The generation and spread of misfolded protein aggregates is common to all
Protein Misfolding Diseases (PMDs) including but not limited to Alzheimer’s,
Parkinson’s and Prion disease. Yet despite this common feature, the early
mechanisms underpinning the apparent perturbation in protein handling and
processing in such conditions remain poorly understood. Research in our lab
has previously shown protein misfolding may be seeded and supported in
different ways in the brain. Mice homozygous for the P101L mutation in murine
PrP (101LL) are capable of supporting protein misfolding and amyloid plaque
formation after challenge with pre-formed PrP fibrils and wild type mice of the
same genetic background do not, and appear to be capable of curtailing this
accumulation of abnormal protein in vivo. Thus, both mouse lines provided
ideal tools to investigate early cellular and molecular responses to abnormal
PrP fibril challenge. PMDs occur in vivo with a regional dependant progression
however; such investigations are more amenable to controlled initiation and
visualisation in vitro. Therefore, primary neuronal hippocampal cell cultures
were generated from each line (101LL and WT) and were thoroughly assessed
at the cellular level. Comparative molecular analysis of these cultures and in
vivo hippocampal tissue from both WT and 101LL lines was carried out and
transcriptome analysis confirmed a broadly comparable global expression
profile at a basal level between in vivo, in vitro and genotype. Interestingly, this
suggests that the genotype specific differential protein misfolding responses
are therefore likely to be the result of regulatory cascades initiated after
inoculation. To examine this at the cellular level, cultures were then challenged
with fluorescently labelled recombinant PrP fibrils.
Fibrils were directly interacting with PrPC on neuronal surfaces but did not
cause neurotoxicity. However, post-synaptic marker PSD-95 was significantly
reduced in 101LL cultures suggestive that 101LL neurons were more
susceptible to fibril insult than WT. Microglial cells were activated post
fibril challenge in both genotypes. This generic response was possibly to limit
neuronal damage and promote fibril degradation through phagocytosis.
Transcriptome analyses supported these observations and showed an
increase in expression of genes associated with phagocytosis was occurring
after fibril challenge in both genotypes. Hypertrophic astrocytes and an
increase in expression of genes associated with astrocyte differentiation and
development was only evident in WT fibril-challenged cultures indicating
differential glial responses were occurring between genotypes. Intracellular
labelling showed localisation with endosomes and lysosomes in WT cells
indicating an endolysosomal pathway was operative however; localisation in
101LL cells was primarily in endosomes indicating an impairment in abnormal
protein degradation pathways was occurring in 101LL fibril-challenged
cultures. Transcriptomic analysis identified reduced gene expression of
lysosomal associated genes Laptm5 and Ctsz in 101LL fibril-challenged
cultures, supporting endolysosomal processing was reduced in this genotype.
Additional transcriptomic profiles obtained post-fibril challenge showed an
increase in gene expression in WT cultures associated with endocytosis,
macrophage engulfment, immune and defence response and ER-associated
misfolded protein catabolic processes none of which were induced in 101LL
fibril-challenged cultures. These analyses suggest a dysfunction of multiple
mechanisms may be associated with an inability to clear misfolded protein.
These data provide key information on pathways and cellular mechanisms
involved in either clearance of misfolded protein or initiation of amyloid
formation. Identifying these pathways will provide a number of possible test
targets with the potential of impacting diagnosis and/or intervention in PMD’s
such as AD, PD and prions
