greiner Bio-One) pre-coated with 25 μg/ml fibrin. For GGT inhibition, BMDMs or human macrophages were pre-incubated with 5 μM acivicin or GGsTop for 1 h, and the cells were plated on fibrin-coated 96-well plates as described above. PBS was used as vehicle control. Cells were then incubated for 24-48 h and fixed with 4% PFA for 10 min. The DHE fluorescence was detected at an excitation/emission wavelength of 518 nm/605 nm using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.).
Real-time imaging of glutathione. The real-time GSH dynamics in living BMDMs was determined with the fluorescent GSH probe, RealThiol RT-AM as described (37). Cells were plated on 2-well chamber slide (Nunc) pre-coated with 25 μg/ml fibrin. Cells were stimulated with fibrin with or without 5 μM acivicin or GGsTop. The GSH synthesis inhibitor, buthionine sulfoximine (BSO, Sigma-Aldrich) was used as positive control for reducing intracellular GSH levels at 100 μM. After 24 h incubation, cells were loaded with 1 μM RT-AM probe and were incubated for 5 min before imaging. The cells were kept at 37° C. during the entire experiment. Following incubation, fluorescence emissions after sequential excitation at 405 nm and 488 nm was acquired using sequential confocal laser scanning microscopy (Olympus FV1000; Olympus, Tokyo, Japan) with a 10× objective and 2× optical magnification. For each independent experiment, laser power and detector settings were set using the control/untreated cells incubated with RT-AM alone as reference. All settings were kept constant throughout the experiment. The ratio bound intracellular RT-AM:unbound intracellular RT-AM for each treatment condition was calculated by subtracting the 488-nm fluorescence signal from the 405 nm fluorescence signal from 30-50 cells/treatment for each independent experiment. The ratio calculated for each treatment was expressed as percentage from that of the untreated cells (control).
Blood sampling and hematological analysis. Hematological analysis was carried out for mice treated with saline or acivicin for 10 days. Blood samples were collected from the heart of each anesthetized mouse via cardiac puncture. The complete blood cell counts were measured with an automated blood count analyzer (Hemavet, Drew Scientific Inc).
Protein carbonyl content assay. Blood was isolated from EAE mice via terminal cardiac puncture, and serum levels of protein carbonylation were measured with OxiSelect™ Protein Carbonyl ELISA (Cell Biolabs) according to the manufacturer's protocol. The absorbance at 450 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.).
Real time qPCR. Total RNA was isolated from fibrin- or LPS-stimulated BMDMs (Ggt1dwg/dwg or Ggt1+/+ mice) with the RNAeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time qPCR analysis was performed on spinal cord tissues prepared from MOG35-55 EAE mice. SYBR green-based qPCR was performed using murine primers to Cxcl10, Nos2, Cxcl3, Ccl5, Il1b, or Il12b. Results were analyzed with the Opticon 2 Software and the comparative CT method. Gene expression was normalized to Gapdh and presented as fold change relative to control.
GGT activity assay. GGT activity was measured using the MaxDiscovery gamma-Glutamyl Transferase (GGT) Enzymatic Assay Kit (Bioo Scientific) according to the manufacturer's protocol. BMDMs were pre-incubated for 1 h with 10 μM GGT inhibitor acivicin (Santa Cruz Biotechnology) before plating cells on 25 μg/ml fibrin-coated 6-well culture plates that were prepared as described (67). For in vivo GGT activity assay, spinal cord tissues from the onset of MOG35-55 EAE or LPS-injected substantia nigra area at 12 h were prepared. Tissues were homogenized in 0.1 M Tris-HCl and centrifuged at 13,000 g for 30 min at 4° C. The supernatant was collected and subsequently assessed for GGT activity. The absorbance at 405 nm was detected with a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.). The GGT activity in IU/1 was calculated following the manufacturer's protocol by multiplying the average increase in absorbance at 405 nm over 10 min GGT activity was also measured using a fluorescent probe, ProteoGREEN-gGlu (Goryo Chemical, Hokkaido, Japan), according to the manufacturer's protocol. Cultured BMDMs were plated on 96-well, black μ-clear-bottom microtiter plates (Greiner Bio-One) pre-coated with 25 μg/ml fibrin. BMDMs were incubated with ProteoGREEN-gGlu with acivicin or GGsTop (diluted at threefold concentrations from 0.01 μM to 8.3 μM). The fluorescence intensity (excitation/emission filter pairs of 488 nm/520 nm) was measured using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies).
