Patent Application
20220096515
The present disclosure relates to methods of treatment and pharmaceutical formulations to treating and/or prevent psychiatric disorders in utero.
The global burden of mental health disorders and their consequences have been steadily increasing. While some studies have established an autonomous link between blood vessels and the developmental roots of psychiatric disease, no treatment has been identified to prevent the onset of psychiatric symptoms and/or diseases. (See e.g., S. Li, et al., Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior. Cell research 28, 221 (February, 2018); C. Won, et al., Autonomous vascular networks synchronize GABA neuron migration in the embryonic forebrain. Nat Commun 4, 2149 (2013); S. Li, K. et al. Endothelial VEGF sculpts cortical cytoarchitecture. The Journal of neuroscience: the official journal of the Society for Neuroscience 33, 14809 (Sep. 11, 2013); A. Vasudevan, et al., Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain. Nat Neurosci 11, 429 (April, 2008); and Y. K. Choi and A. Vasudevan, Mechanistic insights into autocrine and paracrine roles of endothelial GABA signaling in the embryonic forebrain. Scientific reports 9, 16256 (Nov. 7, 2019); the disclosures of which are incorporated herein by reference in their entireties.)
By investigating the importance of novel GABA related gene expression in embryonic forebrain endothelial cells, past studies selectively modulated components of the endothelial GABA signaling pathway in vivo. This modulated approach rendered endothelial GABAA receptors dysfunctional and affected GABA release from endothelial cells. The disruption of autocrine and paracrine mechanisms of endothelial cell-mediated GABA signaling had far reaching consequences for brain development, network formation, and subsequently for postnatal behavior. These studies provided novel understanding of how endothelial cell-specific GABA and its receptors signaling shapes neurovascular interactions during embryonic development, and how alterations in this select pathway lead up to psychiatric disease. For instance, embryonic forebrain (telencephalic) angiogenesis was significantly affected and failed to provide physical and chemoattractive guidance for long-distance migration, and final distribution of GABAergic interneurons. It caused a reduction in vascular densities in the embryonic brain, that persisted in the adult brain, with morphological changes in blood vessels indicative of functional changes, accompanied by concurrent GABAergic neuronal cell deficits. This resulted in behavioral dysfunction that was characterized by impaired social recognition, reduced social interactions, communication deficits, increased anxiety and depression and resulted in a new mouse model of psychiatric disorder—the Gabrb3 endothelial cell knockout)(Gabrb3ECKO) mice. These findings are of high significance as they emphasize that the exclusive focus on neuropsychiatric illnesses from a neuronal perspective needs to be broadened to include intrinsic defects within the vasculature that may be the actual trigger for pathophysiological changes.
Currently, psychiatric diseases and/or disorders are treated upon diagnosis or onset of symptoms, which occur after development of physiologic structures, such as blood vessels. However, there is no current treatment for root causes of psychiatric diseases and/or disorders to prevent the onset of any symptoms.
This summary is meant to provide examples and is not intended to be limiting of the scope of the invention in any way. For example, any feature included in an example of this summary is not required by the claims, unless the claims explicitly recite the feature. Also, the features described can be combined in a variety of ways. Various features and steps as described elsewhere in this disclosure can be included in the examples summarized here.
In one embodiment, method for preventing a psychiatric disorder includes providing a therapeutically effective amount of an angiogenesis pathway regulator to an individual.
In a further embodiment, the angiogenesis pathway regulator can cross a utero-placental barrier.
In another embodiment, the angiogenesis pathway regulator is selected from the group consisting of NAD+, GABA, VEGF, and FGF.
In a still further embodiment, the angiogenesis pathway regulator is NAD+.
In still another embodiment, NAD+ is administered at a dose of between 10 mg/kg to 40 mg/kg.
In a yet further embodiment, the administering step is performed orally, nasally, inhalationally, parentally, intravenously, intraperitoneally, subcutaneously, intramuscularly, intradermally, topically, rectally, intracerebrally, intraventricularly, intracerebroventricularly, intrathecally, intracisternally, intraspinally, or perispinally.
In yet another embodiment, the administering step is performed intraperitoneally.
In a further embodiment again, the individual is pregnant.
In another embodiment again, the offspring of the pregnant individual is susceptible to a psychiatric disorder.
In a further additional embodiment, the psychiatric disorder is selected from the group consisting of autism, epilepsy, schizophrenia, OCD, anxiety, and depression.
In another additional embodiment, the method further includes identifying the individual to be treated.
In a still yet further embodiment, identifying the individual to be treated includes identifying a neurological malformation in the individual.
In still yet another embodiment, the neurological malformation is identified by a CT scan or MRI.
In a still further embodiment again, the individual is identified by measuring NAD+ levels in the individual.
In still another embodiment again, a pharmaceutical formulation for the prevention of a psychiatric disorder, includes a therapeutically effective amount of an angiogenesis pathway regulator.
