Rescuing defective vesicular trafficking protects against alpha-synuclein toxicity in cellular and animal models of Parkinson's disease

Studies in yeast are providing critical insights into the mechanisms of neurodegeneration in Parkinson's disease (PD). A recent study shows that disruption of vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi, caused by the overexpression and/or aggregation of alpha-synuclein, is linked to degeneration of dopamine neurons. Overexpression of proteins that are known to enhance ER-to-Golgi transport rescue defective trafficking in yeast, worm, fly, and cellular models of PD

A century-old debate on protein aggregation and neurodegeneration enters the clinic

The correlation between neurodegenerative disease and protein aggregation in the brain has long been recognized, but a causal relationship has not been unequivocally established, in part because a discrete pathogenic aggregate has not been identified. The complexity of these diseases and the dynamic nature of protein aggregation mean that, despite progress towards understanding aggregation, its relationship to disease is difficult to determine in the laboratory. Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered presymptomatically, drastically reduce the incidence of these diseases. The clinical trials could also settle the century-old debate about causality

Discovery of a novel aggregation domain in the huntingtin protein: implications for the mechanisms of Htt aggregation and toxicity

Aggravating aggregation: an N-terminal domain that is in close proximity to the polyQ domain in the huntingtin protein, htt105-138, is shown to be highly aggregation prone. Potential cross-talk between this domain and the polyQ region may play a central role in regulating the aggregation and toxicity of Htt-N-terminal fragments

Amyloids go genomic: insights regarding the sequence determinants of prion formation from genome-wide studies

[Image: see text] The availability of fully sequenced genomes provides a useful starting point for identifying putative amyloid and prion forming sequences through genome-wide scans. With an inventory in hand, one can assess the amyloid forming potential and the functional consequences of amyloid formation for each sequence. Thus, advancing our understanding of how cells process and utilize deleterious and functional aggregates, respectively

Molecular electron microscopy approaches to elucidating the mechanisms of protein fibrillogenesis

Electron microscopy (EM) has played a central role in our current understanding of the mechanisms underlying the pathogenesis of several amyloid diseases, including Alzheimer's disease, Parkinson's disease, and prion diseases. In this chapter, we discuss the application of various EM techniques to monitor and characterize quaternary structural changes during amyloid fibril formation in vitro and the potential of extending some of these techniques to characterizing ex vivo material. In particular, we would like to bring to the attention of the reader two very powerful molecular EM techniques that remain under utilized by researchers in the amyloid community, namely scanning transmission electron microscopy and single particle molecular averaging EM. An overview of the strength and limitations of these techniques as tools for elucidating the structural basis of amyloid fibril formation will be presented

Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer's disease research

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-beta (Abeta) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Abeta aggregation contributes to neurodegeneration in AD and to the identity of the toxic Abeta species. In this paper, we describe methods and detailed protocols for reproducibly preparing Abeta aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Abeta aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Abeta aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to "catalyze" the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Abeta) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Abeta. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Abeta was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Abeta in a concentration-dependent but ATP-independent manner. Inhibition of Abeta fibrillization by Hsp104 is observable up to Hsp104/Abeta stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Abeta amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Abeta protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Abeta. Together, these findings suggest that the strong inhibition of Abeta fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation