This document relates to methods and materials for treating a neuromyelitis optica (NMO) spectrum disorder such as NMO. For example, one or more tetracycline antibiotics can be administered to a mammal having, or at risk of developing, a NMO spectrum disorder to treat the mammal.
NMO is a debilitating and sometimes fatal neurological autoimmune condition characterized by preferential demyelination of optic nerves and the spinal cord. While NMO symptoms can be mitigated, this disease has no cure and eventually all patients experience impairments. Thus, there is unmet medical need to identify novel and specific therapeutics for the treatment of NMO attacks and the prevention of NMO relapses. An IgG autoantibody specific for the astrocytic AQP4 water channel (NMO-IgG or AQP4-IgG) is the primary pathogenic effector of NMO (Lennon et al., Lancet 364:2106-2112 (2004); Lennon et al., J. Exp. Med. 202:473 (2005); and Roemer et al., Brain 130:1194-1205 (2007)). AQP4 is highly concentrated at astrocyte end-feet which embrace capillaries, glutamatergic synapses, nodes of Ranvier, ventricle walls and pia-glial interfaces (Szu et al., Front. Integr. Neurosci. 10:8 (2016); Hinson et al., Proc. Natl. Acad. Sci. USA 109: 1245-1250 (2012); Guo et al., Acta Neuropathol. 133:597-612 (2017); and Misu et al., Brain 130:1224-1234 (2007)). NMO-IgG mainly targets astrocytic AQP4 (Hinson et al., Proc. Natl. Acad. Sci. USA 114:5491-5496 (2017); Hinson et al., Neurology 69:2221-2231 (2007); and Hinson et al., Arch. Neurol. 66:1164-1167 (2009)).
This document provides methods and materials related to treating a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO. For example, this document provides methods and materials for using one or more tetracycline antibiotics (e.g., minocycline) to treat a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO. In some cases, a mammal having, or at risk of developing, a NMO spectrum disorder can be administered a composition including one or more tetracycline antibiotics to treat the mammal.
Current treatments for NMO, include intravenous corticosteroid (such as methylprednisolone), plasma exchange, and other antibody-depleting therapies. These treatments can be non-specific and can induce adverse complications (Scott et al., Neurology 77:2128-2134 (2011); and Drozdowicz et al., Mayo Clin. Proc. 89:817-834 (2014)). As demonstrated herein, astrocyte-microglia interaction drives pathogenesis of NMO, and minocycline can reverse NMO-IgG (e.g., AQP4-IgG) induced motor dysfunction and can reduce NMO-IgG induced microglia-astrocyte interactions. The identification of a previously unrealized role for microglia in NMO pathogenesis, provides a unique target for treating mammals having, or at risk of developing, NMO. For example, a mammal having, or at risk of developing, NMO can be treated by administering minocycline to reduce or eliminate microglia activation.
In general, one aspect of this document features methods for treating a mammal having a NMO spectrum disorder. The methods can include, or consist essentially of, administering a composition including a tetracycline antibiotic to a mammal having a NMO spectrum disorder to reduce or eliminate a motor function impairment in the mammal. The method can include identifying the mammal as being in need of a treatment for the NMO spectrum disorder. The mammal can be a human. The NMO spectrum disorder can be NMO. The motor function impairment can be decreased visual acuity, visual field defects, loss of color vision, muscle weakness, reduced sensation, perverted sensation, loss of bladder control, loss of bowel control, paraparesis, quadriparesis, neuroinflammation, vomiting, hiccups, bladder dysfunction, bowel dysfunction, confusion, seizures, coma, respiratory failure, or cognitive impairment. The tetracycline antibiotic can target microglia in the mammal. The tetracycline antibiotic can target C3a receptor (C3aR) polypeptides on the microglia. The tetracycline antibiotic can be minocycline. The composition can include from about 50 µg to about 300 µg of the minocycline.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials related to treating a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder such as NMO. For example, this document provides methods and materials for using one or more tetracycline antibiotics (e.g., minocycline) to treat a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO. In some cases, a mammal having, or at risk of developing, a NMO spectrum disorder can be administered a composition including one or more tetracycline antibiotics to treat the mammal.
