RESUMEN
Amyloid-ß oligomers may cause cognitive deficits in Alzheimer's disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-ß oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer's disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-ß-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapia , Cognición/fisiología , Receptor EphB2/deficiencia , Receptor EphB2/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Línea Celular , Células Cultivadas , Giro Dentado/metabolismo , Modelos Animales de Enfermedad , Humanos , Potenciación a Largo Plazo , Memoria/fisiología , Ratones , Ratones Transgénicos , Plasticidad Neuronal , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Receptor EphB2/química , Receptor EphB2/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.
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Anticuerpos Biespecíficos , Linfocitos T , Humanos , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Neutralizantes/inmunologíaRESUMEN
Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.
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Productos Biológicos , Humanos , Anticuerpos , Desarrollo de Medicamentos , Medición de RiesgoRESUMEN
Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.
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Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Azul de Metileno/farmacología , Azul de Metileno/uso terapéutico , Proteínas del Tejido Nervioso/metabolismo , Análisis de Varianza , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Drosophila , Embrión de Mamíferos , Antagonistas de Aminoácidos Excitadores/toxicidad , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Ácido Quinurénico/toxicidad , Ratones , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica , Mutación/genética , Proteínas del Tejido Nervioso/genética , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/prevención & control , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Desempeño Psicomotor , Ratas , Prueba de Desempeño de Rotación con Aceleración Constante , Transfección , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.
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Biomarcadores/metabolismo , Enfermedad de Parkinson/diagnóstico , alfa-Sinucleína/química , Animales , Anticuerpos/metabolismo , Bioquímica/métodos , Estudios de Cohortes , Diseño de Fármacos , Femenino , Transferencia Resonante de Energía de Fluorescencia/métodos , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , Inmunoensayo/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , ARN Interferente Pequeño/metabolismo , alfa-Sinucleína/líquido cefalorraquídeoRESUMEN
Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.
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Enfermedades Neurodegenerativas/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Péptidos/química , Péptidos/toxicidad , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Epítopos/química , Epítopos/inmunología , Epítopos/toxicidad , Células HEK293 , Humanos , Cuerpos de Inclusión/química , Peso Molecular , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Péptidos/inmunología , Relación Estructura-Actividad , Expansión de Repetición de TrinucleótidoRESUMEN
ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.
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Inmunoglobulina G , Neoplasias , Humanos , Ratones , Animales , Interleucina-2 , Ratones Transgénicos , Leucocitos Mononucleares , InmunoterapiaRESUMEN
The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.
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Anticuerpos , HumanosRESUMEN
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
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Receptores Quiméricos de Antígenos , Vacunas , Biomarcadores/análisis , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Activa , Reacción en Cadena de la PolimerasaRESUMEN
Huntington disease (HD) is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis.
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Proteínas Mutantes/química , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Péptidos/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Humanos , Proteína Huntingtina , Técnicas In Vitro , Ratones , Microscopía de Fuerza Atómica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos/química , Expansión de Repetición de TrinucleótidoRESUMEN
Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.
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Adenosina Trifosfato/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Cuerpos de Inclusión/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Adenosina Trifosfato/genética , Animales , Bovinos , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteína Huntingtina , Cuerpos de Inclusión/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Células PC12 , Ratas , SolubilidadRESUMEN
Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.
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Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Choque Térmico HSP72/deficiencia , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/genética , Factores de Edad , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas del Choque Térmico HSP72/clasificación , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/mortalidad , Cuerpos de Inclusión/patología , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Trastornos del Movimiento/etiología , Trastornos del Movimiento/genética , Proteínas del Tejido Nervioso/metabolismo , Examen Neurológico/métodos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Expansión de Repetición de Trinucleótido/genética , Pérdida de Peso/genéticaRESUMEN
Given the expanding number of complex therapeutic protein drugs and advanced therapy medicinal products that are being developed, improving our ability to assess the potential immunogenicity of biologics is critical to ensuring treatment efficacy and patient safety. In this context, the European Immunogenicity Platform annual meeting provides opportunities for experts from industry and academia, regulators and clinicians to convene and discuss immunogenicity assessment methods and tools. This report summarizes the key messages on immunogenicity testing, prediction, clinical relevance and advanced therapy medicinal products discussed at the 11th Open Scientific European Immunogenicity Platform Symposium on Immunogenicity of Biopharmaceuticals, Lisbon, Portugal, 17-19 February 2020.
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Biofarmacia/métodos , Inmunogenética/métodos , Europa (Continente) , HumanosRESUMEN
A pathological hallmark of many protein-misfolding diseases is the formation of insoluble aggregates. Quantitative methods are needed to better resolve and define the formation, aggregation, and temporal dynamics of soluble misfolded proteins in native settings. In this book chapter we describe simple and sensitive detection methods to characterize high ordered aggregates (AGERA) and subsets of distinct soluble aggregates (SEC-FRET) of mutant huntingtin protein in biological samples.
