Patent Application
20180280472
The invention involves prevention, amelioration or treatment of learning disabilities or other neurological disorders or diseases, such as those associated with fetal alcohol syndrome (FAS), by administering a SK2 channel or other SK channel inhibitor. Exposure of the brain, such as a developing fetal brain, to alcohol or other harmful agents or conditions, causes learning disabilities that persist and can present life-long challenges for an individual so exposed. The inventors have found that such learning disabilities caused by stressors like alcohol exposure in utero, can be treated by administering a SK channel blocker, antagonist, inhibitor or modifier, such as the SK2 channel blocker tamapin which is a short peptide found in scorpion venom.
The CDC estimates that 0.2 to 1.5 infants for every 1,000 live births have fetal alcohol syndrome (FAS) caused by exposure of the fetus in utero to alcohol. It estimates the lifetime cost for one individual with FAS to be $2 million dollars and the total cost to the U.S. to be $4 billion annually. Maternal alcohol consumption is the most commonly identifiable non-genetic cause of mental retardation or learning disability and damage to the brain associated with FAS. Ethanol is a common environmental toxin known to have age dependent effects on brain development and behavior. The cardinal features of intrauterine fetal exposure to EtOH include microcephaly, dysmorphic features, intellectual disability, and executive and behavioral dysfunction. In view of the significant consequences and costs, there is a need to identify prophylactic agents that ameliorate ethanol associated damage the brain and nervous system of a fetus as well as to treat such damage once it occurs, for example, in a neonate or child.
With this objective in mind, and based on earlier work involving heat shock protein expression described in U.S. Provisional Application No. 62/240,561 (herein incorporated by reference), the inventors sought to investigate brain injury acquired during fetal development and early life. They investigated mechanisms of injury, whether particular injuries correlate with learning disabilities, as well as specific factors or agents that might modify or mitigate injury or serve as post-injury interventions or treatments.
Tamapin is a short polypeptide toxin isolated from the Indian Red Scorpion (Mesobuthus tamalus) and is known to be a selective blocker of SK2 channels. A SK2 channel, also known as a KCNN2 or KCa2.2 channel, is a potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2. SK2 is an ion channel protein that is activated before membrane hyperpolarization and is thought to regulate neuronal excitability by contributing to the slow component of synaptic afterhyperpolarization (AHP); see KCNN2 potassium calcium-activated channel subfamily N member 2, Gene ID: 3781, full report, updated 9 Oct. 2016, available at: https://_www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSearch=37 81 (last accessed Oct. 11, 2016) which is hereby incorporated by reference.
The SK2 channel has not previously been associated with learning disabilities caused by fetal exposure to alcohol.
As demonstrated herein, the inventors have found that compounds, such as tamapin, that block, antagonize, inhibit or modify SK receptor numbers or SK receptor activity in the nervous system can protect and treat a subject, such as a fetus in utero or a neonate from the negative effects of exposure to ethanol or other stressors. Non-limiting aspects and applications of these findings include the following.
A method for treating a subject having, or at risk of acquiring a brain injury during fetal development comprising administering an inhibitor of at least one SK channel, such as tamapin or a tamapin analog that blocks, antagonizes, inhibits or modifies the SK2 channel. The subject may be fetus, such as a first, second, or third trimester fetus in utero, a pregnant woman, a preterm infant, neonate, child or adult at risk of acquiring injury to the brain or nervous system, especially, a fetus, preterm infant, or child whose brain is growing or developing and thus is susceptible to disruptions to growth or development associated with over-expression or over-activity of a SK channel compared to a corresponding normal individual. A SK channel blocker can physically block a channel comprising a SK protein; a SK channel inhibitor or antagonist, which may also be a channel blocker, inhibits or antagonizes activity associated with a SK channel such as ion transport or signal transduction; a SK channel modifier modifies the structure of a SK channel, e.g, by allosteric effects, or modifies at least one activity associated with a SK channel. These compounds may selectively or predominantly block or act on one kind of SK channel or act block or act on different SK channels, such as on channels comprising SK1 (KCNN1) and/or SK2 (KCNN2) and/or SK3 (KCNN3) and/or SK4 (KCNN4) proteins.
