RESUMEN
The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein-ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a ß-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.
Asunto(s)
Descubrimiento de Drogas , Espectroscopía de Resonancia Magnética/métodos , Receptor trkA/química , Amitriptilina/metabolismo , Sitios de Unión , Diseño de Fármacos , Factor de Crecimiento Nervioso/metabolismo , Estructura Terciaria de Proteína , Receptor trkA/metabolismo , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75(NTR), the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development.
Asunto(s)
Diferenciación Celular , Neuronas Colinérgicas/citología , Proteínas Hedgehog/metabolismo , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Transducción de Señal , Acetilcolinesterasa/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Calcio/metabolismo , Línea Celular , Linaje de la Célula , Neuronas Colinérgicas/metabolismo , Neuronas Colinérgicas/trasplante , Femenino , Humanos , Células Madre Pluripotentes/metabolismo , Ratas , Ratas Endogámicas Lew , Trasplante HeterólogoRESUMEN
Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Precursores de Proteínas/metabolismo , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Animales Recién Nacidos , Caspasa 3/metabolismo , Supervivencia Celular , Células Cultivadas , Corteza Cerebral/citología , Humanos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación/fisiología , Factor de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Fosforilación , Unión Proteica , Precursores de Proteínas/genética , Ensayo de Unión Radioligante/métodos , Ratas , Receptores de Factores de Crecimiento , Proteínas Recombinantes , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
The cholinergic neurons of the basal forebrain are amongst the first to degenerate in Alzheimer's disease. These neurons are unique in the brain, expressing the tyrosine kinase receptor TrkA, together with the common neurotrophin receptor p75NTR; both of which bind nerve growth factor. Activation of the TrkA receptor is important in the maintenance of cell viability, whereas the p75NTR receptor has been implicated in apoptosis. Mutations in the gene for presenilin 1, a multi-transmembrane aspartyl protease, are known to cause familial Alzheimer's disease. This is thought to be due to their effect on gamma-secretase-dependent processing of amyloid precursor protein and subsequent formation of amyloid. Since p75NTR was recently shown to undergo gamma-secretase regulated intramembrane proteolysis, this study examines the effect of familial Alzheimer mutations on processing of p75NTR. PC12 cells were stably transfected with familial mutations M146V, A246E and deltaE9 and wild-type presenilin 1 and were examined here for gamma-secretase-dependent proteolysis of p75NTR. Overexpression of wild-type presenilin 1 did not increase gamma-secretase-mediated cleavage of p75NTR. However, by contrast, the presence of the M146V mutation was shown to significantly increase cleavage of p75NTR compared with the other mutations. Survival of cholinergic neurons will depend on the balance between the receptors TrkA and p75NTR, and their respective signalling pathways. Thus alterations in p75NTR proteolysis may influence this equilibrium. The novel finding that a mutation may increase processing of p75NTR may have implications for the pathogenic outcome in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Presenilina-1/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Enfermedad de Alzheimer/metabolismo , Análisis de Varianza , Animales , Hidrólisis , Mutación , Células PC12 , Presenilina-1/genética , RatasRESUMEN
Amphipods of Diporeia spp. have declined considerably during the last decade in the Great Lakes. We examined the possibility that disease may be affecting these populations. A histological survey assessed the parasites in species of Diporeia within Lakes Huron and Michigan, USA, and the host response to some of them and to unknown factors. Amphipods were found to have an intranuclear inclusion body, and were hosts to a rickettsia-like organism, fungi, a haplosporidian, a microsporidian, epibiotic ciliates, a gregarine, a cestode, acanthocephalans and nodule formations. Epibiotic ciliates were most common (37% prevalence of infection), but a microsporidian (3.8%), a rickettsia-like organism (1.6%), fungi, including a yeast-like organism (1.3%), worms (1.3%), and a haplosporidian (0.7%) are likely associated with mortalities or detrimental effects on the host. The role these agents may have played in the decline of Diporeia spp. in the Great Lakes over the last decade is not clear. Interrelationships with the dynamics of various physical and biological factors such as high sedimentation, diminished food supplies, and virulent parasites could synergistically cause the decline in Diporeia spp. populations in Lakes Michigan and Huron.
Asunto(s)
Anfípodos/parasitología , Parásitos/fisiología , Parásitos/patogenicidad , Anfípodos/anatomía & histología , Análisis de Varianza , Animales , Agua Dulce , Great Lakes Region , Interacciones Huésped-Parásitos , Prevalencia , Estaciones del Año , Coloración y EtiquetadoRESUMEN
TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.