In a still further additional embodiment, the angiogenesis pathway regulator is selected from the group consisting of NAD+, GABA, VEGF, and FGF.
In still another additional embodiment, the angiogenesis pathway regulator can cross a utero-placental barrier.
In a yet further embodiment again, the angiogenesis pathway regulator is NAD+.
In yet another embodiment again, NAD+ is at a dose of between 10 mg to 40 mg.
In a yet further additional embodiment, NAD+ is at a dose of 10 mg in 100 μL of saline.
In yet another additional embodiment, the pharmaceutical formulation further includes at least one of the following: a buffer, a stabilizer, a balancer, a flavor, a filler, a disintegrant, a lubricant, a glidant, or a binder.
In a further additional embodiment again, the angiogenesis pathway regulator is formulated for administration orally, nasally, inhalationally, parentally, intravenously, intraperitoneally, subcutaneously, intramuscularly, intradermally, topically, rectally, intracerebrally, intraventricularly, intracerebroventricularly, intrathecally, intracisternally, intraspinally, or perispinally.
In another additional embodiment again, the angiogenesis pathway regulator is NAD+ and is formulated for intraperitoneal administration.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention.
FIGS. 5A1-5E illustrate control animals receiving NAD+ in the prenatal period also depicted the MGE specific target location in accordance with various embodiments of the invention.
Turning now to the drawings and data, embodiments of the invention are generally directed to methods of treating and/or preventing psychiatric diseases and/or disorders as well as pharmaceutical formulations configured to treat and/or prevent psychiatric diseases and/or disorders. In many embodiments, the methods and formulations use a regulator of angiogenesis pathways. In several embodiments, the regulators rescue angiogenesis and neurovascular interactions. Specifically, some embodiments rescue telencephalic angiogenesis at prenatal stages to restore downstream neurovascular interactions, normalize brain development, and ameliorate postnatal behavioral symptoms. Many embodiments use at least one of NAD+, GABA, VEGF, and FGF as the angiogenesis pathway regulator. Various embodiments treat a psychiatric disease and/or disorder selected from autism, epilepsy, schizophrenia, OCD, anxiety, and depression.
Intrinsic defects within forebrain blood vessels from the earliest developmental time points can be a major cause for the origin of psychiatric diseases. Embryonic forebrain angiogenesis precludes neuronal development and provides valuable guidance cues for neurogenesis and neuronal migration. Many embodiments rescue prenatal forebrain angiogenesis to trigger a rescue of downstream neurovascular interactions. Such rescue provides significant benefits for brain repair during this critical developmental phase.
A natural physiological molecule that can serve to improve cell proliferation and migration would be ideal for in vivo use, during this sensitive gestational time frame. Various embodiments provide the angiogenesis pathway regulator (e.g., NAD+) during a window of prenatal development that can serve to rescue angiogenesis and neurovascular interactions in the embryonic telencephalon. NAD+ is a co-enzyme found in all living cells and is able to cross the utero-placental barrier. (See e.g., G. J. Burton and A. L. Fowden, The placenta: a multifaceted, transient organ. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 370, 20140066 (Mar. 5, 2015); the disclosure of which is incorporated by reference herein in its entirety.) Additionally, NADPH oxidase in endothelial cells has been reported to generate reactive oxygen species that stimulate angiogenic factors like VEGF, with implications for postnatal angiogenesis in vivo. (See e.g., M. Ushio-Fukai, Redox signaling in angiogenesis: role of NADPH oxidase. Cardiovascular research 71, 226 (Jul. 15, 2006); the disclosure of which is incorporated by reference herein in its entirety.). Additionally, NAD+ precursors have been used in the context of aging, Alzheimer's disease, or to relieve postpartum metabolic stress. However, there are no reports of NAD+ use and impact in the prenatal developmental period. (See e.g., A. Das et al., Impairment of an Endothelial NAD(+)-H2S Signaling Network Is a Reversible Cause of Vascular Aging. Cell 173, 74 (Mar. 22, 2018); N. Braidy, R. Grant, P. S. Sachdev, Nicotinamide adenine dinucleotide and its related precursors for the treatment of Alzheimer's disease. Current opinion in psychiatry 31, 160 (March, 2018); and P. H. Ear et al., Maternal Nicotinamide Riboside Enhances Postpartum Weight Loss, Juvenile Offspring Development, and Neurogenesis of Adult Offspring. Cell reports 26, 969 (Jan. 22, 2019); the disclosures of which are incorporated herein by reference in their entireties.)
Turning to
Several embodiments are directed towards compounds and their use as therapeutics to treat and/or prevent psychiatric diseases and/or disorders in an individual. NAD+ in accordance with various embodiments has shown an ability to rescue angiogenesis and morphological malformations or defects, including congenital malformations and defects, in defective telencephalon in vitro as well as to promote GABAergic neuronal development and migration with prenatal treatment. As such, various embodiments utilize NAD+ and/or similar angiogenesis regulators to treat and/or prevent psychiatric diseases and/or disorders. Many of these embodiments use NAD+ as an angiogenesis regulator. However, additional embodiments use GABA, VEGF, and/or FGF (alone or in combination with one or more of the listed compounds).
