Monitoring alpha-synuclein aggregation and transmission in an in vivo model of Parkinson’s disease

Prasad, Vibha; Schulz, Jörg B. (Thesis advisor); Marquardt, Till (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease, after Alzheimer’s disease. The pathological hallmark of PD is the presence of Lewybodies (LBs), cytoplasmic inclusions predominantly formed by the accumulation ofthe synaptic protein - alpha-synuclein. Alpha-synuclein aggregates are present assoluble oligomers, which continue to aggregate into insoluble fibrils. These fibrils accumulate to eventually form LBs. Aggregates of alpha-synuclein occur in pathological situations and it has been shown that the protein pathology spread sthroughout the brain. As presence of aggregated alpha-synuclein is suggested to induce aggregation of monomeric alpha-synuclein, a mechanism of cell-to-celltransfer of pathogenic alpha-synuclein has been suggested.Aims1) To establish a system to monitor changes in alpha-synuclein aggregation, using Bimolecular Fluorescence Complementation system (BiFC).2) To evaluate the influence of risk factors implicated in PD, on alpha-synuclein aggregation in flies.3) To investigate the cell-to-cell transfer of alpha-synuclein in flies. MethodsWe generated transgenic alpha-synuclein flies that employed the Bimolecular Fluorescence Complementation system (BiFC). The BiFC alpha-synuclein systeminvolved the reassembly of two non-fluorescent Venus (YFP) portions, thus generating a fluorescent Venus protein. The reassembly of Venus is triggered byantiparallel dimerization of two alpha-synuclein monomers. According to this, the stoichiometry between alpha-synuclein oligomerization and Venus signal intensity is given. We exposed flies with pan neural expression of BiFC tagged alpha-synuclein to numerous treatments (risk factors implicated in PD) for an established time period of five days. We then processed the fly head lysates to collect the soluble alphasy nuclein fraction (supernatant) and the insoluble alpha-synuclein fraction (pellet).The soluble alpha-synuclein fraction was analyzed by the Venus fluorescence measurement. The insoluble alpha-synuclein fraction was extracted with urea buffer Abstract 4and analyzed by Filter Retardation Assay (FRA). The abundance of alpha-synucleinin both fractions provided insights on how the different treatments influenced alphasynuclein aggregation. Regarding the investigation of transmission of alphasynuclein, we restricted the expression of BiFC tagged alpha-synuclein to PigmentDispersing Factor (PDF) neurons of flies. After aging the flies for a ten-day timeperiod, the fly heads were dissected and analyzed by confocal microscopy.Results among the agents administered to flies, alterations within the cellular protein degradation mechanism (proteasome, autophagy) resulted in gross changes in the abundance of insoluble/soluble alpha-synuclein. In contrary, administration of metalions did not/only mildly changed the aggregation properties of alpha-synuclein. Finally, we increased alpha-synuclein phosphorylation at serine 129, which causedan increased aggregation of alpha-synuclein. Interestingly, this increasedaggregation correlated with an enhancement of alpha-synuclein induced toxicity, asevident by impaired fly locomotion. Regarding transmission of alpha-synuclein in flies, we demonstrated the presence ofBiFC alpha-synuclein outside the PDF neurons. Detailed analysis indicated thatalpha-synuclein is released at the axonal end of the PDF neurons in our flies. As PDFneurons regulate the circadian rhythm of flies, we exposed the BiFC alpha-synucleinflies to 12 hours of light and 12 hours of darkness (LD cycle) and its fly siblings to 24hours of darkness (DD cycle). Subsequent analysis showed that presence oflight/PDF neuronal activity is required for efficient transmission of alpha-synuclein. LDflies showed prominent alpha-synuclein signal outside the PDF neurons, whereas DDflies almost completely lacked alpha-synuclein outside the PD neurons. ConclusionsOur experimental set-up to analyze alterations in alpha-synuclein aggregation in fliesis fast, sensitive and inexpensive. This method is an ideal tool to test potential aggregation inhibitors and might be extended to other aggregopathies. Abstract 5We successfully established an in vivo model for the investigation of alpha-synucleintransmission. Our results prove the requirement of light for neuronal activity thatadvocates alpha-synuclein transmission, from the PDF neurons to the surroundingneurons in the fly brain via connected neural network. As PDF neurons are easy togenetically manipulate in our system, we suggest blocking the key cellularcomponents involved in protein/vesicle transport and analyzing the impact of suchalterations on alpha-synuclein transmission. This should help rule out the pathway required for the transmission of alpha-synuclein, at least for those cells concerned with alpha-synuclein release.