Investigating pathogenic mechanisms associated with prion protein and α-synuclein misfolding

Abstract

Neuronal loss and the aggregation of misfolded prion protein (PrPSc) and α-synuclein (αsyn) in the central nervous system are hallmarks of prion and synucleinopathy disorders (such as Parkinson’s disease, multiple system atrophy, dementia with Lewy body), respectively. PrPSc is unusual because it is the major component of prions; ‘proteinacious infectious particles’ that are the causative agent in prion disease. Many features of prion disease have been directly attributed to PrPSc including its ability to propagate, causing disease in susceptible animals and be transmissible. Another feature of PrPSc is strain variability; where the conformation of the misfolded species ascribes the clinical profile of disease that develops. Neurotoxicity is also intimately associated with the generation of PrPSc, however, the precise mechanisms underlying its development in prion disease are not resolved. Mounting evidence suggests that αsyn shares similar pathogenic mechanisms to PrP in terms of its ability to misfold, propagate and cause disease in susceptible animals. Like PrP, the mechanisms associated with the toxicity of misfolded αsyn are not well defined. This thesis studied the pathogenic mechanisms of PrP and αsyn misfolding with particular interest in how these proteins cause neurotoxicity through the development of in vitro and ex vivo systems that model pathogenic aspects of their respective disorders. A potential neurotoxic mechanism of misfolded αsyn was found using the protein misfolding cyclic amplification (PMCA) assay to produce a heterogeneous population of misfolded species and their pathogenicity examined in cultured neuronal cells. Misfolded αsyn was found to bind to the lipid cardiolipin that is virtually exclusively expressed in mitochondria. Extensive analysis revealed misfolded αsyn causes hyperactive respiration in the mitochondria of live cells without causing any functional deficit. This robust data gives strong support for the mitochondrion as a target for misfolded αsyn and reveals a potential up-stream pathogenic pathway of neuronal cell loss in Parkinson’s disease. Next, a model of prion disease was established using organotypic brain slice cultures. Upon infection with a mouse-adapted human prion strain, brain slices were shown to propagate protease resistant PrP and develop neurotoxicity. The relevance of protein misfolding in these systems was assessed using the small molecule Anle138b; which has been previously reported to alter misfolding of PrP and αsyn, and be efficacious to prion infected animals and animal models of PD. This molecule was found to modulate the misfolding of both proteins, which in the case of PrP, led to a rescue of prion-induced neuronal loss in organotypic brain slice cultures. Collectively this work robustly shows misfolded αsyn and PrP can cause substantial alterations to neurons they are exposed to. Findings from this work contribute to our general understanding on these proteins in disease and highlight both similarities and differences in their misfolding and pathogenicity.