In some cases, a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder such as NMO can be administered one or more tetracycline antibiotics (e.g., minocycline) to reduce or eliminate one or more NMO spectrum disorder impairments (e.g., NMO-IgG induced impairments) and/or one or more symptoms of a NMO spectrum disorder. For example, one or more tetracycline antibiotics (e.g., minocycline) can be administered to a mammal (e.g., a human) as described herein to reduce the severity of one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. NMO spectrum disorder impairments can be vision impairments and/or motor function impairments. Examples of NMO spectrum disorder impairments and symptoms of a NMO spectrum disorder include, without limitation, decreased visual acuity, visual field defects, loss of color vision, muscle weakness, reduced sensation, perverted sensation, loss of bladder control, loss of bowel control, paraparesis, quadriparesis, neuroinflammation, vomiting, hiccups, bladder dysfunction, bowel dysfunction, confusion, seizures, coma, respiratory failure, and cognitive impairment.
In some cases, a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder such as NMO can be administered one or more tetracycline antibiotics (e.g., minocycline) to reduce or eliminate one or more NMO spectrum disorder pathologies (e.g., NMO-IgG induced pathologies). For example, one or more tetracycline antibiotics (e.g., minocycline) can be administered to a mammal (e.g., a human) as described herein to reduce the severity of one or more NMO spectrum disorder pathologies by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Examples of NMO spectrum disorder pathologies include, without limitation, inflammatory demyelination, inflammatory lesions (e.g., with or without demyelination), inflammatory cell invasions, and blood-brain barrier damage.
Any appropriate mammal having, or at risk of developing, a NMO spectrum disorder can be treated as described herein (e.g., by administering one or more tetracycline antibiotics). Examples of mammals having, or at risk of developing, a NMO spectrum disorder that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, rat, and rabbit. In some cases, a human having, or at risk of developing, a NMO spectrum disorder can be treated by administering one or more tetracycline antibiotics to the human.
Any appropriate NMO spectrum disorder can be treated as described herein (e.g., by administering one or more tetracycline antibiotics). In some cases, a NMO spectrum disorder can be a NMO spectrum disorder accompanied by the presence of anti-AQP4 autoantibodies (e.g., NMO-IgG or AQP4-IgG). In some cases, a NMO spectrum disorder can be a NMO spectrum disorder accompanied by the presence of anti-myelin oligodendrocyte glycoprotein (MOG) autoantibodies (e.g., MOG-IgG). In some cases, a NMO spectrum disorder can be a NMO spectrum disorder can lack autoantibodies. Examples of NMO spectrum disorders that can be treated as described herein can include, without limitation, AQP4-IgG-positive NMO (also referred to as Devic’s disease), limited forms of Devic’s disease (e.g., single events of longitudinally extensive myelitis, recurrent events of longitudinally extensive myelitis, bilateral simultaneous optic neuritis, and bilateral recurrent optic neuritis), Asian optic-spinal MS, longitudinally extensive myelitis, optic neuritis (e.g., optic neuritis associated with systemic autoimmune disease), myelitis associated with lesions in the brain (e.g., in specific brain areas such as the hypothalamus, periventricular nucleus, and brainstem), and seronegative NMO (e.g., NMO lacking an autoantibody). In some cases, a mammal (e.g., a human) having, or at risk of developing, NMO can be treated by administering one or more tetracycline antibiotics to the mammal.
