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Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells.
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BACKGROUND: The Tau58/2 and Tau58/4 mouse lines expressing 0N4R tau with a P301S mutation mimic aspects of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In a side-by-side comparison, we report the age-dependent development of cognitive, motor, and behavioral deficits in comparison with the spatial-temporal evolution of cellular tau pathology in both models. METHODS: We applied the SHIRPA primary screen and specific neuromotor, behavioral, and cognitive paradigms. The spatiotemporal development of tau pathology was investigated immunohistochemically. Levels of sarkosyl-insoluble paired helical filaments were determined via a MesoScale Discovery biomarker assay. RESULTS: Neuromotor impairments developed from age 3 months in both models. On electron microscopy, spinal cord neurofibrillary pathology was visible in mice aged 3 months; however, AT8 immunoreactivity was not yet observed in Tau58/4 mice. Behavioral abnormalities and memory deficits occurred at a later stage (>9 months) when tau pathology was fully disseminated throughout the brain. Spatiotemporally, tau pathology spread from the spinal cord via the midbrain to the frontal cortex, while the hippocampus was relatively spared, thus explaining the late onset of cognitive deficits. CONCLUSIONS: Our findings indicate the face and construct validity of both Tau58 models, which may provide new, valuable insights into the pathologic effects of tau species in vivo and may consequently facilitate the development of new therapeutic targets to delay or halt neurodegenerative processes occurring in tauopathies.
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BACKGROUND: Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. OBJECTIVE: We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. METHODS: Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. RESULTS: Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. CONCLUSION: The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.
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Enfermedad de Alzheimer , Tauopatías , Ratones , Humanos , Animales , Proteínas tau/metabolismo , Tauopatías/patología , Enfermedad de Alzheimer/patología , Ratones Transgénicos , Modelos Animales de Enfermedad , Anticuerpos Monoclonales , Encéfalo/patologíaRESUMEN
BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.
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Barrera Hematoencefálica , Transcitosis , Barrera Hematoencefálica/metabolismo , Transcitosis/fisiología , Receptores de Transferrina , Transporte Biológico/fisiología , AnticuerposRESUMEN
INTRODUCTION: Diagnosis of Alzheimer's disease (AD) based on amyloid beta (A), pathologic tau (T), and neurodegeneration (N) biomarkers in peripheral fluids promises to accelerate clinical trials and intercept disease earlier. METHODS: Qualification of a Simoa plasma p217+tau assay was performed, followed by clinical utility evaluation in a cohort of 227 subjects with broad A and T spectrum. RESULTS: The p217+tau plasma assay was accurate, precise, dilution linear, and highly sensitive. All measured samples were within linear range of the assay, presented higher concentration in AD versus healthy controls (P < .0001), and plasma and cerebrospinal fluid levels correlated (r2 = 0.35). The plasma p217+tau results were predictive of central T and A status (area under the curve = 0.90 and 0.90, respectively) with low false +/- rates. DISCUSSION: The assay described here exhibits good technical performance and shows potential as a highly accurate peripheral biomarker for A or T status in AD and cognitively normal subjects.
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BACKGROUND: Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. METHODS: Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. RESULTS: We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. CONCLUSIONS: This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.
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Enfermedad de Alzheimer/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Epítopos , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Unión Proteica , Proteínas Recombinantes , Sonicación , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.
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Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales/metabolismo , Descubrimiento de Drogas/métodos , Proteínas tau/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas tau/químicaRESUMEN
BACKGROUND: Early and accurate detection and staging is critical to managing Alzheimer's disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-ß peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. OBJECTIVE: Develop an assay for measuring p217tau in CSF. METHODS: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217â+âtau, using the PT3 antibody. RESULTS: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217â+âtau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217â+âtau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217â+âtau-targeting immunotherapeutics. CONCLUSION: The assay for measuring p217â+âtau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.
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Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Proteínas tau/análisis , Proteínas tau/metabolismo , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquídeo , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.
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Encéfalo/fisiopatología , Red Nerviosa/fisiopatología , Vías Nerviosas/fisiopatología , Agregación Patológica de Proteínas/fisiopatología , Proteínas tau/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Ratones , Red Nerviosa/patología , Vías Nerviosas/patología , Agregación Patológica de Proteínas/patologíaRESUMEN
We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.
