Utilisation of in-vitro models to facilitate the study of early mechanisms of misfolded protein formation, accumulation and clearance

1 Roslin Institute, University of Edinburgh, UK; 2 Centre for Integrative Physiology, University of Edinburgh

A common feature of neurodegenerative diseases is the progressive accumulation of damaged proteins which are converted into disordered aggregated structures. Alzheimer’s, Parkinson’s and Prion diseases are all examples of Protein Misfolding Diseases (PMD’s). Prion diseases are characterised by the conversion of a host-encoded cellular isoform of prion protein (PrPC) into a misfolded pathologic PrP isoform. The relationship between misfolded prion protein, infection and neurotoxicity is still not clearly understood. Inoculation of pre-formed amyloid seeds into transgenic mice expressing a proline to leucine mutation at PrP codon 101 (101LL) resulted in the formation of PrP amyloid plaques in the brain in the absence of prion disease. WT control mice were all negative for plaque deposition. We hypothesise that in the absence of this point mutation the “healthy” brain can maintain homeostasis and efficiently clear abnormal protein accumulations. Further analysis of these intricate mechanisms is crucial to fully understand protein misfolding particularly for the development of potential therapeutics.

To aid with these investigations in vitro models were developed which included primary hippocampal neuronal cultures and organotypic brain slice models. Both systems were characterised using ICC in conjunction with IMARIS software analysis, LDH viability assays and SDS-PAGE. Phenotypically both WT and 101LL cultures were differentiated into highly branched neuronal networks supported by astrocyte and microglial populations, all of which express PrPC. Furthermore the detection of pre-synaptic synapsin 1 and post-synaptic PSD-95 indicated normal formation and maturation of excitatory synapses. Transcriptome analysis (Affymetrix microarray) using Partek (FDR, Bonferroni) indicated no gross differences between 101LL and WT cultures and no changes in Prnp expression. Interestingly using unadjusted P-Value of <0.01 a small group of genes were identified of which four were associated with protein misfolding and up-regulated in the 101LL’s (Midline 1, Lumican, TGFBI and LAMA1). Microarray findings will be used as a baseline for further transcriptomic analysis post fibril challenge.

Uptake and processing of fluorescently labelled recombinant PrP fibrils was also examined in neuronal cultures. Preliminary challenge studies indicate fibril interactions with host PrPC on neuronal surfaces, along with astrocytes and microglia. Initial intracellular labelling showed localisation of fibrils with lipid rafts and endosomes indicating an endocytic pathway is operative. Cells from WT and 101LL mice will be compared to determine if enhanced plaque formation is due to altered cellular localisation of fibrils.

References

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5. Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC, PLoS One (2012).

Funded by: BBSRC

* entered into the PhD student poster competition