RESUMO
The aggregation of α-synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super-resolution and atomic force microscopy imaging of individual α-synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α-synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.
Assuntos
Amiloide/metabolismo , Encéfalo/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Microscopia de Força Atômica , Tamanho da Partícula , Agregados Proteicos , Termodinâmica , alfa-Sinucleína/análiseRESUMO
Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site-specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA-antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic-labelled oligonucleotides prepared with this reagent can be site-specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA-antibody bioconjugates was confirmed by a new LC-MS protocol and the bioconjugate probes were used in fluorescence or super-resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site-specific, well-defined and plasma-stable DNA-antibody bioconjugates for biological applications.
Assuntos
Acrilatos/química , Anticorpos/química , Benzoatos/química , DNA/química , Oligonucleotídeos/química , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Proteostasis, or protein homeostasis, encompasses the maintenance of the conformational and functional integrity of the proteome and involves an integrated network of cellular pathways. Molecular chaperones, such as the small heat shock proteins (sHsps), are key elements of the proteostasis network that have crucial roles in inhibiting the aggregation of misfolded proteins. Failure of the proteostasis network can lead to the accumulation of misfolded proteins into intracellular and extracellular deposits. Deposits containing fibrillar forms of α-synuclein (α-syn) are characteristic of neurodegenerative disorders including Parkinson's disease and dementia with Lewy bodies. Here we show that the sHsp Hsp27 (HSPB1) binds to α-syn fibrils, inhibiting fibril growth by preventing elongation. Using total internal reflection fluorescence (TIRF)-based imaging methods, we show that Hsp27 binds along the surface of α-syn fibrils, decreasing their hydrophobicity. Binding of Hsp27 also inhibits cytotoxicity of α-syn fibrils. Our results demonstrate that the ability of sHsps, such as Hsp27, to bind fibrils represents an important mechanism through which they may mitigate cellular toxicity associated with aberrant protein aggregation. Fibril binding may represent a generic mechanism by which chaperone-active sHsps interact with aggregation-prone proteins, highlighting the potential to target sHsp activity to prevent or disrupt the onset and progression of α-syn aggregation associated with α-synucleinopathies.
Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Neuroblastoma/patologia , Agregados Proteicos , alfa-Sinucleína/metabolismo , Animais , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Camundongos , Chaperonas Moleculares , Neuroblastoma/metabolismo , Células Tumorais Cultivadas , alfa-Sinucleína/genéticaRESUMO
The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250â nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Neurônios/patologia , Doença de Parkinson/patologia , Agregados Proteicos , alfa-Sinucleína/química , Peptídeos beta-Amiloides/metabolismo , Aptâmeros de Peptídeos/química , Humanos , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismoRESUMO
PTEN-induced kinase 1 (PINK1) impacts cell health and human pathology through diverse pathways. The strict processing of full-length PINK1 on the outer mitochondrial membrane populates a cytoplasmic pool of cleaved PINK1 (cPINK1) that is constitutively degraded. However, despite rapid proteasomal clearance, cPINK1 still appears to exert quality control influence over the neuronal protein homeostasis network, including protein synthesis and degradation machineries. The cytoplasmic concentration and activity of this molecule is therefore a powerful sensor that coordinates aspects of mitochondrial and cellular health. In addition, full-length PINK1 is retained on the mitochondrial membrane following depolarisation, where it is a powerful inducer of multiple mitophagic pathways. This function is executed primarily through the phosphorylation of several ubiquitin ligases, including its most widely studied substrate Parkin. Furthermore, the phosphorylation of both pro- and anti-apoptotic proteins by mitochondrial PINK1 acts as a pro-cellular survival signal when faced with apoptotic stimuli. Through these varied roles PINK1 directly influences functions central to cell dysfunction in neurodegenerative disease.
