RESUMO
The prevalence of dementia in the Western world in people over the age of 60 has been estimated to be greater than 5%, about two-thirds of which are due to Alzheimer's disease. The age-specific prevalence of Alzheimer's disease nearly doubles every 5 years after age 65, leading to a prevalence of greater than 25% in those over the age of 90 (ref. 3). Here, to search for low-frequency variants in the amyloid-ß precursor protein (APP) gene with a significant effect on the risk of Alzheimer's disease, we studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. We found a coding mutation (A673T) in the APP gene that protects against Alzheimer's disease and cognitive decline in the elderly without Alzheimer's disease. This substitution is adjacent to the aspartyl protease ß-site in APP, and results in an approximately 40% reduction in the formation of amyloidogenic peptides in vitro. The strong protective effect of the A673T substitution against Alzheimer's disease provides proof of principle for the hypothesis that reducing the ß-cleavage of APP may protect against the disease. Furthermore, as the A673T allele also protects against cognitive decline in the elderly without Alzheimer's disease, the two may be mediated through the same or similar mechanisms.
Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Transtornos Cognitivos/genética , Transtornos Cognitivos/fisiopatologia , Mutação/genética , Alelos , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/prevenção & controle , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Ácido Aspártico Endopeptidases/metabolismo , Cognição/fisiologia , Transtornos Cognitivos/prevenção & controle , Predisposição Genética para Doença , Células HEK293 , Humanos , Placa Amiloide/genética , Placa Amiloide/metabolismoRESUMO
Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the ß-secretase recognition sequence and is part of the amyloid-ß (Aß) peptide cleavage product (position 2 of Aß). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aß(1-42) aggregation with A2T Aß peptides, an observation not conserved in Aß(1-40) peptides. When combined in a ratio of 1:9 Aß(1-42)/Aß(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aß(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aß peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aß aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Alelos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Catálise , DNA Complementar/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Heterozigoto , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Mutação , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Ligação ProteicaRESUMO
In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3ß, we show that the ß isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3ß in axon degeneration.
Assuntos
Axônios/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Degeneração Neural/enzimologia , Degeneração Neural/patologia , Neurônios/patologia , Fosfotransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletroporação , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Degeneração Neural/tratamento farmacológico , Degeneração Neural/prevenção & controle , Fator de Crescimento Neural/deficiência , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Fosforilação/fisiologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Vermelha FluorescenteRESUMO
Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Imunoglobulina G/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Pessoa de Meia-Idade , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Quimiocinas/genética , Estatísticas não Paramétricas , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.
Assuntos
Neuropilinas/química , Semaforina-3A/química , Fator A de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Anticorpos/química , Sítios de Ligação , Cristalografia por Raios X/métodos , Dimerização , Conformação Molecular , Dados de Sequência Molecular , Neuropilinas/fisiologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Semaforina-3A/metabolismo , Semaforinas/metabolismo , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Accumulation of amyloid ß (Aß) in the brain is proposed as a cause of Alzheimer's disease (AD), with Aß oligomers hypothesized to be the primary mediators of neurotoxicity. Crenezumab is a humanized immunoglobulin G4 monoclonal antibody that has been shown to bind to synthetic monomeric and aggregated Aß in vitro; however, less is known about the binding characteristic in vivo. In this study, we evaluated the binding patterns of crenezumab to synthetic and native forms of Aß both in vitro and in vivo. METHODS: Crenezumab was used to immunoprecipitate Aß from synthetic Aß preparations or brain homogenates from a PS2APP mouse model of AD to determine the forms of Aß that crenezumab interacts with. Following systemic dosing in PS2APP or nontransgenic control mice, immunohistochemistry was used to localize crenezumab and assess its relative distribution in the brain, compared with amyloid plaques and markers of neuritic dystrophies (BACE1; LAMP1). Pharmacodynamic correlations were performed to investigate the relationship between peripheral and central target engagement. RESULTS: In vitro, crenezumab immunoprecipitated Aß oligomers from both synthetic Aß preparations and endogenous brain homogenates from PS2APP mice. In vivo studies in the PS2APP mouse showed that crenezumab localizes to regions surrounding the periphery of amyloid plaques in addition to the hippocampal mossy fibers. These regions around the plaques are reported to be enriched in oligomeric Aß, actively incorporate soluble Aß, and contribute to Aß-induced neurotoxicity and axonal dystrophy. In addition, crenezumab did not appear to bind to the dense core region of plaques or vascular amyloid. CONCLUSIONS: Crenezumab binds to multiple forms of amyloid ß (Aß), particularly oligomeric forms, and localizes to brain areas rich in Aß oligomers, including the halo around plaques and hippocampal mossy fibers, but not to vascular Aß. These insights highlight a unique mechanism of action for crenezumab of engaging Aß oligomers.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Encéfalo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Placa Amiloide/metabolismo , Ligação ProteicaRESUMO
Assessing BACE1 (ß-site APP cleaving enzyme 1) knockout mice for general health and neurological function may be useful in predicting risks associated with prolonged pharmacological BACE1 inhibition, a treatment approach currently being developed for Alzheimer's disease. To determine whether BACE1 deletion-associated effects in mice generalize to another species, we developed a novel Bace1-/- rat line using zinc-finger nuclease technology and compared Bace1-/- mice and rats with their Bace1+/+ counterparts. Lack of BACE1 was confirmed in Bace1-/- animals from both species. Removal of BACE1 affected startle magnitude, balance beam performance, pain response, and nerve myelination in both species. While both mice and rats lacking BACE1 have shown increased mortality, the increase was smaller and restricted to early developmental stages for rats. Bace1-/- mice and rats further differed in body weight, spontaneous locomotor activity, and prepulse inhibition of startle. While the effects of species and genetic background on these phenotypes remain difficult to distinguish, our findings suggest that BACE1's role in myelination and some sensorimotor functions is consistent between mice and rats and may be conserved in other species. Other phenotypes differ between these models, suggesting that some effects of BACE1 inhibition vary with the biological context (e.g. species or background strain).
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Deleção de Genes , Reflexo de Sobressalto/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Peso Corporal/genética , Peso Corporal/fisiologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Atividade Motora/fisiologia , Inibição Pré-Pulso/genética , Inibição Pré-Pulso/fisiologia , Ratos , Reflexo de Sobressalto/fisiologia , Especificidade da EspécieRESUMO
Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.
Assuntos
Zíper de Leucina , MAP Quinase Quinase Quinases/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Transdução de Sinais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos Transgênicos , Neuroproteção , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Medula Espinal/enzimologia , Medula Espinal/patologia , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/imunologia , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/imunologia , Encéfalo/metabolismo , Feminino , Infusões Intraventriculares , Macaca fascicularisRESUMO
We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including ß-amyloid (Aß), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain.
Assuntos
Doença de Alzheimer/genética , Neurônios/metabolismo , Receptores de Superfície Celular/genética , Receptores de Fator de Crescimento Neural/genética , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides , Animais , Região CA3 Hipocampal/citologia , Morte Celular/genética , Feminino , Predisposição Genética para Doença , Ácido Glutâmico , Células HEK293 , Humanos , Masculino , Camundongos , Receptores de Netrina , Ratos , EstaurosporinaRESUMO
Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ß-secretase (BACE1) can cross the BBB and reduce brain amyloid-ß (Aß) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aß in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aß both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.