A subject may also be one who is at risk, who has been diagnosed to be at risk, or who has fetal alcohol syndrome or damage to the brain or nervous system associated with exposure to alcohol, drugs or other agents or conditions that increase SK channel protein expression or activity. A subject also, specifically, includes a pregnant woman who carries a subject in need of treatment. A subject is preferably a human; however, the invention also includes treatment of other mammalian or animal subjects who express SK or SK-like channel proteins, including canines, felines, equines, simians and other valuable or commercially raised animals.
Advantageously, the method comprises administering tamapin or another SK channel blocker, antagonist, inhibitor or modifier to the subject, optionally, along with a carrier or excipient. Other active ingredients may be coadministered before, at the same time, or after the SK channel inhibitor. One or more SK inhibitors may be administered or a SK inhibitor that inhibits more than one type of SK channel may be selected.
The invention also involves a method for treating a learning disability associated with fetal alcohol syndrome or for treating another learning disability, neurological disease, disorder or condition, comprising administering tamapin or a tamapin analog or at least one other SK channel inhibitor to a subject in need thereof. A subject may be one having a learning disability such as cognitive dysfunction, intellectual disability, dyspraxia, or mental retardation. A subject may also have another disease, disorder or condition associated with fetal alcohol syndrome, a fetal alcohol spectrum disorder, have been exposed in utero to a drug or other toxic agent, such as one inducing or triggering the expression of at least one heat shock protein. In one embodiment the subject is a fetal subject exposed to alcohol or agent(s) or condition(s) that increase the expression or activity of SK2 (KCNN2) channel or another SK channel protein such as SK1, SK3, or SK4 in utero.
SK channel blockers, antagonists, inhibitors and modifiers include tamapin, Lei-dab7, Apamin, Scyllatoxin or analog(s) thereof. Other SK channel blockers include Dequalinium, d-Tubocurarine, UC1-1684, UCL-1848, Cyproheptadine, Fluoxetine, NS8593, Scyllatoxin (Leiurotoxin-I), Lei-Dab7, N-methyl-laudanosine, N-Me-bicuculline, Pancuronium, Atracurium, 1-ethyl-1H-benzo[d]imidazol-2(3H)-on, 6,7-dichloro-3-(hydroxyimino)indolin-2-one, N-cyclohexyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4-amine, and (R)—N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1H-benzo[d]imidazol-2-amine. Such blockers, antagonists, inhibitors or modifiers may act reversibly or irreversibly.
In one embodiment methods according to the invention will administer tamapin or a tamapin analog to a subject, such as a subject in utero or to another subject in need thereof. Tamapin or a tamapin analog may be administered to a fetus who has been exposed to alcohol, ischemia or to at least one agent or condition that increases the expression or activity of SK2 channel or another SK channel in cells of the nervous system compared to those in a normal subject not exposed to alcohol, ischemia, or said at least one agent. A therapeutic amount of tamapin or tamapin analog within a suitable therapeutic range for a particular subject may be selected by one skilled in the art. For example, a dosage sufficient to expose SK receptors in neurons or other cells of the nervous system to a concentration of 24 pM to 1 nM tamapin or tamapin analog may be administered.
In some embodiments of the invention, tamapin analogs will be administered to a subject in need thereof. Such analogs include Tamapin isotype 2, peptides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions or additions to a native tamapin sequence as well as the specific analogs described in Table 1. Preferably, a tamapin derivative or analog will retain at least one functional property of native tamapin, such as the ability to block, antagonize, inhibit, or modify a SK channel or SK channel activity, block in a reversible manner SK2 channels with selectivity for SK2 channels over SK1 channels, block SK2 channels with higher affinity than SK3 channels and SK4 channels (affinity for SK2 channel>SK3>SK1>SK4), exhibit activity that is not voltage dependent, or induce cellular uptake, inactivation, recycling or destruction of SK channels.
