RESUMO
Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Piperidinas/farmacologia , Quinolinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Xenoenxertos , Humanos , Hidroxiquinolinas/farmacologia , MAP Quinase Quinase 4/metabolismo , Camundongos , Transplante de Neoplasias , Pinocitose/efeitos dos fármacos , Vacúolos/metabolismo , Peixe-ZebraRESUMO
Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.
Assuntos
Melanócitos/citologia , Células de Schwann/citologia , Pele/inervação , Animais , Diferenciação Celular , Movimento Celular , Proteínas de Homeodomínio , Camundongos , Neuroglia , Receptor ErbB-3/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismoRESUMO
Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain. Stem Cells 2018;36:1736-1751.
Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Perception of the environment relies on somatosensory neurons. Mechanosensory, proprioceptor and many nociceptor subtypes of these neurons have specific mechanosensitivity profiles to adequately differentiate stimulus patterns. Nevertheless, the cellular basis of differential mechanosensation remains largely elusive. Successful transduction of sensory information relies on the recruitment of sensory neurons and mechanosensation occurring at their peripheral axonal endings in vivo. Conspicuously, existing in vitro models aimed to decipher molecular mechanisms of mechanosensation test single sensory neuron somata at any one time. Here, we introduce a compartmental in vitro chamber design to deliver precisely controlled mechanical stimulation of sensory axons with synchronous real-time imaging of Ca(2+) transients in neuronal somata that reliably reflect action potential firing patterns. We report of three previously not characterized types of mechanosensitive neuron subpopulations with distinct intrinsic axonal properties tuned specifically to static indentation or vibration stimuli, showing that different classes of sensory neurons are tuned to specific types of mechanical stimuli. Primary receptor currents of vibration neurons display rapidly adapting conductance reliably detected for every single stimulus during vibration and are consistently converted into action potentials. This result allows for the characterization of two critical steps of mechanosensation in vivo: primary signal detection and signal conversion into specific action potential firing patterns in axons.
Assuntos
Axônios/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Camundongos , VibraçãoRESUMO
The versatility of somatosensation arises from heterogeneous dorsal root ganglion (DRG) neurons. However, soma transcriptomes of individual human DRG (hDRG) neurons-critical in-formation to decipher their functions-are lacking due to technical difficulties. Here, we developed a novel approach to isolate individual hDRG neuron somas for deep RNA sequencing (RNA-seq). On average, >9,000 unique genes per neuron were detected, and 16 neuronal types were identified. Cross-species analyses revealed remarkable divergence among pain-sensing neurons and the existence of human-specific nociceptor types. Our deep RNA-seq dataset was especially powerful for providing insight into the molecular mechanisms underlying human somatosensation and identifying high potential novel drug targets. Our dataset also guided the selection of molecular markers to visualize different types of human afferents and the discovery of novel functional properties using single-cell in vivo electrophysiological recordings. In summary, by employing a novel soma sequencing method, we generated an unprecedented hDRG neuron atlas, providing new insights into human somatosensation, establishing a critical foundation for translational work, and clarifying human species-species properties.
RESUMO
Transduction of pain following noxious stimuli is mediated by the activation of specialized ion channels and receptors expressed by nociceptive sensory neurons. A common early nociceptive sublineage expressing the nerve growth factor receptor TrkA diversifies into peptidergic and non-peptidergic nociceptors around birth. In this process, peptidergic neurons maintain TrkA expression, while non-peptidergic neurons downregulate TrkA and upregulate the common glial-derived neurotrophic factor family ligand receptor Ret and bind the isolectin B4 (IB4). Although Ret can have profound impacts on the molecular and physiological properties of nociceptive neurons, its role is not fully understood. Here we have deleted Ret in small- and medium-size sensory neurons, bypassing the early lethality of the full Ret knockout. We identify that Ret is expressed in two distinct populations of small-medium sized non-peptidergic neurons, an IB4(+) and an IB4(-) population. In these neurons, Ret is a critical regulator of several ion channels and receptors, including Nav1.8, Nav1.9, ASIC2a, P2X3, TrpC3, TrpM8, TrpA1, delta opioid receptor, MrgD, MrgA1 and MrgB4. Ret-deficient mice fail to respond to mustard oil-induced neurogenic inflammation, have elevated basal responses and a failure to terminate injury-induced sensitization to cold stimuli, hypersensitivity to basal but not injury-induced mechanical stimuli, while heat sensation is largely intact. We propose that elevated pain responses could be contributed by GPR35, which is dysregulated in adult Ret-deficient mice. Our results show that Ret is critical for expression of several molecular substrates participating in the detection and transduction of sensory stimuli, resulting in altered physiology following Ret deficiency.