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder, also can include identifying a mammal as having, or as being at risk of developing, a NMO spectrum disorder. Any appropriate method can be used to identify a mammal as having, or as being at risk of developing, a NMO spectrum disorder. For example, neurological examinations (e.g., neurological examinations for muscle strength, coordination, sensation, cognitive functions such as memory and thinking, and vision and speech), neurological imaging (e.g., magnetic resonance imaging (MRI) to detect lesions or damaged areas of the brain, optic nerves and spinal cord), blood tests (e.g., blood tests looking for the presence of autoantibodies such as NMO-IgG (e.g., AQP4-IgG)), lumbar punctures (e.g., to test the amounts and types of leukocytes, proteins, and/or antibodies in the spinal fluid), stimuli response tests (e.g., to learn how well the brain responds to stimuli such as sounds, sights, touch, and/or memory) can be used to identify a mammal as having, or as being at risk of developing, a NMO spectrum disorder.
Once identified as having, or as being at risk of developing, a NMO spectrum disorder, a mammal (e.g., a human) can be administered, or instructed to self-administer, one or more tetracycline antibiotics. A tetracycline antibiotic can be any appropriate tetracycline antibiotic (e.g., any antibiotic in the tetracycline family of antibiotics). As used herein, a tetracycline antibiotic has a linear fused tetracyclic nucleus (rings designated A, B, C and D) to which a variety of functional groups (designated as R groups; e.g., chloride, methyl, and hydroxyl groups) are attached as shown below.
In some cases, a tetracycline antibiotic can be a broad-spectrum tetracycline antibiotic. In some cases, a tetracycline antibiotic can be a second-generation tetracycline antibiotic. In some cases, a tetracycline antibiotic can target (e.g., can selectively target) microglia (e.g., microglial C3aR polypeptides). For example, a tetracycline antibiotic can selectively target C3aR polypeptides on microglia to reduce or eliminate microglia activation. In some cases, a tetracycline antibiotic can cross the blood-brain barrier. Examples of tetracycline antibiotics that can be used to treat a mammal having, or at risk of developing, a NMO spectrum disorder as described herein include, without limitation, minocycline, tetracycline-3, and doxycycline. In some cases, a tetracycline antibiotic that can be used as described herein can be minocycline. A chemical formula for a minocycline can be as follows.
For example, a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder such as NMO can be treated by administering minocycline to the mammal. In some cases, tetracycline antibiotics that can be used to treat a mammal having, or at risk of developing, a NMO spectrum disorder as described herein include, without limitation, those described in Robert et al., Nat. Neurosci. 18: 1081-1083 (2015); Sharma et al., Circ Res. 124:727-736 (2019); and Sultan et al., Front. Neurosci. 7:31 (2013).
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder, can include administering to the mammal one or more agents that can deplete microglia. Examples of agents that can be used as described herein to deplete microglia include, without limitation, plexxikon compounds (see, e.g., Elmore et al., Neuron 82:380-397 (2014)).
In some cases, one or more tetracycline antibiotics (e.g., minocycline) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO. For example, one or more tetracycline antibiotics can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein include, without limitation, saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol (PEG; e.g., PEG400), sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, and corn oil.
In some cases, when a composition containing one or more tetracycline antibiotics (e.g., minocycline) is administered to a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO, the composition can be designed for oral or parenteral (including, without limitation, subcutaneous, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, or intraperitoneal (i.p.) injection) administration to the mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
A composition containing one or more tetracycline antibiotics (e.g., minocycline) can be administered to a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO in any appropriate amount (e.g., any appropriate dose). Effective amounts can vary depending on the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. An effective amount of a composition containing one or more tetracycline antibiotics can be any amount that can treat a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO without producing significant toxicity to the mammal. For example, an effective amount of minocycline can be from about 50 micrograms (µg) to about 300 µg (e.g., from about 50 µg to about 250 µg, from about 50 µg to about 200 µg, from about 50 µg to about 150 µg, from about 50 µg to about 100 µg, from about 100 µg to about 300 µg, from about 150 µg to about 300 µg, from about 200 µg to about 300 µg, from about 250 µg to about 300 µg, from about 100 µg to about 250 µg, from about 150 µg to about 200 µg, or from about 100 µg to about 200 µg) per day. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the NMO spectrum disorder in the mammal being treated may require an increase or decrease in the actual effective amount administered.
