RESUMEN
Tau protein accumulation drives toxicity in several neurodegenerative disorders. To better understand the pathways regulating tau homeostasis in disease, we investigated the role of ubiquilins (UBQLNs)-a class of proteins linked to ubiquitin-mediated protein quality control (PQC) and various neurodegenerative diseases-in regulating tau. Cell-based assays identified UBQLN2 as the primary brain-expressed UBQLN to regulate tau. UBQLN2 efficiently lowered wild-type tau levels regardless of aggregation, suggesting that UBQLN2 interacts with and regulates tau protein under normal conditions or early in disease. Moreover, UBQLN2 itself proved to be prone to accumulation as insoluble protein in male and female tau transgenic mice and the human tauopathy progressive supranuclear palsy. Genetic manipulation of UBQLN2 in a tauopathy mouse model demonstrated that a physiological UBQLN2 balance is required for tau homeostasis. UBQLN2 overexpression exacerbated phosphorylated tau pathology and toxicity in mice expressing P301S mutant tau, whereas P301S mice lacking UBQLN2 showed significantly reduced phosphorylated tau. Further studies support the view that an imbalance of UBQLN2 perturbs ubiquitin-dependent PQC and autophagy. We conclude that changes in UBQLN2 levels, whether because of pathogenic mutations or secondary to disease states, such as tauopathy, contribute to proteostatic imbalances that exacerbate neurodegeneration.SIGNIFICANCE STATEMENT We defined a role for the protein quality control protein Ubiquilin-2 (UBQLN2), in age-related neurodegenerative tauopathies. This group of disorders is characterized by the accumulation of tau protein aggregates, which differ when UBQLN2 levels are altered. Given the lack of effective disease-modifying therapies for tauopathies and the function of UBQLN2 in handling various disease-linked proteins, we explored the role of UBQLN2 in regulating tau. We found that UBQLN2 reduced tau levels in cell models but behaved differently in mouse brain, where it accelerated mutant tau pathology and tau-mediated toxicity. A better understanding of the diverse functions of regulatory proteins like UBQLN2 can elucidate some of the causative factors in neurodegenerative disease and outline new routes to therapeutic intervention.
Asunto(s)
Enfermedades Neurodegenerativas , Tauopatías , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Tauopatías/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.
Asunto(s)
Amiloide/metabolismo , Anticuerpos Monoclonales/inmunología , Encéfalo/patología , Amiloide/antagonistas & inhibidores , Amiloide/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Encéfalo/inmunología , Estudios de Casos y Controles , Humanos , Ratones , Modelos Moleculares , Conformación ProteicaRESUMEN
Divergent protein context helps explain why polyglutamine expansion diseases differ clinically and pathologically. This heterogeneity may also extend to how polyglutamine disease proteins are handled by cellular pathways of proteostasis. Studies suggest, for example, that the ubiquitin-proteasome shuttle protein Ubiquilin-2 (UBQLN2) selectively interacts with specific polyglutamine disease proteins. Here we employ cellular models, primary neurons and mouse models to investigate the potential differential regulation by UBQLN2 of two polyglutamine disease proteins, huntingtin (HTT) and ataxin-3 (ATXN3). In cells, overexpressed UBQLN2 selectively lowered levels of full-length pathogenic HTT but not of HTT exon 1 fragment or full-length ATXN3. Consistent with these results, UBQLN2 specifically reduced accumulation of aggregated mutant HTT but not mutant ATXN3 in mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3), respectively. Normally a cytoplasmic protein, UBQLN2 translocated to the nuclei of neurons in HD mice but not in SCA3 mice. Remarkably, instead of reducing the accumulation of nuclear mutant ATXN3, UBQLN2 induced an accumulation of cytoplasmic ATXN3 aggregates in neurons of SCA3 mice. Together these results reveal a selective action of UBQLN2 toward polyglutamine disease proteins, indicating that polyglutamine expansion alone is insufficient to promote UBQLN2-mediated clearance of this class of disease proteins. Additional factors, including nuclear translocation of UBQLN2, may facilitate its action to clear intranuclear, aggregated disease proteins like HTT.