Assuntos
Nociceptores/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Comportamento Animal/fisiologia , Biomarcadores/metabolismo , Feminino , Gânglios Espinais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptores/citologia , Medição da Dor , Fenótipo , Proteínas Proto-Oncogênicas c-ret/genética , Receptor trkA/genética , Receptor trkA/metabolismo , Transdução de Sinais/fisiologia , Temperatura , Tato/fisiologia , Percepção do Tato/fisiologiaRESUMO
Distinct types of dorsal root ganglion sensory neurons may have unique contributions to chronic pain. Identification of primate sensory neuron types is critical for understanding the cellular origin and heritability of chronic pain. However, molecular insights into the primate sensory neurons are missing. Here we classify non-human primate dorsal root ganglion sensory neurons based on their transcriptome and map human pain heritability to neuronal types. First, we identified cell correlates between two major datasets for mouse sensory neuron types. Machine learning exposes an overall cross-species conservation of somatosensory neurons between primate and mouse, although with differences at individual gene level, highlighting the importance of primate data for clinical translation. We map genomic loci associated with chronic pain in human onto primate sensory neuron types to identify the cellular origin of chronic pain. Genome-wide associations for chronic pain converge on two different neuronal types distributed between pain disorders that display different genetic susceptibilities, suggesting both unique and shared mechanisms between different pain conditions.
Assuntos
Dor Crônica/genética , Dor Crônica/metabolismo , Células Receptoras Sensoriais/metabolismo , Transcriptoma , Animais , Feminino , Gânglios Espinais , Expressão Gênica , Humanos , Macaca mulatta , Masculino , Camundongos , Neurônios , PrimatasRESUMO
Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.
Assuntos
Encéfalo/enzimologia , Corpos de Inclusão/enzimologia , Corpos de Lewy/enzimologia , Degeneração Neural/enzimologia , Degeneração Neural/genética , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma/deficiência , Animais , Encéfalo/patologia , Feminino , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Corpos de Lewy/genética , Corpos de Lewy/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Degeneração Neural/patologia , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/fisiologiaRESUMO
An essential prerequisite for the survival of an organism is the ability to detect and respond to aversive stimuli. Current belief is that noxious stimuli directly activate nociceptive sensory nerve endings in the skin. We discovered a specialized cutaneous glial cell type with extensive processes forming a mesh-like network in the subepidermal border of the skin that conveys noxious thermal and mechanical sensitivity. We demonstrate a direct excitatory functional connection to sensory neurons and provide evidence of a previously unknown organ that has an essential physiological role in sensing noxious stimuli. Thus, these glial cells, which are intimately associated with unmyelinated nociceptive nerves, are inherently mechanosensitive and transmit nociceptive information to the nerve.
Assuntos
Percepção da Dor/fisiologia , Células de Schwann/fisiologia , Pele/inervação , Animais , Feminino , Masculino , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/fisiologia , Optogenética , Limiar da Dor , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/metabolismo , Termorreceptores/fisiologiaRESUMO
Growth differentiation factor-1 (GDF1), a TGF-beta superfamily member, participates in early embryo patterning. Later functions are implied by the Gdf1 expression in the peripheral and central nervous system. Such roles of the gene have been difficult to study, because Gdf1 null mice die during late embryogenesis. Here, we report the production of a mouse carrying a conditional Gdf1 allele, with exon 2 flanked by loxP sites. Crossing these mice with CaMKIIalpha-Cre mice resulted in Gdf1 ablation in the forebrain postnatally. Such mice displayed no behavioral changes or altered expression levels in a set of hippocampal genes examined. However, excision of the floxed Gdf1 exon caused increased expression of the remaining part of the bicistronic Uog1-Gdf1 transcript in the hippocampus. This indicates that the transcript level is regulated by a negative feedback-loop, sensing presence of either the protein or the mRNA region encoded by Gdf1 exon 2.
