RESUMO
Anti-IgLON5 (IgLON5-IgG)-associated disease is a newly defined clinical entity. This literature review aims to evaluate its pathogenesis, which remains a pivotal question. Features that favour a primary neurodegenerative mechanism include the non-inflammatory tauopathy neuropathological signature and overrepresentation of microtubule-associated protein tau (MAPT) H1/H1 genotype as seen in other sporadic tauopathies. In contrast, the cell-surface localisation of IgLON5, capability of anti-IgLON5 antibodies to exert direct in vitro pathogenicity and disrupt IgLON5 interactions with its binding partners, human leukocyte antigen (HLA)-DRB1*10:01 and HLA-DQB1*05:01 allele preponderance with high affinity binding of IgLON5 peptides, and responsiveness to immunotherapy favour a primary autoimmune process. The presentation and course of anti-IgLON5-associated disease is heterogenous; hence, we hypothesise that a multitude of immune mechanisms are likely simultaneously operational in this disease cohort.
Assuntos
Moléculas de Adesão Celular Neuronais , Imunoglobulina G , Humanos , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Tauopatias/imunologia , Tauopatias/genética , Tauopatias/metabolismo , Animais , Proteínas tau/imunologia , Proteínas tau/metabolismo , Proteínas tau/genética , Autoanticorpos/imunologiaRESUMO
Tauopathies are a group of neurodegenerative diseases characterized by the presence of tau inclusions. We have developed over fifty anti-tau single-domain antibodies (sdAbs) derived from phage display libraries of a llama immunized with recombinant and pathological tau immunogens. We examined the therapeutic potential of four of these sdAbs in a Drosophila tauopathy model following their transgenic expression either in all neurons or neuronal subtypes. Three of these sdAbs showed therapeutic potential in various assays, effectively clearing pathological tau and attenuating or preventing tau-induced phenotypes that typically manifest as defects in neuronal axonal transport, neurodegeneration, functional impairments, and shortened lifespan. Of these three, one sdAb was superior in every assay, which may at least in part be attributed to its tau-binding epitope. These findings support its development as a gene therapy for tauopathies.
Assuntos
Anticorpos de Domínio Único , Tauopatias , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/imunologia , Animais , Tauopatias/imunologia , Tauopatias/patologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/farmacologia , Humanos , Modelos Animais de Doenças , Drosophila , Animais Geneticamente Modificados , Neurônios/metabolismo , Neurônios/patologia , Camelídeos Americanos/imunologia , Drosophila melanogaster/imunologiaRESUMO
Prior to the formation of amyloid fibrils, the pathological hallmark in tau-related neurodegenerative disease, tau monomers aggregate into a diverse range of oligomers. Granular tau oligomers, consisting of approximately 40 tau protein molecules, are present in the prefrontal cortex of patients at Braak stages I-II, preclinical stages of Alzheimer's disease (AD). Antibodies to granular tau oligomers as antigens have not been reported. Therefore, we generated new rat monoclonal antibodies by immunization with granular tau oligomers. Three antibodies from different hybridoma clones showed stronger immunoreactivity to granular tau oligomers and tau fibrils compared with monomeric tau. Of the three antibodies, 2D6-2C6 showed 3000-fold greater immunoreactivity in P301L-tau transgenic (rTg4510) mice than in non-transgenic mice, while MC1 antibody, which detects pathological conformations of tau, showed a 5.5-fold increase. These results suggest that 2D6-2C6 recognizes aggregates more specifically than MC1. In AD subjects, 2D6-2C6 recognized neurofibrillary tangles and pretangles, and co-localized within AT8-positive cells containing phosphorylated tau aggregates. The epitope of 2D6-2C6 is the 423-430 amino acid (AA) sequence of C-terminal regions. Taken together, a novel monoclonal antibody, 2D6-2C6, generated by immunization with granular tau oligomers binds to tau aggregates at the 423-430 AA sequence.
