- feedback (low n number might be a concern)
- 2022 Aug 11: full proposal due
- 2022 Nov/Dec: Decision on funding
- 2023 Jan/Feb: Funding
- Monday June 6th - Jaewon sends Jessica/Christina total approximate budget and how many months for project (usually about 24 months)
- Friday, June 10th - MJFF sends Jaewon email invitation to submit RFP
- Monday, June 13th - Jaewon sends 1-2 page Letter of Intent, Christina/Jessica share this with Jamie Eberling (for reference), the Clinical Team and External Reviewers for their consideration
- Christina and Jessica gather any feedback as soon as possible
- If feedback is positive, Jaewon submits full proposal in MJFF system on Thursday, August 11th
- If more time is needed, we can look to the next RFP cycle ~3 months later
- Study title: MC1 PET [18F]BCPP-EF imaging in Parkin-PD patients
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MC1 PET in FA
Eugenii A. Rabiner) MC1 in FA & HV https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/mc1-in-friedreichs-ataxia-and-healthy-volunteers/
FA is a rare disease that causes damage to the nervous system and movement problems. One of the characteristics of this disease is damage to the mitochondria. We hope to measure the amount of MC1 in the heart in HV & FA patients in this study. We’re interested in the amount of MC1 in the heart as most FA have cardiac problems. Previously we have measured the amount of MC1 in the brain using a PET radiotracer called [18F]BCPP-EF. PET (positron emission tomography) is a medical imaging method, which uses marker molecules labelled with a small radioactive tag (called PET tracers) to measure the quantities of various molecules in the human body. We would now like to see if it is possible to use [18F]BCPP-EF to measure MC1 in the heart. We hope to use MC1 as a measure of the effectiveness of therapies to treat FA in the future.
HV & FA patients will have 1 screening visit. Eligible participants (up to 26 healthy men and women and up to 12 patients, aged 18-65) will receive either 1 or 2 PET scans depending on which part of the study they are taking part in. The patients will also complete 1 MRI (magnetic resonance imaging) scan.
Participants will be exposed to radiation levels equivalent to just over 3 years of naturally occurring background radiation.
- QTS synapsis review on 2/11/20
implantation of a microdialysis probe can alter tissue morphology resulting in disturbed microcirculation, rate of metabolism or integrity of physiological barriers, such as the blood-brain barrier.[33] While acute reactions to probe insertion, such as implantation traumas, require sufficient recovery time, additional factors, such as necrosis, inflammatory responses,[21] or wound healing processes have to be taken into consideration for long-term sampling.
Microdialysis has a relatively low temporal and spatial resolution compared to, for example, electrochemical biosensors. While the temporal resolution is determined by the length of the sampling intervals (usually a few minutes), the spatial resolution is determined by the dimensions of the probe. The probe size can vary between different areas of application and covers a range of a few millimeters (intracerebral application) up to a few centimeters (subcutaneous application) in length and a few hundred micrometers in diameter.[
- Animal model
(2019 iarc2019-completo-AF)
Results: SUVR relative to plasma were significantly reduced by ~50% in the heart and 57% in skeletal muscle in MCK mice compared to WT mice with no difference in other tissues measured (Fig.1). Ex-vivo dissection and tissue radioactivity estimation was also performed.
[18F]BCPP-EF PET imaging shows reduced MC1 density in frataxin deficient tissues in the MCK mouse model of FA.
We are currently evaluating [18F]BCPP-EF PET as a measure of MC1 density in the heart and brain of healthy volunteers (HV) and FA patients, in an open-label, single center, PET imaging study. The first goal of this work is to determine if [18F]BCPP-EF, which has been demonstrated to be suitable for the quantification of brain MC1 density, can be used for the same purpose in the heart, in 6 HVs.
If this is successful, we will proceed to compare cardiac MC1 density between adult FA patients and HVs.
Microdialysis
- Concept
- extracellular levels of a substance, that changes can reflect clearance and/or release
- the source of the change is not necessarily neuronal (although K induces changes in dopamine, GABA, serotonin, glutamate, 특히 K causes glial cells to release GABA) (Dopamine 도 그럴 염려 있나?)