In many embodiments, NAD+ is utilized as the compound for treatment, as some individuals cannot manufacture or synthesize NAD+ innately from food, vitamins, or other sources. As such, NAD+ precursors may not be successful to treat an individual.
Provided herein are various embodiments of pharmaceuticals for use in a treatment or preventative of psychiatric diseases and/or disorders, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other active ingredients. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. Pharmaceutical compositions may be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared utilizing the various method embodiments as described herein.
The term “active ingredient” refers to a compound, which is administered, alone or in combination with one or more pharmaceutically acceptable excipients or carriers, to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder. In various embodiments, active ingredients include one or more of NAD+, GABA, VEGF, and FGF.
The compounds disclosed herein can exist as therapeutically acceptable salts. The term “therapeutically acceptable salt,” as used herein, represents salts or zwitterionic forms of the compounds disclosed herein which are therapeutically acceptable as defined herein. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound with a suitable acid or base. Therapeutically acceptable salts include acid and basic addition salts. For a more complete discussion of the preparation and selection of salts, refer to “Handbook of Pharmaceutical Salts, Properties, and Use,” Stah and Wermuth, Ed., (Wiley-VCH and VHCA, Zurich, 2002) and Berge et al, J. Pharm. Sci. 1977, 66, 1-19.
Numerous coating agents can be used in accordance with various embodiments of the invention. In some embodiments, the coating agent is one which acts as a coating agent in conventional delayed release oral formulations, including polymers for enteric coating. Examples include hypromellose phthalate (hydroxy propyl methyl cellulose phthalate; HPMCP); hydroxypropylcellulose (HPC; such as KLUCEL®); ethylcellulose (such as ETHOCEL®); and methacrylic acid and methyl methacrylate (MAA/MMA; such as EUDRAGIT®).
Various embodiments of formulations also include at least one disintegrating agent. In some embodiments, a disintegrating agent is a super disintegrant agent. In many embodiments, disintegrants are combined with a resin. Additional disintegrating agents include, but are not limited to, agar, calcium carbonate, maize starch, potato starch, tapioca starch, alginic acid, alginates, certain silicates, and sodium carbonate. Suitable super disintegrating agents include, but are not limited to crospovidone, croscarmellose sodium, AMBERLITE (Rohm and Haas, Philadelphia, Pa.), and sodium starch glycolate.
Several embodiments of a formulation further utilize other components and excipients. For example, sweeteners, flavors, buffering agents, and flavor enhancers to make the dosage form more palatable. Sweeteners include, but are not limited to, fructose, sucrose, glucose, maltose, mannose, galactose, lactose, sucralose, saccharin, aspartame, acesulfame K, and neotame. Common flavoring agents and flavor enhancers that may be included in the formulation of the present invention include, but are not limited to, maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.
Multiple embodiments of a formulation also include a surfactant. In certain embodiments, surfactants are selected from the group consisting of Tween 80, sodium lauryl sulfate, and docusate sodium.
Various embodiments of a formulation also include a lubricant. In certain embodiments, lubricants are selected from the group consisting of magnesium stearate, stearic acid, sodium stearyl fumarate, calcium stearate, hydrogenated vegetable oil, mineral oil, polyethylene glycol, polyethylene glycol 4000-6000, talc, and glyceryl behenate.
Modes of administration, in accordance with multiple embodiments, include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intrauterine, intraperitoneal, or transmucosal (e.g., sublingual, nasal, vaginal or rectal). The actual amount of drug needed will depend on factors such as the size, age and severity of disease in the afflicted individual. The actual amount of drug needed will also depend on the effective concentration ranges of the various active ingredients. Vehicles of administration, in accordance with various embodiments, include ointments, solutions, gels, creams, suppositories, implants, tablets, or capsules, as appropriate.
In some embodiments, active ingredients are administered in a therapeutically effective amount as part of a course of treatment. As used in this context, to “treat” means to ameliorate and/or prevent at least one symptom of a disease and/or disorder to be treated or to provide a beneficial physiological effect. For example, one such amelioration of a symptom could be vascularization the telencephalon.
A therapeutically effective amount can be an amount sufficient to prevent, reduce, ameliorate, and/or or eliminate the symptoms of at least one psychiatric disease and/or disorder susceptible to such treatment.
Dosage, toxicity and therapeutic efficacy of the compounds can be determined, e.g., by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to other tissue and organs and, thereby, reduce side effects.
Data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. If the pharmaceutical is provided systemically, the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration or within the local environment to be treated in a range that includes the ED50 as determined in cell culture or animal models. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by mass spectrometry.
