The accumulation of amyloid β peptide(1–42) (Aβ(1–42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1–42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1–42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1–42) aggregates entered the cells at low nanomolar concentration of Aβ(1–42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1–42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1–42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1–42) uptake and cytotoxicity. The accumulation of amyloid β peptide(1–42) (Aβ(1–42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1–42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1–42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1–42) aggregates entered the cells at low nanomolar concentration of Aβ(1–42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1–42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1–42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1–42) uptake and cytotoxicity.

Amyloid (mycology)

10.1074/jbc.m115.691840

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TDP-43 oligomers detected as initial intermediate species during aggregate formation

bioRxiv (Cold Spring Harbor Laboratory) (2018)

Rachel L. French Ashley N. Reeb Himani Aligireddy Niraja Kedia Dhruva Dhavale

ABSTRACT Aggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALS-linked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

Stress granule

Pathogenesis

10.1101/499343

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Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation

Journal of Biological Chemistry (2019)

Rachel L. French Zachary R. Grese Himani Aligireddy Dhruva Dhavale Ashley N. Reeb

Stress granule

Oligomer

10.1074/jbc.ra118.005889

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Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

bioRxiv (Cold Spring Harbor Laboratory) (2020)

George R. Nahass Yuanzi Sun Yong Xu Mark Batchelor M. Reilly

ABSTRACT Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aβ) and α-syn, prompting our inquiry in its mechanism of action. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that is distinct from other polyphenols and is also distinct from its effect on Aβ. Brazilin preserves the natively unfolded state of α-syn by stabilizing the compact conformation of the α-syn monomer over the aggregation-competent extended conformation. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-β sheet structure of α-syn fibrils. Brazilin eliminates seeding competence of α-syn assemblies from Parkinson’s disease patient brain tissue, and treatment of pre-formed fibril assemblies with Brazilin significantly reduces their toxicity in primary neurons. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson’s Disease. Highlights - The natural polyphenol Brazilin binds to monomeric, oligomeric and fibrillar α-syn - Brazilin shifts the equilibrium away from aggregation-competent monomer conformations - Brazilin inactivates seeding-competent α-syn isolated from Parkinson patients’ brains - Brazilin detoxifies α-syn aggregation intermediates and stabilizes mature amyloid fibrils Graphical Abstract

Fibrillogenesis

10.1101/2020.09.29.318220

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Spectroscopic study on encapsulation of two structurally similar donor–acceptor dyes in model cyclodextrin nanocavity

Journal of Molecular Structure (2014)

Amrita Sarkar Niraja Kedia Pradipta Purkayastha Sanjib Bagchi

Acceptor

Encapsulation

Stoichiometry

10.1016/j.molstruc.2014.04.027

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