RESUMO
BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection.
RESUMO
Medulloblastoma is one of the most common malignant brain tumors of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signaling pathway. EYA1, a haloacid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). Benzarone and benzbromarone have been identified as allosteric inhibitors of EYA proteins. Using benzarone as a point of departure, we developed a panel of 35 derivatives and tested them in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signaling. DS-1-38 inhibited SHH-MB growth in vitro and in vivo, showed excellent brain penetrance, and increased the lifespan of genetically engineered mice predisposed to fatal SHH-MB. These data suggest that EYA inhibitors represent promising therapies for pediatric SHH-MB. SIGNIFICANCE: Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.
Assuntos
Benzobromarona/análogos & derivados , Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Camundongos , Humanos , Criança , Proteínas Hedgehog , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Neoplasias Cerebelares/tratamento farmacológicoRESUMO
Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Axônios/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Degeneração Neural/patologia , Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Canais de Cálcio/metabolismo , ADP-Ribose Cíclica/antagonistas & inibidores , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Ratos Sprague-DawleyRESUMO
The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.
Assuntos
Diferenciação Celular/genética , Crista Neural/citologia , Neuroglia/metabolismo , Células de Schwann/metabolismo , Análise de Sequência de RNA , Animais , Sequência de Bases/genética , Diferenciação Celular/fisiologia , Camundongos Transgênicos , Neurônios/metabolismo , Análise de Sequência de RNA/métodosRESUMO
Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
Assuntos
Transporte Axonal , Axônios/metabolismo , Cinesinas/metabolismo , Fator de Processamento Associado a PTB/metabolismo , RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Grânulos Citoplasmáticos/metabolismo , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos , Mitocôndrias/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismoRESUMO
During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.
Assuntos
Divisão Celular/fisiologia , Cerebelo/metabolismo , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Camundongos , Camundongos Mutantes , Células-Tronco Neurais/citologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options. METHODS: Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo. RESULTS: A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitorâresistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro. CONCLUSIONS: Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Proteínas Hedgehog/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Meduloblastoma/tratamento farmacológico , Anilidas , Animais , Compostos de Bifenilo , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Meduloblastoma/metabolismo , Camundongos , Proteínas/genética , Piridinas , Proteínas Repressoras/genética , Transdução de Sinais , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismoRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP3 receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IP3R1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to the Bclw BH4 domain interact with axonal IP3R1 and prevent paclitaxel-induced degeneration, while Bcl2 and BclxL cannot do so. Together these data identify a Bclw-IP3R1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.
Assuntos
Axônios/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Paclitaxel/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/toxicidade , Axônios/efeitos dos fármacos , Axônios/patologia , Células Cultivadas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to SMO inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling. Paradoxically, we find that cilia loss protects tumor cells from susceptibility to SMO inhibitors and maintains a "persister" state that depends on continuous low output of the Hedgehog program. Persister cells can serve as a reservoir for further tumor evolution, as additional alterations synergize with cilia loss to generate aggressive recurrent tumors. Together, our findings reveal patterns of resistance and provide mechanistic insights for the role of cilia in tumor evolution and drug resistance.Significance: Using a transposon screen and clinical datasets, we identified mutations in ciliogenesis genes as a new class of resistance to SMO inhibitors. Mechanistically, cilia-mutant tumors can either grow slowly in a "persister" state or evolve and progress rapidly in an "aggressive" state. Cancer Discov; 7(12); 1436-49. ©2017 AACR.See related commentary by Goranci-Buzhala et al., p. 1374This article is highlighted in the In This Issue feature, p. 1355.
Assuntos
Cílios/genética , Proteínas Hedgehog/genética , Retroelementos/genética , Receptor Smoothened/antagonistas & inibidores , Animais , Humanos , Camundongos , Transdução de SinaisRESUMO
To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (splicing factor, poly-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts in nuclei, cytoplasm and axons to regulate functionally related mRNAs essential for axon survival. Notably, SFPQ is required for coassembly of LaminB2 (Lmnb2) and Bclw (Bcl2l2) mRNAs in RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability.
