RESUMO
Tauopathies, such as Alzheimer's disease (AD), are neurodegenerative disorders characterized by the deposition of hyperphosphorylated tau aggregates. Proteopathic tau seeds spread through the brain in a temporospatial pattern, indicative of transsynaptic propagation. It is hypothesized that reducing the uptake of tau seeds and subsequent induction of tau aggregation could be a potential approach for abrogating disease progression in AD. Here, we studied to what extent different endosomal routes play a role in the neuronal uptake of preformed tau seeds. Using pharmacological and genetic tools, we identified dynamin-1, actin, and Rac1 as key players. Furthermore, inhibition of PIKfyve, a protein downstream of Rac1, reduced both the trafficking of tau seeds into lysosomes and the induction of tau aggregation. Our work shows that tau aggregates are internalized by a specific endocytic mechanism and that their fate once internalized can be pharmacologically modulated to reduce tau seeding in neurons.
Assuntos
Hipocampo/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Patológica de Proteínas , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Transporte Proteico , Tauopatias/patologiaRESUMO
The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticorpos Monoclonais/administração & dosagem , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Imunização Passiva/métodos , Fragmentos de Peptídeos/antagonistas & inibidores , Placa Amiloide/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Ácido Aspártico Endopeptidases/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Resultado do TratamentoRESUMO
The ß-secretase ß-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Transdução de Sinais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Peixe-Zebra , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Humanos , Camundongos , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/enzimologia , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Development of the cerebellum involves a coordinated program of neuronal process outgrowth and migration resulting in a foliated structure that plays a key role in motor function. Neuron navigator 2 (Nav2) is a cytoskeletal-interacting protein that functions in neurite outgrowth and axonal elongation. Herein we show that hypomorphic mutant mice lacking the full-length Nav2 transcript exhibit ataxia and defects in cerebellar development. At embryonic day (E)17.5, the mutant cerebellum is reduced in size and exhibits defects in vermal foliation. Reduction in cell proliferation at early times (E12.5 and E14.5) may contribute to this size reduction. The full-length Nav2 transcript is expressed in the premigratory zone of the external granule layer (EGL). Granule cells in the germinal zone of the EGL appear to proliferate normally, however, due to the reduction in cerebellar circumference there are fewer total BrdU-labeled granule cells in the mutants, and these fail to migrate normally toward the interior of the cerebellum. In Nav2 hypomorphs, fewer granule cells migrate out of cerebellar EGL explants and neurite outgrowth from both explants and isolated external granule cell cultures is reduced. This suggests that the formation of parallel axon fibers and neuronal migration is disrupted in Nav2 mutants. This work supports an essential role for full-length Nav2 in cerebellar development, including axonal elongation and migration of the EGL neurons.
Assuntos
Ataxia/embriologia , Cerebelo/anormalidades , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Animais , Ataxia/genética , Ataxia/fisiopatologia , Axônios/metabolismo , Axônios/ultraestrutura , Sequência de Bases , Movimento Celular , Proliferação de Células , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/fisiologia , Neurogênese , Equilíbrio Postural/genética , Equilíbrio Postural/fisiologia , Gravidez , Células de Purkinje/citologia , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
