RESUMO
Deposition of α-synuclein into Lewy bodies and Lewy neurites is the hallmark of Parkinson's disease (PD). It is hypothesized that α-synuclein pathology spreads by a "prion-like" mechanism (i.e., by seeded aggregation or templated misfolding). Therefore, various extracellular α-synuclein conformers and/or posttranslational modifications may serve as biomarkers of disease or potential targets for novel interventions. To explore whether the antibody repertoires of PD patients contain anti-α-synuclein antibodies that can potentially be used as markers or immunotherapy, we interrogated peripheral IgG+ memory B cells from PD patients for reactivity to α-synuclein. In total, ten somatically mutated antibodies were recovered, suggesting the presence of an ongoing antigen-driven immune response. The three antibodies that had the highest affinity to recombinant full-length α-synuclein, aSyn-323.1, aSyn-336.1 and aSyn-338.1, were characterized further and shown to recognize epitopes in the C terminus of α-synuclein with binding affinities between 0.3 and 2.8 µM. Furthermore, all three antibodies were able to neutralize the "seeding" of intracellular synuclein aggregates in an in vitro α-synuclein seeding assay. Finally, differential reactivities were observed for all three human anti-α-synuclein antibodies across tissue treatment conditions by immunohistochemistry. Our results suggest that the memory B-cell repertoire of PD patients might represent a potential source of biomarkers and therapies.
Assuntos
Anticorpos/metabolismo , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Doença de Parkinson/imunologia , alfa-Sinucleína/metabolismo , Idoso , Anticorpos/isolamento & purificação , Linfócitos B/imunologia , Células HEK293 , Humanos , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Pessoa de Meia-Idade , Mutação , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/metabolismoRESUMO
Several reports have described the presence of antibodies against Alzheimer's disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.
Assuntos
Doença de Alzheimer/imunologia , Epitopos/imunologia , Memória Imunológica/imunologia , Emaranhados Neurofibrilares/imunologia , Proteínas tau/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos/fisiologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Epitopos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Fosforilação , Adulto JovemRESUMO
BACKGROUND: Amyloid-related imaging abnormalities (ARIA) have been reported with some anti-amyloid-ß (Aß) immunotherapy trials. They are detected with magnetic resonance imaging (MRI) and thought to represent transient accumulation of fluid/edema (ARIA-E) or microhemorrhages (ARIA-H). Although the clinical significance and pathophysiology are unknown, it has been proposed that anti-Aßimmunotherapy may affect blood-brain barrier (BBB) integrity. OBJECTIVE: To examine vascular integrity in aged (12-16 months) PDAPP and wild type mice (WT), we performed a series of longitudinal in vivo MRI studies. METHODS: Mice were treated on a weekly basis using anti-Aßimmunotherapy (3D6) and follow up was done longitudinally from 1-12 weeks after treatment. BBB-integrity was assessed using both visual assessment of T1-weighted scans and repeated T1 mapping in combination with gadolinium (Gd-DOTA). RESULTS: A subset of 3D6 treated PDAPP mice displayed numerous BBB disruptions, whereas WT and saline-treated PDAPP mice showed intact BBB integrity under the conditions tested. In addition, the contrast induced decrease in T1 value was observed in the meningeal and midline area. BBB disruption events occurred early during treatment (between 1 and 5 weeks), were transient, and resolved quickly. Finally, BBB-leakages associated with microhemorrhages were confirmed by Perls'Prussian blue histopathological analysis. CONCLUSION: Our preclinical findings support the hypothesis that 3D6 leads to transient leakage from amyloid-positive vessels. The current study has provided valuable insights on the time course of vascular alterations during immunization treatment and supports further research in relation to the nature of ARIA and the utility of in vivo repeated T1 MRI as a translational tool.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Peptídeos beta-Amiloides/administração & dosagem , Precursor de Proteína beta-Amiloide/biossíntese , Barreira Hematoencefálica/diagnóstico por imagem , Imunoterapia/métodos , Imageamento por Ressonância Magnética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Feminino , Gadolínio , Camundongos , Camundongos TransgênicosRESUMO
Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794.
