RESUMEN
It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.
Asunto(s)
Antidepresivos/farmacología , Receptor trkB/metabolismo , Animales , Antidepresivos/química , Antidepresivos/metabolismo , Sitios de Unión , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular , Colesterol/metabolismo , Embrión de Mamíferos , Fluoxetina/química , Fluoxetina/metabolismo , Fluoxetina/farmacología , Hipocampo/metabolismo , Humanos , Ratones , Modelos Animales , Simulación de Dinámica Molecular , Dominios Proteicos , Ratas , Receptor trkB/química , Corteza Visual/metabolismoRESUMEN
The netrin-1/DCC ligand/receptor pair has key roles in central nervous system (CNS) development, mediating axonal, and neuronal navigation. Although expression of netrin-1 and DCC is maintained in the adult brain, little is known about their role in mature neurons. Notably, netrin-1 is highly expressed in the adult substantia nigra, leading us to investigate a role of the netrin-1/DCC pair in adult nigral neuron fate. Here, we show that silencing netrin-1 in the adult substantia nigra of mice induces DCC cleavage and a significant loss of dopamine neurons, resulting in motor deficits. Because loss of adult dopamine neurons and motor impairments are features of Parkinson's disease (PD), we studied the potential impact of netrin-1 in different animal models of PD. We demonstrate that both overexpression of netrin-1 and brain administration of recombinant netrin-1 are neuroprotective and neurorestorative in mouse and rat models of PD. Of interest, we observed that netrin-1 levels are significantly reduced in PD patient brain samples. These results highlight the key role of netrin-1 in adult dopamine neuron fate, and the therapeutic potential of targeting netrin-1 signaling in PD.
Asunto(s)
Receptor DCC/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , Enfermedad de Parkinson/genética , Sustancia Negra/citología , Animales , Muerte Celular , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Regulación hacia Abajo , Femenino , Silenciador del Gen , Humanos , Masculino , Ratones , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Ratas , Transducción de Señal , Sustancia Negra/metabolismoRESUMEN
The hair bundle of cochlear hair cells comprises specialized microvilli, the stereocilia, which fulfil the role of mechanotransduction. Genetic defects and environmental noise challenge the maintenance of hair bundle structure, critically contributing to age-related hearing loss. Stereocilia fusion is a major component of the hair bundle pathology in mature hair cells, but its role in hearing loss and its molecular basis are poorly understood. Here, we utilized super-resolution expansion microscopy to examine the molecular anatomy of outer hair cell stereocilia fusion in mouse models of age-related hearing loss, heightened endoplasmic reticulum stress and prolonged noise exposure. Prominent stereocilia fusion in our model of heightened endoplasmic reticulum stress, Manf (Mesencephalic astrocyte-derived neurotrophic factor)-inactivated mice in a background with Cadherin 23 missense mutation, impaired mechanotransduction and calcium balance in stereocilia. This was indicated by reduced FM1-43 dye uptake through the mechanotransduction channels, reduced neuroplastin/PMCA2 expression and increased expression of the calcium buffer oncomodulin inside stereocilia. Sparse BAIAP2L2 and myosin 7a expression was retained in the fused stereocilia but mislocalized away from their functional sites at the tips. These hair bundle abnormalities preceded cell soma degeneration, suggesting a sequela from stereociliary molecular perturbations to cell death signalling. In the age-related hearing loss and noise-exposure models, stereocilia fusion was more restricted within the bundles, yet both models exhibited oncomodulin upregulation at the fusion sites, implying perturbed calcium homeostasis. We conclude that stereocilia fusion is linked with the failure to maintain cellular proteostasis and with disturbances in stereociliary calcium balance. KEY POINTS: Stereocilia fusion is a hair cell pathology causing hearing loss. Inactivation of Manf, a component of the endoplasmic reticulum proteostasis machinery, has a cell-intrinsic mode of action in triggering outer hair cell stereocilia fusion and the death of these cells. The genetic background with Cadherin 23 missense mutation contributes to the high susceptibility of outer hair cells to stereocilia fusion, evidenced in Manf-inactivated mice and in the mouse models of early-onset hearing loss and noise exposure. Endoplasmic reticulum stress feeds to outer hair cell stereocilia bundle pathology and impairs the molecular anatomy of calcium regulation. The maintenance of the outer hair cell stereocilia bundle cohesion is challenged by intrinsic and extrinsic stressors, and understanding the underlying mechanisms will probably benefit the development of interventions to promote hearing health.
