Function (of normal aSyn), Grading/Scoring of LB pathology, Homology, In vitro studies of aSyn matrix, In vitro pharmacology assays
Function (of normal aSyn)
| Pathologic condition | |||
|---|---|---|---|
| 2019 Schaser | It is normally localized in the nucleus. | When DNA damage occurs → serine-129 phosphorylated alpha-syn is rapidly recruited to DNA damage sites | that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-syn reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss of function (PMID 30728653) |
| (Mealla et al. 2018, PMID 4 #1267) | that all functions to promote membrane curvature, thereby contributing to synaptic trafficking and vesicle budding [3]: alpha-syn protein sticks the glue to the inner face of the plasma membrane of nerve cells AS in stabilizes the docking of SVs on the plasma membrane by establishing a dynamic link between the two membranes (28); All-them holding affinity for SVs as rate-and strongly cofounders with SVs co-presynaptic in the presence of calcium pre-cell → Upon the binding of SVs alpha-Synuclein-strong-curvature cytoplasmic / spinal vesicle aggregation 일컬 (28); 1] the rotary recruitment to the synaptic membranes | ||
| (Schaser, 2019 #1755) | Alpha-syn is a DNA binding protein that modulates DNA repair → the affinity of α5 to 19 PM is considerably influenced | ||
Grading/Scoring of LB pathology
LB Consortium (McKeith, 2005 #1377): method = Asyn staining
Stage:
- I: Sparse LBs or LNs
- II: Sparse LBs or LNs
- III: 1 LB per high power field and gland LB
- IV: LBs and scattered LNs in low power field
- V: Numerous LBs and LNs
LB inclusion observed throughout in 50% of patients with PD and 80-100% of patients with DLB.
Homology
- Human and mouse share 95% sequence identity, but several regulatory regions influencing SNCA expression levels and transcript isoforms differ between species246
In vitro studies of aSyn
| Preparation | evaluation | Cell assay | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Protein digestion | Dynamic light scattering | Fluorescence polarization | SPR | Sedimentation analysis | ThT assay | TEM | Negative EM | CryoEM | (Overexpression model: human, x-tagged with αGFP using the BAC system) | ||
| Principle | Sonication: αSyn filaments size eg. AD/PD 2023 poster (656) | (On day of surgery PFFs are sonicated to break up the fibrils into fragments close to 50 nm in length, this lessens variability between preparations) (Polinski, 2018) | by photon correlation spectroscopy (or quasi-elastic light scattering) | To look at αSyn aggregation kinetics, easy to measurement of size change vs time | To look at αSyn convey size of αSyn into immobile species | applying a heavy metal salt stain | CryoEM is time consuming and limits its accumulation, miracellular cryolated | nu fast-tagged with αGFP using the BAC system 처음 다는 매 다 만들지 측정 difficult | aSyn signal but normalized to cytoplasm in neurons!? Need to be more careful | Should be most important | (WT) - aSyn derived rat DA neurons or aSyn from human brain samples (PD, MSA) |
| Example | PD_NewDevelopments_Ulysses_faded a_20092023.pdf; Janalsy 2023 (60). NDU Extended meeting; AC immune; AD/PD 2023 (poster (1035)); HEK2 cells overexpressing using BAC system; α-syn lattices recruited from human brain samples (PD, MSA); -Primary neuron eg rat cortical neurons (Seeds) | ||||||||||
In vitro pharmacology assays
unique set of biophysical assays to identify potent inhibitors of α-syn aggregation
| ThT and PFF protein assays | • six compounds capable of preventing α-syn aggregation in either way |
| α-syn ADPP HER assay | • six compounds capable of preventing α-syn aggregation in pAyn |
| Primary neuron sample | • six compounds capable of preventing α-syn aggregation either way primary neuron |
| Binding to human samples | • What is the binding affinity of compounds in human aggregation 일정 |
| hard fibril coupling | • six compounds binding specifically to aggregated own monomers α-syn? |
Summary 노 표가 일컬: Below red missing with Pravdrol
| Cit | GFP-tagged Synuclein (over)expression to PFFs ie GFP fluorescent | Seeded Cell assay (X) so on (X) 일정 (X) primary neuron Human brain Human brain tissue extract | |||
|---|---|---|---|---|---|
| 1 | Matrix? | ||||
| 2 | What aSyn? | Monomer or PFF synthetic/recombinant | Human brain tissue extract | ||
| 3 | Readout | Binding D to αSyn (AC immune) | ThT fluorescence | quick visual inspection at homogeneity (is the protein(s) like in one state or is in pieces and following appearance), dispersibility is often the case (e.g. friends?), size and shape of the protein/complex (are these features on the sample or is it a dot or extended chains and the quantify of protein on the sample on the grid (no covered or can be modified species and clearly identified and selected). [caution] reporter particles are often labeled and compactly folded than the αSyn protein itself e.g. ASYT or ASBP are sufficient to confirm Pdspectrally to acquire aberrant functions with even small changes it is desirable to make detailed attempts to verify findings to validate the study's observation via using non-fluorescently modified species | |
| PLA staining for oligomer | |
| ELISA for oligomer | |
| Staining for ANY aggregation but 너희 wish a sediment via assay results |