Assuntos
Antibacterianos/farmacologia , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bases de Dados Factuais/estatística & dados numéricos , Humanos , Microglia/fisiologia , Minociclina/uso terapêuticoRESUMO
INTRODUCTION: In Alzheimer's disease (AD), pathologic amyloid-beta (Aß) is synaptotoxic and impairs neuronal function at the microscale, influencing brain networks at the macroscale before Aß deposition. The latter can be detected noninvasively, in vivo, using resting-state functional MRI (rsfMRI), a technique used to assess brain functional connectivity (FC). METHODS: RsfMRI was performed longitudinally in TG2576 and PDAPP mice, starting before Aß deposition to determine the earliest FC changes. Additionally, the role of pathologic Aß on early FC alterations was investigated by treating TG2576 mice with the 3D6 anti-Aß-antibody. RESULTS: Both transgenic models showed hypersynchronized FC before Aß deposition and hyposynchronized FC at later stages. Early anti-Aß treatment in TG2576 mice prevented hypersynchronous FC and the associated synaptic impairments and excitatory/inhibitory disbalances. DISCUSSION: Hypersynchrony of FC may be used as a new noninvasive read out of early AD and can be recovered by anti-Aß treatment, encouraging preventive treatment strategies in familial AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Doença de Alzheimer/diagnóstico por imagem , Animais , Autoanticorpos/farmacologia , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Circulação Cerebrovascular/fisiologia , Sincronização Cortical/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Estudos Longitudinais , Imageamento por Ressonância Magnética , Camundongos Transgênicos , Vias Neurais/diagnóstico por imagem , Vias Neurais/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Oxigênio/sangue , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/fisiopatologia , Placa Amiloide/prevenção & controle , Sintomas Prodrômicos , DescansoRESUMO
While histological changes in microglia have long been recognized as a pathological feature of Alzheimer's disease (AD), recent genetic association studies have also strongly implicated microglia in the etiology of the disease. Coding and noncoding polymorphisms in several genes expressed in microglia-including APOE, TREM2, CD33, GRN, and IL1RAP-alter AD risk, and therefore could be considered as entry points for therapeutic intervention. Furthermore, microglia may have a substantial effect on current amyloid ß (Aß) and tau immunotherapy approaches, since they are the primary cell type in the brain to mediate Fc receptor-facilitated antibody effector function. In this review, we discuss the considerations in selecting microglial therapeutic targets from the perspective of drug discovery feasibility, and consider the role of microglia in ongoing immunotherapy clinical strategies. GLIA 2016;64:1710-1732.
Assuntos
Doença de Alzheimer , Imunoterapia/métodos , Microglia/fisiologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos/uso terapêutico , Humanos , Receptores Fc/imunologia , Proteínas tau/imunologiaRESUMO
Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD). A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid ß-protein (Aß), self-associates to form soluble assemblies loosely referred to as "oligomers" and that these are primary mediators of synaptic dysfunction. As such, Aß, and specifically Aß oligomers, are targets for disease modifying therapies. Currently, the most advanced experimental treatment for AD relies on the use of anti-Aß antibodies. In this study, we tested the ability of the monomer-preferring antibody, m266 and a novel aggregate-preferring antibody, 1C22, to attenuate spatial reference memory impairments in J20 mice. Chronic treatment with m266 resulted in a ~70-fold increase in Aß detected in the bloodstream, and a ~50% increase in water-soluble brain Aß--and in both cases Aß was bound to m266. In contrast, 1C22 increased the levels of free Aß in the bloodstream, and bound to amyloid deposits in J20 brain. However, neither 1C22 nor m266 attenuated the cognitive deficits evident in 12month old J20 mice. Moreover, both antibodies failed to alter the levels of soluble Aß oligomers in J20 brain. These results suggest that Aß oligomers may mediate the behavioral deficits seen in J20 mice and highlight the need for the development of aggregate-preferring antibodies that can reach the brain in sufficient levels to neutralize bioactive Aß oligomers. Aside from the lack of positive effect of m266 and 1C22 on cognition, a substantial number of deaths occurred in m266- and 1C22-immunized J20 mice. These fatalities were specific to anti-Aß antibodies and to the J20 mouse line since treatment of wild type or PDAPP mice with these antibodies did not cause any deaths. These and other recent results indicate that J20 mice are particularly susceptible to targeting of the APP/Aß/tau axis. Notwithstanding the specificity of fatalities for J20 mice, it is worrying that the murine precursor (m266) of a lead experimental therapeutic, Solanezumab, did not engage with putatively pathogenic Aß oligomers.
