RESUMO
Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.
Assuntos
Proteína C9orf72 , Expansão das Repetições de DNA , Instabilidade Genômica , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Modelos Animais de Doenças , Quebras de DNA de Cadeia Dupla , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Técnicas de Introdução de Genes , Instabilidade Genômica/genética , Proteína 2 Homóloga a MutS/genéticaRESUMO
Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-ß-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.
Assuntos
Proteína Morfogenética Óssea 2/genética , Anormalidades Craniofaciais/genética , Deficiências do Desenvolvimento/genética , Nanismo/genética , Haploinsuficiência/genética , Cardiopatias Congênitas/genética , Animais , Osso e Ossos/embriologia , Criança , Pré-Escolar , Cromossomos Humanos Par 20/genética , Fissura Palatina/genética , Modelos Animais de Doenças , Feminino , Coração/embriologia , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Fator de Crescimento Transformador beta/genéticaRESUMO
Known examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells. The sex reversal is a transient trait that is not transmitted to the F1 progeny. Growth media with low osmolality and reduced sodium bicarbonate, maintained throughout the gene targeting process, enhance the yield of XY females. As a practical application of the induced sex reversal, we demonstrate the generation of homozygous mutant mice ready for phenotypic studies by the breeding of F0 XY females with their isogenic XY male clonal siblings, thereby eliminating one generation of breeding and the associated costs.
Assuntos
Transtornos do Desenvolvimento Sexual/genética , Fertilidade/genética , Disgenesia Gonadal 46 XY/genética , Processos de Determinação Sexual , Animais , Células-Tronco Embrionárias/citologia , Feminino , Marcação de Genes , Masculino , Camundongos , Microinjeções , MutaçãoRESUMO
The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.
Assuntos
Doença de Alzheimer , Tauopatias , Doença de Alzheimer/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vγ TCR+ IELs, however, the mechanisms that control γδ IEL maintenance within discrete intestinal segments are unclear. Here, we show that Btnl2 suppressed homeostatic proliferation of γδ IELs preferentially in the ileum. High throughput transcriptomic characterization of site-specific Btnl2-KO γδ IELs reveals that Btnl2 regulated the antimicrobial response module of ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ repertoire diversity of ileal γδ IELs. During DSS-induced colitis, Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ IEL frequencies and TCR specificities in response to site-specific homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.
Assuntos
Butirofilinas/genética , Íleo/imunologia , Imunidade Inata/genética , Imunidade nas Mucosas/genética , Linfócitos Intraepiteliais/metabolismo , Animais , Butirofilinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD28 , Humanos , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1RESUMO
Previously we reported the identification of a homozygous COL27A1 (c.2089G>C; p.Gly697Arg) missense variant and proposed it as a founder allele in Puerto Rico segregating with Steel syndrome (STLS, MIM #615155); a rare osteochondrodysplasia characterized by short stature, congenital bilateral hip dysplasia, carpal coalitions, and scoliosis. We now report segregation of this variant in five probands from the initial clinical report defining the syndrome and an additional family of Puerto Rican descent with multiple affected adult individuals. We modeled the orthologous variant in murine Col27a1 and found it recapitulates some of the major Steel syndrome associated skeletal features including reduced body length, scoliosis, and a more rounded skull shape. Characterization of the in vivo murine model shows abnormal collagen deposition in the extracellular matrix and disorganization of the proliferative zone of the growth plate. We report additional COL27A1 pathogenic variant alleles identified in unrelated consanguineous Turkish kindreds suggesting Clan Genomics and identity-by-descent homozygosity contributing to disease in this population. The hypothesis that carrier states for this autosomal recessive osteochondrodysplasia may contribute to common complex traits is further explored in a large clinical population cohort. Our findings augment our understanding of COL27A1 biology and its role in skeletal development; and expand the functional allelic architecture in this gene underlying both rare and common disease phenotypes.
Assuntos
Anormalidades Múltiplas/genética , Colágenos Fibrilares/genética , Efeito Fundador , Luxação do Quadril/genética , Escoliose/genética , Anormalidades Múltiplas/patologia , Adolescente , Animais , Desenvolvimento Ósseo , Criança , Pré-Escolar , Consanguinidade , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Colágenos Fibrilares/metabolismo , Frequência do Gene , Luxação do Quadril/patologia , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Linhagem , Escoliose/patologia , SíndromeRESUMO
T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.
Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária/imunologia , Macaca fascicularis , Camundongos , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pro- and mature brain-derived neurotrophic factor (BDNF) activate two distinct receptors: p75 neurotrophin receptor (p75(NTR)) and TrkB. Mature BDNF facilitates hippocampal synaptic potentiation through TrkB. Here we report that proBDNF, by activating p75(NTR), facilitates hippocampal long-term depression (LTD). Electron microscopy showed that p75(NTR) localized in dendritic spines, in addition to afferent terminals, of CA1 neurons. Deletion of p75(NTR) in mice selectively impaired the NMDA receptor-dependent LTD, without affecting other forms of synaptic plasticity. p75(NTR-/-) mice also showed a decrease in the expression of NR2B, an NMDA receptor subunit uniquely involved in LTD. Activation of p75(NTR) by proBDNF enhanced NR2B-dependent LTD and NR2B-mediated synaptic currents. These results show a crucial role for proBDNF-p75(NTR) signaling in LTD and its potential mechanism, and together with the finding that mature BDNF promotes synaptic potentiation, suggest a bidirectional regulation of synaptic plasticity by proBDNF and mature BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Precursores de Proteínas/farmacologia , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Receptor de Fator de Crescimento Neural , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Fator de Crescimento Neural/deficiência , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.
Assuntos
Autoanticorpos/biossíntese , Autoimunidade , Glomerulonefrite Membranoproliferativa/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Autoanticorpos/sangue , Proteína C9orf72 , Citocinas/sangue , Feminino , Glomerulonefrite Membranoproliferativa/sangue , Glomerulonefrite Membranoproliferativa/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Tecido Linfoide/patologia , Macrófagos/imunologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/imunologia , Análise de Sequência de RNA , TranscriptomaRESUMO
Microglia are the immunocompetent cells of the CNS, and their activation is thought to play an important neurotoxic role in many diseases modeled by glutamate-induced excitotoxicity. One molecule whose expression is upregulated after excitotoxic injury is tissue plasminogen activator (tPA), a serine protease with dual roles in the CNS. The catalytic activity of tPA, which converts plasminogen into plasmin, leads to neuronal death during excitotoxicity. Via a nonproteolytic mechanism, tPA also mediates microglial activation. We show here in culture studies that stimulated wild-type neurons and microglia can release the tPA that elicits the activation, and that tPA acts in combination with other factors. We also show that the finger domain of tPA is necessary to trigger the activation and identify annexin II as its probable binding partner-receptor. Together, these findings suggest that tPA released by either neurons or microglia can act as a neural cytokine, signaling through annexin II to activate microglia in settings of disease and injury. Developing methods to inhibit the interaction of tPA with annexin II would offer a new and selective approach to interfere with microglial activation for therapeutic purposes.
Assuntos
Anexina A2/metabolismo , Microglia/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Western Blotting , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Comunicação Parácrina/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Tissue plasminogen activator (tPA) plays important roles in the brain after excitotoxic injury. It is released by both neurons and microglia and mediates neuronal death and microglial activation. Mice lacking tPA are resistant to excitotoxicity and show very limited microglial activation. Activated microglia are neurotoxic in culture, but this phenomenon is not well documented in vivo. To further understand the sequence of events through which tPA mediates microglial activation and neurodegeneration, we have generated mice that exhibit restricted expression of tPA through introduction of tPA transgenes under the control of neuronal- or microglial-specific promoters into tPA-deficient mice. Neither strain of transgenic mice shows abnormal brain morphology or inflammation in the absence of injury, and unilateral intrahippocampal kainate injections into the transgenic mice induced excitotoxicity and microglial activation reminiscent of wild-type mice. However, there are differences in the kinetics of the resulting pathology. The neuronal tPA-expressing mice exhibit accelerated microglial activation compared with wild-type or microglial tPA-expressing mice. However, microglial tPA-expressing mice exhibit greater neurodegeneration. These data suggest a model in which tPA plays different roles after kainate injection depending on whether it is released by neurons or microglia. We propose that tPA, initially secreted from injured neurons, acts as a cytokine to activate microglia at the site of injury. These activated microglia then secrete additional tPA, which promotes extracellular matrix degradation, neurodegeneration, and self-proliferation. We suggest that an approach to attenuate microglia-mediated neuronal death in vivo might be to pharmacologically prevent microglial activation.
