Patent Application
20170327789
The present invention generally relates to cell differentiation, and more particularly relates to modulation of dopamine receptors to promote neural cell differentiation and to treat neurodegenerative disease.
Neurotransmitters are endogenous chemical messengers that mediate the synaptic function of differentiated neural cells in the mature CNS. Recent studies suggest an important role of neuro chemicals, for example gamma-aminobutyric acid (GABA) and glutamate, in regulating NSC fate both in early development and in adult neurogenesis. GABA regulates adult mouse hippocampal NSCs by maintaining their quiescence through the GABAA receptor, yet can also promote embryonic NSC proliferation, suggesting context specific functions. These effects may reflect influences of local or more distant neuronal activity on the NSC niche. Consistent with this idea, dopamine afferents project to neurogenic zones and depletion of dopamine decreases the proliferation of progenitor cells in the adult subventricular zone (SVZ) through D2-like receptors. Dopamine is also present in early neuronal development in the lateral ganglionic eminence (LGE) and modulates LGE progenitor cell proliferation. Serotonin signaling similarly contributes to the SVZ NSC niche.
It would be desirable to develop a method of differentiating cells which may be useful to treat pathogenic conditions.
It has now been determined that dopamine receptor D4 (DRD4) antagonists have utility with respect to differentiation of neural stein cells.
Thus, in one aspect of the present invention, a method of promoting mammalian neural stem cell differentiation is provided comprising exposing neural stem cells to a DRD4 antagonist.
In another aspect of the invention, a method of promoting differentiation of neural stem cells into glutamatergic cells is provided comprising exposing neural stem cells to a DRD4 antagonist.
In a further aspect of the invention, a method of promoting differentiation of neural stem cells into GABAergic cells is provided comprising exposing neural stem cells to a DRD4 antagonist.
In a further aspect of the invention, a method of treating a neurodegenerative disease in a mammal is provided, comprising administering to the mammal a DRD4 antagonist.
These and other aspects of the invention are described herein by reference to the following figures.
In one aspect of the invention, a method of promoting mammalian neural stern cell differentiation is provided comprising exposing mammalian neural stern cells to a DRD4 antagonist.
The term “DRD4”, or dopamine receptor D4, is a G protein-coupled receptor. As with other dopamine receptor subtypes, the D4 receptor is activated by the neurotransmitter dopamine. The D4 receptor is D2-like in that the activated D4 receptor inhibits the enzyme adenylate cyclase, thereby reducing intracellular cAMP. The D4 receptor is encoded by the DRD4 gene (e.g.
Antagonists of the dopamine D4 receptor include compounds that inhibit or prevent the activity of the D4 receptor, for example, by inhibiting the interaction, such as binding interaction at a binding or active site, of the receptor with its endogenous ligand or substrate. Examples of dopamine D4 receptor antagonists include, but are not limited to, A-381393, L-745,870, L-750,667, L-741,742, S 18126, fananserin, clozapine, buspirone, FAUC 213, sonepiprazole, PD 168568 dihydrochloride and PNU 96415E. Preferred antagonists include antagonists which are specific for DRD4 such as L-741,742 and PNU 96415E.
The term “neuronal” or “neural” stem cell refers to self-renewing, multipotent cells that generate the main phenotype of the nervous system. Neural stem cells primarily differentiate into neurons, astrocytes, and oligodendrocytes. Neurons are further classified by the neurotransmitter with which they interact, e.g. glutamate, GABA (gamma-aminobutyric acid), dopamine, serotonin and acetylcholine.
As one of skill in the art will appreciate, dopamine D4 receptor antagonists may be formulated for use in accordance with the present invention. Thus, the selected antagonist may be formulated by combination with a pharmaceutically acceptable carrier. The expression “pharmaceutically acceptable” means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable. As one of skill in the art will appreciate, the selected carrier will vary with the administrable route used. In this regard, the selected antagonist may be administered by any suitable route. In one embodiment, the selected antagonist is formulated for administration by infusion or injection, either subcutaneously or intravenously, and thus, may accordingly be utilized in combination with a medical-grade carrier, such as an aqueous solution in sterile and pyrogen-free form, optionally buffered or made isotonic. Thus, suitable carriers include distilled water or, more desirably, a sterile carbohydrate-containing solution (e.g. sucrose or dextrose, such as a 5% dextrose solution) or a sterile saline solution comprising sodium chloride and optionally buffered. Suitable sterile saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0.45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater). Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer's solution, e.g. lactated or acetated Ringer's solution, phosphate buffered saline (PBS), TRIS (hydroxymethyl) aminomethane hydroxymethyl) aminomethane)-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS) and Gey's balanced salt solution (GBSS).
In other embodiments, the selected antagonist may be formulated for administration by routes including, but not limited to, oral, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will be combined with appropriate carriers in each case. For example, compositions for oral administration via tablet, capsule or suspension may be prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, anti-oxidants, preservatives, colouring agents and flavouring agents may also be present. Compositions for topical application may be prepared including appropriate carriers. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents may be added to the composition to prevent microbial growth over prolonged storage periods.
In the present method of utilizing a dopamine D4 receptor antagonist for neuronal stem cell differentiation, the selected antagonist is administered to mammalian neuronal stem cells, in vitro, ex vivo or in vivo, in a therapeutically effective amount to promote differentiation of the stem cells. The term “mammal” and “mammalian” is used herein to encompass human and non-human mammals, including domesticated animals such as dogs, cats, horses and the like; an undomesticated animals. The term “therapeutically effective amount” is an amount of DRD4 antagonist required to promote neuronal stem cell differentiation, while not exceeding an amount which may cause significant adverse effects to the stem cells, i.e. undesirable cell changes or effect on cell growth, or cell death. DRD4 antagonist dosages that are therapeutically effective may vary with the neuronal stem cells to be treated, and the type of differentiated cell to be achieved. In one embodiment, dosages within the range of about 0.1-100 mg/m2 is appropriate for use to promote differentiation of neuronal stein cells into glutamergic or GABAergic cells, for example, 0.5-50 mg/m2, e.g. 1-10 mg/m2.
The present method of promoting differentiation of neuronal stein cells may be therapeutically applied to treat conditions in a mammal involving a defect in a particular type of neuron. The terms “treat”, “treating” or “treatment” are used herein to refer to methods that favorably alter the target pathological condition, including those that moderate, reverse, reduce the severity of, or protect against, the progression of the target disorder. For example, use of dopamine D4 receptor antagonists to promote differentiation of glutamatergic neurons is useful to treat pathologic conditions involving these neurons, e.g. neurodegenerative disorders, including but not limited to, Parkinson's and Alzheimer's, traumatic brain injury (including mechanical head injury and surgery), ischemic or hemorrhagic stroke, seizures and metabolic or congenital disorders resulting from deficiencies in glutamatergic neurons.
In another embodiment, dopamine D4 receptor antagonists to promote differentiation of GABAergic neurons is useful to treat pathologic conditions in a mammal involving these neurons, e.g. anxiety, obsessive compulsive, and mood disorders including major depressive disorder, bipolar disorder and seasonal affective disorder, neurodegenerative disorders including Parkinson's disease (PD) and PD-related disorders, Alzheimer's disease and other dementias, Huntington's disease, motor neurone disease, spinocerebellar ataxia, autism and spinal muscular atrophy, epilepsy, seizures, traumatic brain injury, and ischemic stroke.
Embodiments of the present invention are illustrated in the following specific example which is not to be construed as limiting.
Differentiation medium. Step-I medium—Neurocult NS-A basal medium supplemented with 1×B27 and 1×N2 with basic fibroblast growth factor (FGF2-5 ng/ml) and no epidermal growth factor (EGF). Step-II medium—Neurocult NS-A basal media mixed with Neurobasal media (1:1) with N2 (¼ amount) and B27 (no EGF and FGF).
High content screen. Human fetal NSC (hf5205) cells (cells derived from fetal brain tissues approximately 10 weeks old) were seeded at 1000 cells/well in a 384 well plate coated with Poly-L-ornithine (PLO) and laminin. Neuroactive compounds from a compound library were added to each well at a concentration of 5 μM in the step-I medium (without EGF) and incubated at 37° C. and 5% CO2. At day 4 of the incubation, the medium was changed to step-II medium (without FGF) and the cells were incubated with the same test compound for another 4 days. At day 8, cells were fixed with 4% paraformaldehyde and immunostained to identify a neuronal marker (Beta III tubulin), astrocytic marker (Glial Fibrillary Acidic Protein—GFAP) and DAPI. Images were taken using Evotek Opera system. 30 images were taken from different areas of each well and the data was analyzed using Acapella Script program using average signal intensity with set cut-offs and DAPI as a reference for a cell. Bone morphogenetic protein (BMP4) and BIO (Glycogen synthase kinase-3 inhibitor) were used as positive controls for differentiation.
