Mechanisms Of alpha-Synuclein Neurotoxicity In Parkinson’s Disease: A Foundation For Developing New Intervention Strategies

Parkinson’s disease (PD) is a neurological disorder that involves the degeneration of brain regions associated with motor as well as non-motor functions. Clinically, the hallmark motor abnormalities of PD include rigidity, bradykinesia (slowness in movement), postural instability, and resting tremor. These symptoms are associated with basal ganglia dysfunction arising from a loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. The causes of PD are not completely understood, but it is evident that age-related cellular changes play a significant role in the initiation and progression of the disease. Besides age-related factors, mutations in certain genes are associated with an increased risk of PD and an early onset of disease. One such gene, SNCA, encodes the protein alpha-synuclein (aSyn), which forms aggregates that are one of the major components of Lewy body inclusions characteristic of PD and other synucleinopathy diseases including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Multiple factors have been shown to promote the aggregation of aSyn in neurons, including the interaction of aSyn with membranes, post-translational modifications (PTMs), oxidative stress, and dysfunctional cellular clearance mechanisms. Furthermore, studies have revealed that aSyn aggregates (‘seeds’) can propagate from one region of the brain to another via anatomically connected neuronal pathways, thereby leading to the spread of pathology. However, mechanistic insight into the cascade of events that lead to seed-induced pathology, as well as therapeutic intervention strategies to stop the disease progression, remain unknown. Hence, there is a critical need for a more in-depth understanding of the mechanism of aSyn aggregation in PD, as well as downstream consequences of this aggregation. This thesis entitled ‘Mechanisms of ??-synuclein neurotoxicity in Parkinson’s disease: a foundation for developing new intervention strategies’ addresses multiple aspects of this problem.

In the studies outlined in Chapter 3, we examined cellular changes associated with aSyn aggregation in the rat cortex. Cortical dysfunction plays a crucial role in non-motor symptoms associated with PD and other synucleinopathies. Recent studies have reported functional changes in cortical circuitry in pre-clinical models of PD, but without any mechanistic insight. Therefore, in search of causative or predictive factors, we hypothesized that aSyn aggregation leads to alterations of the cellular proteome and lipidome. We utilized an in vivo model of aSyn aggregation involving the injection of aSyn preformed fibrils (PFFs) in rat brain to study the downstream effects of aggregation. In this model, we showed that striatal injection of PFFs leads to the presence of aSyn aggregates that stain positive for the phosphorylated form of serine residue 129 (pSer129) in the sensorimotor cortex, SNpc, and other anatomically connected brain regions. Results from the proteomics study indicate that intrastriatally injected aSyn PFFs do not induce significant changes in the global proteome of the sensorimotor cortex compared to injected aSyn monomer 3 months post-injection. Similarly, no changes were observed in 13 lipid classes, including lipids that play a central role in synaptic composition and cellular signaling. However, analysis of the phosphoproteome of the sensorimotor cortex revealed significant differences between the PFF and monomer groups 3 months post-injection. Gene ontology analysis of the phosphoproteomic changes suggested that aSyn PFF administration led to perturbations in synaptic transmission.

A loss of proteasomal activity is detrimental to neurons, and dysfunction of the ubiquitin-proteasome system has been implicated in an array of neurogenerative diseases including PD. Previous studies revealed that aggregated aSyn can directly interact with proteasomal complexes and inhibit their function. However, the fact that the earlier data were obtained using simple in vitro systems of uncertain disease relevance and were contradicted by subsequent findings suggested a need for further investigation using intact cell models. Therefore, in the studies outlined in Chapter 4, we examined the mechanism of aSyn aggregation-dependent proteasomal impairment. As a starting point for this study, we characterized PFF-induced aSyn aggregation in a rat primary neuronal culture model. We observed that aSyn fibrils are taken up by primary cortical neurons and induce the aggregation of endogenous aSyn in the recipient cell. A decrease in proteasomal activity was observed in neurons treated with aSyn PFFs compared to monomer in an intact cell assay, but not assays carried out in cell lysates. Moreover, no significant down-regulation of active proteasomal subunit was observed, implying that an intracellular inhibitory species related to the aSyn PFFs was involved in lowering the proteasomal activity. To explore a potential link between PFF-mediated, seeded aSyn aggregation and proteasome inhibition, we extended our studies of proteasomal activity to primary hippocampal and midbrain cultures, given that the different types of cultures have variable aSyn expression levels and show variable amounts of pSer129-aSyn pathology (hippocampal > cortical > midbrain in both cases). To our surprise, all three types of cultures showed a decrease in proteasomal activity following incubation with aSyn PFFs versus monomer control. Therefore, we concluded that the extent of pSer129-aSyn pathology arising from PFF-mediated seeded aSyn aggregation was not sufficient to account for PFF-dependent proteasomal impairment. We hypothesize that oligomeric species generated from externally added PFFs —e.g. potentially oligomers that are formed in the endolysosomal compartment and escape into the cytosol are playing a role in the impairment. Further experiments are needed to gain a better mechanistic understanding.