A composition containing one or more tetracycline antibiotics (e.g., minocycline) can be administered to a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO in any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having, or at risk of developing, a NMO spectrum disorder without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about once a month, from about twice a month to about once a month, or from about once a day to about once a week. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and route of administration may require an increase or decrease in administration frequency.
A composition containing one or more tetracycline antibiotics (e.g., minocycline) can be administered to a mammal having, or at risk of developing, a NMO spectrum disorder such as NMO for any appropriate duration. An effective duration for administering or using a composition containing one or more tetracycline antibiotics can be any duration that can treat a mammal having, or at risk of developing, a NMO spectrum disorder without producing significant toxicity to the mammal. For example, the effective duration can vary from several months to several years or to a lifetime. In some cases, the effective duration can range in duration from about 10 years to about a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and route of administration.
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder, can include administering to the mammal one or more tetracycline antibiotics (e.g., minocycline) as the sole active ingredient to treat the mammal. For example, a composition containing one or more tetracycline antibiotics can include the one or more tetracycline antibiotics as the sole active ingredient in the composition that is effective to treat a mammal having, or at risk of developing, a NMO spectrum disorder.
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a NMO spectrum disorder, can include administering to the mammal one or more tetracycline antibiotics (e.g., minocycline) and also administering to the mammal one or more (e.g., one, two, three, four, five or more) additional treatments that are effective against one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder to treat the mammal. Examples of treatments for one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder in a mammal include, without limitation, administering to the mammal one or more active agents (e.g., therapeutic agents) that are effective against one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder such as immunosuppressants (e.g., azathioprine, mycophenolate mofetil, mitoxantrone, intravenous immunoglobulin (IVIG), and cyclophosphamide), corticosteroids (e.g., methylprednisolone, and prednisone), agents that deplete B cells (e.g., rituximab), immunomodulators such as agents that neutralize or deplete complement components (e.g., anti-C5 antibodies such as eculizumab), subjecting the mammal to plasmapheresis, and/or subjecting the mammal to hematopoietic stem cell transplantation (HSCT). In some cases, the treatments for one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder can be performed together with the administration of the one or more tetracycline antibiotics (e.g., minocycline). For example, a composition containing one or more tetracycline antibiotics also can include one or more additional active agents that are effective against one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder. In some cases, the one or more treatments for one or more NMO spectrum disorder impairments and/or one or more symptoms of a NMO spectrum disorder can be performed independent of the administration of the one or more tetracycline antibiotics (e.g., minocycline). When the one or more treatments for one or more symptoms of a NMO spectrum disorder are performed independent of the administration of the one or more tetracycline antibiotics, the one or more tetracycline antibiotics can be administered first, and the one or more treatments for one or more symptoms of a NMO spectrum disorder performed second, or vice versa.