Asunto(s)
Ataxina-3/genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Machado-Joseph/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Relacionadas con la Autofagia/genética , Modelos Animales de Enfermedad , Exones , Heterogeneidad Genética , Humanos , Ratones , Neuronas/metabolismo , Neuronas/patología , Péptidos/genética , Complejo de la Endopetidasa ProteasomalRESUMEN
UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Agregación Patológica de Proteínas , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas Relacionadas con la Autofagia , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Mutación , Neuronas , Conformación Proteica , Dominios Proteicos , UbiquitinaRESUMEN
Antibodies that recognize amyloidogenic aggregates with high conformational and sequence specificity are important for detecting and potentially treating a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, these types of antibodies are challenging to generate because of the large size, hydrophobicity, and heterogeneity of protein aggregates. To address this challenge, we developed a method for generating antibodies specific for amyloid aggregates. First, we grafted amyloidogenic peptide segments from the target polypeptide [Alzheimer's amyloid-ß (Aß) peptide] into the complementarity-determining regions (CDRs) of a stable antibody scaffold. Next, we diversified the grafted and neighboring CDR sites using focused mutagenesis to sample each WT or grafted residue, as well as one to five of the most commonly occurring amino acids at each site in human antibodies. Finally, we displayed these antibody libraries on the surface of yeast cells and selected antibodies that strongly recognize Aß-amyloid fibrils and only weakly recognize soluble Aß. We found that this approach enables the generation of monovalent and bivalent antibodies with nanomolar affinity for Aß fibrils. These antibodies display high conformational and sequence specificity as well as low levels of nonspecific binding and recognize a conformational epitope at the extreme N terminus of human Aß. We expect that this systematic approach will be useful for generating antibodies with conformational and sequence specificity against a wide range of peptide and protein aggregates associated with neurodegenerative disorders.
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Péptidos beta-Amiloides , Regiones Determinantes de Complementariedad , Anticuerpos de Cadena Única , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Humanos , Mutagénesis Sitio-Dirigida , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
The ubiquitin-binding proteasomal shuttle protein UBQLN2 is implicated in common neurodegenerative disorders due to its accumulation in disease-specific aggregates and, when mutated, directly causes familial frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). Like other proteins linked to FTD/ALS, UBQLN2 undergoes phase separation to form condensates. The relationship of UBQLN2 phase separation and accumulation to neurodegeneration, however, remains uncertain. Employing biochemical, neuropathological and behavioral assays, we studied the impact of overexpressing WT or mutant UBQLN2 in the CNS of transgenic mice. Expression of UBQLN2 harboring a pathogenic mutation (P506T) elicited profound and widespread intraneuronal inclusion formation and aggregation without prominent neurodegenerative or behavioral changes. Both WT and mutant UBQLN2 formed ubiquitin- and P62-positive inclusions in neurons, supporting the view that UBQLN2 is intrinsically prone to phase separate, with the size, shape and frequency of inclusions depending on expression level and the presence or absence of a pathogenic mutation. Overexpression of WT or mutant UBQLN2 resulted in a dose-dependent decrease in levels of a key interacting chaperone, HSP70, as well as dose-dependent profound degeneration of the retina. We conclude that, at least in mice, robust aggregation of a pathogenic form of UBQLN2 is insufficient to cause neuronal loss recapitulating that of human FTD/ALS. Our results nevertheless support the view that altering the normal cellular balance of UBQLN2, whether wild type or mutant protein, has deleterious effects on cells of the CNS and retina that likely reflect perturbations in ubiquitin-dependent protein homeostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Modelos Animales de Enfermedad , Degeneración Nerviosa/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Relacionadas con la Autofagia/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Proteostasis/fisiologíaRESUMEN
The culmination of many years of increasing research into the toxicity of tau aggregation in neurodegenerative disease has led to the consensus that soluble, oligomeric forms of tau are likely the most toxic entities in disease. While tauopathies overlap in the presence of tau pathology, each disease has a unique combination of symptoms and pathological features; however, most study into tau has grouped tau oligomers and studied them as a homogenous population. Established evidence from the prion field combined with the most recent tau and amyloidogenic protein research suggests that tau is a prion-like protein, capable of seeding the spread of pathology throughout the brain. Thus, it is likely that tau may also form prion-like strains or diverse conformational structures that may differ by disease and underlie some of the differences in symptoms and pathology in neurodegenerative tauopathies. The development of techniques and new technology for the detection of tau oligomeric strains may, therefore, lead to more efficacious diagnostic and treatment strategies for neurodegenerative disease. [Formula: see text].