Examples of tamapin derivatives are peptides of not more than 50 amino acids in length that have the ability to bind to SK2-type channels and affect the ability of those channel classes to transport ions, for example, to decrease their ability to transport ions. Tamapin derivatives and analogs include those which show specific and definable binding properties to all and/or certain subclasses of SK2-type channels. Such analogs or derivatives also include peptides whose sequences can be generated by using any combination of subparts of the peptides listed below using recombinant DNA techniques or chemical synthesis (e.g., peptide synthesis).
The native tamapin peptide, tamapin isotype 2, as well as tamapin derivatives or analogs in which amino acid changes can be engineered based on combinatorial fusion/shuffling of the amino acid sequences described in Table 1. For example, a shuffled variant can comprise residues (1 to n) of a first sequence selected from Table 1 and residues n+1 to 31) of a second sequence selected from Table 1, where n is 2 to 30. Similar shuffling among 3 or more variants may also be used to derive a new variant or a longer peptide construct comprising a new variant.
The analogs described in Table 1 as well as combinatorial variants thereof may comprise additional amino acid residues or other moieties at the N or C termini, for example, sequences or moieties that improve stability of tamapin or its analog in the blood or its other pharmacokinetic properties or sequences or moieties which target or facilitate passage of tamapin or its analogs into the brain or nervous system tissues. Combinatorial fusion of these sequences may be performed suing standard molecular biology techniques or by chemical synthesis such as peptide synthesis. Further modifications to tamapin or its derivatives or analogs include addition of linker peptides, effector moieties, or other covalent modifications, such as insertion or addition of non-natural amino acids (e.g., D-amino acids, or D- or L-amino acids other than the conventional twenty amino acids), use of modified or functionalized amino acids, or replacement of amino acids in the sequence with other chemical compounds.
Another aspect of the invention involves a method for treating a neurological disease, disorder or condition, comprising administering tamapin or a tamapin analog or at least one other SK channel inhibitor to a subject in need thereof. Such a method may, but need not be, directed to a subject having or at risk of having a learning disability. For example, such a method may be practiced with a subject having Alzheimer's disease or other dementia, neurofibromatosis, Angelman syndrome or another neurological disease, disorder or condition associated with aberrant expression of SK channels. Disease, disorders or conditions associated with stress, injury, insult or ischemia may also be mentioned when associated with the over-expression or over-activity of at least one SK channel in the cells of the nervous system compared to those of a normal individual. Such channels include SK1, SK2, SK3 and SK4 type channels.
A subject who has been exposed to (or is at risk of exposure to) agent(s) or condition(s) that increase the expression or activity of SK2 channel or another SK channel in cells of the nervous system compared to those of a normal individual may be selected for treatment. A fetal subject or a subject of any age who has been exposed to alcohol, drugs, toxins, poisons, or other chemical agents that increase the expression or activity of SK2 channel or another SK channel in cells of the nervous system compared to those of a normal individual may be selected. A fetal subject or subject of any age who has been exposed to prions, viruses, bacteria, yeast, fungi or other microbes, immunogens, allergens, or autoantigens that increase the expression or activity of SK2 channel or another SK channel in cells of the nervous system compared to those of a normal individual may be selected. A subject who has undergone surgery, injury, trauma or ischemia that that increases the expression or activity of a SK2 channel or another SK channel in cells of the nervous system compared to those of a normal or control individual may be selected.
Such subjects may be administered one or more channel blockers, inhibitors or modifiers for a SK channel including those for a SK1, SK2, SK3, and/or SK4 channel, advantageously in a form that reaches a target tissue expressing SK channels. In some embodiments, tamapin, a tamapin analog or one or more SK2 channel blockers, inhibitors or modifiers will be administered. Such SK2 channel blockers, inhibitors or modifiers may be selective for, or predominantly block SK2 channels. Alternatively, they may also block other kinds of SK channels.
