MMP (Mitochondrial Membrane Potential, ΔΨm)
Together with the proton gradient (ΔpH), ΔΨm forms the transmembrane potential (ΔΦm)
- the gradient of the electric potential on the inner mitochondrial membrane generated by proton pumps (Complexes I, III and IV)
- results from redox transformations associated with the activity of the Krebs cycle
- serves as an intermediate form of energy storage which is used by ATP synthase to make ATP
- form the transmembrane potential of hydrogen ions
- a driving force for transport of ions (other than H+) and proteins which are necessary for healthy mitochondrial functioning
- is also a factor determining viability of mitochondria participating in a process of elimination of disabled mitochondria
[Measurement]: detected using cationic (positively-charged) fluorescent dyes (eg. TMRM) that accumulate in the negatively-charged mitochondrial matrix.
[JC-1]
In normal cells ΔΨm is highly negative, -180 mV, JC-1 dye monomer forms red fluorescent (emission at ~590 nm) “J-aggregates” where it has accumulated within the mitochondria.
Collapsed mitochondria: ↑ green (emission at ~529 nm) & ↓ red JC-1
the more negative the ΔΨm, the more dye (eg. TMRM) accumulates.
JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a green fluorescence emission at (~529 nm) for the monomeric form of the probe, which shifts to red (~590 nm) with a concentration-dependent formation of red fluorescent J-aggregates (see Figure 1). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
The ratio of green to red fluorescence depends only on the mitochondrial membrane potential and not on other factors such as mitochondrial size, shape, and density.
These dyes can be used qualitatively in fluorescence microscopy or quantitatively in flow cytometry or microplate spectrophotometry.
[MitoTracker® dyes]
Enter mito accumulated depending on MMP like JC-1 (unlike TMRM & rhodamine 123), its mildly thiol-reactive chloromethyl moiety forms a covalent bond with thiols on proteins and peptides, which traps MitoTracker dyes within mitochondria., even when mito loses MMP, MitoTracker dyes still exist in the mito, thus it is not a marker of ‘functional’ mito {Kholmukhamedov, 2013 #1050}
Rhodamine 123 assay
PHRET used JC1
Korean inline (상식): 불기능 (Barksdale, 2010 #1502) mt fraction 에서 PMI 7.5h 까지만 측정가능 (ie CCCP 칠 때 반응이 있다는 의미이며. 죽기 전 비교시 유지된다는 의미 아님), mouse 뇌: PMI 10h 까지는 PMI 0h 와 유사
[2018 Cleeter]
- CBE → ↓ ΔΨm
- Rotenone (1 complex 1) → ↓ ΔΨm
- CCCP / FCCP → ↑ proton permeability across the mitochondrial inner membrane → ↓ ΔΨm
Mito (Circulating cell-free mitochondria)
{Chou, 2017 #1048} mitochondria can be released into extracellular space, and transferred from cell to cell
- Neurons can release damaged mitochondria and transfer them to astrocytes for disposal and recycling. (Hayakawa, 2016 #1052), and vice versa, Here we show that astrocytes in mice can also release functional mitochondria that enter neurons.
- Quantification: flow cytometry for all mitochondrial particles labeled with MitoTracker Deep Red FM (functional mitochondria 인지는 모르는 것임). These CSF mito originated from astrocyte.
- extracellular mitochondria tend to be contained within membranous particles.
(Choong, 2020 #1193)
[cf: cellular Origin?]
Markers: GLAST (astrocyte), vWF (endothelial cells), CD45 (microglia/microphage), and CD41/61 (platelet) [1].
(Hayakawa, 2016 #1052) neuronal mt by MAP2, astrocytic mt is CD38 (In the brain, CD38 is mainly expressed in glial cells)
[Mitochondrial integrity]
(Genç, 2021 #1297) Betz cells from patients, EM, Mitochondria with intact outer and inner membrane and with intact cristae were considered healthy (fig2)
[MT in exosome]
Mitochondrial mass
Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle 9, is localized in the mitochondrial matrix, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass
mito mass (porin and GRP75) (integral membrane protein, js does porin include VDAC?) (Grünewald, 2016 #935)
porin and adenine nucleotide transporter (ANT)
(Schapira, 1990 #877) n=9, SN
MC1 activity
[ubiquinone & rotenone 사용 (Rotenone-sensitive assay)]
2008 Mortiboys (primary is Birch-Machin 1994): measures NADH oxidation before and after rotenone addition (ie rotenone-sensitive assay)
This kit uses decylubiquinone, an analog of ubiquinone (=CoQ10), as an electron acceptor that gets converted to decylubiquinol through the catalytic activity of Complex I. The Complex I dye that absorbs light at A600 nm in its oxidized form is used as a terminal electron acceptor that accepts electrons from decylubiquinol. Complex I activity is determined colorimetrically by recording the change in absorbance of reduced Complex I dye at A600 nm. Specific Complex I activity is obtained by subtracting the activity in presence of rotenone (이것도 rotenone-sensitive assay) from total activity. This kit can detect as low as 0.1 mU/well and is linear up to 7 mU/well.
[ubiquinone & rotenone 사용 × (Rotenone-insensitive assay)]
이게 PhReT것인듯, i) oxidation of NADH to NAD+ ii) FMN Fe-S로의 전달 ii) ubiquinone (CoQ10)으로의 전자전달의 3단계 중 첫번째만 보는 것이네.
ab109721: Colorimetric, Samples are added to the microplate wells which have been precoated with a specific capture antibody. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. This activity is not dependant on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay.