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Amiloide/metabolismo , Agregación Patológica de Proteínas/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Proteínas Priónicas/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/patología , Tauopatías/patologíaRESUMEN
Recent findings suggest that tau oligomers, which form before neurofibrillary tangles (NFTs), are the true neurotoxic tau entities in neurodegenerative tauopathies, including Alzheimer's disease (AD). Studies in animal models of tauopathy suggest that tau oligomers play a key role in eliciting behavioral and cognitive impairments. Here, we used a novel tau oligomer-specific monoclonal antibody (TOMA) for passive immunization in mice expressing mutant human tau. A single dose of TOMA administered either intravenously or intracerebroventricularly was sufficient to reverse both locomotor and memory deficits in a mouse model of tauopathy for 60 d, coincident with rapid reduction of tau oligomers but not phosphorylated NFTs or monomeric tau. Our data demonstrate that antibody protection is mediated by extracellular and rapid peripheral clearance. These findings provide the first direct evidence in support of a critical role for tau oligomers in disease progression and validate tau oligomers as a target for the treatment of AD and other neurodegenerative tauopathies.
Asunto(s)
Enfermedad de Alzheimer/terapia , Inmunización Pasiva , Ovillos Neurofibrilares/inmunología , Tauopatías/terapia , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Ovillos Neurofibrilares/genética , Ovillos Neurofibrilares/metabolismo , Fosforilación , Tauopatías/genética , Tauopatías/inmunología , Tauopatías/metabolismo , Proteínas tau/genéticaRESUMEN
The key protein implicated in Parkinson's disease and other synucleinopathies is α-synuclein, and a post-translationally modified form of the protein, phosphorylated at serine 129 (pS129), is a principal component in Lewy bodies, a pathological hallmark of PD. While altered proteostasis has been implicated in the etiology of Parkinson's disease, we still have a limited understanding of how α-synuclein is regulated in the nervous system. The protein quality control protein Ubiquilin-2 (UBQLN2) is known to accumulate in synucleinopathies, but whether it directly regulates α-synuclein is unknown. Using cellular and mouse models, we find that UBQLN2 decreases levels of α-synuclein, including the pS129 phosphorylated isoform. Pharmacological inhibition of the proteasome revealed that, while α-synuclein may be cleared by parallel and redundant quality control pathways, UBQLN2 preferentially targets pS129 for proteasomal degradation. Moreover, in brain tissue from human PD and transgenic mice expressing pathogenic α-synuclein (A53T), native UBQLN2 becomes more insoluble. Collectively, our studies support a role for UBQLN2 in directly regulating pathological forms of α-synuclein and indicate that UBQLN2 dysregulation in disease may contribute to α-synuclein-mediated toxicity.
Asunto(s)
Enfermedad de Parkinson , Sinucleinopatías , Ratones , Animales , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Sinucleinopatías/metabolismo , Cuerpos de Lewy/metabolismo , Ratones Transgénicos , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.
RESUMEN
After natural menopause in women, androstenedione becomes the primary hormone secreted by the residual follicle-depleted ovaries. In two independent studies, in rodents that had undergone ovarian follicular depletion, we found that higher endogenous serum androstenedione levels correlated with increased working memory errors. This led to the hypothesis that higher androstenedione levels impair memory. The current study directly tested this hypothesis, examining the cognitive effects of exogenous androstenedione administration in rodents. Middle-aged ovariectomised rats received vehicle or one of two doses of androstenedione. Rats were tested on a spatial working and reference memory maze battery including the water-radial arm maze, Morris water maze (MM) and delay match-to-sample task. Androstenedione at the highest dose impaired reference memory as well as the ability to maintain performance as memory demand was elevated. This was true for both high temporal demand memory retention of one item of spatial information, as well as the ability to handle multiple items of spatial working memory information. We measured glutamic acid decarboxylase (GAD) protein in multiple brain regions to determine whether the gamma-aminobutyric acid (GABA) system relates to androstenedione-induced memory impairments. Results showed that higher entorhinal cortex GAD levels were correlated with worse MM performance, irrespective of androstenedione treatment. These findings suggest that androstenedione, the main hormone produced by the follicle-depleted ovary, is detrimental to working memory, reference memory and memory retention. Furthermore, while spatial reference memory performance might be related to the GABAergic system, it does not appear to be altered with androstenedione administration, at least at the doses used in the current study.