Assuntos
Axônios/fisiologia , Fator de Processamento Associado a PTB/fisiologia , RNA/metabolismo , Regulon/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Transporte Axonal/fisiologia , Sobrevivência Celular/fisiologia , Gânglios Espinais/metabolismo , Técnicas de Silenciamento de Genes , Lamina Tipo B/metabolismo , Camundongos , Camundongos Knockout , Fator de Processamento Associado a PTB/genética , Proteínas/genética , Proteínas/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.
Assuntos
Neoplasias Encefálicas/patologia , Técnicas de Cocultura/métodos , Técnicas de Cultura de Órgãos/métodos , Animais , Astrocitoma/patologia , Corantes Fluorescentes/química , Camundongos , Microscopia de Fluorescência/métodos , Microesferas , Microambiente TumoralRESUMO
Axon guidance relies on precise translation of extracellular signal gradients into local changes in cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here, we show that during embryonic development in growing axons, a low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A levels specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo, leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance.
Assuntos
Axônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Peptidilprolil Isomerase de Interação com NIMA , Proteínas do Tecido Nervoso/genética , Peptidilprolil Isomerase/genética , Fosforilação , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Sonic hedgehog (Shh) signaling is critical in development and oncogenesis, but the mechanisms regulating this pathway remain unclear. Although protein phosphorylation clearly affects Shh signaling, little is known about phosphatases governing the pathway. Here, we conducted a small hairpin RNA (shRNA) screen of the phosphatome and identified Eya1 as a positive regulator of Shh signaling. We find that the catalytically active phosphatase Eya1 cooperates with the DNA-binding protein Six1 to promote gene induction in response to Shh and that Eya1/Six1 together regulate Gli transcriptional activators. We show that Eya1, which is mutated in a human deafness disorder, branchio-oto-renal syndrome, is critical for Shh-dependent hindbrain growth and development. Moreover, Eya1 drives the growth of medulloblastoma, a Shh-dependent hindbrain tumor. Together, these results identify Eya1 and Six1 as key components of the Shh transcriptional network in normal development and in oncogenesis.
Assuntos
Carcinogênese/patologia , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Meduloblastoma/patologia , Proteínas Nucleares/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Superfície Celular/genética , Rombencéfalo/citologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinogênese/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rombencéfalo/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de ZincoRESUMO
The olfactory system relies on precise circuitry connecting olfactory sensory neurons (OSNs) and appropriate relay and processing neurons of the olfactory bulb (OB). In mammals, the exact correspondence between specific olfactory receptor types and individual glomeruli enables a spatially precise map of glomerular activation that corresponds to distinct odors. However, the mechanisms that govern the establishment and maintenance of the glomerular circuitry are largely unknown. Here we show that high levels of Sonic Hedgehog (Shh) signaling at multiple sites enable refinement and maintenance of olfactory glomerular circuitry. Mice expressing a mutant version of Shh (Shh(Ala/Ala)), with impaired binding to proteoglycan co-receptors, exhibit disproportionately small olfactory bulbs containing fewer glomeruli. Notably, in mutant animals the correspondence between individual glomeruli and specific olfactory receptors is lost, as olfactory sensory neurons expressing different olfactory receptors converge on the same glomeruli. These deficits arise at late stages in post-natal development and continue into adulthood, indicating impaired pruning of erroneous connections within the olfactory bulb. In addition, mature Shh(Ala/Ala) mice exhibit decreased proliferation in the subventricular zone (SVZ), with particular reduction in neurogenesis of calbindin-expressing periglomerular cells. Thus, Shh interactions with proteoglycan co-receptors function at multiple locations to regulate neurogenesis and precise olfactory connectivity, thereby promoting functional neuronal circuitry.