APP.V717I and Tau.P301L transgenic mice develop Alzheimer's disease pathology comprising important aspects of human disease including increased levels of amyloid peptides, cognitive and motor impairment, amyloid plaques and neurofibrillary tangles. The combined model, APP.V717I×Tau.P301L bigenic mice (biAT mice) exhibit aggravated amyloid and tau pathology with severe cognitive and behavioral defects. In the present study, we investigated early changes in synaptic function in the CA1 and CA3 regions of acute hippocampal slices of young APP.V717I, Tau.P301L and biAT transgenic animals. We have used planar multi-electrode arrays (MEA) and improved methods for simultaneous multi-site recordings from two hippocampal sub-regions. In the CA1 region, long-term potentiation (LTP) was severely impaired in all transgenic animals when compared with age-matched wild-type controls, while basal synaptic transmission and paired-pulse facilitation were minimally affected. In the CA3 region, LTP was normal in Tau.P301L and APP.V717I but clearly impaired in biAT mice. Surprisingly, frequency facilitation in CA3 was significantly enhanced in Tau.P301L mice, while not affected in APP.V717I mice and depressed in biAT mice. The findings demonstrate important synaptic changes that differ considerably in the hippocampal sub-regions already at young age, well before the typical amyloid or tau pathology is evident.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Eletrodos , Hipocampo/patologia , Hipocampo/fisiopatologia , Sinapses/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Biofísica , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Técnicas In Vitro , Isoleucina/genética , Potenciação de Longa Duração/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Sinapses/fisiologia , Fatores de Tempo , Valina/genética , Proteínas tau/genéticaRESUMO
Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral separation resulted in the 40 nM inhibitor, (-)-37, which showed in vivo reduction of amyloid beta peptides. The crystal structure of 18a showed a binding mode driven by interaction with the catalytic aspartate dyad and distribution of the biaryl amide decoration towards S1 and S3 pockets.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Piperazinas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Sítios de Ligação , Simulação por Computador , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Piperazinas/síntese química , Piperazinas/farmacologia , Estrutura Terciária de Proteína , EstereoisomerismoRESUMO
Mammalian microtubule plus-end tracking proteins (+TIPs) specifically associate with the ends of growing microtubules. +TIPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TIPs, consistent with a previous study on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005;28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TIPs, such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding sites on microtubule ends overlap with those of the CAP_GLY-motif proteins. In interphase cells, mammalian Navigators also prominently localize to centrosomes, a localization that does not depend on an intact microtubule network. Fluorescence recovery after photobleaching (FRAP) experiments indicate that NAV1 associates with intracellular structures other than microtubules or centrosomes. Expression of GFP-tagged Navigators induces the formation of neurite-like extensions in non-neuronal cells, showing that Navigators can dominantly alter cytoskeletal behavior. For NAV1 this function depends on its ATPase activity; it is not achieved by a classical type of MT bundling and stabilization. Combined our data suggest that Navigators are +TIPs that can reorganize the cytoskeleton to guide cell shape changes. Our data are consistent with a role for Navigators in neurite outgrowth.
Assuntos
Citoesqueleto/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Neuritos/ultraestrutura , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células HeLa , Humanos , Camundongos , Proteínas de Neoplasias/fisiologia , Proteínas de Neurofilamentos/fisiologia , Estrutura Terciária de ProteínaRESUMO
Although very different in etiology and symptoms, numerous neurodegenerative diseases can be classified as proteinopathies. More so, evidence indicates that the key misfolded proteins at the basis of different neuropathies might share common mechanisms of propagation. As such, the prion-like spreading of protein aggregates through the neural network is subject of intensive research focus and requires adequate models. Here, we made use of the well-defined architecture and large accessibility of the visual system, of which the retinotopic connections represent a simple route of anterograde signaling and an elegant model to investigate transsynaptic, prion-like spreading. In two independent studies, uptake and seeding of alpha-synuclein and tau were examined after intravitreal injection of preformed fibrils. However, extracellular matrix components in the vitreous space and at the vitreoretinal surface appeared to act as a barrier for the entry of both fibrils into the retina. These results show that further experimental refinement is needed to fully realize the potential of the visual system as a model for studying the molecular and cellular mechanisms of anterograde, transsynaptic spreading of prion-like proteins.