In certain instances, a course of treatment can be monitored. In some cases, methods described herein also can include monitoring the severity or progression of a NMO spectrum disorder such as NMO in a mammal. Any appropriate method can be used to monitor the severity or progression of a NMO spectrum disorder in a mammal. In some cases, one or more NMO spectrum disorder impairments (e.g., NMO-IgG induced impairments) can be assessed using any appropriate methods and/or techniques, and can be assessed at different time points. For example, physical examinations (e.g., eye examinations and/or motor function testing) can be used to assess NMO spectrum disorder vision impairments (e.g., decreased visual acuity, visual field defects, and loss of color vision). For example, ambulation status, coordination analysis, and/or gait analysis can be used to assess NMO spectrum disorder motor function impairments (e.g., muscle weakness, reduced sensation, perverted sensation, loss of bladder control, loss of bowel control, paraparesis, and quadriparesis). In some cases, one or more NMO spectrum disorder pathologies (e.g., NMO-IgG induced pathologies such as inflammatory demyelination, inflammatory lesions (e.g., with or without demyelination), inflammatory cell invasions, blood-brain barrier damage, loss of AQP4, and/or loss of one or more glutamate transporters (e.g., EAAT2 and/or EAAT1)) can be assessed using any appropriate methods and/or techniques, and can be assessed at different time points. For example, laboratory tests, imaging techniques, and/or biopsies can be used to assess NMO spectrum disorder pathologies (e.g., inflammatory demyelination, inflammatory lesions (e.g., with or without demyelination), inflammatory cell invasions, blood-brain barrier damage, loss of AQP4, and/or loss of one or more glutamate transporters (e.g., EAAT2 and/or EAAT1)). In some cases, one or more symptoms of a NMO spectrum disorder such as NMO can be assessed using any appropriate methods and/or techniques, and can be assessed at different time points. For example, neurological examinations (e.g., neurological examinations for muscle strength, coordination, sensation, cognitive functions such as memory and thinking, and vision and speech), neurological imaging (e.g., magnetic resonance imaging (MRI) to detect lesions or damaged areas the brain, optic nerves and spinal cord), blood tests (e.g., blood tests looking for the presence of autoantibodies such as NMO-IgG (e.g., AQP4-IgG)), lumbar punctures (e.g., to test the levels of immune cells, proteins, and/or antibodies in the spinal fluid), stimuli response tests (e.g., to learn how well the brain responds to stimuli such as sounds, sights, touch, and/or memory) can be used to assess one or more symptoms of a NMO spectrum disorder (e.g., neuroinflammation, vomiting, hiccups, bladder dysfunction, bowel dysfunction, confusion, seizures, coma, respiratory failure, and cognitive impairment).
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
To investigate what signals drive microglial activation in NMO and how microglia may participate in the pathology, an informative in vivo murine model of NMO was developed that utilizes chronic intrathecal infusion of NMO patient-derived or monoclonal AQP4-specific IgGs. Using microglial depletion approaches combined with genetic knockouts, it was found that microglia participate in NMO pathophysiology by interacting with astrocytes in a complement C3 dependent manner. These results reveal unexpected complement-mediated astrocyte-microglia crosstalk in NMO pathogenesis, which can be targeted for therapeutic interceptions.
To investigate the cellular and molecular mechanisms underlying NMO pathology, a murine model was developed that utilizes IgG purified from NMO patient serum (NMO-IgG) or monoclonal AQP4-specific IgG. IgG collected from healthy individuals, or from healthy control subjects (control-IgG), or a control monoclonal IgG of irrelevant specificity was used in each experiment. NMO-IgG and control-IgG was purified by protein G adsorption (
To determine AQP4 involvement in our NMO model, motor dysfunction following NMO-IgG infusion was compared in wild-type (WT) mice and AQP4 knockout (AQP4-/-) mice. Deficiency of AQP4 was verified via post-mortem CNS tissue immunostaining (
AQP4 immunostaining was performed on both longitudinal and transverse spinal cord sections from mice infused with NMO-IgG. At day 5 of infusion, spinal cord AQP4 immunoreactivity was significantly reduced, particularly in the region surrounding NMO-IgG infusion (
Immunostaining was performed using the neuronal marker NeuN. Significant loss of NeuN staining was observed in both dorsal and ventral horns of NMO-IgG recipient mice when compared to control-IgG recipients (
To examine microglial activation in our murine model of NMO, genetic labeling of microglia (CX3CR1GFP/+) and immunostaining for the microglial marker Iba1 were used. Dramatic microglial activation was found in NMO-IgG recipient mice when compared with controls (
