Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

Ferreira, Elisabete S.C.; Almeida, Zaida L.; Cruz, Pedro F.; Sousa, Marta Silva E; Verı́ssimo, Paula; Brito, Rui M. M.

doi:10.3390/ijms23010391

Cited by 6 publications

(4 citation statements)

References 60 publications

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“…Human wtTTR was aggregated in vitro into thioflavin T-positive aggregates according to published protocols. 46 , 50 The absorbance, emission, and excitation profiles of AMDX-9101 were then measured in reactions containing 4 µM of AMDX-9101 in solution with and without 5 µM of TTR aggregates, and the fold increase in fluorescence induced by TTR was calculated. The peak fluorescence emission of AMDX-9101 (549 nm) was enhanced approximately 27-fold upon incubation with aggregated TTR, compared to fluorescence in the absence of TTR, while excited at 450 nm ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

Pilotte,

Huang,

Khoury

et al. 2024

Trans. Vis. Sci. Tech.

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Background Transthyretin amyloidosis (ATTR) is a significant cause of cardiomyopathy and other morbidities in the elderly and Black Americans. ATTR can be treated with new disease-modifying therapies, but large shortfalls exist in its diagnosis. The objective of this study was to test whether TTR amyloid can be detected and imaged in the conjunctiva using a novel small-molecule fluorescent ocular tracer, with the implication that ATTR might be diagnosable by a simple eye examination. Methods Three approaches were used in this study. First, AMDX-9101 was incubated with in vitro aggregated TTR protein, and changes in its excitation and emission spectra were quantified. Second, a cadaver eye from a patient with familial amyloid polyneuropathy type II TTR mutation and a vitrectomy sample from an hATTR patient were incubated with AMDX-9101 and counterstained with Congo Red and antibodies to TTR to determine whether AMDX-9101 labels disease-related TTR amyloid deposits in human conjunctiva and eye. Last, imaging of in vitro aggregated TTR amyloid labeled with AMDX-9101 was tested in a porcine ex vivo model, using a widely available clinical ophthalmic imaging device. Results AMDX-9101 hyper-fluoresced in the presence of TTR amyloid in vitro, labeled TTR amyloid deposits in postmortem human conjunctiva and other ocular tissues and could be detected under the conjunctiva of a porcine eye using commercially available ophthalmic imaging equipment. Conclusions AMDX-9101 enabled detection of TTR amyloid in the conjunctiva, and the fluorescent binding signal can be visualized using commercially available ophthalmic imaging equipment. Translational Relevance AMDX-9101 detection of TTR amyloid may provide a potential new and noninvasive test for ATTR that could lead to earlier ATTR diagnosis, as well as facilitate development of new therapeutics.

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Section: Resultsmentioning

confidence: 99%

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

Pilotte,

Huang,

Khoury

et al. 2024

Trans. Vis. Sci. Tech.

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“…Pure microglial cell cultures were obtained as previously described using a technique that relies on the preferential adhesion of microglial cells to PEI [31,33]. Briefly, brains from postnatal day 1 C57BL/6J mouse pups (Janvier LABS) were removed by dissection, and the meninges were stripped away, after which brain tissue samples were dissociated by repeated pipetting in L15 Leibovitz medium.…”

Section: Neuronal and Glial Cell Culturesmentioning

confidence: 99%

The Pesticide Chlordecone Promotes Parkinsonism-like Neurodegeneration with Tau Lesions in Midbrain Cultures and C. elegans Worms

