A Rapid Cellular FRET Assay of Polyglutamine Aggregation Identifies a Novel Inhibitor

Pollitt, S; Pallos, Judit; Shao, Jieya; Desai, Urvee A.; K., Aye Aye; Thompson, Leslie M.; Marsh, J. Lawrence; Diamond, Marc I.

doi:10.1016/s0896-6273(03)00697-4

Cited by 146 publications

(143 citation statements)

References 39 publications

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“…Our laboratory has previously used FRET to quantify protein aggregation in cultured cells (13,21,22). The use of a plate reader for measuring FRET has certain disadvantages, and thus we created a facile, next-generation system to detect seeding activity with ultrahigh sensitivity and specificity.…”

Section: Resultsmentioning

confidence: 99%

Proteopathic tau seeding predicts tauopathy in vivo

Holmes

Furman

Mahan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

539

792

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Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼300 fM) and synuclein (∼300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.amyloid | neuropathology | dementia | aging P rotein aggregation characterizes many neurodegenerative disorders, including Alzheimer's disease (AD) and the related tauopathies. These disorders feature the accumulation of fibrillar deposits of the microtubule-associated protein tau with progressive deterioration of the central nervous system. Tau pathology and its associated brain atrophy do not appear randomly throughout the brain, but rather progress along distinct neural networks (1-5). This aspect suggests a role for transcellular spread of a pathogenic agent via neural connections. Our laboratory and others have previously hypothesized that tau aggregates-or seeds-serve as this agent of spread, transmitting the aggregated state from cell to cell via prion-like mechanisms (6-15).Mounting fundamental insights support this hypothesis. Tau seeds applied to the outside of cells bind the cell surface by attaching to heparan sulfate proteoglycans, triggering uptake by macropinocytosis (13). Upon internalization, tau seeds nucleate the fibrillization of endogenous tau monomer via templated conformational change, or seeding (8, 10). Tau seeding requires a critical unit of size for activity, as only particular species propagate the aggregated state (16). In vivo studies have described tau protein spreading from local sites to distant regions, presumably via transsynaptic movement (11,12,(17)(18)(19). Finally, our laboratory and another recently demonstrated that tau propagates discrete amyloid conformations through the brains of animals that give rise to unique neuropathologies (18,20).Despite this evidence, it remains unclear wheth...

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Section: Resultsmentioning

confidence: 99%

Proteopathic tau seeding predicts tauopathy in vivo

Holmes

Furman

Mahan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

539

792

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“…This method of measuring protein aggregation by FRET has reliably allowed detection of subtle changes in response to pharmacologic (22) as well as genetic (23) manipulations of androgen receptor and huntingtin protein aggregation that were corroborated by visual and biochemical analyses. Because the relative amount of spectral FRET measured depends on the ratio of acceptor to donor, we use a constant ratio of 3:1 when RD-YFP and RD-CFP are co-expressed within the same cell.…”

Section: Fret Assaysmentioning

confidence: 95%

“…Fluorescence Plate Reader-Spectral FRET measurements (FRET/donor) were obtained using a TecanM1000 fluorescence plate reader according to methods described previously (22). When donor and acceptor are not fused to the same protein, spectral FRET measurements depend on careful control for the relative amount of donor and acceptor proteins expressed within the cell.…”

Section: Fret Assaysmentioning

confidence: 99%

Trans-cellular Propagation of Tau Aggregation by Fibrillar Species

Kfoury

Holmes

Jiang

et al. 2012

Journal of Biological Chemistry

506

477

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Background: Trans-cellular propagation of aggregation may be important in neurodegeneration, but mechanisms are unknown. Results: Tau fibrils are secreted into the extracellular space, where they directly trigger aggregation in recipient cells by contacting native protein. Conclusion:Trans-cellular movement of Tau fibrils seeds subsequent aggregation. Significance: Therapies that block trans-cellular movement, including antibodies, may have an important role in neurodegenerative diseases.

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“…Autophagy Function Is Impaired in ␣-Syn Aggregate-bearing Cells-To further substantiate that ␣-syn aggregates impair autophagy function, we examined the degradation of the estab- lished autophagy substrate, YFP-tagged huntingtin exon 1 fragment, with 72 glutamine repeats (Htt Gln-72), which forms highly insoluble aggregates that are degraded by autophagy (13,45). Htt Gln-72 and p-␣-syn did not associate with each other when examined by IF in Pff-td cells (Fig.…”

Section: ␣-Syn Aggregates Also Interact With Autophagy and Proteasomementioning

confidence: 99%

Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy

Tanik

Schultheiss²,

Volpicelli‐Daley³

et al. 2013

Journal of Biological Chemistry

279

250

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Background: ␣-Synuclein aggregates and macroautophagy are associated with neurodegeneration. Results: Modulation of macroautophagic activity does not affect ␣-synuclein aggregate levels, although these aggregates cause accumulation of immature autophagosomes. Conclusion: ␣-Synuclein aggregates are resistant to degradation and impair autophagy by delaying autophagosome maturation. Significance: Understanding the impact of ␣-synuclein aggregates on autophagy may help elucidate therapies for ␣-synucleinmediated neurodegeneration.

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A Rapid Cellular FRET Assay of Polyglutamine Aggregation Identifies a Novel Inhibitor

Cited by 146 publications

References 39 publications

Proteopathic tau seeding predicts tauopathy in vivo

Proteopathic tau seeding predicts tauopathy in vivo

Trans-cellular Propagation of Tau Aggregation by Fibrillar Species

Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy

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