Gene-set-based analysis identified two pathways (A total of xx genes constituting these pathways)
the more genes for any given pathway are identified, the greater the confidence that this pathway should be prioritized over others (gene set enrichment analysis)
- concentrations of proteins in the same complex are less noisy compared with proteins that are not within one complex 31
Protein
We need single cell (RNA & Proteomics) since The same test (for example, changes in protein concentration over time) may produce entirely different results when focusing on a single cell vs the population.
We need perturbed system since ‘response to stress/damage/pathologic/dynamic chane’, not steady-state. Especially perturbed systems reveal large differences between protein and mRNA abundance changes 28, 29.
Broad scale assessment, molecular profiling
[Targeted approach]
Issues: resource in MBM,
Could we develop a BM for screening and selecting the patients, using blood BM (eg NDE TOM40/20 or any others?)
- Short term (pilot): compare Parkin-PD and sPD and HC for blood BMs using existing methods (eg. ps65-UB, EV mito)
- Juntendo univ (Dr. Hattori) for Parkin-PD blood samples.
- Parkin-PD vs HC: what markers are altered in the patients who has mitochondrial dysfunction?
- sPD: How many sPD patients have mitochondrial dysfunction as measured on identified BMs?
- CSF 도 법이 correlation 보면 좋은데 Juntendo univ doesn’t have CSF, NCNP has CSF samples but doesn’t have sufficient Parkin-PD patients (ie 200-300 PD patients in total)
- NDE 단계로 가면서 걸러지면 1단계에서 많은 BM을 검사해야 할 텐데, 종국에 NDE 아닌 Blood 사용도 option 일 것임
- Long term:
- develop plasma-derived NDE-based BM and characterise in sPD patients
- We would need Natural Hx STUDY (?)
Question
- Clinical trial feasibility in terms of cost? (eg. if the prevalence of high-risk sPD is 20%, we will need to screen 500 patients to get 100 patients)
Genes Mitochondria
MitoCarta3.0, mito-protein database
PRS Mitochondria
(Billingsley, 2019 #1190), for sPD
Method:
- primary mito gene (n=178): nuclear genes implicated in genetic mito disorders, ⓒ secondary mito genes (n=1328): nuclear genes implicated in mito function
- 이 연구가 GWAS 자체는 아니며, 기존의 GWAS를 leverage 한 것임.
[PD DEVELOPMENT Risk] primary and secondary genelists are significantly associated with increased PD risk
[Progression] the primary mitochondrial gene list was not significantly associated with AAO of disease, but the secondary gene list was correlated with later AAO, (each 1SD increase in PRS → a 0.55 year increase in the AAO), suggesting disease causation and progression are genetically separable processes in PD
(mRNA level을 보는 eQTL을 도구변수로 사용한 Mendelian randomization)
Further analysis suggested that the 14 identified genes are not only involved in mitophagy, but implicate new mitochondrial processes (ie mitochondrial bioenergetics and proteostasis pathways)
Following two are mi-ribosome-related genes whose expressions are altered in sPD: ↑ MRPS34 (blood) ↑ MRPL43 (blood)
(GWAS는 leverage된 것)
OWAS leverage된 것
MC1 PET
[18F]BCPP-EF (MC1) PET tracer
Mechanism
BCPP compounds specifically inhibit NADH-driven proton transport by binding to MC-I. (Kazami, 2019 #874). These results strongly suggest that BCPP compounds are MC-I inhibitors that share the same binding site as rotenone on MC-I
Fig. 5. Model of inhibitory effects of respiratory chain complexes by BCPP compounds. BCPP compounds specifically inhibit NADH-driven proton transport by binding to MC-I.
- may also have potential as a reporter for mitochondrial density.
근거: Rotenone competes with each other (in the living brain)
- Rotenone || compete with each other (in the living brain)
- BCPP compounds (Fig. 1) bound to the site shared with rotenone using PET imaging in the living brain (Harada et al., 2013; Tsukada et al., 2014a,b). (2019 Kazami)
- BCPP compounds inhibited the binding of 3H-dihydrorotenone to MC-I and the proton pumping activity of MC-I in vitro
- In vitro studies on rat brain slices allowed higher doses of rotenone and signal intensity of 18F-BCPP-EF was decreased by 96% upon maximum block (McCluskey, 2020 #484),
- The IC50 value of BCPP-EF for NADH-driven proton pumping activity was 26.1 nM. Compared to our previous study, the dose of BCPP-EF in PET imaging for rats (Harada et al., 2013; Tsukada et al., 2014a) and monkeys (Tsukada et al., 2014b,c,d, 2016; Kanazawa et al., 2017) were lower than this value by one thousandth. Moreover, it is one ten thousandth lower for humans (data not shown). → 따라서 pet probe로서는 physiologically inactive and does not inhibit MC1.