RESUMO
BACKGROUND & AIMS: Ghrelin is an orexigenic peptide with gastroprokinetic effects. Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying, and increased plasma ghrelin levels. We investigated the causative role of ghrelin herein by comparing changes in ghrelin receptor knockout (growth hormone secretagogue receptor [GHS-R](-/-)) and wild-type (GHS-R(+/+)) mice with STZ-induced diabetes. METHODS: Gastric emptying was measured with the [(13)C]octanoic acid breath test. The messenger RNA (mRNA) expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin was quantified by real-time reverse-transcription polymerase chain reaction. Neural contractions were elicited by electrical field stimulation in fundic smooth muscle strips. RESULTS: Diabetes increased plasma ghrelin levels to a similar extent in both genotypes. Hyperphagia was more pronounced in GHS-R(+/+) than in GHS-R(-/-) mice between days 12 and 21. Increases in NPY and AgRP mRNA expression were less pronounced in diabetic GHS-R(-/-) than in GHS-R(+/+) mice from day 15 on, whereas decreases in proopiomelanocortin mRNA levels were similar in both genotypes. Gastric emptying was accelerated to a similar extent in both genotypes, starting on day 16. In fundic smooth muscle strips of diabetic GHS-R(+/+) and GHS-R(-/-) mice, neuronal relaxations were reduced, whereas contractions were increased; this increase was related to an increased affinity of muscarinic and tachykinergic receptors. CONCLUSIONS: Diabetic hyperphagia is regulated by central mechanisms in which the ghrelin-signaling pathway affects the expression of NPY and AgRP in the hypothalamus. The acceleration of gastric emptying, which is not affected by ghrelin signaling, is not the cause of diabetic hyperphagia and probably involves local contractility changes in the fundus.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Esvaziamento Gástrico/fisiologia , Grelina/sangue , Hiperfagia/fisiopatologia , Receptores de Grelina/genética , Acetilcolina/farmacologia , Proteína Relacionada com Agouti/genética , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Colinérgicos/farmacologia , Diabetes Mellitus Experimental/metabolismo , Ingestão de Alimentos/fisiologia , Fundo Gástrico/inervação , Fundo Gástrico/fisiologia , Grelina/genética , Hiperfagia/metabolismo , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Neuropeptídeo Y/genética , Neurotransmissores/farmacologia , Pró-Opiomelanocortina/genética , RNA Mensageiro/metabolismo , Receptores de Grelina/metabolismo , Substância P/farmacologiaRESUMO
Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/farmacologia , Receptores Acoplados a Proteínas G/fisiologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Feminino , Trato Gastrointestinal/metabolismo , Genes fos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Despite several years of research, only a handful of ß-secretase (BACE) 1 inhibitors have entered clinical trials as potential therapeutics against Alzheimer's disease. The intrinsic basic nature of low molecular weight, amidine-containing BACE 1 inhibitors makes them far from optimal as central nervous system drugs. Herein we present a set of novel heteroaryl-fused piperazine amidine inhibitors designed to lower the basicity of the key, enzyme binding, amidine functionality. This study resulted in the identification of highly potent (IC50 ≤ 10 nM), permeable lead compounds with a reduced propensity to suffer from P-glycoprotein-mediated efflux.
RESUMO
Lithium has been the standard pharmacological treatment for bipolar disorder over the last 50 years; however, the molecular targets through which lithium exerts its therapeutic effects are still not defined. We characterized the phenotype of mice with a dysfunctional IMPA1 gene (IMPA1-/-) to study the in vivo physiological functions of IMPA1, in general, and more specifically its potential role as a molecular target in mediating lithium-dependent physiological effects. Homozygote IMPA1-/- mice died in utero between days 9.5 and 10.5 post coitum (p.c.) demonstrating the importance of IMPA1 in early embryonic development. Intriguingly, the embryonic lethality could be reversed by myo-inositol supplementation via the pregnant mothers. In brains of adult IMPA1-/- mice, IMPase activity levels were found to be reduced (up to 65% in hippocampus); however, inositol levels were not found to be altered. Behavioral analysis of the IMPA1-/- mice indicated an increased motor activity in both the open-field test and the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures, the latter supporting the idea that IMPA1 represents a physiologically relevant target for lithium. In conclusion the IMPA1-/- mouse represents a novel model to study inositol homeostasis, and indicates that genetic inactivation of IMPA1 can mimic some actions of lithium.