Asunto(s)
Androstenodiona/sangre , Trastornos de la Memoria/sangre , Animales , Corteza Entorrinal/metabolismo , Femenino , Glutamato Descarboxilasa/metabolismo , Aprendizaje por Laberinto , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Memoria a Corto Plazo , Menopausia/sangre , Menopausia/fisiología , Ovariectomía , Ratas , Ratas Endogámicas F344 , Retención en PsicologíaRESUMEN
The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.
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Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , Agregado de Proteínas , Células HEK293 , HumanosRESUMEN
BACKGROUND: The coexistence of α-synuclein and tau aggregates in several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, raises the possibility that a seeding mechanism is involved in disease progression. METHODS: To further investigate the role of α-synuclein in the tau aggregation pathway, we performed a set of experiments using both recombinant and brain-derived tau and α-synuclein oligomers to seed monomeric tau aggregation in vitro and in vivo. Brain-derived tau oligomers were isolated from well-characterized cases of progressive supranuclear palsy (n = 4) and complexes of brain-derived α-synuclein/tau oligomers isolated from patients with Parkinson's disease (n = 4). The isolated structures were purified and characterized by standard biochemical methods, then injected into Htau mice (n = 24) to assess their toxicity and role in tau aggregation. RESULTS: We found that α-synuclein induced a distinct toxic tau oligomeric strain that avoids fibril formation. In vivo, Parkinson's disease brain-derived α-synuclein/tau oligomers administered into Htau mouse brains accelerated endogenous tau oligomer formation concurrent with increasing cell loss. CONCLUSIONS: Our findings provide evidence, for the first time, that α-synuclein enhances the harmful effects of tau, thus contributing to disease progression.
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Encéfalo/metabolismo , Encéfalo/patología , Enfermedad de Parkinson/metabolismo , Parálisis Supranuclear Progresiva/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Ratones , Proteínas RecombinantesRESUMEN
Purpose: Retinal ganglion cell (RGC) death following axonal injury occurring in traumatic optic neuropathy (TON) causes irreversible vision loss. GRP78 is a molecular chaperone that enhances protein folding and controls activation of endoplasmic reticulum (ER) stress pathways. This study determined whether adeno-associated virus (AAV)-mediated gene transfer of GRP78 protected RGCs from death in a mouse model of TON induced by optic nerve crush (ONC). Methods: ONC was induced by a transient crush of optic nerve behind the eye globe. AAV was used to deliver genes into retina. Molecules in the ER stress branches, tau oligomers, and RGC injury were determined by immunohistochemistry or Western blot. Results: Among tested AAV serotypes, AAV2 was the most efficient for delivering genes to RGCs. Intravitreal delivery of AAV2-GRP78 markedly attenuated ER stress and RGC death 3 days after ONC, and significantly improved RGC survival and function 7 days after ONC. Protein aggregation is increased during ER stress and aggregated proteins such as tau oligomers are key players in neurodegenerative diseases. AAV2-GRP78 alleviated ONC-induced increases in tau phosphorylation and oligomerization. Furthermore, tau oligomers directly induced RGC death, and blocking tau oligomers with tau oligomer monoclonal antibody (TOMA) attenuated ONC-induced RGC loss. Conclusion: These data indicate that the beneficial effect of AAV2-GRP78 is partially mediated by the reduction of misfolded tau, and provide compelling evidence that gene therapy with AAV2-GRP78 or immunotherapy with TOMA offers novel therapeutic approaches to alleviate RGC loss in TON.
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Dependovirus/genética , Estrés del Retículo Endoplásmico/fisiología , Proteínas de Choque Térmico/genética , Traumatismos del Nervio Óptico/prevención & control , Células Ganglionares de la Retina/metabolismo , Transfección , Proteínas tau/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Electrorretinografía , Chaperón BiP del Retículo Endoplásmico , Expresión Génica/fisiología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Compresión Nerviosa , Traumatismos del Nervio Óptico/metabolismo , Agregado de Proteínas , Daño por Reperfusión/prevención & control , Tomografía de Coherencia ÓpticaRESUMEN
BACKGROUND: We have evaluated the efficacy of targeting the toxic, oligomeric form of tau protein by passive immunotherapy in a mouse model of synucleinopathy. Parkinson's disease and Lewy body dementia are two of the most common neurodegenerative disorders and are primarily characterized by the accumulation of α-synuclein in Lewy bodies. However, evidence shows that smaller, oligomeric aggregates are likely the most toxic form of the protein. Moreover, a large body of research suggests that α-synuclein interacts with tau in disease and may act in a synergistic mechanism, implicating tau oligomers as a potential therapeutic target. METHODS: We treated seven-month-old mice overexpressing mutated α-synuclein (A53T mice) with tau oligomer-specific monoclonal antibody (TOMA) and a control antibody and assessed both behavioral and pathological phenotypes. RESULTS: We found that A53T mice treated with TOMA were protected from cognitive and motor deficits two weeks after a single injection. Levels of toxic tau oligomers were specifically decreased in the brains of TOMA-treated mice. Tau oligomer depletion also protected against dopamine and synaptic protein loss. CONCLUSION: These results indicate that targeting tau oligomers is beneficial for a mouse model of synucleinopathy and may be a viable therapeutic strategy for treating diseases in which tau and α-synuclein have a synergistic toxicity.
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Anticuerpos Monoclonales/farmacología , Encéfalo/efectos de los fármacos , Inmunoterapia/métodos , alfa-Sinucleína , Proteínas tau/antagonistas & inhibidores , Animales , Encéfalo/patología , Humanos , Inmunización Pasiva , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/patologíaRESUMEN
Tau oligomers have been shown to be the main toxic tau species in a number of neurodegenerative disorders. In order to study tau oligomers both in vitro and in vivo, we have established methods for the reliable preparation, isolation, and detection of tau oligomers. Methods for the seeding of tau oligomers, isolation of tau oligomers from tissue, and detection of tau oligomers using tau oligomer-specific antibodies by biochemical and immunohistochemical methods are detailed below.
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Tauopatías/metabolismo , Proteínas tau/química , Proteínas tau/aislamiento & purificación , Amiloide/metabolismo , Animales , Humanos , InmunohistoquímicaRESUMEN
It is well-established that inflammation plays an important role in Alzheimer's disease (AD) and frontotemporal lobar dementia (FTLD). Inflammation and synapse loss occur in disease prior to the formation of larger aggregates, but the contribution of tau to inflammation has not yet been thoroughly investigated. Tau pathologically aggregates to form large fibrillar structures known as tangles. However, evidence suggests that smaller soluble aggregates, called oligomers, are the most toxic species and form prior to tangles. Furthermore, tau oligomers can spread to neighboring cells and between anatomically connected brain regions. In addition, recent evidence suggests that inspecting the retina may be a window to brain pathology. We hypothesized that there is a relationship between tau oligomers and inflammation, which are hallmarks of early disease. We conducted immunofluorescence and biochemical analyses on tauopathy mice, FTLD, and AD subjects. We showed that oligomers co-localize with astrocytes, microglia, and HMGB1, a pro-inflammatory cytokine. Additionally, we show that tau oligomers are present in the retina and are associated with inflammatory cells suggesting that the retina may be a valid non-invasive biomarker for brain pathology. These results suggest that there may be a toxic relationship between tau oligomers and inflammation. Therefore, the ability of tau oligomers to spread may initiate a feed-forward cycle in which tau oligomers induce inflammation, leading to neuronal damage, and thus more inflammation. Further mechanistic studies are warranted in order to understand this relationship, which may have critical implications for improving the treatment of tauopathies.