Assuntos
Mitofagia , Doenças Neurodegenerativas , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A central need for neurodegenerative diseases is to find curative drugs for the many clinical subtypes, the causative gene for most cases being unknown. This requires the classification of disease cases at the genetic and cellular level, an understanding of disease aetiology in the subtypes and the development of phenotypic assays for high throughput screening of large compound libraries. Herein we describe a method that facilitates these requirements based on cell morphology that is being increasingly used as a readout defining cell state. In patient-derived fibroblasts we quantified 124 morphological features in 100,000 cells from 15 people with two genotypes (SPAST and SPG7) of Hereditary Spastic Paraplegia (HSP) and matched controls. Using machine learning analysis, we distinguished between each genotype and separated them from controls. Cell morphologies changed with treatment with noscapine, a tubulin-binding drug, in a genotype-dependent manner, revealing a novel effect on one of the genotypes (SPG7). These findings demonstrate a method for morphological profiling in fibroblasts, an accessible non-neural cell, to classify and distinguish between clinical subtypes of neurodegenerative diseases, for drug discovery, and potentially for biomarkers of disease severity and progression.
Assuntos
Genótipo , Preparações Farmacêuticas , Análise de Célula Única/métodos , Paraplegia Espástica Hereditária/patologia , ATPases Associadas a Diversas Atividades Celulares/genética , Progressão da Doença , Humanos , Aprendizado de Máquina , Metaloendopeptidases/genética , Mutação , Índice de Gravidade de Doença , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/genética , Espastina/genéticaRESUMO
Neuroserpin is a secreted protease inhibitor known to inhibit amyloid formation by the Alzheimer's beta peptide (Aß). To test whether this effect was constrained to Aß, we used a range of in vitro assays to demonstrate that neuroserpin inhibits amyloid formation by several different proteins and protects against the associated cytotoxicity but, unlike other known chaperones, has a poor ability to inhibit amorphous protein aggregation. Collectively, these results suggest that neuroserpin has an unusual chaperone selectivity for intermediates on the amyloid-forming pathway. Bioinformatics analyses identified a highly conserved 14-residue region containing an α helix shared between neuroserpin and the thyroxine-transport protein transthyretin, and we subsequently demonstrated that transthyretin also preferentially inhibits amyloid formation. Last, we used rationally designed neuroserpin mutants to demonstrate a direct involvement of the conserved 14-mer region in its chaperone activity. Identification of this conserved region may prove useful in the future design of anti-amyloid reagents.
RESUMO
In addition to increased aberrant protein aggregation, inflammation has been proposed as a key element in the pathogenesis and progression of Alzheimer's disease. How inflammation interacts with other disease pathways and how protein aggregation increases during disease are not clear. We used single-molecule imaging approaches and membrane permeabilization assays to determine the effect of chronic exposure to tumour necrosis factor, a master proinflammatory cytokine, on protein aggregation in human-induced pluripotent stem cell-derived neurons harbouring monogenic Alzheimer's disease mutations. We report that exposure of Alzheimer's disease neurons, but not control neurons, to tumour necrosis factor induces substantial production of extracellular protein aggregates. Aggregates from Alzheimer's disease neurons are composed of amyloid-ß and α-synuclein and induce significant permeabilization of lipid membranes in an assay of pathogenicity. These findings provide support for a causal relationship between two crucial processes in Alzheimer's disease pathogenesis and suggest that targeting inflammation, particularly tumour necrosis factor, may have beneficial downstream effects on ameliorating aberrant protein aggregation and accumulation.