Assuntos
Antimaníacos/farmacologia , Desenvolvimento Embrionário/genética , Carbonato de Lítio/farmacologia , Agonistas Muscarínicos/farmacologia , Monoéster Fosfórico Hidrolases/genética , Pilocarpina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cromatografia Gasosa , Ingestão de Líquidos/efeitos dos fármacos , Inositol/deficiência , Inositol/farmacologia , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mutagênese , Monoéster Fosfórico Hidrolases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Natação/psicologiaRESUMO
The interaction between CC chemokine receptor 2 (CCR2) with monocyte chemoattractant proteins, such as MCP-1, regulates the activation and recruitment of inflammatory leukocytes. In this study, we characterized (S)-3-[3,4-difluoro-phenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester (JNJ-27141491) as a noncompetitive and orally active functional antagonist of human (h)CCR2. JNJ-27141491 strongly suppressed hCCR2-mediated in vitro functions, such as MCP-1-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate binding; MCP-1, -3, and -4-induced Ca(2+) mobilization; and leukocyte chemotaxis toward MCP-1 (IC(50) = 7-97 nM), whereas it had little or no effect on the function of other chemokine receptors tested. The inhibition of CCR2 function was both insurmountable and reversible, consistent with a noncompetitive mode of action. JNJ-27141491 blocked the binding of (125)I-MCP-1 to human monocytes (IC(50) = 0.4 microM), but it failed to affect MCP-1 binding to mouse, rat, and dog cells (IC(50) > 10 microM). Therefore, transgenic mice, in which the mouse (m)CCR2 gene was replaced by the human counterpart, were generated for in vivo testing. In these mice, oral administration of JNJ-27141491 dose-dependently [5-40 mg/kg q.d. (once daily) or b.i.d.] inhibited monocyte and neutrophil recruitment to the alveolar space 48 h after intratracheal mMCP-1/lipopolysaccharide instillation. Furthermore, treatment with JNJ-27141491 (20 mg/kg q.d.) significantly delayed the onset and temporarily reduced neurological signs in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Taken together, these results identify JNJ-27141491 as a noncompetitive, functional antagonist of hCCR2, capable of exerting oral anti-inflammatory activity in transgenic hCCR2-expressing mice.
Assuntos
Imidazóis/farmacologia , Receptores CCR2/antagonistas & inibidores , Administração Oral , Sequência de Aminoácidos , Animais , Células CHO , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacologia , Cricetinae , Cricetulus , Cães , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/prevenção & controle , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Receptores CCR2/metabolismoRESUMO
Probe-level data from Affymetrix GeneChips can be summarized in many ways to produce probe-set level gene expression measures (GEMs). Disturbingly, the different approaches not only generate quite different measures but they could also yield very different analysis results. Here, we explore the question of how much the analysis results really do differ, first at the gene level, then at the biological process level. We demonstrate that, even though the gene level results may not necessarily match each other particularly well, as long as there is reasonably strong differentiation between the groups in the data, the various GEMs do in fact produce results that are similar to one another at the biological process level. Not only that the results are biologically relevant. As the extent of differentiation drops, the degree of concurrence weakens, although the biological relevance of findings at the biological process level may yet remain.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The Mus81-Eme1 endonuclease is implicated in the efficient rescue of broken replication forks in Saccharomyces cerevisiae and Schizosaccharomyces pombe. We have used gene targeting to study the function of the Mus81-Eme1 endonuclease in mammalian cells. Mus81-deficient mice develop normally and are fertile. Surprisingly, embryonic fibroblasts from Mus81(-/-) animals fail to proliferate in vitro. This proliferation defect can be rescued by expression of the papillomavirus E6 protein that promotes degradation of p53. When grown in culture, Mus81(-/-) cells have elevated levels of DNA damage, acquire chromosomal aberrations, and are hypersensitive to agents that generate DNA cross-links. In contrast to the situation in yeast, murine Mus81 is not required for replication restart following camptothecin treatment. Mus81(-/-) mice and cells are hypersensitive to DNA cross-linking agents. Cross-link-induced double-strand break formation is normal in Mus81(-/-) cells, but the resolution of repair intermediates is not. The persistence of Rad51 foci in Mus81(-/-) cells suggests that Mus81 acts at a late step in the repair of cross-link-induced lesions. Despite these defects, Mus81(-/-) mice do not show increased predisposition to lymphoma or any other malignancy in the first year of life.
Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Endonucleases/deficiência , Endonucleases/metabolismo , Instabilidade Genômica/genética , Animais , Camptotecina/farmacologia , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Aberrações Cromossômicas , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos , Camundongos , Rad51 Recombinase , Proteínas de Saccharomyces cerevisiaeRESUMO
ß-Site APP-cleaving enzyme 1 (BACE1) inhibition is considered one of the most promising therapeutic strategies for Alzheimer's disease, but current BACE1 inhibitors also block BACE2. As the localization and function of BACE2 in the brain remain unknown, it is difficult to predict whether relevant side effects can be caused by off-target inhibition of BACE2 and whether it is important to generate BACE1-specific inhibitors. Here, we show that BACE2 is expressed in discrete subsets of neurons and glia throughout the adult mouse brain. We uncover four new substrates processed by BACE2 in cultured glia: vascular cell adhesion molecule 1, delta and notch-like epidermal growth factor-related receptor, fibroblast growth factor receptor 1, and plexin domain containing 2. Although these substrates were not prominently cleaved by BACE2 in healthy adult mice, proinflammatory TNF induced a drastic increase in BACE2-mediated shedding of vascular cell adhesion molecule 1 in CSF. Thus, although under steady-state conditions the effect of BACE2 cross-inhibition by BACE1-directed inhibitors is rather subtle, it is important to consider that side effects might become apparent under physiopathological conditions that induce TNF expression.
RESUMO
Lithium is used as treatment for bipolar disorder with particular efficacy in the treatment of mania. Lithium inhibits glycogen synthase kinase 3beta (GSK-3beta) directly or indirectly via stimulation of the kinase Akt-1. We therefore investigated the possibility that transgenic mice overexpressing GSK-3beta could be of relevance to model bipolar disorder. Transgenic mice showed hypophagia, an increased general locomotor activity, and decreased habituation as assessed in an open field, an increased acoustic startle response, and again decreased habituation. The forced swim test revealed a reduced immobility in transgenic mice, but this is probably related to the hyperactivity of the animals. There were no differences in baseline and stress-induced increases of plasma adrenocorticotrophic hormone and corticosterone levels. Molecular analysis suggests compensatory mechanisms in the striatum of these transgenic mice for the overload of active GSK-3beta by dimming the endogenous GSK-3beta signaling pathway via upregulation of Akt-1 expression. Brain-derived neurotrophic factor protein levels were increased in the hippocampus of the transgenic mice. This suggests some kind of compensatory mechanism to the observed reduction in brain weight, which has been related previously to a reduced size of the somatodendritic compartment. Together, in mice overexpressing GSK-3beta, specific intracellular signaling pathways are affected, which is accompanied by altered plasticity processes and increased activity and reactivity, whereas habituation processes seem to be decreased. The behavioral observations led to the suggestion that the model at hand recapitulates hyperactivity as observed in the manic phase of bipolar disorder.
Assuntos
Transtorno Bipolar/etiologia , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/metabolismo , Hipercinese/etiologia , Hormônio Adrenocorticotrópico/sangue , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corticosterona/sangue , DNA/biossíntese , Ingestão de Líquidos/fisiologia , Ingestão de Alimentos/fisiologia , Feminino , Glicogênio Sintase Quinase 3 beta , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora , Fotoperíodo , Reflexo de Sobressalto , Transdução de Sinais , NataçãoRESUMO
Lithium is a potent mood-stabilizing medication in bipolar disorder. Despite 50 years of clinical use, the mechanism of action is unknown. Multiple effects have been attributed to lithium including the uncompetitive inhibition of inositol monophosphatase (IMPase). IMPA2, one of the genes that encode IMPase, is located in a region with linkage to bipolar disorder. Owing to the role of IMPase in cell signaling and the possibility that this enzyme is a target for mood-stabilizing drugs, we generated IMPA2(-/-) mice. Possible involvement of IMPase in complex behaviors related to affective disorders was assessed by monitoring the behavior of the IMPA2(-/-) mice in the forced swim test, the tail suspension test (TST), the elevated zero-maze and open field test. It has been described that chronically lithium-treated mice exhibit reduced immobility time in the forced swim test and decreased exploratory behavior. We found increased rearing of IMPA2(-/-) mice in the open field, suggesting an increased exploratory behavior. Although immobility time of IMPA2(-/-) female but not male mice in the forced swim test was reduced, no difference was found between male and female IMPA2(-/-) and IMPA2(+/+) mice in the TST and overall there was no clear effect of the deletion of IMPA2 on depression-like behavior. Frontal cortex IMPase activity and inositol levels in the IMPA2(-/-) mice did not differ from IMPA2(+/+) mice, but kidney inositol levels were reduced. In conclusion, phenotypic characterization of the IMPA2(-/-) mouse indicates that deleting IMPA2 does not mimic the effects of lithium treatment.
