Main function of mitochondria is ATP synthesis Electrochemical - - PowerPoint PPT Presentation

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Main function of mitochondria is ATP synthesis Electrochemical - - PowerPoint PPT Presentation

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A NOVEL BIOLUMINESCENT PROBE FOR NON - INVASIVE QUANTIFICATION OF MITOCHONDRIAL MEMBRANE POTENTIAL 1 Main function of mitochondria is ATP synthesis Electrochemical proton gradient is the driving force for ATP synthesis 2 Electron transport

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SLIDE 1

A NOVEL BIOLUMINESCENT PROBE FOR NON-INVASIVE QUANTIFICATION

OF MITOCHONDRIAL MEMBRANE POTENTIAL

1

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SLIDE 2

Main function of mitochondria is ATP synthesis

Electrochemical proton gradient is the driving force for ATP synthesis

2

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SLIDE 3

Electron transport chain (ETC)

3 Nicholls, D. G. & Ferguson, S. J. Bioenergetics. Fourth edition / edn, (Academic Press, Elsevier, 2013).

Energy of proton gradient: βˆ†π» = πΊβˆ†πœ” RT ln 𝐼

  • 𝐼
  • = πΊβˆ†πœ” 2.3RT Ξ”π‘žπΌ

Ξ”Οˆ – electric potential, MMP

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SLIDE 4

4 Figures are adapted from Cell Metabolism (2015) 22, 204-206

  • Mol. Cell Bio. (2005) 6, 318-327

Intracellular signaling

  • Change of gene expression

profile

  • Adaptation to stress
  • Apoptosis

Biosynthetic function

  • Long chain fatty acids
  • Branched chain amino acids
  • Nucleotide biosynthesis

ATP synthesis is not the only role of mitochondria in a cell

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SLIDE 5

5

Monitoring of mitochondria function is important to understand progression of these pathologies

Mitochondrial dysfunction is involved in many processes

MMP dysregulation is implicated in :

  • Aging
  • Alzheimer disease
  • Parkinson’s disease
  • Diabetes mellitus 2 type
  • Cardiomyopathy
  • Ischemia

Figure is adapted from Cell (2013) 153, 1194-1217

Mitochondrial membrane potential (MMP) is one of the key parameters that characterizes function of mitochondria

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SLIDE 6

Quantification of MMP relies on fluorescent dyes

Cytosol

30-60 mV

Mitochondrion

180-200 mV

O O O N N

TMRM

O O O N N

TMRM

Accumulation Decrease of MMP will lead to decrease

  • f

fluorescent signal.

6

Intensity

  • f

the signal is proportional to the amount of TMRM that was accumulated βˆ†πœ” = π‘†π‘ˆ 𝑨𝐺 ln π‘ˆπ‘π‘†π‘ π‘ˆπ‘π‘†π‘

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SLIDE 7

Drawbacks of fluorescent dyes for measuring MMP

  • None of them are suitable to be used in living animals

(mammals)

Methods, (2008), 46, 304-311

7

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SLIDE 8

Measurement of MMP in vivo is extremely important

8

  • Aging
  • Alzheimer disease
  • Parkinson’s disease
  • Diabetes mellitus 2 type
  • Cardiomyopathy
  • Ischemia

Impossible to create relevant disease model in cell culture

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SLIDE 9

Strategy for non-invasive imaging of mitochondrial membrane potential (MMP)

Bio-orthogonal reaction

+

Bioluminescent imaging

MMP

in a context of various diseases

9

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SLIDE 10

PPh2 R' N3 Phosphine Azide Ph P Ph N R' N2 Aza-ylide

+

H2O PPh2 O R' NH2

+

Amine Phosphine oxide

The Staudinger reaction

Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635. Hermann Staudinger

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SLIDE 11

The Staudinger ligation – the first bioorthogonal reaction

Science (2000) 287, 2007

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SLIDE 12

Using the Staudinger ligation to activate luciferin

O OCH3 P Ph Ph + N3 R' N2 O OCH3 P Ph Ph N R'

Aza-ylide

CH3OH O N P Ph Ph R' H2O O N H P Ph Ph O R'

phosphine Ligation product

S N S N CO2H O R R = H light R = not H no light

Nat Chem Biol (2005) 1, 13

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SLIDE 13

Using TPP cation to deliver reagents to mitochondria

  • M. Murphy, Biochimica et Biophysica Acta, 1777 (2008), 1028-1031

Edward J. Gane, et al. Liver international (2010), 1019-1026

Cytosol

P

X

30-60 mV

3-10 X 100-500 X

Mitochondrion

180-200 mV TPP

13

  • MitoQ (CoQ10)

In Phase 2 was proved to protect against liver damage during hepatitis C

O O O O H

6-10

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SLIDE 14

Design of mitochondria-activated luciferin (MAL) probe

14

MMP DEPENDENT ACCUMULATION MMP DEPENDENT RATE OF LUCIFERIN RELEASE How to make luciferin uncaging MMP dependent?

  • 1. Take two reagents that release luciferin upon reaction
  • 2. Target them to mitochondria
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SLIDE 15

15

Potentiation of reaction rate – 105 fold Only mitochondrial uncaging is observed

The bioluminescent signal intensity depends on MMP

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SLIDE 16

Reaction rate enhancement

16

𝑆 = π‘ˆπ‘„π‘„

  • π‘ˆπ‘„π‘„
  • β‰ˆ π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„
  • π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„
  • = π‘ˆπ‘„π‘„π·π‘€

π‘ˆπ‘„π‘„π·π‘€ = 10

βˆ† .,

πœ‘ πœ‘ = 𝑙 π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„

π‘ˆπ‘„π‘„π·π‘€

𝑙 π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„

π‘ˆπ‘„π‘„π·π‘€

= 𝑆 π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„

π‘ˆπ‘„π‘„π·π‘€

π΅π‘¨π‘—π‘’π‘π‘ˆπ‘„π‘„

π‘ˆπ‘„π‘„π·π‘€

= 𝑆 = 10

βˆ† . = 10 βˆ† .,

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SLIDE 17

The bioluminescent signal intensity depends on MMP

17

Intact mitochondria Depolarized mitochondria

High rate of luciferin release High signal Low rate of luciferin release Low signal

FCCP, valinomycin

Valinomycin

Nigericin

FCCP Nigericin

Hyperpolarized mitochondria

Very high rate of release Extremely high signal

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SLIDE 18

18

HT-1080 luc2 cells

With MAL depolarization is detectable down to 0.5 uM Mitochondria activated luciferin (MAL) TMRM

Detection of valinomycin induced depolarization in cells

O O O N N

vs

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SLIDE 19

19

HT-1080 luc2 cells

Mitochondria activated luciferin-3 (MAL3) TMRM

Detection of valinomycin induced depolarization in cells

O O O N N

vs

Total photon flux [p/(cm2*sr)] RFU

Response of MAL3 assay is more non-linear

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SLIDE 20

20

HT-1080 luc2 cells

Mitochondria activated luciferin-3 (MAL3) TMRM

Detection of hyperpolarization in cells

O O O N N

vs

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SLIDE 21

21

Monitoring mitochondrial potential in vivo