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Encéfalo/metabolismo , Microglía/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Ratones Transgénicos , Microglía/patología , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Tauopatías/patologíaRESUMEN
Aggregation of tau protein and spreading of tau aggregates are pivotal pathological processes in a range of neurological disorders. Accumulating evidence suggests that immunotherapy targeting tau may be a viable therapeutic strategy. We have previously described the isolation of antibody CBTAU-22.1 from the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to specifically bind a disease-associated phosphorylated epitope in the C-terminus of tau (Ser422) and to be able to inhibit the spreading of pathological tau aggregates from P301S spinal cord lysates in vitro, albeit with limited potency. Using a combination of rational design and random mutagenesis we have derived a variant antibody with improved affinity while maintaining the specificity of the parental antibody. This affinity improved antibody showed greatly enhanced potency in a cell-based immunodepletion assay using paired helical filaments (PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the affinity improved antibody limits the in vitro aggregation propensity of full length tau species specifically phosphorylated at position 422 produced by employing a native chemical ligation approach. Together, these results indicate that in addition to being able to inhibit the spreading of pathological tau aggregates, the matured antibody can potentially also interfere with the nucleation of tau which is believed to be the first step of the pathogenic process. Finally, the functionality in a P301L transgenic mice co-injection model highlights the therapeutic potential of human antibody dmCBTAU-22.1.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Anticuerpos/farmacología , Encéfalo/metabolismo , Serina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Afinidad de Anticuerpos/efectos de los fármacos , Autopsia , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Epítopos/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Mutagénesis , Mutación/genética , Fosforilación/fisiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/terapiaRESUMEN
The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.
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Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/metabolismo , Encéfalo/metabolismo , Proteínas tau/inmunología , Animales , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Resonancia por Plasmón de Superficie , Proteínas tau/deficiencia , Proteínas tau/genéticaRESUMEN
Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.
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Linfocitos B/metabolismo , Inmunoglobulina G/farmacología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Cristalización , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Epítopos Inmunodominantes/metabolismo , Masculino , Microglía/metabolismo , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Agregado de Proteínas , Adulto JovenRESUMEN
Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aß) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer's Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aß-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aß, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aß can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aß-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aß provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aß, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aß-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in AD.
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Péptidos beta-Amiloides/metabolismo , Proteínas Priónicas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Ratones Transgénicos , Mutación/genética , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Priónicas/ultraestructura , Agregación Patológica de Proteínas/inducido químicamente , Agregación Patológica de Proteínas/patología , Tauopatías/genética , Transfección , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, but the precise physiological function of the protein remains ill-defined. Recently, our group proposed a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses in the Drosophila melanogaster neuromuscular junctions.Flies harbor only one Lrrk gene, which might encompass the functions of both mammalian LRRK1 and LRRK2. We therefore studied the role of LRRK2 in mammalian synaptic function and provide evidence that knockout or pharmacological inhibition of LRRK2 results in defects in synaptic vesicle endocytosis, altered synaptic morphology and impairments in neurotransmission. In addition, our data indicate that mammalian endophilin A1 (EndoA1,also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73 and S75, two residues in the BAR domain. Hence, our results indicate that LRRK2 kinase activity has an important role in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequent neurotransmission at the synapse.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis/genética , Proteínas Serina-Treonina Quinasas/genética , Transmisión Sináptica/genética , Vesículas Sinápticas/genética , Animales , Células Cultivadas , Clatrina/metabolismo , Drosophila melanogaster , Dinamina I/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Hipocampo/citología , Hidrazonas/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Long-Evans , Sacarosa/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.
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Tauopatías , Proteínas tau/administración & dosificación , Proteínas tau/genética , Factores de Edad , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Ovillos Neurofibrilares/metabolismo , Tauopatías/inducido químicamente , Tauopatías/genética , Tauopatías/patología , Proteínas tau/químicaRESUMEN
After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson's disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme's activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways.
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Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Western Blotting , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Ratas , Ratas Long-Evans , Sensibilidad y EspecificidadRESUMEN
This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, ß and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1µM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.
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Extractos Vegetales/farmacología , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Thymelaeaceae/química , Factores de TiempoRESUMEN
LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2(G2019S) gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Drosophila/metabolismo , Endocitosis/fisiología , Unión Neuromuscular/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Aciltransferasas/genética , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/metabolismo , Células CHO , Calcio/metabolismo , Clatrina/metabolismo , Cricetinae , Drosophila , Proteínas de Drosophila/genética , Endocitosis/genética , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Espectrometría de Masas , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutación/genética , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Serina/genética , Serina/metabolismo , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/genética , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/fisiología , TransfecciónRESUMEN
CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon. Involvement of CRF(1) receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G(i)-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.