An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day, as determined to be beneficial. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments. For example, several divided doses may be administered daily, one dose, or cyclic administration of the compounds to achieve the desired therapeutic result.
Preservatives and other additives, like antimicrobial, antioxidant, chelating agents, and inert gases, can also be present. (See generally, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005.)
Several embodiments are directed towards treatments of individuals with compounds or derivatives thereof to treat and/or prevent psychiatric diseases and/or disorders. In some embodiments, compounds or derivatives thereof are administered to an individual having a psychiatric disease and/or disorder, while certain embodiments administer compounds or derivatives thereof to an individual susceptible to having a psychiatric disease and/or disorder. Further embodiments administer compounds or derivatives thereof to a pregnant individual, where the child is susceptible to developing a psychiatric disease and/or disorder or a neurological malformation or defect. In some embodiments, the pregnant individual possesses a psychiatric disease and/or disorder, thus making the child susceptible to developing a psychiatric disease and/or disorder. Certain embodiments treat a child in utero (e.g., a fetus), while some embodiments treat a child shortly after birth, such as a premature child.
Turning to
At 104, certain embodiments treat individuals for a structural malformation and/or at risk for a neurological and/or psychiatric disorder by providing a therapeutically effective amount of an angiogenesis regulator. As used in this context, to “treat” means to ameliorate at least one symptom of the disorder to be treated or to provide a beneficial physiological effect. For example, amelioration of a symptom could be formation or development of typical or natural neurological formations. A therapeutically effective amount can be an amount sufficient to prevent, reduce, ameliorate, or eliminate a neurologic malformation.
Various embodiments utilize NAD+ and/or similar angiogenesis regulators to treat and/or prevent neurological and/or psychiatric disorder. Many of these embodiments use NAD+ as an angiogenesis regulator. However, additional embodiments use GABA, VEGF, and/or FGF (alone or in combination with one or more of the listed compounds including NAD+). Various compounds and formulations for treatment are described elsewhere herein that can be used to treat an in individual in accordance with various embodiments.
Various embodiments provide the compound via one or more suitable methods, such as orally, nasally, inhalationally, parentally, intravenously, intraperitoneally, subcutaneously, intramuscularly, intradermally, topically, rectally, intracerebrally, intraventricularly, intracerebroventricularly, intrathecally, intracisternally, intraspinally, perispinally, transdermally, and/or combinations thereof.
In some embodiments, treatment occurs during a period commensurate with neuronal proliferation and migration, which can occur during a time period between approximately 10 weeks and approximately 25 weeks of gestation.
Certain embodiments provide the compound one time, while other compounds provide the compound periodically, such as weekly, monthly, bimonthly, once per trimester, or any other timing to effectively treat an individual. In some embodiments, treatment is performed in the first trimester, while certain embodiments perform treatment in the second trimester, and further embodiments perform treatment in the third trimester. Some embodiments treat throughout the pregnancy at regular intervals (e.g., daily, weekly, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, etc.). Some embodiments treat preterm or premature children after birth, where brain formation may still be affected by administration of an angiogenesis pathway regulator.
Certain embodiments treat an individual at a dosage of approximately 10 mg/kg—for example, for a 60-70 kg human, a dosage would be approximately 600-700 mg. Some embodiments treat at a dosage higher than 10 mg/kg, such as up to 40 mg/kg. In many embodiments excess NAD+ may be removed from the body without deleterious effects.
Returning to
Although the following embodiments provide details on certain embodiments of the inventions, it should be understood that these are only exemplary in nature, and are not intended to limit the scope of the invention.
The various examples described herein use one or more of the following procedures to generate the described results.
Animals: Timed pregnant CD1 mice were purchased from Charles River laboratories, MA. Colonies of GAD65-GFP mice were maintained in our institutional animal facility. Tie2-cre mice and Gabrb3 floxed (Gabrb3fl/fl) mice were obtained from Jackson Labs. The Tie2-cre transgene is known for uniform expression of cre-recombinase in endothelial cells during embryogenesis and adulthood. To selectively delete Gabrb3 in endothelial cells, Tie2-cre transgenic mice (males) were crossed to Gabrb3fl/fl mice (females) to generate Tie2-cre; Gabrb3fl/+ mice (males). These were further crossed with Gabrb3fl/fl mice (females) to obtain the Gabrb3 conditional knock-outs (Tie2-cre; Gabrb3fl/fl mice). The day of plug discovery was designated embryonic day 0 (E0). Animal experiments were in full compliance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the HMRI and McLean Institutional Animal Care Committees.