The expression of CD68, a marker for microglial activation, was examined and it was largely increased in microglia following NMO-IgG exposure (
Microglial activation after NMO-IgG infusion indicates a potential role for microglia in NMO pathogenesis. To test this idea, microglia ablation approaches were utilized to directly examine the requirement of microglia in NMO-induced motor deficits. This was accomplished by using CX3CR1CreER/+: R26iDTR/+ mice, which are induced by tamoxifen treatment (150 mg/Kg, i.p.) to express diphtheria toxin receptor (DTR) in microglia. Spinal microglia were mostly depleted 1 to 3 days after administering diphtheria toxin (DT, 50 µg/Kg, i.p.) and these microglia gradually repopulated 5 to 7 days after DT (
To test the effects of microglia depletion and repopulation on NMO-IgG induced motor dysfunction DT was injected to deplete microglia prior to NMO-IgG infusion. It was found that microglial ablation strongly suppressed the onset of motor dysfunction in the mouse NMO model (
These results demonstrate a critical role for microglia in NMO pathogenesis. However, the question remains as to what cellular and molecular mechanisms underlie microglial function in NMO. This investigation of spinal astrocyte and microglia activation revealed an intriguing coalescence of astrocytes and microglia following NMO-IgG infusion (
To further investigate this unusual microglia-astrocyte interaction, in vivo 2-photon imaging of microglia (labeled with CX3CR1GFP/+) and astrocytes (labeled with SR101) in the spinal cord as performed. In response to NMO-IgG infusion, significant microglial process extension towards astrocytes was observed (
Astrocytes exposed in culture to NMO-IgG increase their production and secretion of all complement components, except C1q, which is made by activated microglia (see, Howe et al., Glia 62:692-708 (2014)). In this murine NMO model, a dramatic increase of C3 expression was observed in GFAP+ astrocytes following NMO-IgG exposure (
The cleavage of the complement component C3 produces two signaling molecules, C3a and C3b. Immunostaining revealed that C3a receptor (C3aR) is exclusively expressed on microglia and is upregulated after NMO-IgG infusion (
To further investigate the role of C3-dependent complement signaling in NMO-IgG-induced pathology and motor dysfunction, NMO-IgG was infused into C3-/- and C3aR-/- mice (
Female mice (6-8 weeks old) were used in accordance with institutional guidelines as approved by the animal care and use committee at Mayo Clinic. C57BL/6J (Charles River) and CX3CR1GFP/+ mice were used as wild-type animals. AQP4 null mice were as described elsewhere (Lennon et al., J. Exp. Med. 202:473 (2005)). C3 null mice (B6;129S4-C3tm1Crr/J) and C3aR null mice (C. 129S4-C3ar1tm1Cge/J) were purchased from Jackson lab. CX3CR1CreER-EYFP/+ mice were as described elsewhere (Christopher et al., Cell 155: 1596-1609 (2013)). These mice were crossed with R26iDTR/+ (bought from Jackson Laboratory) to obtain CX3CR1CreER/+:R26iDTR/+ mice. Mice were assigned to experimental groups randomly within a litter. Experimenters were blind to drug treatments.
A 3.5 cm polyurethane-silicone catheter (Alzet, CA) was inserted at the condylar canal to accesses the subarachnoid space at the cisterna magna and extended to lumbar level of spinal cord. Five days later an osmotic mini-pump delivery system, containing either NMO-IgG or control-IgG, was placed subcutaneously over the right shoulder. IgG was delivered continuously for 5-7 days (1-10 µg/day) (
TM (Sigma) was administered as a solution in corn oil (Sigma) to mice over 4 weeks of age via i.p. injection. Animals received four doses of TM (150 mg kg-1, 20 mg mL-1 in corn oil) in 48 hour intervals. For total CX3CR1+ cell ablation, two doses of DT (Sigma, Catalogue #D0564, 50 mg kg-1, 2.5 mg mL-1 in PBS) were given at 3 and 5 days after the last TM treatment. Mice administered with DT only (without TM) were used as control for all ablation experiments.
The rotarod tests were performed using a five-lane Rotarod apparatus (Med Assocaites Inc). The rotarod speed started from 4 rounds per minute and uniformly accelerated to 40 rounds per minute over 5 minutes. Each mouse was tested for 3 times with 15 minute intervals. For gait analysis, mouse fore and hind limbs were covered by different color ink and allowed to walk freely across a narrow strip of paper. Stride length of hind limbs was reported as the mean of 5 sequential steps.