aSyn fibrils can vary in their atomic-level assembly, giving rise to structural polymorphisms. Ample evidence from experiments carried out in cellular and animal models with different recombinant aSyn proteoforms or with aSyn aggregates isolated from the brains of individuals with different synucleinopathy disorders indicate that these structural differences can lead to differences in disease manifestation by altering the seeding properties of the fibrillar material. Therefore, in studies outlined in Chapter 5, we focused on identifying cellular factors that influence the formation of aSyn fibrils with various structural polymorphisms. Using a bottom-up approach, we showed that different cellular factors (i.e., ionic strength and membrane interaction) and aSyn modifications (adenylylation, mutations, and N- and C- terminal truncations) can give rise to variations in fibril morphology. To obtain additional mechanistic insights, we examined several aSyn variants (human, mouse, and chimeric aSyn) for differences in aggregation kinetics and the ability to induce pathology in different cellular models of PD. We found that mouse aSyn, which differs from human WT aSyn by 7 mismatches, including 2 mismatches in the C-terminal domain, formed fibrils more rapidly than the human WT protein but with similar kinetics compared to the human familial mutant, A53T aSyn. In addition, the fibrils formed by mouse aSyn and human A53T aSyn formed fibrils with similar structures that differed from the human WT aSyn fibril structure. Fibrils formed from mouse aSyn and human A53T aSyn also had similar abilities to induce seeded aSyn aggregation, and both had a greater seeding propensity than fibrillar human WT aSyn in cell culture models. Although these findings highlight the ability of aSyn variants with different sequences or PTM profiles to form distinct fibrillar strains, a limitation of these studies is that fibrils prepared from recombinant aSyn are structurally different from fibrils derived from the brains of MSA patients. Efforts to characterize aSyn strains isolated directly from patients’ brains are limited by the fact that the amplification of brain-derived fibrils via seeded aggregation in recombinant aSyn solutions yields fibrils with different structures compared to the input seed material. Accordingly, some aspect of the cellular environment appears to be essential for the formation of aSyn fibrils structurally similar to the fibrils that exist in the brains of patients. To address this hypothesis, we examined cellular and animal models of seeded aSyn aggregation as potential vehicles for the high-fidelity amplification of aSyn fibrils derived from patient brain samples.

In chapter 6, I highlight future directions that stem from the results outlined above. In light of our discovery of phosphoproteome changes associated with seeded aSyn aggregation in rat cortex, I propose directions for a larger-scale study involving longer aggregation times and analyses of circuit-level changes across multiple brain regions. The mechanism by which internalized seeds escape from the endocytic compartment remains a key puzzle in understanding the prion-like propagation of aSyn PFFs. Therefore, I discuss new approaches that could be used to study endosomal escape in primary neuronal cultures and in vivo. Lastly, based on the collective findings in this thesis, I discuss potential therapeutic intervention strategies and screening assays to slow the progression of pathology by preventing the formation of new aSyn aggregates in the remaining healthy neurons.

In summary, the results presented herein have yielded key mechanistic insights into prion-like propagation of aSyn, especially the role of seed polymorphism, and downstream effects of aSyn aggregation.