Histology, immunohistochemistry, and microscopic analysis: Paraffin immunohistochemistry (IHC) was performed on embryonic brains only while frozen section IHC was used on both embryonic and adult brains. Briefly, for paraffin IHC-E18 brains were fixed in zinc fixative (BD Biosciences PharMingen) for 24 h and processed for paraffin histology. Histological stainings with hematoxylin (Vector Laboratories) and eosin (Sigma) were performed on 8 μm coronal sections. Lectin histochemistry (with biotinylated isolectin B4, 1:50, Sigma) as well as IHC was performed on 20 μm sections. Primary antibodies used for IHC were as follows: anti-PHH3 (1:200, Millipore) and anti-NKX2.1, (1:50; Sigma) followed by secondary detection with AlexaFluor conjugates (Invitrogen). DAPI (Vector Laboratories) was used to label nuclei. For frozen section IHC, E18 and P90 brains were removed, fixed in 4% PFA for 24 hours, cryo-protected in sucrose gradient, embedded into OCT medium for frozen blocks; sectioned at 40 μm on a cryostat and immunostained with anti-GABA (1:80, Sigma) and anti -PROX1 (1:80, Millipore) antibodies. Twenty sections from each brain were used for IHC and histology experiments. Uniform penetration of antibodies or stains throughout the section was ascertained and quality of the staining in each digital section was examined. Only those sections which showed uniform labeling were included in further analysis. All low and high-magnification images were obtained from an FSX100 microscope (Olympus).
Morphometry: A stereological point grid was superimposed on digital images of biotinylated isolectin-B4+ vessels using ImageJ software. The ratio between points falling on blood vessels and on brain tissue was calculated for each section, and average values were obtained.
Cell counting: Profiles of GABA+ immunoreactive cells were counted in the prefrontal cortex (at bregma levels 1.5, 0.5 and −1.5) using stereotaxic coordinates from the atlas: The Mouse Brain in Stereotaxic Coordinates, by Paxinos and Franklin. For each area, cells in the strip of cortex from the pial surface to the white-gray matter interface were counted using ImageJ software and plotted.
Primary culture of endothelial cells and endothelial cell staining: Embryonic brains were dissected under a stereo-microscope, after the NAD+ treatment paradigm and the telencephalon was removed. Pial membranes were peeled off. Telencephalon without pial membranes from the periventricular region was pooled. Purity of endothelial cell cultures was established with endothelial cell markers and determined to be one hundred percent. Isolation and culture of endothelial cells were performed according to published methodology. Periventricular endothelial cells were prepared from CD1 (wild type), saline-treated Gabrb3fl/fl, saline-treated Gabrb3ECKO, and NAD+-treated Gabrb3ECKO embryos. Endothelial cells were labeled with anti-biotinylated isolectinB4 (1:100, Sigma), anti-GABA (1:400, Sigma), anti-DAXX (1:100, Santa Cruz Biotechnology) and anti-P2X4 (1:100, Abclonal) followed by secondary detection with AlexaFluor conjugates (Invitrogen). DAPI (Invitrogen) was used to label nuclei. Images were taken on an FSX100 microscope (Olympus). 1 million cells were examined for each immunohistochemistry condition.
Endothelial cell proliferation and long-distance cell migration assay: To test for cell proliferation, CD1 periventricular endothelial cells (1 million cells per experiment) were incubated in the presence of the mitotic marker 5-bromo-2′-deoxyuridine (0.05% BrdU) with or without muscimol, for 1 hour to examine the impact on proliferation of these cells and processed for BrdU immunohistochemistry.
In preparation for long-distance cell migration assays, square culture inserts (ibidi GmbH) were placed at one end of a 35 mm dish. Cultures of CD1 derived endothelial cells, purified from the periventricular plexus, were plated in the insert and allowed to migrate for 24 hours in endothelial cell culture medium. Endothelial cells were labeled with cell trace marker (CellLight Plasma Membrane-RFP, BacMam 2.0, Invitrogen) to visualize endothelial cell morphologies during subsequent imaging. The migration of endothelial cells from one end of the dish to the other spanning a distance of 3.5 cm was imaged and quantified.
Isolation and primary culture of neuronal cells: Primary culture of embryonic GABAergic neurons from CD1 telencephalon or after the saline or NAD+ treatment paradigms were performed using established methods. Briefly, embryonic brains were extracted under a stereo microscope and placed into cold PBS. After removal of the pial membrane, the telencephalon was dissected from each embryonic brain. The telencephalon was minced into 1-2 mm slices in cold PBS. Minced telencephalon was treated with 0.1× trypsin/EDTA at 37° C. for 5 min. Trypsin treatment was stopped by adding FBS-DMEM media and DNase I consecutively. Dissociated cells were filtered with a 40 μm cell strainer and finally filtered cells were cultured in poly-D-lysine coated culture dishes with Neurobasal medium (Life technologies) with 1× B-27 (Life technologies) and 1× Glutamax (Life technologies).