Assuntos
Doença de Alzheimer , Anticorpos Monoclonais , Camundongos Transgênicos , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/imunologia , Proteínas tau/química , Anticorpos Monoclonais/imunologia , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/imunologia , Ratos , Imunização , Feminino , Sequência de Aminoácidos , Epitopos/imunologia , Masculino , Idoso , Agregados Proteicos , Emaranhados Neurofibrilares/metabolismoRESUMO
Amyloid-ß (Aß) plaques and neurofibrillary tangles containing phosphorylated tau protein are major hallmarks of Alzheimer's disease (AD). Drug discovery efforts to target Aß and tau have been the primary focus for several decades. Recently, substantial breakthroughs have been achieved in the clinical development of Aß antibodies; aducanumab was approved under conditional accelerated pathway by Food and Drug Administration (FDA) in the U.S. as the first disease-modifying agent for treating AD, and lecanemab has been granted traditional full approved in the U.S. and Japan. In addition, donanemab met the primary endpoint in a phase 3 study. On the other hand, tau-targeting therapies have failed to show clinical benefit although that increased tau levels show a strong correlation with cognitive impairment relative to Aß depositions. Currently, tau immunotherapies, such as anti-tau antibodies and tau vaccines, have shown functional benefits in clinical trials. Also, clinical trials for combination therapy of Aß and tau antibodies to see their potential are being investigated. In this review, we provide updates on the results of clinical trials of anti-Aß antibodies and anti-tau therapeutics and suggest future directions for these therapeutics.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Proteínas tau , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/imunologia , Humanos , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismo , Proteínas tau/imunologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Imunoterapia , AnimaisRESUMO
Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer's disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents' ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the "oligomeric Tau" T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that "total" Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau "knockout" human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.
Assuntos
Anticorpos , Western Blotting , Encéfalo , Imuno-Histoquímica , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/imunologia , Humanos , Imuno-Histoquímica/métodos , Anticorpos/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Fosforilação , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/imunologia , Reprodutibilidade dos TestesRESUMO
The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.
Assuntos
Doença de Alzheimer , Anticorpos Monoclonais , Proteínas tau , Proteínas tau/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/biossíntese , Humanos , Camundongos , Doença de Alzheimer/imunologia , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/sangue , Ensaio de Imunoadsorção Enzimática , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Hibridomas/imunologia , Camundongos Endogâmicos BALB C , Especificidade de Anticorpos , Domínios Proteicos , Epitopos/imunologiaRESUMO
The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.
Assuntos
Proteínas Intrinsicamente Desordenadas , Anticorpos de Domínio Único , Proteínas tau , Humanos , Epitopos/química , Epitopos/imunologia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/imunologia , Peptídeos/química , Peptídeos/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
The hypothesis that neurodegenerative diseases are proteinopathies due to toxic effect of different underlying proteins, such as amyloid-beta and 3+4R-tau in Alzheimer's disease (AD) and alpha-synuclein in Parkinson's disease (PD), while still controversial is supported by several studies in the literature. This has led to conduct clinical trials attempting to reduce the load of these allegedly toxic proteins by immunotherapy, mostly but not solely based on antibodies against these proteins. Already completed clinical trials have ranged from initially negative results to recently partial positive outcomes, specifically for anti-amyloid antibodies in AD but also albeit to lesser degree for anti-synuclein antibodies in PD. Currently, there are several ongoing clinical trials in degenerative parkinsonisms with anti-synuclein approaches in PD and multiple system atrophy (MSA), as well as with anti-tau antibodies in 4R-tauopathies such as progressive supranuclear palsy (PSP). While it can be argued that expectations that part of these clinical trials will be positive can be hope or hype, it is reasonable to consider the future possibility of "cocktail" combination of different antibodies after the available experimental evidence of cross-talk between these proteins and neuropathological evidence of coexistence of these proteinopathies more frequently than expected by chance. Moreover, such "cocktail" approaches are widespread and accepted common practice in other fields such as oncology, and the complexity of neurodegenerative parkinsonisms makes reasonable the option for testing and eventually applying such combined approaches, should these prove useful separately, in the setting of patients with evidence of underlying concomitant proteinopathies, for example through biomarkers.