Asunto(s)
Cadherinas , Células Ciliadas Auditivas Externas , Mecanotransducción Celular , Estereocilios , Animales , Estereocilios/metabolismo , Estereocilios/patología , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patología , Ratones , Cadherinas/metabolismo , Cadherinas/genética , Estrés del Retículo Endoplásmico , Ratones Endogámicos C57BL , Masculino , Calcio/metabolismo , Miosina VIIa/metabolismo , Femenino , Pérdida Auditiva/patología , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Mutación Missense , Proteínas de Unión al CalcioRESUMEN
AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación , Proteínas de Choque Térmico , Enfermedad por Cuerpos de Lewy , Respuesta de Proteína Desplegada , Regulación hacia Arriba , alfa-Sinucleína , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , alfa-Sinucleína/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Chaperón BiP del Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Choque Térmico/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Ratones , Ratas , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Endorribonucleasas/metabolismo , Endorribonucleasas/farmacología , Endorribonucleasas/uso terapéutico , Superóxido Dismutasa-1/genética , Colina O-Acetiltransferasa/metabolismo , Colina O-Acetiltransferasa/farmacología , Colina O-Acetiltransferasa/uso terapéutico , Dopamina/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Neuronas Motoras/metabolismo , Estrés del Retículo Endoplásmico , Factores de Crecimiento Nervioso/metabolismoRESUMEN
BACKGROUND: Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that protects dopamine neurons and improves motor function in animal models of Parkinson's disease (PD). OBJECTIVE: The primary objectives of this study were to assess the safety and tolerability of both CDNF and the drug delivery system (DDS) in patients with PD of moderate severity. METHODS: We assessed the safety and tolerability of monthly intraputamenal CDNF infusions in patients with PD using an investigational DDS, a bone-anchored transcutaneous port connected to four catheters. This phase 1 trial was divided into a placebo-controlled, double-blind, 6-month main study followed by an active-treatment 6-month extension. Eligible patients, aged 35 to 75 years, had moderate idiopathic PD for 5 to 15 years and Hoehn and Yahr score ≤ 3 (off state). Seventeen patients were randomized to placebo (n = 6), 0.4 mg CDNF (n = 6), or 1.2 mg CDNF (n = 5). The primary endpoints were safety and tolerability of CDNF and DDS and catheter implantation accuracy. Secondary endpoints were measures of PD symptoms, including Unified Parkinson's Disease Rating Scale, and DDS patency and port stability. Exploratory endpoints included motor symptom assessment (PKG, Global Kinetics Pty Ltd, Melbourne, Australia) and positron emission tomography using dopamine transporter radioligand [18 F]FE-PE2I. RESULTS: Drug-related adverse events were mild to moderate with no difference between placebo and treatment groups. No severe adverse events were associated with the drug, and device delivery accuracy met specification. The severe adverse events recorded were associated with the infusion procedure and did not reoccur after procedural modification. There were no significant changes between placebo and CDNF treatment groups in secondary endpoints between baseline and the end of the main and extension studies. CONCLUSIONS: Intraputamenally administered CDNF was safe and well tolerated, and possible signs of biological response to the drug were observed in individual patients. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Asunto(s)
Enfermedad de Parkinson , Animales , Enfermedad de Parkinson/tratamiento farmacológico , Dopamina , Factores de Crecimiento Nervioso/fisiología , Factores de Crecimiento Nervioso/uso terapéutico , Neuronas Dopaminérgicas , Sistemas de Liberación de Medicamentos , Método Doble CiegoRESUMEN
Midbrain dopamine neurons deteriorate in Parkinson's disease (PD) that is a progressive neurodegenerative movement disorder. No cure is available that would stop the dopaminergic decline or restore function of injured neurons in PD. Neurotrophic factors (NTFs), e.g., glial cell line-derived neurotrophic factor (GDNF) are small, secreted proteins that promote neuron survival during mammalian development and regulate adult neuronal plasticity, and they are studied as potential therapeutic agents for the treatment of neurodegenerative diseases. However, results from clinical trials of GDNF and related NTF neurturin (NRTN) in PD have been modest so far. In this review, we focus on cerebral dopamine neurotrophic factor (CDNF), an unconventional neurotrophic protein. CDNF delivered to the brain parenchyma protects and restores dopamine neurons in animal models of PD. In a recent Phase I-II clinical trial CDNF was found safe and well tolerated. CDNF deletion in mice led to age-dependent functional changes in the brain dopaminergic system and loss of enteric neurons resulting in slower gastrointestinal motility. These defects in Cdnf-/- mice intriguingly resemble deficiencies observed in early stage PD. Different from classical NTFs, CDNF can function both as an extracellular trophic factor and as an intracellular, endoplasmic reticulum (ER) luminal protein that protects neurons and other cell types against ER stress. Similarly to the homologous mesencephalic astrocyte-derived neurotrophic factor (MANF), CDNF is able to regulate ER stress-induced unfolded protein response (UPR) signaling and promote protein homeostasis in the ER. Since ER stress is thought to be one of the pathophysiological mechanisms contributing to the dopaminergic degeneration in PD, CDNF, and its small-molecule derivatives that are under development may provide useful tools for experimental medicine and future therapies for the treatment of PD and other neurodegenerative protein-misfolding diseases.