Assuntos
Alelos , Padronização Corporal/genética , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Prosencéfalo/embriologia , Animais , Cruzamentos Genéticos , Primers do DNA/genética , Componentes do Gene , Vetores Genéticos/genética , Fator 1 de Diferenciação de Crescimento , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Nociception is protective and prevents tissue damage but can also facilitate chronic pain. Whether a general principle governs these two types of pain is unknown. Here, we show that both basal mechanical and neuropathic pain are controlled by the microRNA-183 (miR-183) cluster in mice. This single cluster controls more than 80% of neuropathic pain-regulated genes and scales basal mechanical sensitivity and mechanical allodynia by regulating auxiliary voltage-gated calcium channel subunits α2δ-1 and α2δ-2. Basal sensitivity is controlled in nociceptors, and allodynia involves TrkB+ light-touch mechanoreceptors. These light-touch-sensitive neurons, which normally do not elicit pain, produce pain during neuropathy that is reversed by gabapentin. Thus, a single microRNA cluster continuously scales acute noxious mechanical sensitivity in nociceptive neurons and suppresses neuropathic pain transduction in a specific, light-touch-sensitive neuronal type recruited during mechanical allodynia.
Assuntos
Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Neuralgia/genética , Dor/genética , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Mecanorreceptores/fisiologia , Camundongos , MicroRNAs/genética , Nociceptores/fisiologiaRESUMO
Adrenaline is a fundamental circulating hormone for bodily responses to internal and external stressors. Chromaffin cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland, where they detach from the nerve and form postsynaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell type specification. Subsequently, these programs down-regulate SCP-gene and up-regulate chromaffin cell-gene networks. The AM forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development.
Assuntos
Medula Suprarrenal/embriologia , Diferenciação Celular , Células Cromafins/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Neurais/citologia , Células Neuroendócrinas/citologia , Células de Schwann/citologia , Medula Suprarrenal/citologia , Animais , Diferenciação Celular/genética , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Mutantes , Proteína Proteolipídica de Mielina/genética , Crista Neural/citologia , Nervos Periféricos/citologia , Fatores de Transcrição SOXE/genética , Nicho de Células-Tronco/genética , Transcrição GênicaRESUMO
Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.
Assuntos
Neurônios Motores/fisiologia , Músculo Liso/fisiologia , Neurônios/fisiologia , Mamilos/fisiologia , Piloereção/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Gânglios Simpáticos/fisiologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The primary sensory system requires the integrated function of multiple cell types, although its full complexity remains unclear. We used comprehensive transcriptome analysis of 622 single mouse neurons to classify them in an unbiased manner, independent of any a priori knowledge of sensory subtypes. Our results reveal eleven types: three distinct low-threshold mechanoreceptive neurons, two proprioceptive, and six principal types of thermosensitive, itch sensitive, type C low-threshold mechanosensitive and nociceptive neurons with markedly different molecular and operational properties. Confirming previously anticipated major neuronal types, our results also classify and provide markers for new, functionally distinct subtypes. For example, our results suggest that itching during inflammatory skin diseases such as atopic dermatitis is linked to a distinct itch-generating type. We demonstrate single-cell RNA-seq as an effective strategy for dissecting sensory responsive cells into distinct neuronal types. The resulting catalog illustrates the diversity of sensory types and the cellular complexity underlying somatic sensation.
