| probe | Argument / Example | Counterargument |
|---|---|---|
| EGFP-mCherry fusion | The fluorescence of these proteins is differentially sensitive to the lysosomal environment EGFP is sensitive whereas mCherry is not, and this difference is used to monitor lysosomal delivery of the probe | 일된 fixation된 후엔 mitophagy 일어나니므로 mtKeima 넣어봐야 무의미할 것. |
| TOLLES (TOLerance Of Lysosomal EnvironmentS) (Katayama, 2020 #769) | TOLLES: completely resistant to acidity. applicable (lysosome 안에서 유지됨), & neutral pH로 환경 바뀌어도 유지됨 (fixation해도 signal 냄) YPet YFP (Yellow fluorescent protein) irreversibly acid-denatured and degraded by pepsin (lysosome 안에서 없어짐) | Living cell에서 mt-Keima 넣은 후라도 이후에 fixation 이면 pH gradient across lysosomal membrane 이 없어지므로 mt-Keima의 signal 이 없어진다는 의미인 듯 |
| YPet fusion | ||
| mt-SRAI | SRAI: (signal-retaining autophagy indicator). SRAI is engineered to localize into the mitochondrial matrix by fusing a tandem repeat of the cox VIII presequence, (the degree of localization to mitoch is comparable to mt-Keima) Verified using CCCP-induced mitophagy | 일단 fixation 한 후에는 이거 쓸 수 있나? Mitophagy 안 일어나니 당연히 안 되겠지. |
| (SN AAV-mtSRAI) | Use AAV system to express mito-SRAI in neurons in the right SN of mice. mice were transcardially perfused with fixative for histological examination js: 미리 mt-SRAI가 mitoch와 lysosome에 생체내에서 가도록 한 후 fixation imaging 가능함 |
mitophagy in vivo
How to compare WT mice vs PARK2 mice? WT에서는 무엇을 기대하나? CCCP 투여할 수 있나? TR-FRET 에서 Takeshi 처럼 E1, 2 & FRET-donor/acceptor Ub를 living brain에 넣으면 mitophagy 일어나나?
| 후보지들 | Argument / Example | Counterargument |
|---|---|---|
| TR-FRET | Takeshi: I will add E1, E2, and Ub (FRET donor and acceptor) included in this kit to cell/brain lysate in order to measure exogenous functional Parkin in PARK2 KO cells/mice. | Misa: we need to add artificially E1, 2 and FRET-donor/acceptor Ub into biosample (i.e. brain) including parkin. In this assay, we can not understand if exogenous parkin (by AAV) is working in the brain, and mitophagy is induced by exogenous parkin. |
| pS65-Ub | Basal level: should be no different between WT and PARK2 KO Mice. Ability ie with CCCP: PS65UB will not show increases in PARK2 KO Mice, but WT mice will 가능하겠지. | In vivo 에서, AAV-PARK2 와 CCCP 를 동시에 투여할 방법이 없을텐데? |
| Mitoch numbers etc | ||
| MJFF (Padmanabhan, 2019 #820) | mtDNA copy number, transcription, mitochondrial membrane potential and Cori Cycle flux in PBMC In an attempt to determine if mitophagy can be monitored in vivo in preclinical models, at endogenous levels of these proteins, a new mitophagy reporter mouse model was recently generated (45). Surprisingly, deleting PINK1 or PRKN failed to influence basal mitophagy in the SN. | |
xli) Assay Development → Assay Optimization → Assay Validation
xlii) Figure 3. PINK1-Parkin pathway biomarkers. The schematic demonstrates the three essential phases for a biomarker assay: development, optimization and validation. Most biomarker efforts for PINK1 and Parkin fall under the assay development and assay optimization phases. MJFF-funded studies are listed along with the platform used for analysis in parenthesis. The assay development efforts use recombinant proteins while assay optimization efforts are in PBMCs derived from PINK1 and/or PRKN mutation cases and controls.