In embodiments of the invention, a subject may be a fetal subject or a subject of any age, such as first, second or third trimester human fetus, neonate, toddler, child, pre-teen, preadolescent, adolescent or other individual with a developing, growing, reorganizing or remodeling nervous system. Subjects having diseases, disorders or conditions that caused by, are characterized by, or otherwise associated with over-expression or over-activity of SK channel proteins or with epigenetic changes to the nervous system, including obesity, alcohol, drug or other substance abuse or addiction, cardiovascular disease, diabetes, arthritis, and autoimmune diseases may benefit from administration of a SK channel blocker, antagonist, inhibitor or modifier.
Normal subjects who are at risk of, or who expect to be exposed to, alcohol, drugs, inhalants, chemical agents, biological agents, antigens, allergens, toxins, radiation, X-ray, UV, physical agents, physical, mental or psychological stress, post-traumatic stress, athletic or occupational injury or stress, battlefield injury or stress, or other conditions that increase the expression of or activity of, a SK channel in the brain or nervous system may also benefit from administration of a SK channel blocker, antagonist, inhibitor or modifier either prophylactically (before), currently with, or after exposure to said agent or condition in order to prevent or ameliorate the effects of said exposure.
The present invention provides pharmaceutical compositions comprising at least one SK channel blocker, antagonist, inhibitor or modifier which may be admixed with other active ingredients or pharmaceutically acceptable carriers. Such channels include SK1, SK2, SK3 and/or SK4. The ingredients, formulations and forms of such compositions are selected so as to permit delivery of a SK channel blocker, antagonist, inhibitor or modifier to a target tissue, such as to neurons or cells in the nervous system, including the central nervous system, peripheral nervous system, sensory, motor, sympathetic, parasympathetic, autonomic, somatic and other divisions thereof, including the enteric nervous system. Advantageously compositions containing SK channel blocker, antagonist, inhibitor or modifier such as tamapin are formulated to permit their uptake into the blood stream and/or passage into the nervous system.
Such pharmaceutical compositions can be configured for administration to a subject by a wide variety of delivery routes including but not limited to an intravascular delivery route such as by injection or infusion, subcutaneous, intramuscular, intraperitoneal, epidural, or intrathecal delivery routes, or configured for oral, enteral, pulmonary (e.g., via inhalation), intranasal, transmucosal (e.g., by sublingual administration), transdermal or other delivery routes and/or forms of administration known in the art.
The pharmaceutical compositions may be prepared in liquid form, or may be in dried powder form, such as lyophilized form. For oral or enteral use, the pharmaceutical compositions can be configured, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs or enteral formulas.
The compositions of the invention containing at least one SK channel blocker, antagonist, inhibitor or modifier can be prepared in liquid form, or can be in dried powder, such as lyophilized form, implantable sustained release formulations are also useful, as are transdermal or transmucosal formulations. Additionally or alternatively, the invention provides compositions for use in any of the various slow or sustained release formulations or microparticle formulations known to the skilled artisan, for example, sustained release microparticle formulations, which can be administered via pulmonary, intranasal, or subcutaneous delivery routes.
Liquid pharmaceutical compositions of the invention that are sterile solutions or suspensions can be administered to a patient by injection, for example, intramuscularly, intrathecally, epidurally, intravascularly, intravenously, intrarterially, intraperitoneally or subcutaneously. Sterile solutions can also be administered by intravenous infusion. A SK channel blocker, antagonist, inhibitor or modifier can be included in a sterile solid pharmaceutical composition, such as a lyophilized powder, which can be dissolved or suspended at a convenient time before administration to a patient using sterile water, saline, buffered saline or other appropriate sterile injectable medium.
