Do amyloid fibrils disrupt trafficking and function in the endolysosomal pathway?

Introduction and hypothesis
Amyloid fibril formation is associated an array of diseases including Alzheimer's, Parkinson's, type 2 diabetes and dialysis-related amyloidosis (DRA). In these and many other amyloid diseases amyloid fibril formation results in tissue degeneration. The mechanisms of tissue degeneration in amyloid disorders are poorly understood, although recent studies suggest that endocytic trafficking and lysosomal function are impaired. Consistent with this, our data show that amyloid toxicity is enhanced by fibril fragmentation and that this effect is associated with the endocytosis of nanoscale fibrils into lysosomes, reduced protein degradation by lysosomes and increased plasma membrane expression of lysosomal membrane proteins. We therefore hypothesise that amyloid fibrils disrupt protein trafficking to lysosomes resulting in lysosomal dysfunction.

Objective 1. Proteomic analysis of the effect of amyloid fibrils on the localisation of lysosomal proteins.
To determine whether amyloid fibrils disrupt trafficking of proteins in the endolysosomal pathway we will examine whether exposure of cells to fibrils causes lysosomal proteins to be mislocalised to other organelles. However, rather than using traditional cell biological approaches that will restrict this to the analysis of a small number of proteins, we will use organelle proteomics to perform a simultaneous analysis of the hundreds of proteins that constitute the lysosomal proteome. Specifically, we will use subcellular fractionation in combination with stable isotope labeling with amino acids in cell culture (SILAC) proteomics.

The peptide A-beta, the culprit of Alzheimer's, will be expressed in E. coli, purified and assembled into amyloid fibrils. SH-SY5Y neuroblastoma cells differentially labelled with heavy or light SILAC medium will be incubated in the presence and absence of A-beta fibrils. Subcellular fractionation will isolate lysosomes and other membrane fractions. Mass spectrometry will quantify the relative abundance of proteins in fractions from untreated and fibril treated cells. Lysosomal proteins whose level in either the lysosomal or in the other membrane fractions are altered by over/equal to 2-fold will be subjected to bioinformatic analysis. Interrogation of ontology, protein interaction and pathway databases combined with manual curation of the literature, will be used to determine their function and to predict the effect on lysosome function.

Objective 2. Validation of the proteomics data and nalysis of the effect on lysosomal function.
For proteins shown in objective 1 to have reduced levels in lysosomes and/or are increased in other membrane fractions, we will use an array of techniques to validate the proteomics data. Subcellular fractions will be immunoblotted with specific antibodies, proteins will be localised by immunofluorescence microscopy using confocal and super resolution microscopes and cell surface expression measured with flow cytometry. Moreover, in these experiments we will test if nanoscale amyloid fibrils formed from other disease-associated sequences also cause the mislocalisation of these proteins. Finally, the consequences for lysosome function will then be examined e.g the effect on substrate hydrolysis will be assayed for any hydrolases that have reduced levels in lysosomes.

Outcomes
In summary this project will determine whether amyloid fibrils disrupt the trafficking of lysosomal proteins and whether this results in impaired function of this vital organelle.