RESUMO
Soluble aggregates of amyloid-ß (Aß) have been associated with neuronal and synaptic loss in Alzheimer's disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aß that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/diagnóstico , Progressão da Doença , Agregados Proteicos/fisiologia , Idoso , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Biomarcadores/líquido cefalorraquidiano , Camelídeos Americanos , Feminino , Humanos , Masculino , Proteínas tau/líquido cefalorraquidianoRESUMO
The aggregates of the Aß peptide associated with Alzheimer's disease are able to both grow in size as well as generate, through secondary nucleation, new small oligomeric species, that are major cytotoxins associated with neuronal death. Despite the importance of these amyloid fibril-dependent processes, their structural and molecular underpinnings have remained challenging to elucidate. Here, we consider two molecular chaperones: the Brichos domain, which suppresses specifically secondary nucleation processes, and clusterin which our results show is capable of inhibiting, specifically, the elongation of Aß fibrils at remarkably low substoichiometric ratios. Microfluidic diffusional sizing measurements demonstrate that this inhibition originates from interactions of clusterin with fibril ends with high affinity. Kinetic experiments in the presence of both molecular chaperones reveal that their inhibitory effects are additive and noncooperative, thereby indicating that the reactive sites associated with the formation of new aggregates and the growth of existing aggregates are distinct.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Clusterina/metabolismo , Humanos , Cinética , Microfluídica , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aß42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aß42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aß42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aß42 may contribute to the onset and progression of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Bicamadas Lipídicas/metabolismo , Agregação Patológica de Proteínas , Peptídeos beta-Amiloides/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , UltracentrifugaçãoRESUMO
The link between protein aggregation and neurodegenerative disease is well established. However, given the heterogeneity of species formed during the aggregation process, it is difficult to delineate details of the molecular events involved in generating pathological aggregates from those producing soluble monomers. As aberrant aggregates are possible pharmacological targets for the treatment of neurodegenerative diseases, the need to observe and characterise soluble oligomers has pushed traditional biophysical techniques to their limits, leading to the development of a plethora of new tools capable of detecting soluble oligomers with high precision and specificity. In this review, we discuss a range of modern biophysical techniques that have been developed to study protein aggregation, and give an overview of how they have been used to understand, in detail, the aberrant aggregation of amyloidogenic proteins associated with the two most common neurodegenerative disorders, Alzheimer's disease and Parkinson's disease.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Doença de Parkinson/fisiopatologia , Agregados Proteicos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/química , Animais , Humanos , alfa-Sinucleína/química , Proteínas tau/químicaRESUMO
The aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson's disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.
Assuntos
Espaço Extracelular/metabolismo , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade , Interações Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Ligação ProteicaRESUMO
Small oligomers of the protein α-synuclein (αS) are highly cytotoxic species associated with Parkinson's disease (PD). In addition, αS can form co-aggregates with its mutational variants and with other proteins such as amyloid-ß (Aß) and tau, which are implicated in Alzheimer's disease. The processes of self-oligomerization and co-oligomerization of αS are, however, challenging to study quantitatively. Here, we have utilized single-molecule techniques to measure the equilibrium populations of oligomers formed in vitro by mixtures of wild-type αS with its mutational variants and with Aß40, Aß42, and a fragment of tau. Using a statistical mechanical model, we find that co-oligomer formation is generally more favorable than self-oligomer formation at equilibrium. Furthermore, self-oligomers more potently disrupt lipid membranes than do co-oligomers. However, this difference is sometimes outweighed by the greater formation propensity of co-oligomers when multiple proteins coexist. Our results suggest that co-oligomer formation may be important in PD and related neurodegenerative diseases.
Assuntos
Peptídeos beta-Amiloides/biossíntese , alfa-Sinucleína/metabolismo , Proteínas tau/biossíntese , Peptídeos beta-Amiloides/química , Humanos , Modelos Moleculares , Termodinâmica , alfa-Sinucleína/química , Proteínas tau/químicaRESUMO
Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Animais , Técnicas de Cocultura , Células-Tronco Embrionárias/metabolismo , Humanos , Peroxidação de Lipídeos , Poro de Transição de Permeabilidade Mitocondrial , Oxirredução , Técnicas de Patch-Clamp , Permeabilidade , Proteômica , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.
Assuntos
Proteínas de Ligação a DNA/química , Citometria de Fluxo/métodos , Corpos de Inclusão/química , Proteínas Mutantes/química , Agregados Proteicos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Corpos de Inclusão/metabolismo , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.
Assuntos
Clusterina/metabolismo , Clusterina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Síndromes Neurotóxicas/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Clusterina/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estresse do Retículo Endoplasmático/genética , Olho/metabolismo , Olho/ultraestrutura , Hemolinfa/citologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Larva , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios Motores/ultraestrutura , Neuroblastoma/patologia , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/patologia , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
One potential therapeutic strategy for Alzheimer's disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-ß peptide (Aß); and bapineuzumab, a humanized monoclonal antibody raised against Aß, could all reduce the Ca2+ influx caused by synthetic Aß oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.