By Martin Rakusa
EAN eCommunications Board
Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (α-syn). The mechanism of spreading is similar to prions and includes seeding and nucleation, in which initial seeds of α-syn recruit other soluble monomers, Aggregates can be found in the CSF and blood. The concentration of the aggregates can be increased using a protein misfolding cyclic amplification (PMCA) technique. Aggregation is monitored with a specific dye thioflavin T (ThT). Previous studies have found a significant difference in the maximum fluorescence signal of the α-syn-PMCA aggregates in different α-syn. Signals were larger in PD than MSA samples.
Shahnawaz et al. investigated this issue further. The aim of their study was to differentiate between PD and MSA using α-syn-PMCA. They also explored biochemical, biophysical and biological differences between α-syn aggregates from PD and MSA cerebrospinal fluid (CSF) and brain samples.
The study included 94 patients with PD, 75 patients with MSA and 56 control individuals. Most of the CSF samples were obtained from the Mayo Clinic. The patients fulfilled clinical criteria including the UK Brain Bank guidelines.
Frozen samples of frontal cortex from the patients was obtained from the Banner Sun Health Research Institute. Control brain tissue was supplied by National Human Tissue Resource Center.
Purification and characterisation of monomeric α-syn was performed. A pET-21b plasmid carrying the coding DNA sequence for human α-syn was overexpressed in Escherichia coli and extracted from the cells. Protein concentration was determined by bicinchoninic acid (BCA) assay. The purity of the protein was evaluated by silver staining.
Samples of seed-free, monomeric α-syn at a concentration and ThT were placed in well plates to perform α-syn-PMCA. CSF from patients and controls were added. For positive controls, healthy CSF and α-syn oligomeric seeds were used.
The increase in ThT fluorescence was monitored with a microplate spectrofluorometer. Four rounds of amplification were done.
The concentration of the protein after the amplification was measured with three different methods: (1) protein quantity in pellets was measured by silver staining after SDS–PAGE; (2) dot blot analysis of sedimented materials; and (3) BCA measurements of total protein content in the supernatant fraction.
Samples containing α-syn aggregates amplified by PMCA were treated with different concentrations of proteinase K. Afterwards, electrophoresis was performed, and the membranes were probed with the antibodies against α-syn.
The structure of the α-syn-PMCA aggregate was imaged by electron microscopy. Helical models were manually built based on individual fibril density.
Cell cytotoxicity was tested on the rabbit kidney cells with different concentrations of amplified α-syn fibrils originating from CSF samples from patients with MSA and patients with PD.
Results were consistent on samples collected from different cohorts of patients and across several separate experiments. The authors could differentiate between patients with alpha synucleinopathy and controls. Sensitivity for PD was 93.6%, and for MSA 84.6%, specificity was 100% for both diseases. At the end, they combined all samples and were able to differentiate between PD and MSA with an overall sensitivity of 95.4%.
They observed two main differences between samples from patients with PD and MSA during the α-syn-PMCA. First, kinematic for patients with PD was slower than MSA. Second, maximum ThT fluorescence was higher (from 2,000 and 8,000 units) in samples from patients with PD than in samples from patients with MSA (up to 1800 units). Typical differences remained even after the α-syn-PMCA reaction was repeated several times with the diluted product and new α-syn monomers.
To determine whether α-syn was the same in CSF and brain, the authors compared CSF samples with several diluted brain samples from patients with PD or MSA. The kinetics of aggregation and maximum ThT fluorescence was characteristic for PD or MSA regardless of the origin of the sample. These results indicated that the aggregates in the CSF and brain samples were similar. In addition, aggregates residual after proteinase degradation were similar for CSF and brain samples.
The authors also explored structural differences between α-syn aggregates from patients with PD or MSA. Most of the studies were performed with samples from the second cycle of amplification. The amount of aggregates derived from the CSF samples of PD patients were the same as from those from MSA patients. The authors concluded that differences in ThT fluorescence might be due to different structures of aggregates.
When they compared different ThT dyes, two showed specific binding affinities. HS-199 bound preferentially to aggregates of PD patients, while HS-169 had high emission of fluorescence for MSA aggregates. The results were the same for aggregates from CSF and brain samples.
To analyse the biochemical differences between α-syn aggregates, the authors tested resistance to proteolytic degradation and performed epitope-mapping experiments. N-terminal and middle regions of the α-syn aggregates were very resistant to proteinase K activity even after one hour. Aggregates from CSF of patients with PD were larger (four bands, molecular weight 4 to 10 kDa) than from patients with MSA (two bands, 4 and 6 kDa). The signature was maintained after serial replication in vitro by α-syn-PMCA.
With circular dichroism spectroscopy and FTIR spectroscopy, the secondary structure of α-syn aggregates was explored. Both PD and MSA α-syn aggregates predominantly consisted of β-sheets. MSA aggregates had a higher proportion of β-sheet structure than PD aggregates, which could also have an additional α-helix- or random-coil-type structures.
Using cryo-electron tomography (cryo-ET), the authors further explored fibrils structure. They were composed of two protofilaments that intertwined in a left-handed helix. Fibrils from patients with PD were composed of longer stretches of straight filaments with helical twists in comparison to those from patients with MSA.
Finally, biological differences between aggregates from PD and MSA were tested. Highly significant toxicity was demonstrated in the RK13 cells and the neuronal precursor cells from human induced pluripotent stem cells. The results indicated that aggregates from PD were four-times less toxic than those from MSA.
In conclusion, this important paper identified that, whilst PD and MSA are both synucleinopathies, seeds of the misfolded proteins, which behave like a prion, differ chemically and biologically. In the future, PMCA might be used to detect low concentrations of α-syn in the CSF. With disease-specific ThT dyes, it may be possible to differentiate between PD and MSA with high sensitivity and specificity.