Assuntos
Células Receptoras Sensoriais/química , Células Receptoras Sensoriais/classificação , Análise de Sequência de RNA/métodos , Animais , Comportamento Animal , Tamanho Celular , Feminino , Expressão Gênica/fisiologia , Inflamação/fisiopatologia , Inflamação/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prurido/fisiopatologia , Prurido/psicologiaRESUMO
Three genetic mouse models were examined to define effects of bone morphogenetic protein (BMP) signalling on gene expression in normal and injured adult brain. CaMKII-Cre eliminated the BMP receptor Acvr1 (Alk2) and the common TGFbeta superfamily signal mediator Smad4 or activated a constitutively active Acvr1 in postnatal forebrain neurons. All mutants followed mendelian ratios, with no overt phenotypic changes. In situ hybridization demonstrated normal patterns of the dendritic marker MAP2 (Mtap2) throughout cortex despite neuron-specific losses of Acvr1 or Smad4. However, strong up-regulation of Mtap2 transcript in these mice was found by quantitative RT-PCR (qRT-PCR), indicating that Mtap2 is normally suppressed by BMP. Traumatic brain injury (TBI) resulted in increases of histone-associated DNA fragments in both control and Smad4-deficient cortex. Several cell-type-specific transcripts known to be involved in injury-related responses were measured by qRT-PCR. Gfap mRNA was strongly up-regulated in controls as well as in the loss-of-BMP-signalling mutants. Notably, activated Acvr1 signalling gave significantly lower TBI-induced up-regulations of Gfap and Phox2a mRNA levels, indicating reductions in astroglial and neuronal reactions to injury. Strong impairment in injury-induced Timp1 transcript up-regulation was also seen in these mice. In contrast, osteopontin (Spp1) transcript levels in activated microglia were not reduced by Acvr1 signalling. Altogether, the data suggest that BMP signalling is dispensable in adult cortical neurons but that augmented BMP signalling affects molecular changes associated with neuronal lesions.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Lesões Encefálicas/metabolismo , Regulação da Expressão Gênica/fisiologia , Expressão Gênica/fisiologia , Transdução de Sinais/fisiologia , Receptores de Ativinas Tipo I/genética , Análise de Variância , Animais , Comportamento Animal/fisiologia , Peso Corporal/genética , Lesões Encefálicas/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Morte Celular/fisiologia , Proteínas de Fluorescência Verde/biossíntese , História Medieval , Hibridização In Situ/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína Smad4/genéticaRESUMO
Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.
Assuntos
Integrases/metabolismo , Recombinação Genética , Tirosina 3-Mono-Oxigenase/genética , Proteínas Virais/metabolismo , Regiões 3' não Traduzidas , Glândulas Suprarrenais/metabolismo , Animais , Química Encefálica/imunologia , Eletroporação , Feminino , Genes Reporter , Heterozigoto , Imuno-Histoquímica , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco , Distribuição Tecidual/genética , Transgenes , beta-Galactosidase/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.
Assuntos
Diferenciação Celular/fisiologia , Gânglios/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Crescimento Neural/metabolismo , Neurônios/enzimologia , Sistema Nervoso Periférico/enzimologia , Fatores de Transcrição , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Embrião de Galinha , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Gânglios/citologia , Gânglios/crescimento & desenvolvimento , Genes Reguladores/efeitos dos fármacos , Genes Reguladores/genética , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/enzimologia , Neuritos/ultraestrutura , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurotrofina 3/metabolismo , Neurotrofina 3/farmacologia , Células PC12 , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Ratos , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/genética , Proteínas Smad , Proteína Smad1 , Transativadores/metabolismo , Transativadores/farmacologia , Proteínas Elk-1 do Domínio etsRESUMO
We investigated the use of the mouse tyrosine hydroxylase (TH) gene to drive knock-in constructs in catecholaminergic neurons. Two targeting constructs representing truncated forms of either of the BMP receptors ALK-2 or BMPR-II preceded by an internal ribosome entry site (IRES) were introduced into the 3' untranslated region of TH. An frt-flanked neomycin-resistance (neo(r)) cassette was placed in the 3' end of the targeting constructs. Mice homozygous for the knock-in alleles showed various degrees of hypokinetic behavior, depending mainly on whether the neo(r) cassette was removed. In situ hybridization and immunohistochemistry showed that TH mRNA and protein were variously down-regulated in these mouse strains. Reduced levels of dopamine and noradrenalin were found in several brain areas. However, number and morphology of neurons in substantia nigra and their projections to striatum appeared normal in the neo(r)-positive TH hypomorphic mice as examined by markers for L-aromatic amino acid decarboxylase and the dopamine transporter. Elimination of the neo(r) cassette from the knock-in alleles partially restored TH and dopamine levels. The present neo(r)-positive TH hypomorphic mice show that nigrostriatal innervation develops independently of TH and should find use as a model for conditions of reduced catecholamine synthesis, as seen in, for example, L-dihydroxyphenylalanine-responsive dystonia/infantile parkinsonism.