Assuntos
Peptídeos beta-Amiloides/imunologia , Anticorpos/administração & dosagem , Encéfalo/metabolismo , Imunização Passiva , Transtornos da Memória/imunologia , Transtornos da Memória/terapia , Nootrópicos/administração & dosagem , Peptídeos beta-Amiloides/sangue , Animais , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Infusões Parenterais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologiaRESUMO
Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.
Assuntos
Imunidade Inata , Doença de Parkinson/diagnóstico , Doença de Parkinson/imunologia , alfa-Sinucleína/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Citocinas/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fagocitose , Regulação para Cima , alfa-Sinucleína/genéticaRESUMO
A series of potent α4ß1/α4ß7 integrin inhibitors is reported, including an inhibitor 12d with remarkable oral exposure and efficacy in rat models of rheumatoid arthritis and Crohn's disease.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Administração Oral , Animais , Área Sob a Curva , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Modelos Animais de Doenças , Meia-Vida , Humanos , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Células Jurkat , Microssomos Hepáticos/metabolismo , RatosRESUMO
Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.
Assuntos
Integrina alfa4/química , Polietilenoglicóis/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Ésteres , Meia-Vida , Humanos , Injeções Subcutâneas , Integrina alfa4/imunologia , Integrina alfa4/metabolismo , Células Jurkat , RatosRESUMO
Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores Notch/antagonistas & inibidores , Sulfonamidas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Animais , Área Sob a Curva , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cães , Relação Dose-Resposta a Droga , Desenho de Fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Químicos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/farmacocinética , Quinolinas/síntese química , Quinolinas/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Fatores de Tempo , Fatores de Transcrição HES-1RESUMO
A series of (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4-ylamino)-3-(4-(carbamoyloxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative compounds 11b and 11p showed efficacy in multiple in vivo animal models. The in vitro selectivity of 11p is also described.