Parrales-Macias

Michel

Tourville

et al. 2023

Cells

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Chlordecone (CLD) is an organochlorine pesticide (OCP) that is currently banned but still contaminates ecosystems in the French Caribbean. Because OCPs are known to increase the risk of Parkinson’s disease (PD), we tested whether chronic low-level intoxication with CLD could reproduce certain key characteristics of Parkinsonism-like neurodegeneration. For that, we used culture systems of mouse midbrain dopamine (DA) neurons and glial cells, together with the nematode C. elegans as an in vivo model organism. We established that CLD kills cultured DA neurons in a concentration- and time-dependent manner while exerting no direct proinflammatory effects on glial cells. DA cell loss was not impacted by the degree of maturation of the culture. The use of fluorogenic probes revealed that CLD neurotoxicity was the consequence of oxidative stress-mediated insults and mitochondrial disturbances. In C. elegans worms, CLD exposure caused a progressive loss of DA neurons associated with locomotor deficits secondary to alterations in food perception. L-DOPA, a molecule used for PD treatment, corrected these deficits. Cholinergic and serotoninergic neuronal cells were also affected by CLD in C. elegans, although to a lesser extent than DA neurons. Noticeably, CLD also promoted the phosphorylation of the aggregation-prone protein tau (but not of α-synuclein) both in midbrain cell cultures and in a transgenic C. elegans strain expressing a human form of tau in neurons. In summary, our data suggest that CLD is more likely to promote atypical forms of Parkinsonism characterized by tau pathology than classical synucleinopathy-associated PD.

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“…TTR is known to transport thyroid hormones (thyroxine and triiodothyronine) and retinol (vitamin A) through binding to the retinol-binding protein (RBP). Tetramer dissociation and partial unfolding of TTR potentiates aggregation [ 45 ] and, therefore, the formation of amyloid fibrils, causing fatal amyloid diseases [ 46 , 47 , 48 ]. TTR amyloid deposits may be associated with wild-type TTR amyloidosis (ATTRwt), a relatively common cardiac disease of aging, but also to several TTR variants linked to hereditary cardiac and neurodegenerative amyloid diseases (ATTRv).…”

Section: Chaperones In Amyloid Disassemblymentioning

confidence: 99%

“…The amyloid systems tested to date with tetracyclines were amyloid-β (Aβ) [ 154 ], α-synuclein [ 155 , 156 ], transthyretin (TTR) [ 46 , 157 , 158 ], β2-microglobulin (β2m) [ 159 ], islet amyloid polypeptide (IAPP) [ 160 , 161 ], immunoglobulin (Ig) light chain (LC) [ 162 ], prion protein (PrP) [ 163 ], and polyQ [ 164 ]. These experiments revealed that tetracyclines are capable of not only completely disassemble amyloid fibrils, but also of blocking assembly into amyloid fibrils, generating non-toxic species after disaggregation, preventing neuronal death and, in some cases, producing clinical benefits in patients affected by amyloidosis.…”

Section: Chaperones In Amyloid Disassemblymentioning

confidence: 99%

Amyloid Disassembly: What Can We Learn from Chaperones?

Almeida

Brito

2022

Biomedicines

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Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-β structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple tissues. In this cascade of events, soluble oligomeric species are formed, which are among the most cytotoxic molecular entities along the amyloid cascade. The direct or indirect action of these amyloid soluble oligomers and amyloid protofibrils and fibrils in several tissues and organs lead to cell death in some cases and organ disfunction in general. There are dozens of different proteins and peptides causing multiple amyloid pathologies, chief among them Alzheimer’s, Parkinson’s, Huntington’s, and several other neurodegenerative diseases. Amyloid fibril disassembly is among the disease-modifying therapeutic strategies being pursued to overcome amyloid pathologies. The clearance of preformed amyloids and consequently the arresting of the progression of organ deterioration may increase patient survival and quality of life. In this review, we compiled from the literature many examples of chemical and biochemical agents able to disaggregate preformed amyloids, which have been classified as molecular chaperones, chemical chaperones, and pharmacological chaperones. We focused on their mode of action, chemical structure, interactions with the fibrillar structures, morphology and toxicity of the disaggregation products, and the potential use of disaggregation agents as a treatment option in amyloidosis.

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Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

Cited by 6 publications

References 60 publications

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

The Pesticide Chlordecone Promotes Parkinsonism-like Neurodegeneration with Tau Lesions in Midbrain Cultures and C. elegans Worms

Amyloid Disassembly: What Can We Learn from Chaperones?

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