Stereotactic LPS injection and drug treatment. Mice were anaesthetized with isoflurane and placed in a stereotactic apparatus (David Kopf Instruments). LPS (Sigma-Aldrich) was dissolved in endotoxin-free distilled water (HyClone) and was diluted to 1 μg/ml with PBS. LPS (2 μl of 1 μg/ml) or PBS was slowly injected (0.3 μl/min) using a 10-μl Hamilton syringe attached to a 33-G needle into the substantia nigra at coordinates (anteroposterior, 3.0 mm; mediolateral, 1.3 mm; dorsoventral, 4.7 mm from the bregma, according to Paxinos and Watson) Animals received acivicin (5 mg/kg) or saline intraperitoneally beginning 5 days before LPS injection and for 7 days after LPS injection. After 12 h of the LPS injection, the substantia nigra area was immediately collected on ice and kept at −80° C. for later analysis of GGT activity. After 7 d of the LPS injection, mice were perfused with 4% PFA, and the brains were post-fixed in 4% PFA overnight at 4° C. Immunohistochemistry in coronal brain sections (30 μm) were performed using antibodies to tyrosine hydroxylase (TH, 1:2000; P40101, Pel Freez) and Iba-1 (1:1000; 019-19741, Wako). Images were acquired with all-in-one BZ-X700 fluorescence microscope. Images were quantified using NIH ImageJ (version 1.50) by blinded observers.
T-cell proliferation assay. For antigen-specific T cell proliferation, näive CD4+ T cells were magnetically sorted from the TCR-transgenic 2D2 mice using naïve CD4+ T cell isolation kits (Miltenyi Biotec). BMDMs were incubated with 5 μM acivicin, primed with MOG35-55 peptide (10 μg/ml) and then stimulated with LPS (10 ng/ml) for 24 h. CD4 T cells were cultured with MOG35-55 peptide primed BMDMs for 3 days and BrdU incorporation was assessed as described previously (67). For non-antigen-specific T cell proliferation, naïve CD4+ T cells were treated with acivicin and stimulated with mouse T-activator CD3/CD28 Dynabeads (Thermo Fisher Scientific) for 3 d. Cells were then fixed, permeabilized, and stained with FITC-conjugated anti-BrdU (BrdU Flow Kits, BD Biosciences).
Statistical analyses. Statistical analyses were performed with GraphPad Prism (Version 7). Data are presented as mean±s.e.m. No statistical methods were used to predetermine sample size, but sample sizes are similar to those reported previously. Statistical significance was determined with two-sided unpaired student's t-test, or non-parametric, two-sided Mann-Whitney test, or a oneway or two-way ANOVA analysis of variance followed by Bonferroni or Tukey's post-test (multiple comparisons). Mice were age and sex-matched and were randomly assigned to experimental groups. The assignment of EAE scores, histopathological analysis and quantification were done in a blinded manner. The statistical significance of the changes in the mean clinical score for each day of the EAE experiment was estimated using permutation tests (9). The corresponding P values were estimated using 1000 permutations. In each permutation, mice were randomly permuted.