Neuronal migration assay: In preparation for cell migration assays, one well square culture inserts (ibidi GmbH) were placed in the center of a poly-Ornithine/Laminin coated 35 mm dish, to make a small rectangular patch that is labeled outside the dish. Embryonic neurons prepared from the saline and NAD+ treated groups were seeded in the inserts in Neurobasal medium, supplemented with 1× B-27 and 1× Glutamax. After the cells had attached, the inserts were removed to initiate cell migration in all 4 quadrants of the dish. After 24 hours, cells were fixed and labeled with anti-β-Tubulin antibody (1:2000, Biolegend). Neuronal migration was assessed by measuring the distance between the final positions of cells from the border of the rectangular patch was outlined on the first day, using ImageJ software.
Calcium imaging: For Ca2+ assays, periventricular endothelial cells (1 million cells per assay) were incubated with the Ca2+ indicator dye FluoForte AM according to manufacturer's instructions (Enzo Life Sciences), loaded into the chamber of an FSX 100 microscope and imaged continuously before and after αβ-meATP, Bz-ATP and 2Me-SADP application (100 μM). Fluorescence micrographs were digitalized and results were expressed as change in fluorescence over baseline fluorescence.
Gene expression profile analysis: RNA samples were prepared from saline-treated Gabrb3fl/fl, saline-treated Gabrb3ECKO and NAD+-treated Gabrb3ECKO groups from three different brain pools. Total RNA from each of the samples was extracted by using the PicoPure RNA Isolation kit (Arcturus) following the supplier's protocol. Microarray was performed with Mouse Gene 2.0 ST Array (Affymetrix) at the Boston University Microarray & Sequencing Resource, Boston, MA. Mouse Gene 2.0 ST CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package (version 1.36.1) included in the Bioconductor software suite (version 2.11). Principal component analysis (PCA) was analyzed and visualized using Transcriptome Analysis Console 4.0 (Affymetrix). Heatmap visualization and analysis were performed using Morpheus (Broad Institute, Boston, Mass., USA; software.broadinstitute.org/GENE-E/), and ranked by t-test statistics. Violin plot visualization was generated with Z-score using GraphPad Prism v8.0 (GraphPad Software, La Jolla Calif. USA). The gene ontology for gene enrichment study was performed in three GO TERM annotation categories by using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Mouse Genome Informatics (MGI) (GO TERM structure categories: cerebral cortex interneuron migration; GABAergic neuron differentiation; interneuron development; MGE derived cells).
Quantitative real-time PCR: RT was performed by using iScript Reverse Transcription Supermix kit (Bio-Rad). PCR reactions were run on a CFX96 Touch Real Time PCR (Bio-Rad) with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). Primers for qPCR (Daxx, P2x4, P2x7, P2y13, Tek, vwf, F2r, Sirt2, Nos1, Nrp1, Pax6, Gad1, Gad2, Grin2a, Trpm2, Trpc4 and Trpv6) were obtained from Thermo Fisher Scientific. The housekeeping gene Gapdh was used as a reference. The relative gene expression and subsequent fold changes among different samples were determined according to published methodology.
ELISA: Periventricular endothelial cells were prepared after the NAD+ treatment paradigm, or after the treatment with purinergic receptor agonists/antagonists, and seeded in 12 well culture plates at 0.1×106 cells/well. Supernatants from endothelial cell cultures were collected after 96 hours and stored at −80° C. for ELISA. GABA concentrations were quantitatively determined by competitive ELISA according to the manufacturers' protocol (GABA Research ELISA kits, Labor Diagnostica Nord, Germany), and absorbance was measured using a multiplate microplate fluorescence reader (Molecular Devices, CA) at 450 nm.
In vivo imaging of brain microvasculature by multiphoton laser-scanning microscopy: In vivo imaging of the brain vasculature in cranial window bearing mice was performed as described previously. Briefly, a cranial window was implanted by removing circular area of skull and dura. Then, the window was sealed with a 7 mm cover glass glued to the bone. For the measurement of red blood cell (RBC) velocity and blood vessel diameter, we used multiphoton laser-scanning microscopy (MPLSM). To avoid potential tissue/vessel alteration caused by the window implantation procedure, we performed imaging at least 10 days after cranial window implantation. For imaging, mice were anesthetized with ketamine/xylazine, then tetramethylrhodamine (TAMRA)-dextran (MW 500,000) was administrated through retro-orbital injection. Using TAMRA-dextran contrast enhanced angiography, region of interest is first identified. Since the intravenously injected dye labels only the blood plasma, RBCs appear as dark patches moving within the vessel lumen. Centerline RBC velocity was measured by repetitively scanning a line along the central axis of a single blood vessel (x-t) and enabling the tracking of the motion of these dark patches. The space-time image produced by the line-scan contained diagonal dark streaks formed by moving RBCs, with a slope that was inversely proportional to the centerline RBC velocity. This space-time image was then computationally processed using Matlab and Python to extract the gradient of each streak, corresponding to the RBC velocity. Briefly, diagonal filters of varying gradients were tested for each streak until the gradient that fit best was found. This was conducted for each streak in the space-time image, and the mean gradient was taken. For the vessel diameter, an edge filter was applied to determine the blood vessel boundaries, and the blood vessel diameter extracted at the indicated region of interest. The blood flow rate was determined through the following formula:
Where is the final flow rate, r is the vessel radius, and
is the blood flow velocity.