Assuntos
Imunoterapia , alfa-Sinucleína , Proteínas tau , Humanos , alfa-Sinucleína/imunologia , Imunoterapia/métodos , Proteínas tau/imunologia , Animais , Doença de Parkinson/terapia , Doença de Parkinson/imunologia , Doença de Parkinson/tratamento farmacológicoRESUMO
BACKGROUND: PNT001 is a humanized full-length IgG4 S228P monoclonal antibody that binds the cis conformation of the phosphorylated Thr231-Pro232 motif in human full-length (2N4R) tau (cis-pT231 tau) with high selectivity and affinity. It binds selectively to cis-pT231 tau in human tauopathy brain sections, inhibits aggregation of tau, and has shown efficacy in preclinical models of tauopathy. Good Laboratory Practice six-month toxicology studies in cynomolgous monkeys have shown no test article-related findings. OBJECTIVES: To evaluate the safety, tolerability, pharmacokinetics, and immunogenicity of single escalating intravenous doses of PNT001 in healthy volunteers. DESIGN: Phase 1, randomized, double-blind, and placebo-controlled 16-week study. SETTING: Subjects were recruited across three clinical research sites in the United States. PARTICIPANTS: Fifty healthy volunteer subjects enrolled, with 49 receiving the double-blind study drug. INTERVENTION: Six cohorts were administered single escalating doses of PNT001 (33, 100, 300, 900, 2,700, and 4,000 mg). The subjects were randomized 6:2 (PNT001:placebo). MEASUREMENTS: Safety was evaluated by the occurrence of adverse events, electrocardiography, physical examinations, neurological examinations, vital signs, and suicidality. Pharmacokinetics and biomarkers were assessed via serum and cerebrospinal fluid sample analyses. RESULTS: Dose continuation after review of sentinel group data and dose escalation after completion of full cohort data were determined by an external, independent safety board. There were no study pauses or safety concerns identified by the safety board. A total of 49 subjects received the study drugs, with 36 receiving PNT001 and 13 receiving placebo. There were three related non-serious adverse events, each Grade 1, which occurred at the lowest doses and resolved without sequelae. No maximum tolerated dose was identified, and no premature discontinuations, dose reductions, or interruptions due to treatment-related adverse events occurred. One unrelated serious adverse event occurred in a placebo subject with an undisclosed medical condition. No other safety findings were identified. Doses of 900-4,000 mg produced concentrations in the cerebrospinal fluid exceeding the binding affinity constant of PNT001 for cis-pT231 tau (45 ng/mL), indicating that concentrations sufficient for target engagement can be obtained in the cerebrospinal fluid within the tested dose range. The serum pharmacokinetic profile was as expected for a monoclonal antibody. The terminal half-lives ranged from 23.8-33.8 days, and the cerebrospinal fluid exposures were approximately 0.1% of the plasma concentration and dose-proportional. Of the 36 subjects receiving PNT001, one post-baseline positive anti-drug antibody result was observed at Day 112 in a subject who received PNT001 (300 mg). CONCLUSIONS: Single doses of PNT001 were safe and well-tolerated at all dose levels studied, including those doses expected to produce therapeutic benefit. These results support multiple ascending dose trials in patients with neurodegenerative tauopathies for this novel mid-domain tau antibody.
Assuntos
Anticorpos Monoclonais , Tauopatias , Humanos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Método Duplo-Cego , Voluntários Saudáveis , Tauopatias/imunologia , Tauopatias/terapia , Estados Unidos , Proteínas tau/imunologiaRESUMO
BACKGROUND: Soluble oligomeric forms of Tau protein have emerged as crucial players in the propagation of Tau pathology in Alzheimer's disease (AD). Our objective is to introduce a single-domain antibody (sdAb) named 2C5 as a novel radiotracer for the efficient detection and longitudinal monitoring of oligomeric Tau species in the human brain. METHODS: The development and production of 2C5 involved llama immunization with the largest human Tau isoform oligomers of different maturation states. Subsequently, 2C5 underwent comprehensive in vitro characterization for affinity and specificity via Enzyme-Linked Immunosorbent Assay and immunohistochemistry on human brain slices. Technetium-99m was employed to radiolabel 2C5, followed by its administration to healthy mice for biodistribution analysis. RESULTS: 2C5 exhibited robust binding affinity towards Tau oligomers (Kd = 6.280 nM ± 0.557) and to Tau fibers (Kd = 5.024 nM ± 0.453), with relatively weaker binding observed for native Tau protein (Kd = 1791 nM ± 8.714) and amyloid peptide (Kd > 10,000 nM). Remarkably, this SdAb facilitated immuno-histological labeling of pathological forms of Tau in neurons and neuritic plaques, yielding a high-contrast outcome in AD patients, closely mirroring the performance of reference antibodies AT8 and T22. Furthermore, 2C5 SdAb was successfully radiolabeled with 99mTc, preserving stability for up to 6 h post-radiolabeling (radiochemical purity > 93%). However, following intravenous injection into healthy mice, the predominant uptake occurred in kidneys, amounting to 115.32 ± 3.67, 97.70 ± 43.14 and 168.20 ± 34.52% of injected dose per gram (% ID/g) at 5, 10 and 45 min respectively. Conversely, brain uptake remained minimal at all measured time points, registering at 0.17 ± 0.03, 0.12 ± 0.07 and 0.02 ± 0.01% ID/g at 5, 10 and 45 min post-injection respectively. CONCLUSION: 2C5 demonstrates excellent affinity and specificity for pathological Tau oligomers, particularly in their early stages of oligomerization. However, the current limitation of insufficient blood-brain barrier penetration necessitates further modifications before considering its application in nuclear medicine imaging for humans.
Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Animais , Humanos , Camundongos , Doença de Alzheimer/diagnóstico por imagem , Encéfalo/patologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Proteínas tau/química , Proteínas tau/imunologia , Distribuição TecidualRESUMO
Sleep deprivation induces amyloid beta peptide and phosphorylated tau deposits in the brain and cerebrospinal fluid together with altered serotonin metabolism. Thus, it is likely that sleep deprivation is one of the predisposing factors in precipitating Alzheimer's disease (AD) brain pathology. Our previous studies indicate significant brain pathology following sleep deprivation or AD. Keeping these views in consideration in this review, nanodelivery of monoclonal antibodies to amyloid beta peptide (AßP), phosphorylated tau (p-tau), and tumor necrosis factor alpha (TNF-α) in sleep deprivation-induced AD is discussed based on our own investigations. Our results suggest that nanowired delivery of monoclonal antibodies to AßP with p-tau and TNF-α induces superior neuroprotection in AD caused by sleep deprivation, not reported earlier.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais , Encéfalo , Neuroproteção , Privação do Sono , Fator de Necrose Tumoral alfa/imunologia , Sistemas de Liberação de Fármacos por Nanopartículas/química , Sistemas de Liberação de Fármacos por Nanopartículas/farmacologia , Proteínas tau/imunologiaRESUMO
Extracellular deposition of amyloid-ß as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles are two of the characteristic hallmarks of Alzheimer's disease1,2. The regional progression of brain atrophy in Alzheimer's disease highly correlates with tau accumulation but not amyloid deposition3-5, and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. So far, little is known about the extent or role of the adaptive immune response and its interaction with the innate immune response in the presence of amyloid-ß or tau pathology6. Here we systematically compared the immunological milieux in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not those with amyloid deposition developed a unique innate and adaptive immune response and that depletion of microglia or T cells blocked tau-mediated neurodegeneration. Numbers of T cells, especially those of cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the Alzheimer's disease brain. T cell numbers correlated with the extent of neuronal loss, and the cells dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of interferon-γ and PDCD1 signalling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy- and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in Alzheimer's disease and primary tauopathies.
Assuntos
Encéfalo , Microglia , Emaranhados Neurofibrilares , Linfócitos T , Tauopatias , Animais , Camundongos , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Microglia/imunologia , Microglia/metabolismo , Emaranhados Neurofibrilares/imunologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Proteínas tau/imunologia , Proteínas tau/metabolismo , Tauopatias/imunologia , Tauopatias/metabolismo , Tauopatias/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Imunidade InataRESUMO
Neuronal clearance of proteins in the brain proves to be important for immunotherapy.
Assuntos
Anticorpos Monoclonais , Encéfalo , Imunoterapia , Neurônios , Tauopatias , Proteínas tau , Encéfalo/imunologia , Encéfalo/patologia , Neurônios/imunologia , Neurônios/patologia , Tauopatias/terapia , Proteínas tau/antagonistas & inibidores , Proteínas tau/imunologia , Anticorpos Monoclonais/uso terapêutico , Humanos , Animais , CamundongosRESUMO
Aggregates of the protein tau are proposed to drive pathogenesis in neurodegenerative diseases. Tau can be targeted by using passively transferred antibodies (Abs), but the mechanisms of Ab protection are incompletely understood. In this work, we used a variety of cell and animal model systems and showed that the cytosolic Ab receptor and E3 ligase TRIM21 (T21) could play a role in Ab protection against tau pathology. Tau-Ab complexes were internalized to the cytosol of neurons, which enabled T21 engagement and protection against seeded aggregation. Ab-mediated protection against tau pathology was lost in mice that lacked T21. Thus, the cytosolic compartment provides a site of immunotherapeutic protection, which may help in the design of Ab-based therapies in neurodegenerative disease.
