ABSTRACT
BACKGROUND: Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. METHODS: We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. RESULTS: BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+ )-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. CONCLUSION: The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. © 2019 International Parkinson and Movement Disorder Society.
Subject(s)
Dopaminergic Neurons/metabolism , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Parkinson Disease/metabolism , Substantia Nigra/metabolism , Animals , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Dopaminergic Neurons/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Mice , Oxidopamine/pharmacology , Parkinson Disease, Secondary/chemically induced , Substantia Nigra/drug effectsABSTRACT
In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue.
Subject(s)
Drug Design , Genetic Variation/genetics , Neurturin/chemistry , Neurturin/metabolism , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Amphetamine/pharmacology , Animals , CHO Cells , Cricetulus , Disease Models, Animal , Humans , Macaca fascicularis , Male , Models, Molecular , Neurturin/genetics , Oxidopamine/toxicity , Parkinson Disease/complications , Parkinson Disease/etiology , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Rats , Rats, Wistar , Stereotyped Behavior/drug effects , Sympatholytics/toxicity , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-ß signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-ß signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.
Subject(s)
Antineoplastic Agents/pharmacology , Bone Morphogenetic Protein 7/biosynthesis , Idiopathic Pulmonary Fibrosis/drug therapy , Tilorone/pharmacology , Animals , Cell Line, Tumor , Collagen Type I/biosynthesis , Collagen Type I, alpha 1 Chain , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/pathology , Inhibitor of Differentiation Proteins/metabolism , Mice , Neoplasm Proteins/metabolism , Signal Transduction/drug effectsABSTRACT
Neurotrophic factors promote survival, proliferation and differentiation of neurons inducing intracellular signaling via specific receptors. The conventional biochemical methods often fail to reveal full repertoire of neurotrophic factor-receptor interactions because of their limited sensitivity. We evaluated several approaches to study signaling of Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands and found that reporter-gene systems possess exceptionally high sensitivity and a heuristic power to identify novel biologically relevant growth factor-receptor interactions. We identified persephin, a GDNF family member, as a novel ligand for GFRalpha1/RET receptor complex. We confirmed this finding by several independent methods, including neurite outgrowth assay from the explants of sympathetic ganglia expressing Gfralpha1 and Ret mRNA but not persephin's conventional receptor GFRalpha4. As the activation of GFRalpha1/RET was shown to rescue dopaminergic neurons, our results suggest the potential of persephin for the treatment of Parkinson's disease.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Nerve Tissue Proteins/metabolism , Parkinson Disease/therapy , Signal Transduction/physiology , Animals , Biological Assay/methods , Cell Line , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Humans , Mice , Models, Molecular , Nerve Growth Factors/chemistry , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/genetics , Neurons/physiology , Protein Conformation , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Rats , Rats, WistarABSTRACT
In this study, the feasibility of direct infusion electrospray ionization microchip mass spectrometry (chip-MS) was compared to the commonly used liquid chromatography-mass spectrometry (LC-MS) in non-targeted metabolomics analysis of human foreskin fibroblasts (HFF) and human induced pluripotent stem cells (hiPSC) reprogrammed from HFF. The total number of the detected features with chip-MS and LC-MS were 619 and 1959, respectively. Approximately 25% of detected features showed statistically significant changes between the cell lines with both analytical methods. The results show that chip-MS is a rapid and simple method that allows high sample throughput from small sample volumes and can detect the main metabolites and classify cells based on their metabolic profiles. However, the selectivity of chip-MS is limited compared to LC-MS and chip-MS may suffer from ion suppression.
