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Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases.

ABSTRACT: Alzheimer's disease and Parkinson's disease are the two most common neurodegenerative diseases globally. These neurodegenerative diseases have characteristic late-stage symptoms allowing for differential diagnosis; however, they both share the presence of misfolded protein aggregates which appear years before clinical manifestation. Historically, research has focused on the detection of higher-ordered aggregates (or amyloids); however, recent evidence has shown that the oligomeric state of these protein aggregates plays a greater role in disease pathology, resulting in increased efforts to detect oligomers to aid in disease diagnosis. In this review, we summarize some of the exciting new developments towards the development of fluorescent probes that can detect oligomeric aggregates of amyloidogenic proteins present in Alzheimer's and Parkinson's disease patients.

SUBMITTER: Teppang KL

PROVIDER: S-EPMC10766704 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases.

Teppang Kristine L KL Zhao Qilin Q Yang Jerry J

Frontiers in chemistry 20231222

Alzheimer's disease and Parkinson's disease are the two most common neurodegenerative diseases globally. These neurodegenerative diseases have characteristic late-stage symptoms allowing for differential diagnosis; however, they both share the presence of misfolded protein aggregates which appear years before clinical manifestation. Historically, research has focused on the detection of higher-ordered aggregates (or amyloids); however, recent evidence has shown that the oligomeric state of these ...[more]

PMID: 38188930

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Project description:Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or "presequences") that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-β precursor protein (APP) or its cleavage product amyloid-β (Aβ), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aβ, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.

Project description:Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.

Dataset Information

Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases.

Publications

Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases.

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