Implantable sustained release formulations containing at least one SK channel blocker, antagonist, inhibitor or modifier are also useful embodiments of the pharmaceutical compositions of the invention. For example, the pharmaceutically acceptable carrier, being a biodegradable matrix implanted within the body or under the skin of a human or non-human vertebrate, can be a hydrogel. Alternatively, it may be formed from a poly-alpha-amino acid component. Other techniques for making implants for delivery of drugs are also known and useful in accordance with the invention.
Nasal delivery forms. In accordance with the invention, intranasal delivery of a composition containing at least one SK channel blocker, antagonist, inhibitor or modifier is also useful. This mode allows passage of the at least one SK channel blocker, antagonist, inhibitor or modifier to the blood stream directly after administration to the inside of the nose, without the necessity for deposition of the product in the lung. Formulations suitable for intransal administration include those with dextran or cyclodextran, and intranasal delivery devices are known.
Oral dosage forms. An oral dosage form containing at least one SK channel blocker, antagonist, inhibitor or modifier, may be used. If necessary, the composition can be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the molecule itself, where said moiety permits inhibition of proteolysis; and uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached half-life extending moieties in this invention can also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.
In powder forms, the pharmaceutically acceptable carrier is a finely divided solid, which is in admixture with finely divided active ingredient(s), including the inventive composition. For example, in some embodiments, a powder form is useful when the pharmaceutical composition is configured as an inhalant.
Pulmonary delivery forms. Pulmonary delivery of the inventive compositions is also useful. The at least one SK channel blocker, antagonist, inhibitor or modifier is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art may be employed. All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy. A SK channel blocker, antagonist, inhibitor or modifier may be prepared in particulate form with an average particle size of less than 10 microns most preferably 0.5 to 5 microns for effective delivery to the distal lung. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation can also include a buffer and/or simple sugar for protein stabilization and regulation of osmotic pressure. The nebulizer formulation may also contain a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing at least one SK channel blocker, antagonist, inhibitor or modifier suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid can also be used as a surfactant. Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the at least one SK channel blocker, antagonist, inhibitor or modifier and can also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
Transdermal, transmucosal and buccal delivery. In some embodiments, a pharmaceutical composition comprising at least one SK channel blocker, antagonist, inhibitor or modifier is configured as a part of a pharmaceutically acceptable transdermal or transmucosal patch or a troche. Transdermal patch drug delivery systems, for example, matrix type transdermal patches, are known and useful for practicing sonic embodiments of the present pharmaceutical compositions. A variety of pharmaceutically acceptable systems for transmucosal delivery of therapeutic agents is also known in the art and is compatible with the practice of the present invention. Optionally, a transmucosal delivery system can be in the form of a bilayer tablet, in which the inner layer also contains additional binding agents, flavoring agents, or fillers. Some useful systems employ a non-ionic detergent along with a permeation enhancer. Transmucosal delivery devices may be in free form, such as a cream, gel, or ointment, or may comprise a determinate form such as a tablet, patch or troche. For example, delivery of the inventive composition can be via a transmucosal delivery system comprising a laminated composite of, for example, an adhesive layer, a backing layer, a permeable membrane defining a reservoir containing the inventive composition, a peel seal disc underlying the membrane, one or more heat seals, and a removable release liner. These examples are merely illustrative of available transmucosal drug delivery technology and are not limiting of the present invention.
Buccal delivery formulations are known in the art for use with peptides, such as the tamapin peptide. For example, known tablet or patch systems configured for drug delivery through the oral mucosa such as via sublingual mucosa, include some embodiments that comprise an inner layer containing the drug, a permeation enhancer, such as a bile salt or fusidate, and a hydrophilic polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch, gelatin, or other polymers known to be useful for this purpose. This inner layer can have one surface adapted to contact and adhere to the moist mucosal tissue of the oral cavity and can have an opposing surface adhering to an overlying non-adhesive inert layer.