Assuntos
Anticorpos Monoclonais , Imunização Passiva , Ribonucleoproteínas , Tauopatias , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Proteínas tau , Animais , Camundongos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Citosol/metabolismo , Modelos Animais de Doenças , Receptores Fc , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas tau/imunologia , Tauopatias/terapia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoAssuntos
Doença de Alzheimer , Anticorpos Monoclonais , Fármacos Neuroprotetores , Proteínas tau , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Humanos , Fármacos Neuroprotetores/imunologia , Fármacos Neuroprotetores/uso terapêutico , Proteínas tau/antagonistas & inibidores , Proteínas tau/imunologiaRESUMO
Human Tau protein is the most reliable biomarker for the prediction of Alzheimer's disease (AD). However, the assay to detect low concentrations of tau protein in serum is a great challenge for the early diagnosis of AD. This paper reports an electrochemiluminescence (ECL) immunosensor for Tau protein in serum samples. Gold nanostars (AuNSs) decorated on carbon nitride nanosheets (AuNS@g-CN nanostructure) show highly strong and stable ECL activity compared to pristine CN nanosheets due to the electrocatalytic and surface plasmon effects of AuNSs. As a result of the strong electromagnetic field at branches, AuNSs showed a better ECL enhancement effect than their spherical counterpart. For the fabrication of a specific immunosensor, immobilized AuNSs were functionalized with a monoclonal antibody specific for Tau protein. In the presence of Tau protein, the ECL intensity of the immunosensor decreased considerably. Under the optimal conditions, this ECL based immunosensor exhibits a dynamic linear range from 0.1 to 100 ng mL-1 with a low limit of detection of 0.034 ng mL-1. The LOD is less than the Tau level in human serum; thus, this study provides a useful method for the determination of Tau. The fabricated ECL immunosensor was successfully applied to the detection of Tau, the biomarker in serum samples. Therefore, the present approach is very promising for application in diagnosing AD within the early stages of the disease.
Assuntos
Doença de Alzheimer/sangue , Técnicas Biossensoriais/métodos , Nanoestruturas/química , Proteínas tau/sangue , Área Sob a Curva , Técnicas Biossensoriais/instrumentação , Ouro/química , Humanos , Limite de Detecção , Medições Luminescentes , Microscopia Eletrônica de Varredura , Nitrilas/química , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Proteínas tau/imunologiaRESUMO
Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.
Assuntos
Doença de Alzheimer , Imunoterapia , Tauopatias , Proteínas tau , Doença de Alzheimer/metabolismo , Anticorpos/uso terapêutico , Humanos , Imunoterapia/métodos , Neurônios/metabolismo , Tauopatias/tratamento farmacológico , Tauopatias/metabolismo , Tauopatias/patologia , Proteínas tau/imunologia , Proteínas tau/metabolismoRESUMO
One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.
Assuntos
Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Anticorpos Monoclonais/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.
Assuntos
Resinas Acrílicas/química , Separação Celular/métodos , Hidrocarbonetos Fluorados/química , Imunoensaio/métodos , Imagem Individual de Molécula/métodos , Anticorpos/análise , Anticorpos/imunologia , Separação Celular/instrumentação , Escherichia coli , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Imunoensaio/instrumentação , Imagem Individual de Molécula/instrumentação , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
A randomized, double-blind, placebo-controlled, 52-week study (no. NCT03068468) evaluated gosuranemab, an anti-tau monoclonal antibody, in the treatment of progressive supranuclear palsy (PSP). In total, 486 participants dosed were assigned to either gosuranemab (n = 321) or placebo (n = 165). Efficacy was not demonstrated on adjusted mean change of PSP Rating Scale score at week 52 between gosuranemab and placebo (10.4 versus 10.6, P = 0.85, primary endpoint), or at secondary endpoints, resulting in discontinuation of the open-label, long-term extension. Unbound N-terminal tau in cerebrospinal fluid decreased by 98% with gosuranemab and increased by 11% with placebo (P < 0.0001). Incidences of adverse events and deaths were similar between groups. This well-powered study suggests that N-terminal tau neutralization does not translate into clinical efficacy.