Subject(s)
Chromatography, High Pressure Liquid/methods , Induced Pluripotent Stem Cells/metabolism , Metabolome/physiology , Spectrometry, Mass, Electrospray Ionization/methods , Cell Line , Fibroblasts/metabolism , Humans , MetabolomicsABSTRACT
Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), which consist of GDNF, neurturin, artemin and persephin, regulate the development and maintenance of the nervous system. GDNF protects and repairs dopamine-containing neurons, which degenerate in Parkinson's disease, and motoneurons, which die in amyotrophic lateral sclerosis. GDNF and neurturin have shown promise in clinical trials of Parkinson's disease, and artemin is currently undergoing clinical trials for chronic pain treatment. However, the delivery of GFLs into the brain through invasive approaches such as neurosurgery, viral vectors or by the use of encapsulated cells is associated with multiple obstacles. The development of small molecules that specifically activate GFL receptors and that can be applied systemically would overcome most of these problems. The unique nature of the GFL receptors, recent progress in elucidation of the 3D structures of GFLs and GFL-receptor complexes and the use of high-throughput screening have resulted in the development of the first small molecules that mimic the effects of the different GFLs.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/physiology , Glial Cell Line-Derived Neurotrophic Factor/physiology , Nervous System Diseases/drug therapy , Nervous System Diseases/physiopathology , Animals , Chronic Disease , Humans , Molecular Mimicry , Nerve Growth Factors/pharmacologyABSTRACT
CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.
Subject(s)
CRISPR-Associated Protein 9/metabolism , Cellular Reprogramming/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Alu Elements/genetics , Base Sequence , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Kruppel-Like Factor 4 , Male , Nanog Homeobox Protein/metabolism , Neural Stem Cells/metabolism , Nucleotide Motifs/genetics , RNA, Guide, Kinetoplastida/metabolism , Transcription, GeneticABSTRACT
An epidemic of Zika virus (ZIKV) infection associated with congenital abnormalities such as microcephaly, is ongoing in the Americas and the Pacific. Currently there are no approved therapies to treat this emerging viral disease. Here, we tested three cell-directed broad-spectrum antiviral compounds against ZIKV replication using human retinal pigment epithelial (RPE) cells and a low-passage ZIKV strain isolated from fetal brain. We found that obatoclax, SaliPhe, and gemcitabine inhibited ZIKV infections at noncytotoxic concentrations. Moreover, all three compounds prevented production of viral RNA and proteins as well as activation of cellular caspase 8, 3 and 7. However, these compounds differentially affected ZIKV-mediated transcription, translation and posttranslational modifications of cellular factors as well as metabolic pathways indicating that these agents possess different mechanisms of action. Interestingly, combination of obatoclax and SaliPhe at nanomolar concentrations had a synergistic effect against ZIKV infection. Thus, our results provided the foundation for development of broad-spectrum cell-directed antivirals or their combinations for treatment of ZIKV and other emerging viral diseases.
Subject(s)
Amides/pharmacology , Antiviral Agents/pharmacology , Deoxycytidine/analogs & derivatives , Pyrroles/pharmacology , Salicylates/pharmacology , Signal Transduction/drug effects , Zika Virus/drug effects , Brain/cytology , Caspases/metabolism , Deoxycytidine/pharmacology , Drug Combinations , Enzyme Inhibitors/pharmacology , Fetus/anatomy & histology , Humans , Indoles , Metabolic Networks and Pathways/drug effects , RNA, Viral/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/virology , Virus Replication/drug effects , Zika Virus/isolation & purification , Zika Virus/metabolism , GemcitabineABSTRACT
Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.
ABSTRACT
The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/chemistry , Glial Cell Line-Derived Neurotrophic Factor/chemistry , Amino Acid Sequence , Animals , Cell Line , Cysteine/chemistry , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Mutation , Phosphorylation , Protein Binding , Protein Structure, TertiaryABSTRACT
Chikungunya virus (CHIKV) is an arthritogenic arbovirus of the Alphavirus genus, which has infected millions of people after its re-emergence in the last decade. In this study, a BHK cell line containing a stable CHIKV replicon with a luciferase reporter was used in a high-throughput platform to screen approximately 3000 compounds. Following initial validation, 25 compounds were chosen as primary hits for secondary validation with wild type and reporter CHIKV infection, which identified three promising compounds. Abamectin (EC50 = 1.5 µM) and ivermectin (EC50 = 0.6 µM) are fermentation products generated by a soil dwelling actinomycete, Streptomyces avermitilis, whereas berberine (EC50 = 1.8 µM) is a plant-derived isoquinoline alkaloid. They inhibited CHIKV replication in a dose-dependent manner and had broad antiviral activity against other alphaviruses--Semliki Forest virus and Sindbis virus. Abamectin and ivermectin were also active against yellow fever virus, a flavivirus. These compounds caused reduced synthesis of CHIKV genomic and antigenomic viral RNA as well as downregulation of viral protein expression. Time of addition experiments also suggested that they act on the replication phase of the viral infectious cycle.
Subject(s)
Alphavirus/drug effects , Antiviral Agents/pharmacology , Chikungunya Fever/drug therapy , Chikungunya virus/drug effects , Alphavirus/physiology , Animals , Berberine/pharmacology , Cell Line , Cell Line, Tumor , Chikungunya Fever/virology , Chikungunya virus/physiology , Cricetinae , DNA, Viral/antagonists & inhibitors , DNA, Viral/biosynthesis , DNA, Viral/drug effects , Flavivirus/drug effects , Humans , Ivermectin/analogs & derivatives , Ivermectin/pharmacology , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , RNA, Viral/drug effects , RNA, Viral/genetics , Replicon/drug effects , Viral Proteins/antagonists & inhibitors , Viral Proteins/biosynthesis , Viral Proteins/drug effects , Virus Replication/drug effects , Yellow fever virus/drug effectsABSTRACT
UNLABELLED: We present an individualized systems medicine (ISM) approach to optimize cancer drug therapies one patient at a time. ISM is based on (i) molecular profiling and ex vivo drug sensitivity and resistance testing (DSRT) of patients' cancer cells to 187 oncology drugs, (ii) clinical implementation of therapies predicted to be effective, and (iii) studying consecutive samples from the treated patients to understand the basis of resistance. Here, application of ISM to 28 samples from patients with acute myeloid leukemia (AML) uncovered five major taxonomic drug-response subtypes based on DSRT profiles, some with distinct genomic features (e.g., MLL gene fusions in subgroup IV and FLT3-ITD mutations in subgroup V). Therapy based on DSRT resulted in several clinical responses. After progression under DSRT-guided therapies, AML cells displayed significant clonal evolution and novel genomic changes potentially explaining resistance, whereas ex vivo DSRT data showed resistance to the clinically applied drugs and new vulnerabilities to previously ineffective drugs. SIGNIFICANCE: Here, we demonstrate an ISM strategy to optimize safe and effective personalized cancer therapies for individual patients as well as to understand and predict disease evolution and the next line of therapy. This approach could facilitate systematic drug repositioning of approved targeted drugs as well as help to prioritize and de-risk emerging drugs for clinical testing.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Precision Medicine/methods , Antineoplastic Agents/pharmacology , Disease Progression , Drug Repositioning , Gene Expression Profiling , Genome, Human , Humans , Signal Transduction/drug effects , Treatment OutcomeABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL-syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3-dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid-releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/metabolism , Nerve Tissue Proteins/metabolism , Neurturin/metabolism , Syndecan-3/metabolism , Animals , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Movement/drug effects , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Embryo, Mammalian/cytology , Enzyme Activation/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Heparin/metabolism , Humans , Immobilized Proteins/metabolism , Interneurons/cytology , Interneurons/drug effects , Interneurons/metabolism , Ligands , Mice , Models, Biological , Neurites/drug effects , Neurites/metabolism , Protein Binding/drug effects , Protein Transport/drug effects , Rats , Signal Transduction/drug effects , src-Family Kinases/metabolismABSTRACT
HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi(-)(/)(-) mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi(-)(/)(-) MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi(-)(/)(-) cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi(-)(/)(-) compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi(-)(/)(-) but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi(-)(/)(-) mice. By contrast, Hsepi(-)(/)(-) and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.