Assuntos
Antirreumáticos/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/síntese química , Artrite Experimental/tratamento farmacológico , Asma/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Colágeno Tipo II/antagonistas & inibidores , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Fibronectinas/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Esclerose Múltipla/tratamento farmacológico , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese químicaRESUMO
BACKGROUND: Clinical studies of ß-amyloid (Aß) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aß plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aß pathology and after anti-Aß immunotherapy. METHODS: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aß pathology and after anti-Aß immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. RESULTS: The central vasculature displayed morphological abnormalities associated with vascular Aß deposits. Treatment with 3D6 antibody induced clearance of vascular Aß that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aß deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aß accumulation. CONCLUSIONS: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aß. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Vasos Sanguíneos/patologia , Encéfalo/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Aquaporina 4/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestrutura , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hemorragias Intracranianas/etiologia , Meninges/patologia , Meninges/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação/genética , Fatores de TempoRESUMO
Passive immunization with anti-Aß antibodies leads to the reduction of AD-like neuropathology in transgenic mice. Previously we showed that anti-Aß antibodies enter the brain and bind to amyloid plaques. Now using (125)I-labeled 3D6, the mouse parent antibody of the clinical candidate bapineuzumab, we further characterized the pharmacokinetic profile of this antibody in the brain and serum. Our studies demonstrated that following a single intravenous injection, the labeled antibody accumulates and persists in plaque rich regions of the brain in transgenic PDAPP mice. Accumulation was specific to amyloid since it did not occur in non-transgenic animals lacking human APP, could not be measured in transgenic animals prior to plaque deposition, and correlated with the level of plaque burden in aging transgenic mice. After a single intravenous injection, CNS levels of (125)I-labeled 3D6 continued to increase for 14 days even as serum levels of the antibody declined. The calculated half-life of antibody in the circulation was 6 days, while antibody levels in the CNS remained stable for nearly a month. When given at supra-therapeutic levels, unlabeled antibody did not compete with tracer levels of labeled antibody for accumulation in the CNS, indicating that the binding capacity of plaques was very high. Our results demonstrate that even when administered in the periphery at very low (tracer) doses, 3D6 and bapineuzumab cross the blood brain barrier to accumulate in plaque rich regions of the brain. CNS clearance is markedly slower than in the serum and correlates with binding to deposited amyloid in a transgenic model of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Amiloide/imunologia , Anticorpos/análise , Sistema Nervoso Central/imunologia , Envelhecimento/fisiologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Cerebelo/imunologia , Cerebelo/metabolismo , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Hipocampo/imunologia , Hipocampo/metabolismo , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Placa Amiloide/imunologia , Placa Amiloide/patologiaRESUMO
TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.
Assuntos
Plexo Corióideo/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Laminina/fisiologia , Células Th17/fisiologia , Animais , Antígeno CD146/metabolismo , Células CHO , Movimento Celular , Polaridade Celular , Proliferação de Células , Plexo Corióideo/imunologia , Plexo Corióideo/patologia , Cricetinae , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Interleucina-17/metabolismo , Interleucina-1beta/fisiologia , Interleucinas/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Ligação Proteica , Células Th17/metabolismo , Interleucina 22RESUMO
p75(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75(NTR) is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75(NTR), no p75(NTR) signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75(NTR) modulators and studying p75(NTR) function. Stimulation of p75(NTR)-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75(NTR) mutant could not substitute for wild-type p75(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75(NTR) as well as further characterizing downstream signaling events.
Assuntos
Caspase 3/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Células HEK293 , Humanos , Microscopia de Fluorescência , Fator de Crescimento Neural/farmacologia , Ratos , Receptor trkA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , TransfecçãoRESUMO
Several anti-amyloid ß (Aß) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aß antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aß plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aß plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aß species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aß(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aß moieties. Importantly C-terminal anti-Aß antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aß antibodies effectively interact with both soluble and insoluble forms of Aß and therefore appear particularly well suited for testing the Aß hypothesis of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/imunologia , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Epitopos/imunologia , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Animais , Anticorpos Neutralizantes , Sintomas Comportamentais/tratamento farmacológico , Sintomas Comportamentais/etiologia , Sintomas Comportamentais/imunologia , Biotina/metabolismo , Células Cultivadas , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Espinhas Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Embrião de Mamíferos , Epitopos/metabolismo , Medo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/imunologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/imunologia , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Fosforilação , Ligação Proteica/imunologia , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores de AMPA/metabolismo , Solubilidade , Proteína Vesicular 1 de Transporte de Glutamato/imunologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.
Assuntos
Encéfalo/metabolismo , Desenho de Fármacos , Degeneração Neural/prevenção & controle , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/farmacologia , Tiofenos/farmacocinética , Animais , Técnicas de Química Sintética , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Meia-Vida , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease, and non-selective inhibitors are protective in rodent disease models. Because of their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far, and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective, and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.
Assuntos
Desenho de Fármacos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteômica/métodos , Animais , Células Cultivadas , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.