Evaluation of neuropathology and neurodegeneration in experimental models of Parkinson's disease based on alpha synuclein preformed fibrils or 6-hydroxydopamine

Abstract

Evaluation of neuropathology and neurodegeneration in experimental models of Parkinson’s disease based on alpha synuclein preformed fibrils or 6-hydroxydopamine

Submitted by Cindy Chi Ching Pang For the joint degree of Doctor of Philosophy at The University of Hong Kong and King’s College London in August 2020

Parkinson’s disease (PD) is a complex multisystem neurodegenerative disorder with no cure. Many PD patients will develop dementia, referred to as Parkinson’s disease dementia (PDD). The characteristic neuropathological hallmarks of PD are Lewy bodies (LBs) and Lewy neurites (LNs) that are composed primarily of misfolded alpha synuclein. Alpha synuclein pathology spreads to different regions of the brain as disease progresses and the presence of LBs and LNs correlate closely with the clinical features of PD. However, it is not yet understood which events are necessary to cause dementia in PD. With this in mind, it is important to understand events that facilitate the spread of phosphorylated alpha synuclein to neuroanatomically connected regions, and the consequences of this spread on cellular events that lead to altered cognition. In rodents, injection into the brain of alpha synuclein preformed fibrils (PFFs) has previously been shown to induce human-like Lewy body pathology, leading to neurodegeneration after prolonged periods of time, whereas injection of the free radical 6-hydroxydopamine (6-OHDA) induces rapid neurotoxicity of dopaminergic neurons and motor deficits in the apparent absence of alpha synuclein modifications.

To further investigate the brain changes underlying cognitive dysfunction in PD, the PD-related effects of PFFs and 6-OHDA were examined following their unilateral stereotactic injection into the medial forebrain bundle (MFB) of Sprague-Dawley (SD) rats. Behaviour tests to measure motor, executive and visuospatial related cognitive impairments were used. Tissues were collected at 60, 90 and 120 days post injection (d.p.i.) of PFFs or vehicle. Phosphorylated alpha synuclein was examined in regions connected to the MFB, with a focus on the frontal cortex and hippocampal areas that control cognition. These areas were also examined for changes in tau, synaptic proteins, oxidative stress and astrocyte reactivity. Synaptic disruptions underlie cognitive decline. Synaptic fractions were therefore isolated from rat tissues and immunoblotting techniques were used to study changes in synaptic proteins. To investigate the impact of rapid neuronal loss in the substantia nigra (SN) on alpha synuclein spreading and neurodegenerative events important for cognition, a similar experimental approach was taken following unilateral MFB lesions with 6-hydroxydopamine (6-OHDA). Validation tests were conducted at 3 weeks post injection (w.p.i.) to confirm dopaminergic loss within the SN.

Results from this thesis established that injection of alpha synuclein PFFs into rat MFB can lead to phosphorylation of endogenous alpha synuclein and apparent “spreading” of phosphorylated alpha synuclein to regions connected to the MFB. However, alpha synuclein PFF injection did not cause alterations in tau, synapses or cause disruptions to rat behaviour or cognition up to 120 d.p.i., in partial agreement with previous findings. In contrast, 6-OHDA injection into rat MFB resulted in rapid mass neuronal loss in the SN, phosphorylation and “spreading” of phosphorylated alpha synuclein to MFB connected regions that was associated with altered synaptic marker levels, and cognitive impairments. These results extend previously published reports and suggest that rapid neuronal loss in the SN may initiate a cascade of events that facilitates the phosphorylation and/or spread of alpha synuclein, ultimately leading to cognitive deficits.

The study elucidates events that facilitate alpha synuclein modifications and neurodegenerative changes linked to cognitive dysfunction in PD. Oxidative stress-induced rapid neuronal loss may stimulate neuronal alterations that lead to the induction of oxidative stress and the appearance of phosphorylated alpha synuclein in neuroanatomically connected regions, synaptic changes and cognitive abnormalities, whereas alpha synuclein alone does not cause these neurodegenerative changes, at least not at the time points examined here. A future therapeutic intervention to reduce dementia related to alpha synuclein in PD may therefore be an antioxidant or neuroprotective agent, rather than alpha synuclein targeted therapy. Such a therapy could prevent the progression of a full-blown neurodegeneration cascade that results in PDD.

(An abstract of exactly 584 words)