- Limitations
- examples
| Reference | animal | region | readout | |
|---|---|---|---|---|
| (Wegrzynowicz, 2019 #233) | in vivo | MI2 and C57Bl/6S mice at 3, 6 and 12 months of age ( | striatum Str | Striatal dopamine release (following the infusion of 50 mM KCl for 60 min between 40 and 100 min of the experiment.) fig4b [bar-chart figure: 4 panels for 3 / 6 / 9 / 12 months; y-axis DA (fold change); x-axis time (min) 20 - 140; legend C57Bl/6S vs MI2; 50 mM K+ infusion bar covers 40-100 min] |
| (Ding, 2007 #1679) | Js: largely similar to Wegrzynowicz | |||
Mitophagy
MOA of Mitophagy
| Step | In healthy mitochondria | In unhealthy mitochondria | Detail | Note |
|---|---|---|---|---|
| PINK1 is recruited to mitochondria | (In healthy mitochondria) partially through the inner mitochondrial membrane via the TIM complex, so it then spans the inner mitochondrial membrane. The process of import into the inner membrane is associated with the cleavage of PINK1 from 64-kDa into 60-kDa. PINK1 is then cleaved by PARL into 52-kDa. This new form of PINK1 is degraded by proteases within the mitochondria. This keeps the concentration of PINK1 in check in healthy mitochondria.[8]) there is no Parkin recruitment here! | Mitochondria membrane depolarized (This membrane potential is necessary for the TIM-mediated protein import) → PINK1 is no longer imported into the inner membrane, is not cleaved by PARL and PINK1s are accumulated in the outer mitochondrial membrane. | (In healthy mitochondria, PINK1 is imported through the outer membrane via the TOM complex), and | |
| phosphorylates nearby ubiquitin at serine Ser65 (원래 E1 은 ub 를 adenylate 시키는 것이므로 을 E1 이라고 안 부름) → Parkin binds to the pUb → Parkin is recruited to the mitochondria | (directly) phosphorylates parkin (activates) at serine Ser65 in UBL domain → Parkin undergoes dimerization and becomes an 'FULLY' active state | Parkin (substrate specificity 없이 (substrate 여러 MOM protein 들을 닥치는대로) ubiquitylates (eg. mfn1/mfn2 &, VDAC, FIS1, TOMM20, CISD1, mitoNEET) on the cytoplasmic domain → ubiquitin chains are formed on MOM → | ||
| Polyubiquitin chain 모습?: Ubiquitin 이 길게 염주처럼 늘어난게 parkin 에 붙은 모습. | autophagy (=mitophagy) (뿔뿔이 떠돌아다니던) receptors (=mitophagy receptor) (eg TBK1, p62, TAX1BP1 and CALCOCO2,) are recruited to the ubiquitin chain (ie bind to ubiquitin) → Autophagosome 이와서 mito 를 둘러쌈 (LC3 역할함) → mito is degraded by hydrolases | |||
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This process takes only minutes (Harper, 2018 #755), [feedforward process to form (phosphorylated) polyubiquitinated chain for mitophagy] xi) Pink1 phosphorylates ubiquitin (so it can recruits Parkin) and Parkin (so it gets activated) → Parkin is recruited → Parkin adds this ps65-ubiquitin onto substrate) repeatedly (so polyubi chain is extended) Note, 이 ps65-ub chain 의 (애조의 phosphorylation 은 PINK1의 role 이지만), elongation 은 Parkin 의 role | ||||
| Substrate specificity 없으므로 그냥 density of ubiquitin chain 보는게 좋음 (Harper, 2018 #755), eg. 'abundance of K48 or K63 chains' ((Ohtake, 2016 #756) | ||||
readout
- neuronal mitophagy occurs via the PINK1-Parkin pathway.[14]
- other mitophagy pathway
- mitophagy receptors on the outer mitochondrial membrane surface. These receptors include NIX1, BNIP3 and FUNDC1.
(carbonyl cyanide m-chlorophenylhydrazone) CCCP
- a lipid-soluble weak acid and
- a potent mitochondrial uncoupling agent that increases the proton permeability across the mitochondrial inner membranes → dissipate the transmembrane potential → depolarizing the mitochondria → ↑ mitophagy
- (Ding, 2010 #695) (ie ↑ GFP-LC3 punctation and the formation of lipidated LC3 in a dose-dependent (Fig. 1, A-C and supplemental Figs. S1, A and B, and S2, A-C) and time-dependent (supplemental Fig. S2, D and E) manner in HeLa cells (Fig. 1), HCT116 cells (supplemental Fig. S1), and MEF (supplemental Fig. S2). ↑ autophagosome (electron microscopy)
- allows mitochondrial respiration to proceed at the maximal capacity for the components of the electron transport chain without regard for either the capacity of complex V or proton leak. (2004 palacino)
Mitophagy Probes
| For Living samples | In vivo | For Fixed sample | ||
|---|---|---|---|---|
| mt-Keima | applicable | Not Applicable (continues on next photo) |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| MJFF timeline left-side photo / portrait | small headshot photo | A small portrait image appears at the top-left of the timeline column; preserved as evidence not embedded; person identity not legible. |
| MOA of Mitophagy table column boundaries | 5 columns | The MOA table uses many narrow nested cells; column boundaries inferred from the top header row. |
| Wegrzynowicz bar-chart fold-change axis values | DA (fold change) ranges 0-2 / 0-1.6 across panels | The bar-chart inset has small y-axis numerals (0.3 / 0.6 / 0.9 / 1.2 / 1.5 / 1.6 / 2); transcribed from the most legible. |
| MC1 PET FA timeline “(Mortiboys et al. 2008, PMID: 19067348)“ | PMID digits | PMID number 19067348 verified against visible digits; second PMID 31333417. |
| Mitophagy probe table “Not Applicable” trailing text | trailing line clipped | Right edge of “For Fixed sample” column is partially clipped on this capture; full text continues onto next photo. |