Assuntos
Proteínas Hedgehog/metabolismo , Bulbo Olfatório/crescimento & desenvolvimento , Condutos Olfatórios/crescimento & desenvolvimento , Proteoglicanas/metabolismo , Animais , Calbindinas/metabolismo , Proteínas Hedgehog/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos Transgênicos , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese/fisiologia , Neurônios/patologia , Neurônios/fisiologia , Bulbo Olfatório/patologia , Bulbo Olfatório/fisiopatologia , Proteína de Marcador Olfatório/metabolismo , Condutos Olfatórios/patologia , Condutos Olfatórios/fisiopatologia , Tamanho do Órgão , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Proteínas Hedgehog/metabolismo , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Aminopiridinas/administração & dosagem , Animais , Compostos de Bifenilo/administração & dosagem , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Glioblastoma/genética , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/administração & dosagem , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation.
Assuntos
Proliferação de Células , Cerebelo/metabolismo , Glipicanas/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Cerebelo/citologia , Chlorocebus aethiops , Regulação da Expressão Gênica/fisiologia , Glipicanas/genética , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Proteínas do Tecido Nervoso , Células-Tronco Neurais/citologiaRESUMO
Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit.
Assuntos
Transporte Axonal/fisiologia , Degeneração Neural/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Proteínas/genética , Células Receptoras Sensoriais/fisiologia , Proteína bcl-X/genética , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Transporte Axonal/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/fisiologia , Caspase 6/metabolismo , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Humanos , Masculino , Camundongos , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Gravidez , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Células Receptoras Sensoriais/citologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Proteína bcl-X/metabolismoRESUMO
Small fiber sensory neuropathy is a common disorder in which progressive degeneration of small-diameter nociceptors causes decreased sensitivity to thermal stimuli and painful sensations in the extremities. In the majority of patients, the cause of small fiber sensory neuropathy is unknown, and treatment options are limited. Here, we show that Bcl-w (Bcl-2l2) is required for the viability of small fiber nociceptive sensory neurons. Bcl-w(-/-) mice demonstrate an adult-onset progressive decline in thermosensation and a decrease in nociceptor innervation of the epidermis. This denervation occurs without cell body loss, indicating that lack of Bcl-w results in a primary axonopathy. Consistent with this phenotype, we show that Bcl-w, in contrast to the closely related Bcl-2 and Bcl-xL, is enriched in axons of sensory neurons and that Bcl-w prevents the dying back of axons. Bcl-w(-/-) sensory neurons exhibit mitochondrial abnormalities, including alterations in axonal mitochondrial size, axonal mitochondrial membrane potential, and cellular ATP levels. Collectively, these data establish bcl-w(-/-) mice as an animal model of small fiber sensory neuropathy and provide new insight regarding the role of Bcl-w and of mitochondria in preventing axonal degeneration.
Assuntos
Axônios/patologia , Epiderme/inervação , Mitocôndrias/metabolismo , Nociceptores/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Proteínas/metabolismo , Sensação Térmica/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Comportamento Animal , Western Blotting , Contagem de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Camundongos , Fibras Nervosas/patologia , Testes Neuropsicológicos , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Gravidez , Proteínas/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Limiar SensorialRESUMO
Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization.
Assuntos
Dineínas do Citoplasma/genética , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/patologia , Animais , Axônios/metabolismo , Axônios/patologia , Comportamento Animal , Dineínas do Citoplasma/metabolismo , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Junção Neuromuscular/metabolismo , FenótipoRESUMO
Survival and maturation of dorsal root ganglia sensory neurons during development depend on target-derived neurotrophins. These target-derived signals must be transmitted across long distances to alter gene expression. Here, we address the possibility that long-range retrograde signals initiated by target-derived neurotrophins activate a specialized transcriptional program. The transcription factor MEF2D is expressed in sensory neurons; we show that expression of this factor is induced in response to target-derived neurotrophins that stimulate the distal axons. We demonstrate that MEF2D regulates expression of an anti-apoptotic bcl-2 family member, bcl-w. Expression of mef2d and bcl-w is stimulated in response to activation of a Trk-dependent ERK5/MEF2 pathway, and our data indicate that this pathway promotes sensory neuron survival. We find that mef2d and bcl-w are members of a larger set of retrograde response genes, which are preferentially induced by neurotrophin stimulation of distal axons. Thus, activation of an ERK5/MEF2D transcriptional program establishes and maintains the cellular constituents of functional sensory circuits.