The dosage regimen involved in a method for treating the diseases, disorders or conditions described herein will be determined by the attending physician, considering various factors which modify the action of drugs such as the age, condition, body weight, sex and diet of the patient, the severity of disability, disease, or condition, time of administration and other clinical factors. Dosages will depend on the nature of the at least one SK channel blocker, antagonist, inhibitor, or modifier. Representative dosages include dosages in the range of 0.001-1,000 micrograms, 0.01-100, or 0.1-50 micrograms per kilogram of body weight may be selected. Non-limiting dosage ranges for several SK channel protein blockers, antagonists, inhibitors or modifiers are: Lei-dab? (min: 3 nM and max: 1.0 μM), Apamin (min 0.1 g/kg and max 2.0 g/kg) or (min 50 nM and max 1.0 μM), Scyllatoxin (min 100 pM and max 1.0 nM), and Tamapin (min 24 pM and max 1.0 nM). Administered dosages may be selected to provide the above molar concentrations of these agents at the sites of action.
Medical devices suitable for single, metered, depot, or continuous administration of compositions according to the invention are also contemplated. A device may be designed or adapted to control, meter, measure a precise volume or amount of at least one SK channel blocker, antagonist, inhibitor or modifier. It may also be designed or adapted to administer the SK inhibitor to a subject in utero, to a subject at risk of exposure to alcohol, chemical agents or deleterious conditions, or to an injured or a subject undergoing a surgical procedure. Such devices or compositions may be included in a kit that includes packaging materials or instructions for use.
The following Examples describe certain, nonlimiting features of the invention.
Impaired Motor Skill Learning in mice exposed to EtOH in utero. Pregnant mice CD-1 mice purchased from Charles River Company and bred under a light-dark cycle at a constant temperature were injected i.p. with 2 g/kg with either PBS (control) or ethanol (EtOH) thus exposing fetal mice in utero to PBS or EtOH. Thirty days after mice were born (day P30) motor skills of the control and EtOH-treated groups were compared using Rotarod test. The treatment timeline for the mice is shown in
For assessment of motor learning behavior, mice (P30) were carried out using an accelerating Rotarod [5] apparatus (TSE Instruments) over 3 repeated trials (5 minutes per trial)/day for 2 consecutive days before testing phase. The Rotarod testing involves placing the mice on a rotating bar and determining the length of time that they can retain their balance as the rate of rotation is increased (max 80 rpm, for 5 min). On the day of testing, mice were kept in their home cages and acclimated to the testing room for at least 15 min. 2 min acclimation session at 2 rpm one time prior to the test phase was performed. Testing phase consisted of 3 trials per day separated by 30 min each for 2 consecutive days (total 6 trials). Each trail was terminated when mouse fell off or reached maximum 5 min (maximum speed). During the trials, mice that moved 180° (same direction of rotation) were gently turned around, face forward, to opposite direction of rotation. The latency to fall from a rotating rod was scored automatically with infrared sensors in a Rotamex 5 rotarod (Columbus Inst.; Columbus, Ohio).
The learning index of control and EtOH treated male and female based on the Rotarod test are shown in
While initial coordination of PBS- and EtOH-treated mice was not affected by EtOH exposure as shown by
Rotarod terminal speed was determined on trial 1 and again on trial 6. The mean Rotarod terminal speed for control mice was significantly higher than that for EtOH-treated mice as shown by the black lines in
These results show that exposure of fetal mice to EtOH in utero negatively affects learning and motor skills compared to control mice not given EtOH.
Heat Shock Reporter System for Long-term labelling of cells stressed in utero. In utero electroporation (IUE) with a reporter construct and in utero exposure of fetal CD-1 mice to PBS (control) or ethanol (EtOH) was carried out as described by the timeline in
In utero electroporation was carried out as described [1-3]. Briefly, the plasmid for Flippase-FRT based reporter of heat shock factor 1 (Hsf1) activation was co-electroporated with EGFP-f plasmid (Addgene, 2 μg/μl each).
