Primary aSyn species table tail, BDD update 202305 N-terminal α-synuclein, NDE (TAK-341 IDP / ADx + Cooveohe collaborations), Analyt-NIA ELISA SG4, Fragments / Splice variation (C-terminal truncation 10-30%), Assay Truncation (LC-MS isoform characterization)
Primary aSyn species — table tail (continued)
| species | content (visible) |
|---|---|
| Insoluble | (Zhou, 2011 #602) soluble form HMW phase form. Insoluble HMW phosphorylated insoluble cell + Phosphorylated 1-100 J Sym3-N-D-CAB |
| β-pleated sheet | (Wsxman, 2008 #535) Syn506 and Syn514 recognize conformational variants of α-syn |
| pre amyloid (>220 nm) | (a) it is intrinsic of α-syn that has the property of pre-aggregation and aggregation in the cytoplasm of pyramidal neurons; Pansson 2018 reports the parametric crystal of formation of the structure of α-syn in 5-50 nm in length |
| Lewy bodies | Seeding: Polymorphic α-syn fibrils can recruit and convert native α-syn monomers into the fibril state. polymorphic: different fibril forms (e.g. JE 2018 #2522 hostsr ε α-syn 5-7 mm length, structural heterogeneity among α-syn fibrils from PD patients). Because of their rigid structure, the fibrils cannot be degraded inferior to professome and can serve as further temple seeds. (master Tomimatsu 17/3 (1.451)) major slide aSyn fibrils/oligomers 02 [43]; (Rodriguies, 2022 #2290) parautopstic molecular CGAS3-3, binding cross β-sheet motif (Apaiscw 23/123); mature wide-syn fibrils single molecule imaging; mature anti-α-syn fibrils (F2; [43]) |
(BDD monthly update 202305) — BDD - N-terminal α-synuclein (Cterminal)
- [Otake] ELISA, (Chiome Biosciences) 202304: Antibodies recognizing N-terminal domain and NAC domain were generated. However, sandwich ELISA using these clones did not give positive signal using α-syn (1-95). Trouble shooting (e.g. SPR and affinity maturation) is under consideration. (Kentaro Otake) js: exon 2 를 detect해야 하니까.
- [BDD monthly update 202305] SPR assay to estimate Kd value is on going at Chiome to determine the probability of success in . (Kentaro Otake)
NDE (Neuronal-derived exosome)
20181001 TAK-341 IDP vSubmit doc:
Recently, newer reagents that can detect oligomeric and phosphorylated forms of α-syn have become available. MJFF has recently awarded a grant to ADx Neurosciences, to develop an assay that will quantify CSF oligomeric α-syn in a Simoa bioanalysis platform. This project will require ~1.5 years to validate the assay. MJFF and ADx Neurosciences have agreed to keep Takeda Translational Biomarker Research up to date on the progress of this assay validation. Similarly, Takeda has initiated a collaboration with ADx Neurosciences to develop and validate an assay that quantifies oligomeric α-syn in plasma. The base ~1.5 years to validate the assay. These biomarkers can also be added as exploratory biomarkers in the Phase 2/3 program with TAK-341 to determine if reductions in more pathological species of α-syn can be achieved and what the correlation is with clinical response.
Additionally, in a pre-existing collaboration with Cooveohe, α-syn was successfully detected in necessary derived exosomes isolated and enriched from plasma samples. A collaboration with MJFF will be established by the end of FY2018 to conduct further validation work.
Analyt-NIA ELISA (SG4)
HUMAN α-Syn PATHO ELISA is based on a highly-sensitive Sandwich-ELISA using mAbs SG4. SG4 specifically recognizes amino acids 47-52 in β-sheet rich α-Syn oligomers, do not capture normal oligomeric αSyn
| TAK | collaboration with Exosome Dx, α-syn was successful detected in neuronally derived exosomes isolated and enriched from plasma samples |
| IDP | MJFF will be established by the end of FY2018 to conduct further validation work |
Fragments / Splice variation
(TAK-341 IDP)
up to 10-30 (15)% of total αsyn in Lewy bodies (LBs) is truncated 12.
- C-terminal truncation of αSYN is associated with a tendency to aggregate, and these species are enriched in PD brain. TAK-341 may not bind to these species
mechanism:
- C-truncation of α-syn prone fibril has the capacity to initiate misfolding and assembly of α-syn into amyloid fibrils, as loss of the C terms now allows the hydrophobic NAC motif to interact with other α-syn proteins to polymerize
- Once formed, these truncated fibrils may lead to prion-like seeding of endogenous FL α-syn to kick start a complex cycle in which new fibrils may spread from one cell to the next and propagate therein
- Even after FL α-syn fibrils are formed, it is likely that they will come to be truncated as well due to extracellular proteases they are exposed to in intercellular spreading, lysosomal proteases upon endocytic uptake, and cytoplasmic proteases if they manage to escape the lysosome
Assay Truncation
| content | |
|---|---|
| LC-MS methods have been developed in Zetterberg lab and others for syn isoforms/truncations characterization in α brain homogenates (immunoprecipitations coupled with native and digestion based mass spec approaches). Similar approaches can be implemented with our proteomics partners to characterize and quantify a-syn truncation products in CSF. | Cf) Truncation products in CSF (healthy vs disease) have not been profiled. We will also be developing LCMS to look at fragments of α-syn and pS129 (35-141 LCMS); 1) (B+H/M)/MSA CSF Hu profiling (1+18) 31 slides; Brain |
| 02 (?)Apolicable [Practical applicable] | -reactive to rodents? / Applicable to rodent expression human α-syn / -sensitive? → IN window may be large with α-syn rodent expressing human α-syn |
| sensitive | → IN window may be large with α-syn rodent expressing human α-syn; rodent and human have same truncation pattern? → plan microdialysis for ICF |
| Covariance matrix | TAK-341 NPRC, specific project support 등이 internally / TAK 341 no longer has an interested (le AS no interest) |
| 20240117 | ’no budget’ so no progress; + a similarly low priority / Mathilde Bickel Sandvich detection profiling for ? |