Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species

Sahl, Steffen J.; Weiss, Lucien E.; Duim, Whitney C.; Frydman, Judith; Moerner, W. E.

doi:10.1038/srep00895

Cited by 77 publications

(102 citation statements)

References 25 publications

(33 reference statements)

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“…The aptamer is specific to the conformation of the aggregates, so these can be detected even amongst an excess of monomers. Furthermore, for primary and secondary antibodies, the size of a probe can add a linkage error of 15 nm,28 whereas the small size of aptamers enables them to bind at a higher density and at closer proximity to their epitopes; this leads to a higher imaging resolution, as has also been shown with DNA PAINT and non‐antibody binding proteins such as affimers 29. Typically, we achieve a localisation precision of ≈10 nm and a resolution of ≈25 nm (Table S2), with a limit of detection of ≈30 p m of aggregates (see the Supporting Information).…”

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confidence: 99%

“…We used induced pluripotent stem cells (iPSCs) from a PD patient with a triplication of the SNCA gene and from a healthy control unaffected by the disease to generate cortical neurons. Although SR methods have been used to image fibrils in cells, these are typically exogenously added aggregates generated from fluorophore‐labelled protein 28, 43, 44, 45, 46, 47. This is the first case in which a specific probe for aggregates has been used, and it enables the SR imaging of unlabelled, endogenous aberrant protein complexes.…”

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confidence: 99%

“…In the case of the MJF14‐6‐4‐2 antibody, there was nonspecific staining of regions of the cell that did not contain aggregated αS, whereas the aptamer only detected dual‐labelled aggregates. Furthermore, the larger size of antibodies can add a further 10–15 nm between the target and the labelled probe 28. When used with DNA PAINT, the same MJF14‐6‐4‐2 antibody (Figure S11), was unable to resolve individual aggregates, but instead there was diffuse staining in both the SNCA triplication and control cells.…”

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Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

et al. 2018

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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.

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Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

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“…Aggregation-prone HTT fragments also form soluble protein aggregates in neuronal cells [14]. These fibrillar oligomers or protofibrils, are diffusible structures (Fig.…”

Section: Pathogenic Polyq-containing Protein Aggregates In Patients Amentioning

confidence: 99%

Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures

Trepte

Strempel

Wanker

2014

Essays in Biochemistry

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Published in final edited form as:Trepte, P., Strempel, N., Wanker, E.E. Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures. Abstract:Polyglutamine ( This chapter reviews the current literature regarding misfolding and aggregation of polyQ-containing disease proteins. We specifically focus on studies that have investigated the amyloidogenesis of polyQ-containing huntingtin exon 1 (HTTex1) fragments. These protein fragments are disease-relevant and play a critical role in HD pathogenesis. We will outline potential mechanisms behind mutant HTTex1 aggregation and toxicity, as well as proteins and small molecules that can modify HTTex1 amyloidogenesis in vitro and in vivo. The potential implications of such studies for the development of novel therapeutic strategies are discussed.

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“…Thus, with this method, a reasonable degree of time-dependent behavior can be observed, well beyond the diffraction limit. (171). Critical to success of these experiments was targeted photobleaching of the extremely bright inclusion body (IB) before single-molecule imaging of the blinking EYFP.…”

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confidence: 99%

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

Moerner

2015

Rev. Mod. Phys.

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The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90's, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later superresolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized. The early days Introduction and early inspirationsI want to thank the Nobel Committee for Chemistry, the Royal Swedish Academy of Sciences, and the Nobel Foundation for selecting me for this prize recognizing the development of super-resolved fluorescence microscopy. I am truly honored to share the prize with my two esteemed colleagues, Stefan Hell and Eric Betzig. My primary contributions center on the first optical detection and spectroscopy of single molecules in the condensed phase(1), and on the observations of imaging, blinking and photocontrol not only for single molecules at low temperatures in solids, but also for useful variants of the green fluorescent protein at room temperature (2). This lecture describes the context of the events leading up to these advances as well as a portion of the subsequent developments both around the world and in my laboratory.In the mid-1980's, I derived much early inspiration from amazing advances that were occurring around the world where single nanoscale quantum systems were detected and explored for both scientific and technological reasons. Some of these were (i) the spectroscopy of single electrons or ions confined in vacuum electromagnetic traps (3-5), (ii) scanning tunneling microscopy (STM) (6) and atomic force microscopy (AFM) (7), and (iii) the study of ion currents in single membrane-embedded ion channels (8). But why not optical detection and ...

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Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species

Cited by 77 publications

References 25 publications

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

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