Asunto(s)
Neuronas Dopaminérgicas , Enfermedad de Parkinson , Animales , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Mamíferos/metabolismo , Ratones , Factores de Crecimiento Nervioso/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Respuesta de Proteína DesplegadaRESUMEN
Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) display cytoprotective effects in animal models of neurodegenerative diseases. These endoplasmic reticulum (ER)-resident proteins belong to the same protein family and function as ER stress regulators. The relationship between CDNF and MANF function, as well as their capability for functional compensation, is unknown. We aimed to investigate these questions by generating mice lacking both CDNF and MANF. Results showed that CDNF-deficient Manf-/- mice presented the same phenotypes of growth defect and diabetes as Manf-/- mice. In the muscle, CDNF deficiency resulted in increased activation of unfolded protein response (UPR), which was aggravated when MANF was ablated. In the brain, the combined loss of CDNF and MANF did not exacerbate UPR activation caused by the loss of MANF alone. Consequently, CDNF and MANF deficiency in the brain did not cause degeneration of dopamine neurons. In conclusion, CDNF and MANF present functional redundancy in the muscle, but not in the other tissues examined here. Thus, they regulate the UPR in a tissue-specific manner.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Estrés del Retículo Endoplásmico , Factores de Crecimiento Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Neuronas Dopaminérgicas/patología , Retículo Endoplásmico/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Respuesta de Proteína DesplegadaRESUMEN
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Retículo Endoplásmico/genética , Proteínas de Choque Térmico/genética , Factores de Crecimiento Nervioso/genética , Respuesta de Proteína Desplegada , Animales , Apoptosis/genética , Supervivencia Celular , Neuronas Dopaminérgicas/citología , Embrión de Mamíferos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones , Factores de Crecimiento Nervioso/metabolismo , Cultivo Primario de Células , Unión Proteica , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Mapeo de Interacción de Proteínas , Transducción de SeñalRESUMEN
A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.
Asunto(s)
Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Sitios de Unión , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Enfermedad de Parkinson/metabolismo , Fosforilación , Cultivo Primario de Células , Agregado de Proteínas , Unión Proteica , Conformación Proteica , RatasRESUMEN
Cerebral dopamine neurotrophic factor (CDNF) is a neurotrophic factor that has beneficial effects on dopamine neurons in both in vitro and in vivo models of Parkinson's disease (PD). CDNF was recently tested in phase I-II clinical trials for the treatment of PD, but the mechanisms underlying its neuroprotective properties are still poorly understood, although studies have suggested its role in the regulation of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR). The aim of this study was to investigate the mechanism of action of CDNF through analyzing the involvement of UPR signaling in its anti-apoptotic function. We used tunicamycin to induce ER stress in mice in vivo and used cultured primary neurons and found that CDNF expression is regulated by ER stress in vivo and that the involvement of UPR pathways is important for the neuroprotective function of CDNF. Moreover, we used AP-MS and BiFC to perform the first interactome screening for CDNF and report novel binding partners of CDNF. These findings allowed us to hypothesize that CDNF protects neurons from ER-stress-inducing agents by modulating UPR signaling towards cell survival outcomes.
Asunto(s)
Chaperonas Moleculares , Factores de Crecimiento Nervioso , Enfermedad de Parkinson , Animales , Supervivencia Celular , Neuronas Dopaminérgicas/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Ratones , Chaperonas Moleculares/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Enfermedad de Parkinson/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.
Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas Proto-Oncogénicas c-ret/agonistas , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ratones , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Fármacos Neuroprotectores/farmacologíaRESUMEN
The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 µM (compound 2) and Kd = 337 nM; IC50 = 28.9 µM (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m6A demethylase inhibitors in protecting dopamine neurons. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Neuronas Dopaminérgicas/metabolismo , Metiltransferasas/metabolismo , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/antagonistas & inhibidores , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Animales no Consanguíneos , Apoptosis , Desmetilación , Neuronas Dopaminérgicas/fisiología , Diseño de Fármacos , Metiltransferasas/fisiología , Ratones , Simulación del Acoplamiento Molecular , Cultivo Primario de Células , ARN/metabolismoRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) binds the GFRα1 receptor, and the GDNF-GFRα1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFRα1-RET signaling complex, agents that bind and activate RET directly and independently of GFRα1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFRα1 coexpression. However, RET activation by these ligands is constrained by GFRα1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.
Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Naftalenosulfonatos/administración & dosificación , Proteínas Proto-Oncogénicas c-ret/química , Proteínas Proto-Oncogénicas c-ret/metabolismo , Retinitis Pigmentosa/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Ligandos , Ratones , Naftalenosulfonatos/farmacología , Dominios Proteicos , Proteínas Proto-Oncogénicas c-ret/agonistas , Retinitis Pigmentosa/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/administración & dosificaciónRESUMEN
Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf-/-), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf-/- mouse are much greater than in those of Cdnf+/+ mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf-/- mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf-/- male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf-/- mouse brain is altered. The deficiencies of Cdnf-/- mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.
Asunto(s)
Encéfalo/patología , Encéfalo/fisiología , Dopamina/metabolismo , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/fisiopatología , Factores de Crecimiento Nervioso/fisiología , Neuronas/patología , Neuronas/fisiología , Animales , Apoptosis , Autofagia , Femenino , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/genéticaRESUMEN
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.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Dopamina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Ratones , Oxidopamina/farmacología , Enfermedad de Parkinson Secundaria/inducido químicamente , Sustancia Negra/efectos de los fármacosRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are able to promote the survival of multiple neuronal populations in the body and, therefore, hold considerable promise for disease-modifying treatments of diseases and conditions caused by neurodegeneration. Available data reveal the potential of GFLs for the therapy of Parkinson's disease, neuropathic pain and diseases caused by retinal degeneration but, also, amyotrophic lateral sclerosis and, possibly, Alzheimer's disease. Despite promising data collected in preclinical models, clinical translation of GFLs is yet to be conducted. The main reasons for the limited success of GFLs clinical development are the poor pharmacological characteristics of GFL proteins, such as the inability of GFLs to cross tissue barriers, poor diffusion in tissues, biphasic dose-response and activation of several receptors in the organism in different cell types, along with ethical limitations on patients' selection in clinical trials. The development of small molecules selectively targeting particular GFL receptors with improved pharmacokinetic properties can overcome many of the difficulties and limitations associated with the clinical use of GFL proteins. The current review lists several strategies to target the GFL receptor complex with drug-like molecules, discusses their advantages, provides an overview of available chemical scaffolds and peptides able to activate GFL receptors and describes the effects of these molecules in cultured cells and animal models.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/efectos de los fármacos , Péptidos/farmacología , Animales , Células Cultivadas , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Ligandos , Neuralgia/metabolismo , Neuritas/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1005710.].
RESUMEN
Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinson's disease. Because of its well established dopaminotrophic effects, the therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been studied extensively in various disorders with disturbed dopamine homeostasis. However, the outcomes from preclinical and clinical studies vary, highlighting a need for a better understanding of the physiological role of GDNF on striatal dopaminergic function. Nevertheless, the current lack of appropriate animal models has limited this understanding. Therefore, we have generated novel mouse models to study conditional Gdnf deletion in the CNS during embryonic development and reduction of striatal GDNF levels in adult mice via AAV-Cre delivery. We found that both of these mice have reduced amphetamine-induced locomotor response and striatal dopamine efflux. Embryonic GDNF deletion in the CNS did not affect striatal dopamine levels or dopamine release, but dopamine reuptake was increased due to increased levels of both total and synaptic membrane-associated dopamine transporters. Collectively, these results suggest that endogenous GDNF plays an important role in regulating the function of dopamine transporters in the striatum.SIGNIFICANCE STATEMENT Delivery of ectopic glial cell line-derived neurotrophic factor (GDNF) promotes the function, plasticity, and survival of midbrain dopaminergic neurons, the dysfunction of which contributes to various neurological and psychiatric diseases. However, how the deletion or reduction of GDNF in the CNS affects the function of dopaminergic neurons has remained unknown. Using conditional Gdnf knock-out mice, we found that endogenous GDNF affects striatal dopamine homeostasis and regulates amphetamine-induced behaviors by regulating the level and function of dopamine transporters. These data regarding the physiological role of GDNF are relevant in the context of neurological and neurodegenerative diseases that involve changes in dopamine transporter function.
Asunto(s)
Anfetamina/farmacología , Encéfalo/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial/deficiencia , Animales , Encéfalo/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución AleatoriaRESUMEN
AIMS/HYPOTHESIS: There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress. METHODS: Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-ßH1. RESULTS: There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p < 0.05). MANF knockdown in EndoC-ßH1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-κB signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-ß signalling was inhibited (p < 0.01). CONCLUSIONS/INTERPRETATION: Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.