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Encefalitis/etiología , Enfermedades Neurodegenerativas/complicaciones , Retinitis/etiología , Retinitis/metabolismo , Proteínas tau/metabolismo , Factores de Edad , Animales , Astrocitos/metabolismo , Astrocitos/patología , Proteínas de Unión al Calcio/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalitis/metabolismo , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína HMGB1/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Mutación/genética , Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Neuronas/patología , Retinitis/patología , Proteínas tau/genéticaRESUMEN
The importance of vascular contributions to cognitive impairment and dementia (VCID) associated with Alzheimer's disease (AD) and related neurodegenerative diseases is increasingly recognized, however, the underlying mechanisms remain obscure. There is growing evidence that in addition to Aß deposition, accumulation of hyperphosphorylated oligomeric tau contributes significantly to AD etiology. Tau oligomers are toxic and it has been suggested that they propagate in a "prion-like" fashion, inducing endogenous tau misfolding in cells. Their role in VCID, however, is not yet understood. The present study was designed to determine the severity of vascular deposition of oligomeric tau in the brain in patients with AD and related tauopathies, including dementia with Lewy bodies (DLB) and progressive supranuclear palsy (PSP). Further, we examined a potential link between vascular deposition of fibrillar Aß and that of tau oligomers in the Tg2576 mouse model. We found that tau oligomers accumulate in cerebral microvasculature of human patients with AD and PSP, in association with vascular endothelial and smooth muscle cells. Cerebrovascular deposition of tau oligomers was also found in DLB patients. We also show that tau oligomers accumulate in cerebral microvasculature of Tg2576 mice, partially in association with cerebrovascular Aß deposits. Thus, our findings add to the growing evidence for multifaceted microvascular involvement in the pathogenesis of AD and other neurodegenerative diseases. Accumulation of tau oligomers may represent a potential novel mechanism by which functional and structural integrity of the cerebral microvessels is compromised.
RESUMEN
Various neurodegenerative diseases are characterized by the accumulation of amyloidogenic proteins such as tau, α-synuclein, and amyloid-ß. Prior to the formation of these stable aggregates, intermediate species of the respective proteins-oligomers-appear. Recently acquired data have shown that oligomers may be the most toxic and pathologically significant to neurodegenerative diseases such as Alzheimer's and Parkinson's. The covalent modification of these oligomers may be critically important for biological processes in disease. Ubiquitin and small ubiquitin-like modifiers are the commonly used tags for degradation. While the modification of large amyloid aggregates by ubiquitination is well established, very little is known about the role ubiquitin may play in oligomer processing and the importance of the more recently discovered sumoylation. Many proteins involved in neurodegeneration have been found to be sumoylated, notably tau protein in brains afflicted with Alzheimer's. This evidence suggests that while the cell may not have difficulty recognizing dangerous proteins, in brains afflicted with neurodegenerative disease, the proteasome may be unable to properly digest the tagged proteins. This would allow toxic aggregates to develop, leading to even more proteasome impairment in a snowball effect that could explain the exponential progression in most neurodegenerative diseases. A better understanding of the covalent modifications of oligomers could have a huge impact on the development of therapeutics for neurodegenerative diseases. This review will focus on the proteolysis of tau and other amyloidogenic proteins induced by covalent modification, and recent findings suggesting a relationship between tau oligomers and sumoylation.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Proteolisis , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
Harmine is a naturally occurring monoamine oxidase inhibitor that has recently been shown to selectively inhibit the dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A). We investigated the cognitive effects of 1mg (low) Harmine and 5mg (high) Harmine using the delayed-match-to-sample (DMS) asymmetrical 3-choice water maze task to evaluate spatial working and recent memory, and the Morris water maze task (MM) to test spatial reference memory. Animals were also tested on the visible platform task, a water-escape task with the same motor, motivational, and reinforcement components as the other tasks used to evaluate cognition, but differing in its greater simplicity and that the platform was visible above the surface of the water. A subset of the Harmine-high treated animals showed clear motor impairments on all behavioral tasks, and the visible platform task confirmed a lack of competence to perform the procedural components of water maze testing. After excluding animals from the high dose group that could not perform the procedural components of a swim task, it was revealed that both high- and low-dose treatment with Harmine enhanced performance on the latter portion of DMS testing, but had no effect on MM performance. Thus, this study demonstrates the importance of confirming motor and visual competence when studying animal cognition, and verifies the one-day visible platform task as a reliable measure of ability to perform the procedural components necessary for completion of a swim task.