Behavioral Experiments: Mice were housed in our animal facility with a 12-hour light cycle with ad libitum access to food and water. Offspring stayed with their mothers until weaning at PND 21 after which males and females were separated. Before all behavioral testing, mice were acclimated to the testing room for 1 hour. Behavioral assays were performed according to established protocols referenced here: self-grooming, light-dark box, tail suspension test, Y-maze, open field locomotion activity, three chamber social interaction test and nest building with nestlets and with shredded paper. Both males and females were used for all behavioral assays. Experimenters scoring behaviors were blinded to the genotypes and treatment. Sample sizes for each assay are noted in figure legends.
Statistical analysis: For each experiment, samples were collected from either 1 or 2 embryos of the same genotype or postnatal mice from a given litter. Five to 10 litters of mice for each prenatal experiment and 3 to 10 litters of mice for each postnatal experiment were used. Thus, data from 8 to 10 individuals per prenatal and postnatal condition were used. For behavioral experiments, 8 to 16 litters of mice were used. Statistical significance of differences between groups was analyzed by either one-way ANOVA or two-tailed Student's t test (Prism; GraphPad software). Significance was reported at p<0.05.
In one experiment, periventricular endothelial cells were isolated from E15 wildtype (CD1) forebrain and tested the effect of NAD+ addition in vitro. Exogenous addition of 100 μM NAD+ was able to initiate significant endothelial cell proliferation within 2 days of culture and a 35 mm culture dish was confluent within 4-6 days (
In the Gabrb3ECKO telencephalon, labeling with multiple markers of vessel components, isolectin B4 and CD31/PECAM-1 have revealed reductions in vessel density and pattern formation from embryonic day 13 (E13) onward to E18. This vascular deficit in the embryonic telencephalon persisted in the adult cerebral cortex with larger vessel diameters likely correlating with increased perfusion and indicative of functional changes in blood flow in Gabrb3ECKO vessels. A summary of the embryonic and postnatal phenotype highlights (
Furthermore, another finding was made when the morphology of NAD+ treated Gabrb3ECKO telencephalon was examined at E18 (FIGS. 4E1-4G5). In histological stainings, we have previously reported morphological defects in the Gabrb3ECKO medial telencephalon at E18 along with marked ventricular abnormalities, reduced hippocampus and enlarged striatal compartments and these perturbations in anatomical landmarks were consistently observed in saline-treated Gabrb3ECKO telencephalon at all rostro-caudal levels (FIGS. 4F1-4F5). Of importance, NAD+ treatment during E12 to E17 significantly improved the overall morphology of the Gabrb3ECKO telencephalon (FIGS. 4G1-4G5), restored anatomical landmarks and ventricular size, and was similar to saline controls (FIGS. 4E1-4E5). Interestingly, in the H&E staining, a cluster of cells were observed in the medial ganglionic eminence (MGE) of NAD+-treated Gabrb3ECKO mice that were discernible specifically in the middle sections along the rostro-caudal axis. This seemed to indicate that the prenatal NAD+ treatment had a selective target in the MGE, that was quantified (
Encircling the lateral ventricle is a rich tube-like plexus of vessels which serves as a niche for neuronal proliferation and migration. This unique curved profile of vessels can be observed even in 20 μm thick sections from saline controls (
In another experiment, the significance of the prenatal NAD+ treatment on cellular mechanisms in the E18 Gabrb3ECKO telencephalon was evaluated with comparisons to the saline-treated groups. Expression of the homeodomain protein NKX2.1 was tested for, which is specifically expressed by MGE progenitors and is selective to cells of the ventral telencephalon (preoptic area, MGE, globus pallidus, septum and amygdala). Nkx2.1 mutants lack an MGE and have interneuron loss in the cerebral cortex. Nkx2.1 also acts as a cell fate determinant in regulating the differential migration of cortical and striatal GABAergic interneurons. We observed a significant reduction of NKX2.1 expression in the MGE of the saline-treated Gabrb3ECKO telencephalon (
To test if the prenatal NAD+ treatment was able to influence GABAergic neuronal migration, an in vitro neuronal migration assay was performed (
Next this exemplary embodiment tested whether the prenatal rescue of blood vessel densities, angiogenesis and GABAergic neuronal profiles by NAD+ will persist in the adult Gabrb3ECKO cerebral cortex (
Collectively, these results implicated a rescue of endothelial and neuronal cellular mechanisms by prenatal NAD+ treatment in the Gabrb3ECKO forebrain and raised new questions about the molecular mechanisms of NAD+ action and rescue.