HfNS (hf5205) cells was seeded in 384-well black μ clear bottom coated with laminin and poly-L-ornithine (PLO) at a density of 1×103. Compound library was added at approximately 5 μM using Biomek FXP automation workstation with pin tool. Cells were incubated in Step-I medium (N2, B27, 5 ng/ml FGF) without EGF for the first four days and replaced with step-II medium (1:1 Neurobasal:Neurocult, B27, ¼ N2) without FGF for the next four days. At day 8, cells were fixed with 4% paraformaldehyde (PFA) and blocked with 5% normal goat serum (NGS) and incubated with primary antibody against anti-Beta III tubulin (1:100 MAB1637), anti-GFAP (1:1000, DAKO) and DAPI overnight at 4° C. 25 images were taken from different areas of each well by Evotek Opera system and data were analyzed using Acapella software program using average signal intensity with set cut offs using DAPI stain as reference for cells. BMP4 and BIO (GSK3b inhibitor) were used as a positive control for differentiation.
Immunocytochemistry. hfNS cells differentiated on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X100, block with 5% goat serum, and incubated with primary antibody overnight at 4° C. Antibodies included: Beta-III tubulin (1:100 MAB1637), GFAP (1:1000, DAKO), Vglut1 (1:1000, Synaptic system), GABA (1:1000, Sigma). Appropriate fluorescent-conjugated secondary antibodies were used at 1:500 for 1 h at room temperature. Coverslips were mounted with fluorescent mounting medium (DAKO) containing DAPI (1:1000) and cells were imaged using Leica microscope.
Hit validation for differentation. All retests for differentiation were done by a two step growth withdrawal method (as described in Sun et al. Mol. Cell. Neurosci. 38(2008) 245-258) with and without the test compound for 8 days. Test compounds included L-741,742 (3 μM), PNU96415E (15 μM), Ifenprodil tartrate (1.5 μM), McN A-343 (4 μM), and (−) —N-Phenylcarbamoyleseroline (6 μM). For mature neuronal marker analysis, human fetal NSC were differentiated for 3 weeks using L-741,742 (2-4 μM varying at different stages) in the two step growth withdrawal method.
Single Cell qPCR (Fluidigm Analysis). Differentiated hfNS (hf5205) at week 3 was accutased and cells were resuspended in NS medium containing 1 μg per ml of propidium iodide and filtered through a 40 μm nylon cell strainer followed by live single cell sorting into a 96 well qPCR plate on a BD cell sorter. Cells were sorted into 10 μl of preamplification mix containing 40 nM of all primers for the 48 genes, and the following components of CellsDirect One-Step qRT-PCR kit (Invitrogen) as directed in protocol: 2× reaction mix, SuperScript III RT/platinum Taq mix. After sorting, samples were reverse transcribed and preamplified for 18 cycles. Preamplified samples were diluted (2×) with TE buffer and stored at −20° C. Sample and assay primer preparation for Fluidigm Dynamic arrays was done according to the manufacturer's recommendation. Samples were mixed with 2× assay loading reagent (Fluidigm Corp), 20× EvaGreen and TaqMan gene expression master mix. The Fluidigm Dynamic arrays were primed and loaded on the IFC controller and qPCR experiments were run on a Biomark system for genetic analysis.
Fluidigm Analysis. Hf5205 cells were differentiated in the two-step growth withdrawal method, with and without L-741,742 (3.5 μM) for three weeks. The differentiated cells were harvested and filtered through a 40 uM nylon filter and live sort single cell (based on PI staining) into a 96 well qRT-PCR plate containing pre-amplification mix of 48 primers, reverse transcribed and amplified for 48 genes (as described in Pasca et al. Nature Medicine (2011) November 27; 17(12) 1657-62) in one step using a kit from Invitrogen. Samples and primer pairs were prepared for Fluidigm as per protocol. Cells were identified based on presence of RPS18 and GAPDH, Data was analyzed based on population of cells expressing particular genes using different CT value cut off.