Results
Single cell oxidative stress transcriptome of CNS innate immunity: To functionally profile the oxidative stress transcriptome of CNS innate immunity and identify neuroprotective drugs, a strategy for single-cell RNA-seq (scRNA-seq) transcriptional profiling of ROS+ CNS innate immune cells was developed and termed Tox-seq and performed microglia HTS of a small molecule library, followed by network analysis (
CNS innate immune clusters with oxidative stress and antigen presenting signatures: Single cells were overlaid with gene markers from the core oxidative stress signature and combined with unbiased GO analysis of DEGs from microglia and monocyte/macrophage subclusters (
Co-expression of oxidative stress, coagulation, and glutathione pathway genes: Tox-seq identified the Mg5 microglia cluster as a ROS+ CNS innate immune population enriched with oxidative stress genes. The transcriptomic signature of Mg5 showed the highest expression for oxidative stress, coagulation, inflammatory, antigen presenting, and pattern recognition receptor markers and the lowest expression of homeostatic markers (
Selection of acivicin by microglia HTS and oxidative stress gene network analysis: Tox-seq identified the coagulation pathway as mechanistically coupled to oxidative stress (
Table 4 provides a list of all 1907 compounds with labeling Q1, Q2, Q3 and Q4quadrants in
Acivicin suppresses oxidative stress in innate immune cells: Although acivicin has been studied primarily in cancer cells (34), its functions in inflammation and neurological diseases are poorly understood. The effects of acivicin were tested in innate immune cell activation using a series of secondary assays, such as ROS generation, glutathione regulation, gene expression of prooxidant and inflammatory genes, GGT activity, and antigen presentation. Acivicin inhibited fibrin- and LPS-induced microglial activation in a dose-dependent manner, and decreased ROS generation in mouse and human macrophages (
Therapeutic effects of acivicin in neuroinflammation: In patients with MS and neuromyelitis optica, serum GGT levels correlate with clinical disability, BBB disruption, and inflammatory markers (39). GGTLC1, which encodes the GGT light chain 1, is detected in the cortex of MS patients and is associated with oxidative damage and neuronal injury (40). Serum GGT activity positively correlates with dementia risk and glutathione S-transferase alpha in the plasma of AD patients correlates with late-onset AD progression (41,42). The effects of acivicin treatment were tested in autoimmune acute and chronic progressive models of neuroinflammation, as well as in models of microglia-mediated neurodegeneration. Using immunohistochemistry and FACS, the expression of GGT in EAE spinal cord was first tested.
GGT was not detected in healthy spinal cord but increased in microglia and infiltrating monocyte/macrophages in EAE lesions (
Immunization of non-obese diabetic (NOD) mice with MOG35-55 results in an acute neurological impairment followed by a chronic phase of progressive accumulation of disability (43). In chronic NOD MOG35-55 EAE, therapeutic administration of acivicin during the chronic phase even eighty days after EAE induction suppressed progression as indicated by decreased clinical signs (
The study revealed the oxidative stress transcriptome of CNS innate immune cells in neuroinflammation and identified druggable pathways to suppress neurotoxic innate immunity. By developing a functional transcriptomic and drug discovery pipeline consisting of deep sequencing, small molecule screening, and pathway analysis, novel innate immune cell populations involved in oxidative stress were identified and discovered upstream targeting of glutathione metabolism and redox homeostasis as a therapeutic strategy in neuroinflammation. Using Tox-seq, it was discovered that innate immune cells share a core oxidative stress gene signature mechanistically coupled to coagulation, antigen presentation, and glutathione pathways. The findings introduce the concept of distinct molecular circuits governing oxidative stress and immune-mediated neurodegeneration and reveal their molecular signatures. Given that oxidative stress producing resident and infiltrating innate immune cells are players in MS progression (3,12,45), the oxidative stress signature could enable the identification of specific cell subpopulations contributing to neurotoxicity that could be further characterized with cell-fate mapping studies. Molecular convergence of innate immune cells to an oxidative stress core signature is a springboard for the development of therapies to selectively target CNS innate immune populations that promote oxidative cell injury. Given the broad range of diseases with oxidative stress, the findings have implications for a wide range of diseases including MS, AD, and traumatic brain injury.
