Human Hsp70 Disaggregase reverses Parkinson’s-linked α-Synuclein Amyloid Fibrils

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases

C. elegans Models to Study the Propagation of Prions and Prion-Like Proteins

A hallmark common to many age-related neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), is that patients develop proteinaceous deposits in their central nervous system (CNS). The progressive spreading of these inclusions from initially affected sites to interconnected brain areas is reminiscent of the behavior of bona fide prions in transmissible spongiform encephalopathies (TSEs), hence the term prion-like proteins has been coined. Despite intensive research, the exact mechanisms that facilitate the spreading of protein aggregation between cells, and the associated loss of neurons, remain poorly understood. As population demographics in many countries continue to shift to higher life expectancy, the incidence of neurodegenerative diseases is also rising. This represents a major challenge for healthcare systems and patients’ families, since patients require extensive support over several years and there is still no therapy to cure or stop these diseases. The model organism Caenorhabditis elegans offers unique opportunities to accelerate research and drug development due to its genetic amenability, its transparency, and the high degree of conservation of molecular pathways. Here, we will review how recent studies that utilize this soil dwelling nematode have proceeded to investigate the propagation and intercellular transmission of prions and prion-like proteins and discuss their relevance by comparing their findings to observations in other model systems and patients

Dissecting aggregation and seeding dynamics of α\alpha-Syn polymorphs using the phasor approach to FLIM

International audienceSynucleinopathies are a heterogenous group of neurodegenerative diseases characterized by the progressive accumulation of pathological α-synuclein ($\alpha$-Syn). The importance of structural polymorphism of $\alpha$-Syn assemblies for distinct synucleinopathies and their progression is increasingly recognized. However, the underlying mechanisms are poorly understood. Here we use fluorescence lifetime imaging microscopy (FLIM) to investigate seeded aggregation of $\alpha$-Syn in a biosensor cell line. We show that conformationally distinct $\alpha$-Syn polymorphs exhibit characteristic fluorescence lifetimes. FLIM further revealed that $\alpha$-Syn polymorphs were differentially processed by cellular clearance pathways, yielding fibrillar species with increased seeding capacity. Thus, FLIM is not only a powerful tool to distinguish different amyloid structures, but also to monitor the dynamic process of amyloid remodeling by the cellular environment. Our data suggest that the accumulation of highly seeding competent degradation products for particular polymorphs may account for accelerated disease progression in some patients

Protein Quality Control Pathways at the Crossroad of Synucleinopathies

International audienceThe pathophysiology of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and many others converge at alpha-synuclein ($\alpha$-Syn) aggregation. Although it is still not entirely clear what precise biophysical processes act as triggers, cumulative evidence points towards a crucial role for protein quality control (PQC) systems in modulating $\alpha$-Syn aggregation and toxicity. These encompass distinct cellular strategies that tightly balance protein production, stability, and degradation, ultimately regulating $\alpha$-Syn levels. Here, we review the main aspects of $\alpha$-Syn biology, focusing on the cellular PQC components that are at the heart of recognizing and disposing toxic, aggregate-prone $\alpha$-Syn assemblies: molecular chaperones and the ubiquitin-proteasome system and autophagy-lysosome pathway, respectively. A deeper understanding of these basic protein homeostasis mechanisms might contribute to the development of new therapeutic strategies envisioning the prevention and/or enhanced degradation of $\alpha$-Syn aggregates

The cytoprotective sequestration activity of small heat shock proteins is evolutionarily conserved

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family.</p

Spreading of a Prion Domain from Cell-to-Cell by Vesicular Transport in <i>Caenorhabditis elegans</i>

<div><p>Prion proteins can adopt self-propagating alternative conformations that account for the infectious nature of transmissible spongiform encephalopathies (TSEs) and the epigenetic inheritance of certain traits in yeast. Recent evidence suggests a similar propagation of misfolded proteins in the spreading of pathology of neurodegenerative diseases including Alzheimer's or Parkinson's disease. Currently there is only a limited number of animal model systems available to study the mechanisms that underlie the cell-to-cell transmission of aggregation-prone proteins. Here, we have established a new metazoan model in <i>Caenorhabditis elegans</i> expressing the prion domain NM of the cytosolic yeast prion protein Sup35, in which aggregation and toxicity are dependent upon the length of oligopeptide repeats in the glutamine/asparagine (Q/N)-rich N-terminus. NM forms multiple classes of highly toxic aggregate species and co-localizes to autophagy-related vesicles that transport the prion domain from the site of expression to adjacent tissues. This is associated with a profound cell autonomous and cell non-autonomous disruption of mitochondrial integrity, embryonic and larval arrest, developmental delay, widespread tissue defects, and loss of organismal proteostasis. Our results reveal that the Sup35 prion domain exhibits prion-like properties when expressed in the multicellular organism <i>C. elegans</i> and adapts to different requirements for propagation that involve the autophagy-lysosome pathway to transmit cytosolic aggregation-prone proteins between tissues.</p> </div

NMm::YFP and R2E2m::YFP aggregates are highly toxic.

<p>(A) Motility of 1-day-old adults was determined as speed in % relative to the wild-type N2 control. (B) Confocal images of control N2 (I) and R2E2m::YFP (II) expressing nematodes were stained with rhodamine-phalloidin to reveal actin fibers. Scale bars: 10 µm. (C) The average amount of eggs laid within 2.5 hours by the indicated <i>C. elegans</i> lines. The developmental stage of synchronized embryos was determined after 72 h (displayed as % of eggs laid). (D) 20 L1 larvae of <i>C. elegans</i> lines expressing the indicated transgene were grown at 20°C for 4 days before light micrographs were taken. Within this time wild-type N2 animals become adults and have progeny, some of which in turn would have developed into young adults, as seen with RΔ2-5, but not with NM and R2E2 transgenic animals. (E) DIC images of control (I), and R2E2m::YFP (II–IV) expressing animals. Arrows indicate abnormal gonad (II), old cuticle that has failed to shed and remains attached indicating molting defects (III) and disrupted intestinal cells bordering the distended lumen (IV).</p