{van der Merwe, 2014 #722} functional MC1 content was measured by NADH ferricyanide reductase assay, which allows to determine the content of functional complex I): 원문과 달리 이건 function 인 듯
complex I-linked ADP phosphorylation with glutamate/malate as substrate in the presence of a limiting quantity of ADP, which is a substrate for complex V, the ATP synthase. Js: 이게 palacino 에는 O2 consumption 측정이고, cleeter 에는 ADP Phosphorylation 측정이네
Example {Palacino, 2004 #726, mouse brain} {Cleeter, 2013 #533}
MC2 activity
with succinate as substrate in the presence of a limiting quantity of ADP, which is a substrate for complex V, the ATP synthase. Js: 이게 palacino 에는 O2 consumption 측정이고, cleeter 에는 ADP Phosphorylation 측정이네
Example {Palacino, 2004 #726, mouse brain} {Cleeter, 2013 #533}
MC4 activity
With TMPD/ASCORBATE as substrate in the presence of a limiting quantity of ADP, which is a substrate for complex V, the ATP synthase. Js: 이게 palacino 에는 O2 consumption 측정이고, cleeter 에는 ADP Phosphorylation 측정이네
Example {Palacino, 2004 #726, mouse brain} {Cleeter, 2013 #533}
Mito membrane proteins
Outer membrane protein (TOM40, TOMM40는 아마 GENE 이름? Vice versa)
Inner membrane protein (ANT1)
In house: (WB, Plasma), VDAC, PHB2, CD171 were detected
Cf) For western blot analysis of CSF and serum, 500 µL of CSF and serum samples were ultracentrifuged at 60,000 × g for 1 h and the pellets obtained were used for western blot analysis. {Choong, 2020 #1193}
- TOM40 & ANT1 in human CSF (WB) {Choong, 2020 #1193}
- TOM20 in Parkin KO mice serum {Chou, 2017 #1048}
Mito O2 consumption
If there’s one thing that mitochondria thrive on, it’s oxygen. All of it is consumed by cytochrome oxidase, the last enzyme in the electron transport chain which drives ATP production.
equal amounts of mitochondria consumed equal amounts of oxygen (Hoppeler, 1987 #1166)
In oxidative phosphorylation, oxygen must be present to receive electrons from the protein complexes. This allows for more electrons and high energy molecules to be passed along, and maintains the hydrogen pumping that produces ATP.
There was a significant correlation between the volume density of mitochondria and maximal oxygen consumption.
Mito-ID Extracellular O2 sensor kit (Enzo Life Science, ENZ-51045), FIG 4j (Hayakawa, 2016 #1052): liquid sample에 사용, tissue엔 못 쓰는듯
[for discussion with Kamiguchi san’s group]
- assay development
- measurement in CSF
- in vitro lysate and supernatant (btw this reflect CSF?)
- Parkin-pd 에서 본 것 없으니 (mortiboys등 안 봤음), 우리 또 보는 것 의미일 것.
- validation with mouse brain tissue (parkin KO mouse availability 까다로울수도), 동물 별 무의미 아닌지?
- Patient brain tissue?
- Niigata univ. Midbrain 에서 확인 필수일텐데 intracellular OCR 볼 방법 없는 듯 (SEAHORSE 는 Extracellular OCR 봄)
[Cutaneous respirometry] {Harms, 2016 #1092}
mitochondrial oxygen tension (mitoPO2) and oxygen consumption (mitoVO2). It does this by using the oxygen-dependent optical properties of protoporphyrin-IX, a haem precursor synthesized within mitochondria, known as the PpIX-tripletstate lifetime technique. Testing in healthy volunteers is at an early stage, but has confirmed that mitoPO2 and mitoVO2 measurements are viable (Harms et al., 2016).
Oxygen-dependent delayed fluorescence measurements
(Harms, 2016 #1092) HV (N=30), clinical prototype
[Seahorse flux analysis]
- simultaneously measures pericellular pH and (extracellular) oxygen concentration in media as a function of time
- glycolysis도 동시에 잰다는 듯
- used in rat brain section ({Fried, 2014 #1162}, fibroblast {Haylett, 2016 #1155})
Reagents added in sequence: Oligomycin / FCCP / Rotenone + antimycin A
Key parameters:
- basal respiration (basal measurement minus rotenone/antimycin A response);
- ATP-linked OCR (basal measurement minus oligomycin response, the dependence of OCR on ATP synthase activity);
- ATP turnover, OCR due to proton leak (oligomycin response minus rotenone/antimycin A response);
- ATP coupling efficiency (basal mitochondrial OCR divided by ATP-linked OCR);
- maximum OCR (FCCP response minus rotenone/antimycin A response) and spare respiratory capacity (maximum OCR divided by basal mitochondrial OCR), maximal respiration (efficiency of ETC), and nonmitochondrial respiration (rotenone/antimycin A response).
oligomycin, an inhibitor of complex V
[oxygen extraction fraction (OEF)]
defined as the percent of the oxygen removed from the blood by tissue during its passage through the capillary network (the ratio of blood oxygen that a tissue takes from the blood flow), reflect neural activity, cerebral blood flow (perfusion)! 즉 cellular metabolism 보다는 hemodynamics 를 더 reflect 할 것 같음.
(Liang, 2020 #1208) T2-relaxation-under-spin-tagging (TRUST) MRI, (또 150 PET)
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| MMP intro | (∆4m) / AWm | The Greek Ψ (psi) is rendered inconsistently in the source as 4, W, etc.; reconstructed as ΔΨm. |
| Sigma-Aldrich URL | mak359?lang=ja®ion=JP | URL is rendered partially with OCR-confused characters; reading is best-effort. |
| MC complex labels | MC1 / MC2 / MC3 / MC4 activity rows | The leftmost column letters are partly cut by the navigation pane; reconstructed by adjacency to the named complexes I-IV. |