20240722_183455

Assessment of mitochondria

Summary

		AMED	GE (blood)	Priority	Autopsy brain feasibility?	Brain variability	Fibroblast variability	Serum feasibility?-living	Serum feasibility?-postmortem	PBMC	CSF feasibility?-living	CSF feasibility?-postmortem	Blood variability	Blood vs serum?	Evidence in PD
	Ps65-ub	O	O	Not high (only PARK2-relevant)	O (hou)			Under development						Serum OK 겠지
	Parkin	O	O
	Mito membrane proteins (TOM40/20)	O	O (choong mouse serum, WB0	Not high (structure, maybe compensated) not age-related	O (BUT no study)			Choong, mouse Serum, WB						Serum OK
Biogenesis	mtDNA copy number	O	Plasma(: (Al Amir Dache, 2020 #1513		O			Plasma(: (Al Amir Dache, 2020 #1513, (Borsche 2020) ↑serum mtDNA copy number in PRKN/PINK-PD (fig2, vs HC & sPD)			Peng, 2019 #1051			Serum OK
Somatic damage	Mt DNA damage	O	O Plasma(사용논문: (Al Amir Dache, 2020 #1513),	High (rotenone & age-related)	O (sanders 2014)			Blood, qPCR (Greenamyre lab) Plasma(사용논문: (Al Amir Dache, 2020 #1513),						Plasma OK (Detroit)	= in WBC (Davis et al., 2020, PMID: 32190419)
Cardinal,	ATP production		Inhouse & plasma, luciferin-luciferase Assay	High (ultimate output)	X Barksdale 2010 : not feasible	극심		X (Stier)			(HPLC in CSF)		Gorman fig4/table 2 (그닥잖은듯?)	Plasma OK
Glycolysis may affect this	ATP basal				X (severely affected by PMI), (but, animal 은 가능할 것)			Yes (Gorman 2007)			O (HPLC)
function	MMP		C1 in human (SAH) CSF & plasma {Chou, 2017 #1048}	High? (mito health?)	x			Plasma, JC1, Chou, 2017 #1048}			Plasma, JC1, Chou, 2017 #1048			Plasma OK	↓ (Park2-PD Fibrolast)
function	MC1 activity		No paper,	Rotenone-relevant, but ↓ brain-link (?)	분명가능 (schapira)			Cf) WBC 에 선 본 논문 존재		O
	OCR			? STEve 는 이거 ex vivo? 로 된다는 듯.						O	Enzo, Seahorse
	Phosphatidylethanolamine
	N-formyl peptides (NFP),
	Methylated mtDNA							TaqMan PCR 이지 않을가?

good	Not good
Specific and early (linked to causative epathological mechanisms)	Nonspecific or late 9reflecting reactive plasticity or self-perpetuating neurodegenerative cycles) or physiological response to injury
Constant throughout the course of the disease	Unstable ( transient)
Reflect homogenous population
Oxidative phosphorylation	Glycolysis
neuron	Glial
>2SD above or below the mean

js: CSF 에서 change? sPD 에서 조금이라도 change? (lactate: no change at all)

• Rationale:
  • a- Significant heterogeneity in the pathways among different patients with PD? Heterogenous genetics & clinical variability
  • b- Environmental toxin (rotenone, pesticide)와 연결짓자
  • Pesticide의 OR도 상당하다

Δψm		CSF	Blood	Postmortem	In vitro	In vivo
	Together with the proton gradient (ΔpH), Δψm forms the transmembrane potential of hydrogen ions 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.	JC1 in human CSF & plasma {Chou, 2017 #1048}, and rat CSF {Chou, 2013 #1106} JC1 in human (SAH) CSF & plasma {Chou, 2017 #1048}		(상식) 불가능 {Barksdale, 2010 #1502} mt fraction 에서, PMI 7.5h 까지만 측정가능 (ie CCCP 등 줄 때 반응이 있다는 의미이며, 죽기전비교시 유지된다는 의미 아님) mouse 뇌: PMI 10h 까지는 PMI 0h 와 유사	[2018 Cleeter] CBE → ↓ Δψm Rotenone (+ complex 1) → ↓ CCCP / FCCP → ↓ (Spinazzi, 2012 #1052)	CCCP / FCCP → ↑ proton permeability across the mitochondrial inner membrane → ↓ (Hayakawa, 2016 #886)

[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

{Chou, 2017 #1048} mitochondria can be released into extracellular space, and transferred from cell to cell 4-8

Mitochodnria on electron microscopy,

Mitochodnria on Transmission electron microscopy ((Hayakawa, 20.. #10..)

(continues on next photo: “…mitochondria인지는 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]“)

Uncertain Spans

location	transcription	uncertainty
Wide assessment table column header “AMED” / “GE” / “Priority”	column boundaries	The very wide top-row header is partially cut at the very top edge of the page; column placement inferred from cell alignment in subsequent rows.
Mt DNA damage row right-side cell ”= in WBC (Davis et al., 2020, PMID: 32190419)“	PMID 32190419	The PMID number font is small in the rightmost cell; transcribed digits as visible.
Δψm reference table row “[2018 Cleeter]“	exact author / year of citation	The “2018 Cleeter” label appears as a partial bracketed citation; transcribed as seen.
In vivo column “(Hayakawa, 2016 #886)“	year & ref id	Reference number “#886” is partially clipped at the right page edge; transcribed as legible.
Δψm In vitro column “Rotenone (+ complex 1)“	wording	”+ complex 1” preceded by “Rotenone” with parentheses; exact formatting in source slightly shifted.
Mitochodnria on electron microscopy trailing “((Hayakawa, 20.. #10..)”	Hayakawa year and reference id	Last bullet runs off the right edge; year and reference id are partially visible.