Assays:
- Parkin ligase activity (ELISA)
- pS65Ub (MSD)
- Miro (ELISA)
- mtDNA copy number, deletion rate & transcription (Real-time PCR)
- MMP & Cori flux (Flow cytometry & stable-isotope labeling with mass spectrometry)
- Mfn1/2 (Immunoblotting)
Monthly update
GBA-PD GT
| GBA activator | Parkin GT | cGAS | |
|---|---|---|---|
| 202008 NEW | new | new | https://mytakeda.sharepoint.com/sites/cGAS |
| 202009 (예시) | ALL old, GBA mRNA in NDE | Parkin PET OUT NEEDED | No need at all 일단 작업하자 이건 나중에 |
202009 BM timeline update needed,
Monthly report has
- GBA protein in CSF/plasma, (plasma 여긴 없으니 어쩔? LGE slidedeck에 CSF만 있으니 일단 plasma 빼보자)
- GBA mRNA in NDE
- DatScan 지우자
- aSyn 추가하자 (p-aSyn, RT-Quic)
Sebastian’s slide has
- GBA activity CSF (bps sep 2020) (BFA Mar 2021)
- Cathepsins (bps sep 2020) (BFA May 2021)
- GBA protein CSF (bps sep 2020) (BFA Feb 2021: activator PE와 대략 맞네)
- GBA mRNA in NDE 통째 필요함
BMx Milestones project timeline
Gantt chart spanning FY2020 / FY2021 / FY2022 / FY2023 (Q1-Q4) with milestones:
- Project timeline / BMx development / LG / PE / CN / CS / B
- TE: Parkin PET
- PF: Parkin protein CSF
- BPS / BFA / D: pS65-Ub
- BTV / R: MC1 Imaging
- R: DaTSCan
Milestone codes: BPS = BMx Platform Start, BFA = BMx Feasibility Assessment, BTV = BMx Technical Validation, BCS = BMx Candidate Selected.
MRI
Classification
| Group | Sequence | Abbr. | Physics | Main clinical distinctions | Example |
|---|---|---|---|---|---|
| Spin echo | T1 weighted | T1 | Measuring spin-lattice relaxation by using a short repetition time (TR) and echo time (TE). |
• Lower signal for more water content,[4] as in edema, tumor, infarction, inflammation, infection, hyperacute or chronic hemorrhage.[5] • High signal for fat[4][5] • High signal for paramagnetic substances, such as MRI contrast agents[5] Standard foundation and comparison for other sequences | |
| T2 weighted | T2 | Measuring spin-spin relaxation by using long TR and TE times |
• Higher signal for more water content[4] • Low signal for fat[4] - Note that this only applies to standard Spin Echo (SE) sequences and not the more modern Fast Spin Echo (FSE) sequence (also referred to as Turbo Spin Echo, TSE), which is the most commonly used technique today. In FSE/TSE, fat will have a high signal.[6] • Low signal for paramagnetic substances[5] Standard foundation and comparison for other sequences | ||
| Proton density weighted | PD | Long TR (to reduce T1) and short TE (to minimize T2).[7] | Joint disease and injury.[8] • High signal from meniscus tears.[9] (pictured) | ||
| Gradient echo (GRE) | Steady-state free precession | SSFP | Maintenance of a steady, residual transverse magnetisation over successive cycles.[10] | Creation of cardiac MRI videos (pictured).[10] | |
| Effective T2 or "T2-star" | T2* | Spoiled gradient recalled echo (GRE) with a long echo time and small flip angle[11] | Low signal from hemosiderin deposits (pictured) and hemorrhages.[11] | ||
| Susceptibility-weighted | SWI | (GRE), fully flow compensated, long echo time, combines phase image with magnitude image[12] | Detecting small amounts of hemorrhage (diffuse axonal injury pictured) or calcium.[12] | ||
| Inversion recovery | Short tau inversion recovery | STIR | Fat suppression by setting an inversion time where the signal of fat is zero.[13] | High signal in edema, such as in more severe stress fracture.[14] Shin splints pictured. | |
| Fluid-attenuated inversion recovery | FLAIR | Fluid suppression by setting an inversion time … | High signal in lacunar infarction, multiple sclerosis (MS) … [15] | ||
| Double inversion recovery | DIR | Simultaneous suppression of cerebrospinal fluid and white matter by two inversion times.[16] | High signal of multiple sclerosis plaques (pictured).[16] |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| MJFF Figure 3 caption | Mfn1/2 (Immunoblotting) | The Mfn assay platform parenthesis is partly cut at the right edge; preserved as visible. |
| Padmanabhan 2019 #820 | citation #820 | Reference number is small in the source; preserved as visible. |
| 202009 cGAS column | https://mytakeda.sharepoint.com/sites/cGAS | URL is rendered partly stylized in the OCR; preserved as visible. |
| BMx Milestones legend | BMx Milestones BPS-BMx Platform Start, BFA-BMx Feasibility Assessment, BTV-BMx Technical Validation, BCS-BMx Candidate Selected | Several letter codes are partly obscured by the Gantt chart shading; reconstructed by adjacency. |