Assuntos
Microglia/metabolismo , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Neurotoxinas , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Contagem de Células , Células Cultivadas , Progressão da Doença , Expressão Gênica , Genes fms , Predisposição Genética para Doença , Ácido Caínico , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Proteínas de Neurofilamentos/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Experience-dependent plasticity shapes postnatal development of neural circuits, but the mechanisms that refine dendritic arbors, remodel spines, and impair synaptic activity are poorly understood. Mature brain-derived neurotrophic factor (BDNF) modulates neuronal morphology and synaptic plasticity, including long-term potentiation (LTP) via TrkB activation. BDNF is initially translated as proBDNF, which binds p75(NTR). In vitro, recombinant proBDNF modulates neuronal structure and alters hippocampal long-term plasticity, but the actions of endogenously expressed proBDNF are unclear. Therefore, we generated a cleavage-resistant probdnf knockin mouse. Our results demonstrate that proBDNF negatively regulates hippocampal dendritic complexity and spine density through p75(NTR). Hippocampal slices from probdnf mice exhibit depressed synaptic transmission, impaired LTP, and enhanced long-term depression (LTD) in area CA1. These results suggest that proBDNF acts in vivo as a biologically active factor that regulates hippocampal structure, synaptic transmission, and plasticity, effects that are distinct from those of mature BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Transmissão Sináptica/fisiologia , Alelos , Animais , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Técnicas de Introdução de Genes , Hipocampo/anatomia & histologia , Hipocampo/citologia , Depressão Sináptica de Longo Prazo , Camundongos , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptor trkB/metabolismo , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro-nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75(NTR) in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75(NTR) by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75(NTR) in proNGF-expressing mice rescues the phenotype, confirming the importance of p75(NTR)-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75(NTR) limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.
Assuntos
Encéfalo/metabolismo , Microvasos/patologia , Infarto do Miocárdio/metabolismo , Fator de Crescimento Neural/metabolismo , Pericitos/metabolismo , Precursores de Proteínas/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Western Blotting , Primers do DNA/genética , Ecocardiografia , Ensaio de Imunoadsorção Enzimática , Técnicas de Introdução de Genes , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Microvasos/metabolismo , Mutagênese Sítio-Dirigida , Infarto do Miocárdio/patologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF utilize distinct receptors to mediate divergent neuronal actions. Using new tools to quantitate endogenous BDNF isoforms, we found that mouse neurons secrete both proBDNF and mature BDNF. The highest levels of proBDNF and p75 were observed perinatally and declined, but were still detectable, in adulthood. Thus, BDNF actions are developmentally regulated by secretion of proBDNF or mature BDNF and by local expression of p75 and TrkB.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Animais , Anticorpos Monoclonais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/imunologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Hipocampo/citologia , Camundongos , Camundongos Mutantes , Neurônios/citologia , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkB/metabolismoRESUMO
A common single-nucleotide polymorphism in the brain-derived neurotrophic factor (BDNF) gene, a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met), is associated with alterations in brain anatomy and memory, but its relevance to clinical disorders is unclear. We generated a variant BDNF mouse (BDNF(Met/Met)) that reproduces the phenotypic hallmarks in humans with the variant allele. BDNF(Met) was expressed in brain at normal levels, but its secretion from neurons was defective. When placed in stressful settings, BDNF(Met/Met) mice exhibited increased anxiety-related behaviors that were not normalized by the antidepressant, fluoxetine. A variant BDNF may thus play a key role in genetic predispositions to anxiety and depressive disorders.
Assuntos
Ansiedade/genética , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Ansiedade/tratamento farmacológico , Comportamento Animal , Condicionamento Psicológico , Dendritos/ultraestrutura , Giro Denteado/citologia , Medo , Fluoxetina/administração & dosagem , Fluoxetina/farmacologia , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Neurônios/citologia , Neurônios/metabolismo , Tamanho do Órgão , Ratos , Ratos Sprague-Dawley , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Neuronal cell death occurs during development of the central nervous system as well as in pathological situations such as acute injury and progressive degenerative diseases. For instance, granule cells in the developing cerebellum and neuronal precursor cells in the cortex undergo programmed cell death, or apoptosis. There is currently strong debate conceming the mechanism of death in many degenerative events such as ischemia, blunt head trauma, excitotoxicity and neurodegenerative diseases, i.e. Alzheimer's disease. Neurons can die a necrotic death when the initial insult is too great; apoptosis requires "planning." For example, the cell death seen in the core of an ischemic infarct is necrotic, while in the surrounding penumbra region the death is probably apoptotic. Regardless of the degenerative pathway, damaged or dead neurons are a hallmark of many diseases including Alzheimer's, Parkinson's, glaucoma, ischemia and multiple sclerosis. Molecules such as cytokines, chemokines, reactive nitrogen/oxygen species, and proteases play an important role in promoting and/or mediating neurodegeneration. Proteases have been implicated in both physiological and pathological events, suggesting their intervention in key points when things go awry. In this review we will summarize recent findings linking extracellular proteases with neuronal cell death in both human diseases and their animal models.