- (McCluskey, 2020 #485) review
- Although other mitochondrial complex 1 tracers exist, such as 18F-Flurpiridaz used in cardiac imaging [273], only 18F-BCPP-EF has been successfully used for imaging of mitochondrial complex 1 in the brain.
- MIND-MAPS-PD https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/mind-maps-pd/, ‘MIND-MAPS update for PET SC’ slide by Adam Schwarz
MC1 PET studies
| study | cohort | design | results / notes |
|---|---|---|---|
| MJFF Funding Proposal {Wilson, 2020 #902} MIND-MAPS https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/mind-maps-pd/ | PD patients n=12, early PD (disease duration < 2y) & healthy controls | https://www.michaeljfox.org/grant/mitochondrial-complex-1-pet-imaging-parkinsons-disease |
- Cross-sectional: The signal intensity of [18F]BCPP-EF PET was not different between sPD patients and controls - Longitudinal f/u (11 m interval): no significant longitudinal changes 다음 부분 이해 안 감: Volumetric analysis revealed loss of volumen only in the substantia nigra (-26%) (baseline 줄어있다는 의미?) in early drug-naive PD patients compared with healthy controls. Therefore, we cannot fully exclude the possible effect of volume loss on PET measures in the substantia nigra and that the changes observed in SV2A, σ1R, and MC1 within the substantia nigra do not reflect, at least in part, neuronal loss. |
| {Mansur, 2018 #876} | n=12 PD vs 60 Healthy Controls / Drug naive PD | 18F-BCPP-EF + 11C-SA-4503 + 11C-UCBJ | Highest uptake in striatum, ↓ in signal with age, suggesting that striatal mito density could be particularly susceptible to aging. Brainstem Dorsal Substantia Coeruleus 등 in regions of interest. Distribution of regional VT estimates (FIGURE 4). |
| {Mansur, 2021 #2013} | Test-retest variability favorable: 2-7%. it would appear that the centrum semiovale can be used as a pseudo ref region negating the need for arterial sampling which could greatly simplify the clinical protocol. | ||
| {Tsukada, 2016 #734} | MPTP monkeys | Method: MPTP at doses ranging from 0.2 to 0.4 mg/kg of free base in phosphate-buffered saline was injected intravenously once per week over a 4-mo period until a stable parkinsonian syndrome was observed → MPTP administration was halted for 2 mo, from the last MPTP treatment to the first PET measurements (to avoid competitive binding) |
Results: Significant reductions in presynaptic dopamine parameters and MC-I activity were detected in the striatum of MPTP monkeys. The plot of the striatal Vt of 18F-BCPP-EF against the BPND of 11Cb-CFT (DAT PET) revealed a positive correlation (R² = 0.673, P < 0.001). 3종류의 dopamine synthesis PET 과도 correlated, 특히 11C-6MemTyr (11Cb-β-methyl-m-tyrosine) 과는 R²=0.834 Reduced PET signal 원인은? Below is js SN: neuronal loss + ↓ MC1 density Cortex: ↓ MC1 density degeneration of nigrostriatal dopamine neurons was induced by the intravenous administration of MPTP to monkeys over a period of several months |
{Harada, 2013 #878} [Radioligand binding study] no cells
Specific binding of [18F]BCPP-EF, [18F]BCPP-BF, or [11C]BCPP-EM to MC-I at each rotenone concentration was calculated by subtraction of the radioactivity of nonspecific binding determined at 20 mM from the radioactivity at each dose of rotenone.
Method 1: (마취쥐 decapitation → brain removal → slicing with microtome) Living brain slice imaging (Radioactivity was converted to digitalized imaging data), 22t: Fig 4.2.2.
- PET: After continuous infusion of vehicle or rotenone at dose of 0.1 mg/kg/h for 1 h, PET scanning was conducted for 60 min after injection of each PET probe.
(concentrations) with 3H-dihydrorotenone (concentration was fixed at 4.5 nM, if not said).
results (suppl fig) — Concentration vs binding curve.
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Tsukada Vt vs BPND | R² = 0.673, P < 0.001 | Statistical values are partly stylized in the OCR (with leading 50 / 5O); reading reconstructed from context. |
| Billingsley primary gene count | n=178 | The leading digit is partly stylized; preserved as visible. |
| Mansur 2018 cohort | n=12 PD vs 60 Healthy Controls | The 60 Healthy Controls value is partly cut by a column boundary; preserved as visible. |
| McCluskey reference | (McCluskey, 2020 #484) and (McCluskey, 2020 #485) | Two close reference numbers (#484 and #485) appear in the same paragraph; preserved as visible. |