Mice were deeply anaesthetized with isoflurane (5% in O2) and perfused transcardially with 20 mL PBS followed by 20 mL of cold 4% paraformaldehyde (PFA) in PBS. The spinal cord was removed and post-fixed with 4% PFA for 6 hours at 4° C. Samples were then transferred to 30% sucrose in PBS overnight. Sample sections (15 mm in thickness) were prepared on gelatin-coated glass slide with a cryostat (Leica). The sections were blocked with 10% goat serum and 0.3% Triton X-100 (Sigma) in TBS buffer for 60 min, and then incubated overnight at 4° C. with primary antibody for rabbit anti-Iba1 (1:500, Abcam, 178847), rabbit anti-CD68 (1:500, Abcam, 125212), mouse anti-GFAP (1:500, CST, 3670), rabbit anti-AQP4 (1:500, Sigma, A5971), rabbit anti-C3 (1:200, Thermo, 21349), mouse anti-C3aR (1:500, hycultbiotech, 1123), or rat anti-CD31(1:500, BD, 550274). The sections were then incubated for 60 minutes at room temperature, with secondary antibodies (1:500, Alexa Fluor 594, Life Technologies or Alexa Fluor 488, Life Technologies). The sections were mounted with Fluoromount-G (SouthernBiotech) and fluorescent images were obtained with a confocal microscope (LSM510, Zeiss). Cell counting and fluorescent signal intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Hippocampal tissue slices (400 mm thick) were prepared from P14-21 mice and incubated in imaging media. Microglia were visualized by GFP. For each image in the time-series, 15 z-steps spaced 2 mm apart were collected per image (30 mm total depth). Images were taken at 5 minute intervals for up to 1 hour on Scientifica 2-photon microscope with an X20 lens. Image processing and analysis was performed using NIH Image J software.
Image within the spinal cord was recorded by using a 2-photon microscope (Scientifica) with a Ti:sapphire laser tuned to 900 nm (Mai Tai; Spectra Physics), Microglia stained genetically (CX3CR1GFP/+) and astrocytes stained by intrathecal injection of SR101 (5 µL at 25 µM). After performing laminectomy at L5 level, the spinal column was stabilized via clamps (model STS-A; Narishige) to minimize movement artefacts. Video and images are captured 100-150 µm from the surface.
Quantification of Iba1 cells was done with ImageJ software (NIH Image). Data were presented as mean ±SEM. Student’s t-test and Two-way ANOVA were used to establish significance. No statistical methods were used to predetermine sample sizes.
The microglia inhibitor minocycline reversed NMO-IgG induced motor dysfunction and microglia-astrocyte interaction. Intrathecally injection of minocycline (150 µg/day) prevented the NMO-IgG induced motor dysfunction in rotarod test (
Colony stimulating factor-1 (CSF1) receptor inhibitor Pexidartinib (PLX3397) is known to deplete microglia in vivo. Animals were treated with control chow for 7 days then switched to PLX3397 chow. NMO-IgG infusion was started after 7 days of PLX3397 treatment. PLX3397 eliminated most of the microglia in the L4 spinal cord (
Minocycline hydrochloride was dissolved in PBS with 1% DMSO at 30 µg/µL and mixed with 20 µg/µL NMO-IgG to make a mixture of 15 µg/µL minocycline and 10 µg/µL NMO-IgG. 100 µL mixed drug and IgG was uploaded into osmotic pump and connected with the intrathecal infusion catheter. Pumps were implanted under the skin behind animal neck. The pump can infuse 10 µL contained liquid (150 µg minocycline and 100 µg NMO-IgG) every day. As placebo control, PBS with 1% DMSO was used instead of Minocycline.
PLX3397 was bought from Research Diets, Inc. Animals were treated with PLX3397 by feeding them PLX3397 (600 µg/mg) chow.
A human identified as having NMO is administered one or more tetracycline antibiotics (e.g., minocycline). After administration of one or more tetracycline antibiotics, microglia activation in the human is reduced or eliminated.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Serial No. 62/868,053, filed Jun. 28, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/034641 | 5/27/2020 | WO |
Number | Date | Country | |
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62868053 | Jun 2019 | US |