Gene Expression Profiling. Hf5205 cells were differentiated with 3 μM of L-741,742 in a two-step growth factor withdrawal protocol. RNA was extracted using an RNAeasy kit (Qiagen) from control and treated samples at each time point 0 h (NSC), 48 h after treatment (-EGF), 9 days of treatment (-FGF) and 3 weeks of treatment. RNA extracted from the samples was hybridized on Affymetrix Human Gene 1.0 ST arrays using standard protocol (TCAG, Toronto, Ontario, Canada). RMA background correction, quantile normalization and log2 transformation were applied to the CEL files using the Bioconductor ally package (R 3.0.1, affy package version 1.38.1). Batch correction was applied using ComBat function from sva (3.6.0) and gene annotations were retrieved using hugene10sttranscriptcluster.db (8.0.1). Genes were ranked based on the average log fold change (log FC) of the L-741,742 differentiated hfNS and control differentiated hfNS at 48 h, 9 days and 3 weeks. The data were analyzed using GSEA (Subramanian et al., Proc. Natl. Acad. Sci. 2005. 102(43), 15545-15550) with parameters set to 2000 gene-set permutations and gene-sets size between 8 and 500. The gene-sets included in the GSEA analyses were obtained from KEGG, MsigDB-c2, NCI, Biocarta, IOB, Netpath, HumanCyc, Reactome and the Gene Ontology (GO) databases, updated Oct. 14, 2013 (http://baderlab.org/GeneSets). An enrichment map (version 1.2 of Enrichment Map software (Mexico et al., 2010. PLoS One 5(11), e13984) was generated for each comparison using enriched gene-sets with a False Discovery Rate <0.02% and the overlap coefficient set to 0.5.
Identification of compounds that can direct NSC fate. A 680-neuroactive compound library was interrogated in human fetal NSC for differentiation potential. The differentiation screen was conducted in a 8 day time period (4+4) to identify compounds that accelerate the differentiation mechanism compared to a default differentiation method of human fetal NSC which takes 3 weeks in a two-step growth withdrawal medium. Twenty two (22) compounds were identified that showed an increase in beta III tubulin staining indicating neuronal differentiation and 17 compounds were identified that showed GFAP staining indicating astrocytic differentiation (
DRD4 antagonists differentiate human fetal NSC into specific glutamatergic lineages. To determine if the hit compounds have the potential to specify specific neuronal lineages, the human fetal NSC (hf5205) was differentiated with the hit compounds for 3 weeks using the 2-step growth withdrawal methods. Immunocytochemistry was performed for a series of antibodies marking different classes of neurons based on their neurotransmitters including glutamatergic, dopaminergic, cholinergic and serotonergic. Interestingly, three of the compounds including L-741,742, PNU96415E and Ifenprodil tartrate showed Vglut1-positive staining indicating differentiation into glutamatergic neurons. Vglut1 is a vesicular glutamate transporter required for transport of glutamate into synaptic vesicles of glutamatergic neurons. Two of the hit compounds L-741,742 and PNU96415E are dopamine D4 receptor antagonists showing an average of 30-40% of Vglut1 positive staining compared to control (0-4.2%). Differentiation potential of L-741,742 was further validated in two other human fetal NSC lines (hf6562 and hf6539) and similar potential to promote Vglut1 positive glutamatergic neuron differentiation was found (
The differentiated cells were further tested for gene expression of different neuronal lineage markers by RT-qPCR. A series of primers were designed for markers representing both neurotransmitters and domain specific regions as shown in Table 1.
The L-741,742-differentiated cells showed 15-35 fold increase in VGLUT1 expression, 11-fold increase in NEUROG2 expression when compared to the control (
L-741,742-differentiated neurons show cortical layer V marker. L-741,742-differentiated cells show increased expression of VGLUT1 and NEUROG2. NEUROG2 expression is required for promoting excitatory neurons in the dorsal cortical region during development. To test if L-741,742 promotes or specifies specific cortical layer neurons, a series of primers were designed to mark all layers of cortex; Reelin (layer 1), Tbr1 (layer V1), Ctip2 (Layer V), Satb2 (Layer II-III) and Foxg1 (telenchepalic marker) (Table 1). The expression of these markers in L-741,742-differentiated cells was determined by RT-qPCR. An approximately 23-fold increase in CTIP2 expression and 27-fold increase in FOXG1 expression was observed in L-741,742-differentiated cells (
L-741,742-differentiated cells also showed GABA positive neurons. L-741,742-differentiated cells were also tested for GABA staining from week 1-3 and GABA positive cells were present in early in 1 week differentiated cells, showing up to 11% GABA positive neurons in 2 week differentiated cells (
Characterization of L-741,742-differentiated cells by Fluidigm analysis. To further characterize the different population of neurons in L-741,742 differentiated culture, a single cell Fluidigm array was employed that can perform multiple qRT-PCR (48 genes or 96 genes) at a single cell level as shown in
An increase in the expression of MAP2 (marking neurons), EVT1 (marking lower cortical layer V), and CAMKII, VGLUT1, VGLUT2 and VGLUT3 (marking glutamatergic neurons) in the L-741,742-differentiated cells (
Relevant portions of references referred to herein are incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2015/051186 | 11/13/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62079725 | Nov 2014 | US |