The study revealed previously unknown molecular links between coagulation and oxidative stress. In neuroinflammatory lesions, in situ expression of coagulation genes promoting fibrin formation were identified, such as genes encoding coagulation factors IX and X, Vitamin K Dependent Plasma Glycoprotein, and the von Willebrand factor receptors glycoproteins IX and Ib. Intriguingly, coagulation gene expression was differentially increased in ROS+ CNS innate immune cells that co-expressed genes regulating oxidative stress, such as the NADPH oxidase subunit gp91-phox and GGT, and iron metabolism. Dysregulation of the coagulation pathway and fibrin deposition correlates with cortical damage, microglia activation, and neuronal loss in MS and EAE (29,30,46-48). Administration of anti-coagulants or inhibition of the interaction of fibrin with the CD11b-CD18 integrin receptor (also known as Mac-1, complement receptor 3, αMβ2) reduces clinical signs, oxidative stress, and neurodegeneration in MS animal models (9,10,31). Fibrin signaling via the CD11b-CD18 receptor may potentiate the crosstalk of NADPH oxidase with the GGT pathway (49) leading to redox regulation. Indeed, fibrin activates NADPH oxidase (9) and GGT (herein) to promote degradation of glutathione and oxidative stress in innate immune cells. Furthermore, inhibition of fibrin interaction with CD11b-CD18 or inhibition of NADPH oxidase (9,10), or GGT (herein) suppresses fibrin-induced ROS generation. NADPH oxidase activation and degradation of glutathione by fibrin can be a prooxidant mechanisms in diseases with blood-brain barrier disruption and vascular pathology. Thus, it is possible that there is a positive-feedback loop between coagulation, oxidative stress, and the pro-inflammatory response, whereby subpopulations of innate immune cells promote the local synthesis of coagulation factors to increase fibrin deposition and promote oxidative injury. Local increases in coagulation activity by innate immune cell subpopulations as an oxidative stress mechanism could be relevant for other diseases in the brain and periphery with vascular damage associated with fibrin deposition and oxidative injury (5,7,10,31,50,51).
Acivicin was selected as an upstream regulator of glutathione and redox homeostasis. GGT mediated cleavage of glutathione causes iron redox cycling, which stimulates the release of hydroxyl radicals (34). Thus, redox restoration by acivicin may protect against EAE progression by homeostatic regulation of glutathione in oxidative stress-producing innate immune cells. In accordance, pharmacologic inhibition of GGT by GGsTop reduced oxidative stress markers and protected from renal reperfusion injury (52). In EAE, acivicin suppressed inflammatory and prooxidant pathways and decreased axonal damage, demyelination, and peripheral cell recruitment into the CNS. Suppression of oxidative stress and reduction of chemokines that facilitate cell recruitment by acivicin might reduce myeloid cell numbers and decrease lesion size. Acivicin also regulates glutamate metabolism and leukotriene responses with potential effects on immune cell recruitment, neuronal and T cell functions (34,53,54). Cell sorting and scRNA-seq studies may be used to determine the drug selectivity of acivicin at the single-cell level and its effects on oxidative stress resistance and additional prooxidant and inflammatory markers. These studies could decipher mechanisms linking oxidative stress and peripheral cell recruitment into the CNS in EAE and other models of neurologic disease. Glutamine analogues like acivicin exhibit dose limiting toxicity in anti-cancer trials potentially due to interference with recycling of glutamine (34). Given the toxicity of high doses of acivicin in the clinic or the consequences of global depletion of GGT1 in mice (34), identification of safe drugs modulating glutathione metabolism might facilitate the restoration of redox hemostasis in neuroinflammatory disease.
In summary, by generating the first oxidative stress cell atlas of innate immunity, cell populations and molecular mechanisms involved in oxidative injury and neurotoxicity in neuroinflammatory disease were identified. Transcriptional signatures of oxidative stress genes that can be used as a resource and follow-up studies to validate additional gene targets, cell populations, and drugs from the HTS screen were defined. Furthermore, a method, Tox-seq, was advanced, which can be used to determine the functional role of oxidative stress producing cells in a wide range of disease states. Given the multiple roles of ROS in oxidative damage and redox regulation (55), Tox-seq could reveal molecular pathways governing ROS-mediated functions in physiology and pathology. Integration of functional transcriptomics and HTS may prioritize druggable pathways that could enhance cherry-picking or in silico screens to identify compounds of interest for preclinical testing. Thus, oxidative stress transcriptomics and drug discovery approaches could identify and target neurotoxic CNS innate immune populations and lead to the development of selective neuroprotective strategies.
PCT/US2018/052694
All publications, nucleotide and amino acid sequence identified by their accession nos., patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
The specific methods and compositions described herein are representative of embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a polypeptide” includes a plurality of such nucleic acids or polypeptides (for example, a solution of nucleic acids or polypeptides or a series of nucleic acid or polypeptide preparations), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/009,212, filed on Apr. 13, 2020, the benefit of priority of which is claimed hereby, and which is incorporated by reference herein in its entirety.
This invention was made with government support under Grant No. R35 NS097976 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63009212 | Apr 2020 | US |