Assuntos
Comportamento Animal/fisiologia , Expressão Gênica/fisiologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Hormônio Adrenocorticotrópico/sangue , Anfetamina/farmacologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Corticosterona/sangue , Comportamento Exploratório/fisiologia , Lobo Frontal/metabolismo , Lobo Frontal/fisiologia , Elevação dos Membros Posteriores/métodos , Inositol/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , NataçãoRESUMO
The ß-site amyloid-ß protein precursor (AßPP) cleaving enzyme-1 (BACE1) is the rate limiting enzyme in the generation of amyloid-ß peptide (Aß) from AßPP, one of the major pathways in Alzheimer's disease (AD) pathology. Increased BACE1 levels and activity have been reported in the brain of patients with sporadic AD. Therefore, changes of BACE1 levels in the cerebrospinal fluid (CSF) have also been investigated as a possible biomarker of the disease. We analyzed BACE1 levels in CSF of elderly healthy participants before and after chronic treatment with a BACE inhibitor (BACEi) and evaluated the correlation between BACE1 levels and downstream AD markers. Overall, BACE1 CSF levels showed strong correlations to all downstream AD markers investigated. This is the first reported finding that shows BACE1 levels in CSF were well correlated to its end product Aß1 - 42. As previously described, BACE1 levels were strongly correlated to total-tau and phosphorylated tau levels in CSF. Generally, chronic BACE inhibition did not influence BACE1 CSF protein levels. Follow-up studies including early-stage AD pathophysiology and prodromal AD patients will help to understand the importance of measuring BACE1 routinely in daily clinical practice and AD clinical trials.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Inibidores de Proteases/uso terapêutico , Proteínas tau/líquido cefalorraquidiano , Idoso , Doença de Alzheimer/líquido cefalorraquidiano , Apolipoproteína E4/genética , Biomarcadores/líquido cefalorraquidiano , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/farmacocinéticaRESUMO
Slc17a5-/- mice represent an animal model for the infantile form of sialic acid storage disease (SASD). We analyzed genetic and histological time-course expression of myelin and oligodendrocyte (OL) lineage markers in different parts of the CNS, and related this to postnatal neurobehavioral development in these mice. Sialin-deficient mice display a distinct spatiotemporal pattern of sialic acid storage, CNS hypomyelination and leukoencephalopathy. Whereas few genes are differentially expressed in the perinatal stage (p0), microarray analysis revealed increased differential gene expression in later postnatal stages (p10-p18). This included progressive upregulation of neuroinflammatory genes, as well as continuous down-regulation of genes that encode myelin constituents and typical OL lineage markers. Age-related histopathological analysis indicates that initial myelination occurs normally in hindbrain regions, but progression to more frontal areas is affected in Slc17a5-/- mice. This course of progressive leukoencephalopathy and CNS hypomyelination delays neurobehavioral development in sialin-deficient mice. Slc17a5-/- mice successfully achieve early neurobehavioral milestones, but exhibit progressive delay of later-stage sensory and motor milestones. The present findings may contribute to further understanding of the processes of CNS myelination as well as help to develop therapeutic strategies for SASD and other myelination disorders.