By using this method, the cells that had robust prenatal Hsf1 activation were labeled by RFP reporter. This visualizes the neurons into the primary motor cortex at embryonic day 15.5 (E15.5) at which layer II/III neurons are generated by intraperitoneal (i.p.) injection of PBS/ethanol (2.0 mg/kg body weight) at E16.5 and E17.5.
The RFP reporter expression was induced upon Hsf1 activation in a subset of GFP-positive neurons. This alcohol regimen did not induce any obvious effects in brain structure. Around 35% of the electroporated neurons expressed in the reporter positive cells. However, the reporter expression rate and pattern varied among embryos due to the stochastic activation of Hsf1. Pups were screened using an epifluorescence stereomicroscope at postnatal days (P1) that had GFP-positive cells in their primary motor cortex. Further experiments were performed on these mice i.e., single cell sampling, electrophysiology study, immunohistochemistry (IHC) and neurons morphology at P30.
Single cell sampling. As described previously [4], the cell contents of live single neuron from brain tissue were obtained with the following modification. Under the visual guidance, frontal motor cortex layer II/III neurons that expressed the GFP|/RFP| cells and GFP|/RFP− cells in the motor cortex were targeted by a patch electrode. The patch electrode (3-3.5 MΩ) was used to collect the cell content that was mounted on a micromanipulator (Sutter MP 285) and place over the target neuron under visual guidance. Prior to entering into the artificial cerebral spinal fluid (aCSF) positive pressure in the electrode was applied so that the internal air was flowing out during the whole process. After the electrode had approached to the neuron under visual guidance, small negative pressure was applied by 1 ml syringe suction to rupture the cell membrane. To collect the neuronal contents, strong negative pressure was applied with a 1 ml syringe until the soma was completely extracted into the electrode. The complete aspiration of soma content into the patch electrode was visualized under DIC optics by focusing on various Z plane levels. The electrode was rapidly retracted from the bath and the contents expelled into a thin-walled RNA free PCR tube (corning) containing 2 μl lysis buffer (10 U/μl RNase OUT, 10% IGEPAL and 40 U/μl nuclease free water). The collected single cell contents were then immediately frozen on dry ice and stored at −80° C. until further processing for single cell RNA sequencing.
The increase in KCNN2-expressing pyramidal neurons in M1 cortex correlates with the severity of motor learning deficits in mice prenatally exposed to EtOH. In single cell RNA sequencing of reporter+ and reporter− neurons, the inventors found the specific expression of KCNN2 in reporter+ neurons. As shown by
Electrophysiology: Coronal slices containing primary motor cortex (300 μm) were prepared as described above by using a vibrating blade microtome (Leica VT 1000S) from DNA (HSE-FLP0, CAG GFP and RFP FRT, 2 μg/μl each) electroporated mice (P30) brain. The recording chamber was perfused with oxygenated (95% O2/5% CO2) aCSF at 2 ml/min at room temperature. DNA electroporated primary motor cortex neurons were visualized through an infrared charge-coupled device (CCD) camera (C2741-79; Hamamatsu Photonics, Hamamatsu, Japan). The electrodes were filled with internal solution containing (in mM) 130 K-glucose, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, 2 Na2-ATP and 0.3 Na-GTP (pH 7.3; electrode resistances: 4-6 MΩ). Cells were recorded in the whole-cell voltage or current clamp mode with a holding potential of −60 mV using a patch-clamp amplifier (700B; Molecular Devices, Sunnyvale, Calif. USA). Series resistance was compensated. The output of the amplifier was digitized using an A/D converter board (Digidata 1322; Molecular Devices) with a sampling rate of 10 kHz, and recorded on a hard disk by data acquisition software (pCLAMP10; Molecular Devices). For the mAHP, cells were held at −60 mV under current clamp mode and action potentials were evoked through injecting positive current. LTP was induced electrically under voltage clamp mode by applying single 1-sec train (100 Hz, 100 μA, Isoflexm, A.M.P.I; CPI, Carl Pisaturo Standord University). 100 nM Tamapin (Sigma-Aldrich) was dissolved in normal aCSF and perfused whenever required as shown in figure legends.