Subject(s)
Growth Substances/physiology , Heparitin Sulfate/physiology , Iduronic Acid/analysis , Animals , Carbohydrate Conformation , Cell Division , Cell Movement , Embryo, Mammalian , Fibroblast Growth Factor 2/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Heparitin Sulfate/biosynthesis , Heparitin Sulfate/chemistry , Heparitin Sulfate/deficiency , Mice , Mice, Knockout , Models, Molecular , Mutation , Signal TransductionABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor alpha1 (GFRalpha1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Delta38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF+/-HB-GAM and Delta38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 microg wtGDNF, 17 microg HB-GAM+10 microg wtGDNF or 10 microg Delta38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 microg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Delta38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.
Subject(s)
Dopamine/metabolism , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Neurons/metabolism , Parkinson Disease, Secondary/pathology , Parkinson Disease, Secondary/prevention & control , Substantia Nigra/pathology , Animals , Behavior, Animal/drug effects , Carrier Proteins/therapeutic use , Cytokines/therapeutic use , Disease Models, Animal , Dose-Response Relationship, Drug , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Male , Mitogen-Activated Protein Kinase Kinases/metabolism , Motor Activity/drug effects , Neurons/drug effects , Oxidopamine , Parkinson Disease, Secondary/chemically induced , Rats , Rats, Wistar , Sequence Deletion/genetics , Stereotyped Behavior/drug effects , Tissue Distribution/drug effects , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Extracellular matrix is a crucial regulator of development, plasticity and regeneration in the nervous system. We have now found that N-syndecan, the receptor for the extracellular matrix component heparin-binding growth-associated molecule, is required for survival of primary sensory neurons. We demonstrate massive cell death of cultured dorsal root ganglion (DRG) neurons from mice deficient in the N-syndecan gene as compared with wild-type controls. Importantly, this cell death could not be prevented by nerve growth factor - the neurotrophin, which activates multiple antiapoptotic cascades in DRG neurons. The survival deficiency was observed during first postnatal week. In contrast, DRG neurons from young adult N-syndecan knockout mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.
Subject(s)
Ganglia, Spinal/cytology , Neurons, Afferent/physiology , Syndecans/deficiency , Animals , Carrier Proteins/genetics , Cell Death , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cytokines/genetics , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Growth Cones/drug effects , Growth Cones/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Growth Factor/pharmacology , Neurons, Afferent/cytology , Neurons, Afferent/metabolism , Neurturin/pharmacology , Receptor, trkA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/physiology , Syndecans/geneticsABSTRACT
Dopamine is a crucial neurotransmitter responsible for functioning and maintenance of the nervous system. Dopamine has also been implicated in a number of diseases including schizophrenia, Parkinson's disease and drug addiction. Dopamine agonists are used in early Parkinson's disease treatment. Dopamine antagonists suppress schizophrenia. Therefore, molecules modulating dopamine receptors activity are vastly important for understanding the nervous system functioning and for the treatment of neurological diseases. In this study we describe novel computational models that efficiently predict binding affinity of the existing small molecule dopamine analogs to dopamine receptor. The model provides the set of molecular descriptors that can be used for the development of new small molecule dopamine agonists.
Subject(s)
Computer Simulation , Dopamine/physiology , Models, Chemical , Animals , Dopamine/chemistry , Dopamine Agents/chemistry , Dopamine Agents/pharmacokinetics , Nonlinear Dynamics , Predictive Value of Tests , Protein Binding/drug effects , Receptors, Dopamine/physiology , Reproducibility of ResultsABSTRACT
Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/chemistry , Glial Cell Line-Derived Neurotrophic Factor/chemistry , Heparin/chemistry , Animals , Binding Sites , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Humans , Ligands , MAP Kinase Signaling System , Molecular Conformation , Protein Binding , Protein Structure, Quaternary , Proto-Oncogene Proteins c-ret/chemistry , Rats , Sucrose/analogs & derivatives , Sucrose/chemistry , Thrombin/chemistryABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.