Postmortem aSyn detailed staging table tail, Release of α-syn (sulfonylurea/SUR1 channel), Safety for aSyn / KO of α-syn (SNCA KO mice + Vilar 2018 + Markovich 2007), Seed-Amplification Assay (RT-QuIC) Materials/96 well/Real-time readout/Limitation/Advantage, vendor matrix (PPMI/Tichi/Penix/OPDC/Lema-Marambio/Caughey)

Postmortem aSyn — staging table (continued)

(a) (Zhou 2011 #602) c-Asyn: Previous post-mortem studies using semi-quantitative western blotting indicated that insoluble forms of α-syn are increased in PD brains, whereas soluble forms are not significantly changed or even reduced.

soluble HMW (Knhi 2014 #2058) shows soluble monomer; by ELISA Pgs.

Insoluble row: (Wills 2010 #1270) by WB Pg5; Phosphorylated insoluble cell + Phosphorylated soluble cell; (Hg2) normal control 만 0, PD 만 clearly differentiated, 2018 Gundersen.

(Bhattacharjee 2019 #2237) endogenous full length acetylated 1-140 form is the most abundant species of αsyn in all disease cases and does not differ significantly between groups. The phosphorylated αsyn was rarely detected and were marginally significant between PD cases and controls. The levels of asyn forms in PD vs controls (asyn 1- mature is 100 normal control 1) (cf 13 vs control), increased in PD coagulate region compared in controls in the Lewy body enriched α-syn fraction (detergent-insoluble fraction). In the soluble fraction, only DLB α-syn 5 was significantly increased in PD compared to controls.

Release

• Release of α-syn is calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels, a receptor distributed throughout the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release (Emmanouilidou et al., 2016).
• Erythrocytes and platelets are also known to contain appreciable amounts of α-syn.

Safety for aSyn

KO of α-syn

• SNCA KO mice show a reduction of synaptic vesicle cluster, although response to Amphetamine is normal.
• (NRJ 2017 #1370) A target safety review (TSR) has been completed in α-syn community in presynaptic nerve terminals and involved in dopamine modulation and locomotor function.
• Homologous α-syn knockout mice are viable, fertile, and do not display any gross abnormalities or major brain anatomical changes. There are no major morphological defects in DA neurons however Hwer-Chwn-Sn positive neurons in the SN were reported in a constitutive null mutant transgenic CB57s/J mouse (Garcia-Reibossch et al, 2013).
• In adult α-syn knockout mice impaired locomotor activity is but desserine release after registriated terminal stimulation is normal (Vilar Ploni et al., 2018).
• Novel environment locomotor exploration is impaired with significant decrease in the deference-dependent locomotor activity in response to immunoflavin challenge in knockout mice versus WT mice (Markovich et al., 2007).
• As such Functional Observational Battery (FOB) assessments will be performed in rodent and neurological exams in NHP preclinical safety studies.

Seed-Amplification Assay (RT-QuIC)

Substrate: misfolded (pathologic) oligomers capable of seeding aggregation v. ability of pathological α-syn oligomers to seed aggregation. (proteopathic seeds 즈, whether seeds are present or not)

Species: (Lou, 2022 #2397) Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed (soluble) fibrils likely to dominate the response in seeding assays.

Materials	96 well	Real-time readout via fluorescence
Sample (containing aSyn) Substrate (reactive, aggregately suspended recombinant native aSyn)	Shaking heat cycles: repeated cycles of seeding, amplification, and break down [De novo Fibril formation] (Seeds in the species lead monomers to induce their continuous transformation as they are recruited into the growing fibril (Cont. shed: Secondary nucleation) due to assembly and conversion of monomers on the sides of fibrils, contributing along with primary nucleation to the generation of new seeding surfaces.	proteopathic binds to β-sheet-rich structure, thioflavin T binds to amyloid fibrils but not monomers. When it binds to β-sheet-rich structure such as amyloid aggregates, the dye displays enhanced fluorescence and a characteristic red shift of the emission spectrum (~50 nm time emission). Depending on the particular protein and experimental conditions, thioflavin T may (or may not) show a spectroscopic change after binding to precursor monomers, small oligomers, unaggregated material with a high β-sheet content, or even alpha-helic-rich proteins. Conversely, some assayed fibers do not affect thioflavin T fluorescence, resulting in negative results.