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Mutación , Proteínas del Tejido Nervioso , Proteínas Nucleares , Deficiencias en la Proteostasis , Animales , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Inmunoensayo/métodos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patologíaRESUMEN
A common pathological hallmark in many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, is the formation of fibrillar protein aggregates referred to as amyloids. The amyloidogenic aggregates were long thought to be toxic, but mounting evidence supports the notion that a variety of amyloid aggregate intermediates to fibril formation, termed oligomers, may in fact be the primary culprit leading to neuronal dysfunction and cell death. While amyloid formation is a complex, heterogeneous process, aggregates formed by diverse, diseases-related proteins share many conformational similarities, suggesting common toxic mechanisms among these diseases. Ideally, similar therapeutic strategies may be applicable. This review focuses on the potential role of amyloidogenic oligomers in neurodegenerative disease, highlighting some promising therapeutic strategies.
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Proteínas Amiloidogénicas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Amiloidogénicas/química , Animales , Humanos , Lípidos de la Membrana , Chaperonas Moleculares , Conformación Proteica , Interferencia de ARNRESUMEN
A common pathological hallmark of protein-conformational brain diseases is the formation of disease-specific protein aggregates. In Alzheimer's disease, these are comprised of amyloid-ß and Tau as opposed to α-synuclein in Parkinson's disease and N-terminal fragments of mutant huntingtin in Huntington's disease. Most aggregates also sequester molecular chaperones, a protein family that assists in the folding, refolding, stabilization, and processing of client proteins, including misfolded proteins in brain diseases. Molecular chaperone modulation has achieved remarkable therapeutic effects in some cellular and preclinical animal models of protein-conformational diseases. This has raised hope for chaperone-based strategies to combat these diseases. Here, we review briefly the functional diversity and medical significance of molecular chaperones, their therapeutic potential, and common and specific challenges towards clinical application.
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Encéfalo/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/uso terapéutico , Deficiencias en la Proteostasis/terapia , Animales , Humanos , Deficiencias en la Proteostasis/patologíaRESUMEN
The Huntington's disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a ß-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models.
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Amiloide/química , Amiloide/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Animales , Química Encefálica , Línea Celular Tumoral , Humanos , Proteína Huntingtina , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutación/genéticaRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the amplification of a polyglutamine stretch at the N terminus of the huntingtin protein. N-terminal fragments of the mutant huntingtin (mHtt) aggregate and form intracellular inclusions in brain and peripheral tissues. Aggregates are an important hallmark of the disease, translating into a high need to quantify them in vitro and in vivo. We developed a one-step TR-FRET-based immunoassay to quantify soluble and aggregated mHtt in cell and tissue homogenates. Strikingly, quantification revealed a decrease of soluble mHtt correlating with an increase of aggregated protein in primary neuronal cell cultures, transgenic R6/2, and HdhQ150 knock-in HD mice. These results emphasize the assay's efficiency for highly sensitive and quantitative detection of soluble and aggregated mHtt and its application in high-throughput screening and characterization of HD models.
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Enfermedad de Huntington/metabolismo , Inmunoensayo , Proteínas del Tejido Nervioso/análisis , Proteínas Nucleares/análisis , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Sustitución del Gen , Proteína Huntingtina , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington's Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.
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Envejecimiento , Enfermedad de Huntington/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Cromatografía/métodos , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Ratones , Modelos Biológicos , Unión Proteica , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiologíaRESUMEN
Polyglutamine(polyQ)-expanded proteins are potential therapeutic targets for the treatment of polyQ expansion disorders such as Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3). Here, we used an amino-terminal fragment of a mutant Huntingtin protein (Htt-N-82Q) as bait in an unbiased screen of a 60,000 peptoid library. Peptoid HQP09 was selected from the isolated hits and confirmed as a specific ligand of Htt-N-82Q and Atxn3-77Q mutant proteins in biochemical experiments. We identified three critical residues in the HQP09 sequence that are important for its activity and generated a minimal derivative, HQP09_9, which maintains the specific polyQ-binding activity. We demonstrated that HQP09 and HQP09_9 inhibited aggregation of Htt-N-53Q in vitro and exerted Ca(2+)-stabilizing and neuroprotective effects in experiments with primary striatal neuronal cultures derived from HD mice. We further demonstrated that intracerebroventricular delivery of HQP09 to an HD mouse model resulted in reduced accumulation of mutant Huntingtin aggregates and improved motor behavioral outcomes. These results suggest that HQP09 and similar peptoids hold promise as novel therapeutics for developing treatments for HD, SCA3, and other polyglutamine expansion disorders.