Immunohistochemistry: The brains were removed and post fixed in the same fixative (4% PFA) at 4° C. for overnight, followed by 10% and 30% sucrose in PBS for 24 h each. Thereafter, coronaUsagittal sections were prepared (60 μm) on cryostat (Leica). Free floating mouse brain sections were subjected to target retrieval solution (Dako, California) for 30 min around 100° C. and thereafter incubated for 60 min in methanol (MeOH) and hydrogen peroxide (H2O2) (4:1) solution to diminish the endogenous peroxidase. After subsequently rinsing with PBS-T (3×), nonspecific binding sites were blocked with 2% bovine serum albumin (BSA) for 30 min at room temperature (RT) and the primary antibodies {anti-goat KCNN2 (1:500, Abcam), anti-chicken CFP (1:700, Abcam) and anti-rabbit RFP (1:500, Abcam)} were applied overnight at 4° C. Three-time rinses with PBS-T before incubation with the secondary antibody (anti-goat HRP (1:500, Jackson Immunolab), anti-chicken cy2 (1:200, Jackson Immunolab) and biotinylated anti-rabbit (1:200, Jackson Immunolab)} for 3 h at RT. KCNN2 immunoreactivity was visualized by reaction with cy3: ISA (1:500) for 1 h at RT after rinsing (PBS-T, 3×). Thereafter, these section were treated with PBS: H2O2 (30:1) after rinsing (PBS-T, 3×) for 1 h at RT before A:B:C (1:1:100) incubation for 1 h at RT. The RFP staining was visualized with cy5: TSA (1:500) for 1 h at RT after rinsing (PBS-T, 3×). DAPI (1:10,000) solution was used to reveal the nuclei. The sections were analyzed using Olympus confocal microscope (Japan) equipped with Olympus digital camera. Brightness of images was adjusted using image J and Photoshop.
KCNN2 antagonist, tamapin (100 nM), affects the medium duration afterhyperpolarization (mAHP) in reporter-positive pyramidal neurons in M1.
These data suggest that administration of a KCNN2 antagonist can compensate or reverse effects caused by prenatal EtOH exposure.
KCNN2 antagonist improves motor skill learning in mice exposed to EtOH prenatally. Pregnant mice CD-1 mice were injected i.p. with 2 g/kg with either PBS (control) or ethanol (EtOH) thus exposing fetal mice in utero to PBS or EtOH. Mice were tested using the Rotarod test described above on postnatal days 30, 31, 32 and 33 days as shown by the timeline in
As shown by
These data show that treatment with the KCNN2 antagonist, tamapin, significantly reversed effects of fetal exposure to EtOH.
In the Examples above, all statistical data were presented as the mean with standard error. All data comparisons (test and control) were collected at the same time period, and statistical analysis was performed using two-way analysis of variance (ANOVA) or one-way ANOVA followed by Tukey multiple range tests across multiple means. Repetitive measure ANOVA was used to compare the multiple time points among test and control. Pearson Correlation Coefficients (PCC) within the set was also calculated using Microsoft Excel sheet. The two-tail student's test was used for pairwise comparison. A 95% confidence level was used; considered to indicate statistical significance.
The foregoing discussion discloses embodiments in accordance with the present disclosure. As will be understood by those skilled in the art, the approaches, methods, techniques, materials, devices, and so forth disclosed herein may be embodied in additional embodiments as understood by those of skill in the art, it is the intention of this application to encompass and include such variation. Accordingly, this disclosure is illustrative and should not be taken as limiting the scope of the following claims.
This application claims benefit of U.S. Provisional Application No. 62/240,561, filed Oct. 13, 2015 which is hereby incorporated by reference in its entirety.
This invention was made with government support under National Institute of Health grant R00 AA018387/AA/NIAAA. The government may have certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/56835 | 10/13/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62240561 | Oct 2015 | US |