In this exemplary embodiment, to gain deeper insights into NAD+ mediated specific actions in the subcortical telencephalon, a micro-dissection of the MGE and striatal tissue from telencephalic slices of saline-treated Gabrb3fl/fl mice, saline-treated Gabrb3ECKO mice, and NAD+-treated Gabrb3ECKO mice at E18 was performed. RNA was further extracted, and microarray hybridization on Mouse Gene 2.0 ST arrays (Affymetrix) was performed for Subsequent gene expression analysis (
To gain mechanistic insights into NAD+ action on endothelial cells, periventricular endothelial cells were isolated from saline-treated Gabrb3fl/fl, saline-treated Gabrb3ECKO, and NAD+-treated Gabrb3ECKO groups at the end of the treatment paradigm. These cells were tested for notable changes in gene expression, that were observed in the microarray data, by performing quantitative real-time polymerase chain reaction (qRT-PCR). It was found that the prenatal NAD+ treatment had restored the expression of several critical regulators of angiogenesis in Gabrb3ECKO endothelial cells, for instance Tek, vWF, F2r, Sirt2, Nos1 and Pax6 (
Endothelial cell derived GABA plays dual roles in the embryonic forebrain. It not only activates a positive feedback cycle in endothelial cells that stimulates angiogenesis, but also is an essential chemo-attractive and guidance cue for promotion of long-distance migration of GABAergic interneurons. Endothelial cell specific deletion of Gabrb3 significantly decreased GABA expression and secretion in embryonic periventricular endothelial cells. Since, Gad1 and Gad2 were rescued in Gabrb3ECKO endothelial cells by the prenatal NAD+ treatment (
An important aspect that influences endothelial cell proliferation is Ca2+ influx, which is important for cell cycle progression in the neocortex. GABAA receptor activation in Gabrb3fl/fl periventricular endothelial cells leads to an influx of Ca2+ that influences cell proliferation. However, in Gabrb3ECKO periventricular endothelial cells, due to the deletion of the β3 subunit, the GABAA receptors are dysfunctional. So, the autocrine feedback loop of GABA acting on GABAA receptors will not work in these Gabrb3ECKO endothelial cells to cause Ca2+ influx, even if GABA secretion is restored. In the absence of this mechanism, it was questioned whether the prenatal NAD+ treatment was able to activate alternate mechanisms to trigger Ca2+ influx in Gabrb3ECKO endothelial cells. Interestingly, the gene expression profiling analysis (
The consequences of loss of endothelial Gabrb3 in the embryonic brain persisted in the adult brain, reflecting as reduced vascular densities and functional changes in blood vessels as well as a reduction of cortical interneurons. This resulted in multifaceted behavioral deficits which are common to several different psychiatric diseases; with symptoms that included impaired reciprocal social interactions, communication deficits and heightened anxiety. Since the prenatal NAD+ treatment mediated rescue of cellular and molecular aspects of the Gabrb3ECKO embryonic brain (
Therefore, vessel diameters, red blood cell (RBC) velocity, and blood flow were evaluated in the cerebral cortex of saline-treated Gabrb3fl/fl mice, saline-treated Gabrb3ECKO mice, and NAD+-treated Gabrb3ECKO mice (
The prenatal NAD+ treatment did not have any effect on litter size and pups grew normally to adulthood. Therefore, behavioral tests were performed to screen for stress, anxiety, locomotion, cognition, and sociability in saline-treated Gabrb3fl/fl mice, saline-treated Gabrb3ECKO mice and NAD+-treated Gabrb3ECKO mice. Mice from saline and NAD+-treated groups were housed individually in cages containing wood chip bedding and two nestlets (pressed cotton) (
Anxiety was next assessed with the classic light-dark transition test which triggers a struggle between the desires to explore a novel environment versus natural aversion of a brightly illuminated open space. While the saline-treated Gabrb3ECKO mice showed an aversion to brightly lit open space and preferred the dark area (
NAD+-treated Gabrb3ECKO mice also showed a significant improvement in social communication skills. In a three-chamber social communication test, saline-treated Gabrb3ECKO mice showed no preference for a stranger mouse and spent an approximately similar time in investigating the stranger mouse versus an inanimate object signifying impaired sociability. In contrast, NAD+-treated Gabrb3ECKO mice interacted with the stranger mouse for a significantly longer duration than with the inanimate object, similar to saline controls (
Effects of NAD+ addition to periventricular endothelial cells isolated from E15 wildtype (CD1) forebrain was more robust than GABA. (
Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.
Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the components or steps of the present invention may be made within the spirit and scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described herein, but, rather, is defined by the scope of the appended claims.
The current application claims priority to U.S. Provisional Patent Application No. 63/084,230, filed Sep. 28, 2020; the disclosure of which is hereby incorporated by reference in its entirety.
This invention was made in part with government support under Grant Nos. 1R01NS100808-01A1 and 1R01MH110438-01awarded by the National Institutes of Health. The government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63084230 | Sep 2020 | US |