[αSyn ThT] curve description:

• Lag: seeds are growing at sub-detectable levels. Representing initial quantity of proteopathic seeds.
• Plateau once the available monomer is consumed.

Limitation

• Seed amplification assays are inherently non-linear processes, and slight variations in the initial conditions, which include experimental factors, seed concentration, seed conformation, additional proteins or small molecules within the reaction mix, can have large effects on the kinetics of the aggregation reaction or even precise successful aggregation. Even under identical assay conditions, two identical samples may not precisely reproduce the aggregation kinetics due to variability between technical replicates. Unlike polymerase chain reaction (PCR), for instance, the aggregation reaction has the discrepancies do not show a 1:1 relationship between input and output. As such, it is subject to significant variability in the fractioning and formation of new seeds and new seeding surfaces with each cycle. These sources of intrinsic assay variability,
  • 1 sample size multivariate analysis
  • 0 normalization
  • 1 Assay standardization

Advantage

To preclude the detection of certain off-pathway oligomers that do not aggregate further to form β-sheet-rich structures.

Vendor / cohort matrix (start)

citation	cohort	method	findings
Werd 2023 #2587 PPMI	2015 MJF-PD Exchange PPMI	SAA CSF	Cross-sectional, PD n=345, HC n=163, SAA results were only yes/no, no meaningful UPDRS correlation analysis. PD n=375, HC n=163.
2015 from 20230419 MJF-PDRs
2021 Tichi review		RT-QuIC	Ongoing projects include — evaluation of kinetic parameters as predictors of clinical progression with PPMI dataset, evaluation of serum bioluminescence amplifiable system as a SAA and PEHTOR for sensitive detection in plasma. seed strategy α-syn seeds in LBD and MSA CSF as confirmation by Pre-aggregator compound for α-syn promoted PFF dose-dependent seeding activity inducing nonspecific fibrillization within 120 hours assay time. The shown reaction kinetics will be quantified by the kinetic accuracy and precision was not enough to be quantified by an assay strategy by. This RT-QuIC assay can be quasi-quantitative assay.
2021 Penix #1830	4 four out of five PD CSF showed seeding activity, while none of healthy CSF did		This RT-QuIC assay can be quasi-quantitative assay
2023 Parkinson's Disease Centre OPDC and other cohorts including PPMI	The CSF samples from 14 PD, 24 MSA and 45 idiopathic RBD patients underwent 55 HC		• (PD) RT-QuIC showed 89% sensitivity and 96% specificity for Parkinson's disease. There was no correlation between RT-QuIC quantitative parameters and PD clinical scores (e.g., UPDRS motor) • (MSA) The sensitivity in MSA was 75%; iRBD overall sensitivity in idiopathic REM sleep behaviour disorder was 64% • During Cir, 31% iRBD patients converted to synuclein with 40% of converters showing baseline RT-QuIC positivity
(Lema-Marambio 2023 #2589)	113 de novo PD, 64 healthy controls, 29 RBD MSA	CSF, Ampcom	RT-QuIC was reactive in 105 PDS with 98% accuracy. αSyn-seeds were detected in 93% of the iRBD cases. Of the 87 RT-QuIC positive iRBD subjects, 30 (34.5%) were also α-SAA(+). Discrepancies did not preclude differences in PFF amplification parameters whereas significant but weak correlations were observed between TSO and TTT and the maximum and ThT signal between asyn-α-pyloid 1-42, tau, MAPI; the FigG1 TTT vs CSF αsyn rho 0.37 p=0.4, vs MDS-UPDRS-III 0.15 non-significant, no analysis on DatScan
Caughey	We did not otherwise observe significant correlations of kinetic parameters with clinical metrics. • There were some notable correlations between assay parameters and clinical metrics. For instance, disease duration positively correlated to Caughey αSyn-SAA Fmax (r = +0.53, p = 0.007, n = 24), and negatively to Caughey TTT (r = -0.46, p = 0.025, n = 24), and AbbVie αSyn-SAA TTT (r = -0.42, p = 0.039, n = 25). The only significant correlations to motor