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 (Aβ42) 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 Aβ42. 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 Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer's disease.
Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform ...multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high‐throughput assay based on measuring the extent of aggregate‐induced Ca2+ entry ...into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α‐synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Disruptive influence: To quantify and characterize the membrane‐disrupting protein aggregates, associated with neurodegenerative diseases, a high‐throughput assay based on measuring the amount of aggregate‐induced Ca2+ entry into immobilized lipid vesicles has been developed. Picomolar concentrations of Aβ42 oligomers induce Ca2+ influx, which can be inhibited by the addition of a chaperone and a nanobody.
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.
The process of protein misfolding and aggregation is common to an increasing number of diseases, including neurodegenerative disorders. In the review, we have surveyed the latest advanced biophysical techniques that have been developed to investigate oligomer and fibrils formed from the proteins and peptides most commonly associated with Alzhiemer's and Parkinson's disease.
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 (≈250 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‐β. 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.
Described in detail: The aggregates formed in neurodegenerative disorders are of low abundance and have a broad range of sizes and structures, thus making them difficult to fully describe. We have modified an aggregate‐specific aptamer to develop a super‐resolution microscopy technique that can visualise these species within patient‐derived neurons in greater detail than has previously been possible.
Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary ...astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.
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-β peptide (Aβ); and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.
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•Human cerebrospinal fluid (CSF) can permeabilize membranes and induce Ca2+ influx•CSF of control individuals and those with Alzheimer’s disease show similar Ca2+ influx•An extracellular chaperone clusterin and a nanobody Nb3 can inhibit Ca2+ influx•Bapineuzumab reduces Aβ-aggregate-induced Ca2+ influx but is less effective in CSF
Drews et al. develop and implement sensitive in vitro assays to quantitatively measure the Ca2+ influx caused by human cerebrospinal fluid. If a given chaperone, antibody, or nanobody is effective in reducing Ca2+ influx, the authors determine what concentration is needed to prevent Ca2+ influx.
A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits ...factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the ...switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain‐swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X‐ray data collection.
Che proteins are part of the signal transduction pathway between stimulus and response, mediated by the archaellum. The structure of the chemotaxis protein CheY was determined in two different crystal forms. The novel CheF protein that connects chemotaxis signaling to the motility apparatus was expressed and crystallized, and data were acquired by X‐ray diffraction.
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca
entry ...into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca
sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca
influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
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