Mitochondria Research

Science Note

[Apr. 30, 2024]                                                                                                                                                                                                                            Previous Science Note
Inducible and Inhibitory Organelles in Neurodegeneration

In neurodegeneration, dysfunctional mitochondria contribute to oxidative stress and energy deficits in neurons. Lysosomes, responsible for the disposal of cellular waste, may be impaired, leading to the accumulation of damaged organelles, including mitochondria. At the same time, abnormal lipid metabolism and accumulation of lipid droplets have been implicated in neurodegenerative diseases, potentially exacerbating neuronal dysfunction and contributing to disease progression. The intricate relationships between dysfunctional mitochondria, impaired lysosomal function and abnormal lipid metabolism underscore the complex pathophysiology of neurodegeneration. 

Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration
Click here for the original article: Raffaella De Pace, et. al., Nature Neuroscience, 2024.

APOE4/4 is linked to damaging lipid droplets in Alzheimer’s disease microglia
Click here for the original article: Michael S. Haney,et. al., Nature, 2024.

Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis
Click here for the original article: Ye Zhu, et. al., Nature Cell Biology, 2024.

Point of Interest

- Lysosome–kinesin adaptor related complex (BORC) KO depletes axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins.

- This depletion leads to mitochondrial defects and ultimately to axonal degeneration in neurons.

- A mechanistic connection of BORC deficiency may accelerate common neurodegenerative disorders.

Point of Interest

- Lipid droplet-associated enzyme ACSL1-positive microglia was most abundant in patients with AD having the APOE4/4 genotype.

- In microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation depending on APOE.

- Conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity depending on APOE in neurons.

Point of Interest

- WIPI4 deficiency causes β-Propeller protein-associated neurodegeneration, which induces ferroptosis via an autophagy-independent mechanism in cell culture and in zebrafish.

- WIPI4 depletion increases the localization of ATG2A at ER-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria.

- This leads to increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation and ultimately to ferroptosis.
Related Techniques
           Mitochondrial or internal lipid peroxide detection MitoPeDPP, Liperfluo
           Mitochondrial or internal iron detection Mito-FerroGreen, FerroOrange
           Mitophagy or autophagy detection Mitophagy Detection Kit, Autophagic Flux Assay Kit
           Glycolysis/Oxidative phosphorylation Assay Extracellular OCR Plate Assay Kit, Glycolysis/OXPHOS Assay Kit
           Lipid droplets detection Lipi-Blue/Green/Red/Deep Red
           Lysosome staining pH-dependent (pHLys Red)​ and pH resistance (LysoPrime Green/Deep Red) probes
           Lysosomal acidic pH detection

Lysosoml Acidic pH Detection Kit-Green/Red and Green/Deep Red
  
Related Applications

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe2+

Lysosomal Function and Iron Homeostasis

 

Recent reports suggest that lysosomal neutralization can result in iron depletion, consequently leading to the disruption of cell viability. To verify this, HeLa cells were labeled with FerroOrange for Fe2+ detection, and the lysosomal mass and pH were separately detected with LysoPrime DeepRed and pHLys Green (a product currently under development). Co-staining with FerroOrange and Lysosomal dyes demonstrated that Bafilomycin A1 (Baf. A1), an inhibitor of lysosomal acidification, causes iron depletion consistent with the findings reported in the article. Interestingly, the iron chelator, Deferiprone (DFP), did not impact lysosomal pH, suggesting that lysosomal function plays a key role in managing iron homeostasis.

Reference: Ross A Weber, et. al., Mol Cell (2020)

Products in Use
   - FerroOrange
   - pHLys Green*
   - LysoPrime Deep Red

*pHLys Green is included as a component of the "Lysosomal Acidic pH Detection Kit-Green/Deep Red".

 

Lysosomal Function and Mitochondrial ROS

 

CCCP and Antimycin are recognized inducers of mitochondrial ROS, linked to the loss of mitochondrial membrane potential. Recent studies have shown that CCCP induces not only mitochondrial ROS but also lysosomal dysfunction. To observe mitochondrial ROS, HeLa cells were labeled with MitoBright ROS Deep Red for Mitochondrial Superoxide Detection, and the lysosomal mass and pH were independently detected with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes revealed that CCCP, unlike Antimycin, triggers concurrent lysosomal neutralization and mitochondrial ROS induction.

Reference: Benjamin S Padman, et. al., Autophagy (2013)

Products in Use
   - LysoPrime Green
   - pHLys Red
   - Lysosomal Acidic pH Detection Kit
   - MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Related Products
   - Mitophagy Detection Kit and Mtphagy Dye

Selection guide for mitochondria-related reagents

Mitochondria research is very multi-faceted, because the multi-functional organelle is not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.

Selection Guide of Reagents

The following table lists reagents for mitochondrial research designed to stain and detect mitochondria (MitoBright LT, MitoTracker, etc.), mitochondrial membrane potential (JC-1, TMRM, TMRE, etc.), reactive oxygen species AKA ‘ROS’ (MitoBright ROS, MitoSOX, etc.), mitophagy, and lipid peroxides.

Mitophagy

Mitophagy
Reagent Mtphagy Dye Keima-Red
Principle Mtphagy Dye (included in Mitophagy Detection Kit) is a pH-sensitive fluorescent probe that accumulates in mitochondria and emits red fluorescence due to acidic conditions in a lysosome. A pH-sentitive ratiometric fluorescent protein. The excitation spectrum changes accoring to pH. This protein shows high fluorescence ratio (Ex. 550 nm/440 nm) values in a lysozome.
Fixed cell staining – –
Live-cell staining Yes Yes
Fixation after live-cell staining – –
Staining time > 30 min –
Ex / Em 530 / 700 440, 550 / 620
Product code MD01, MT02 –

Lipophilic peroxide / Singlet oxygen / Superoxide

  Lipophilic peroxide Singlet oxygen Superoxide Superoxide
Reagent MitoPeDPP Si-DMA MitoBright ROS Deep Red MitoSOX
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with lipophilic peroxides in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with singlet oxigen generated in mitochondria to emit red fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit red fluorescence.
Fixed cell staining – – – –
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining – – – –
Staining time > 15 min > 45 min > 10 min > 10 min
Ex / Em 452 / 470 644 / 670 540 / 670 510 / 590
Product code M466 MT05 MT16 –

Membrane potential

Membrane potential
Reagent JC-1 MT-1 TMRM, TMRE
Principle A cell-permeant ratiometric fluorescent dye that accumulates in intact mitochondria due to the membrane potential. The excitation spectrum changes according to the mitochondria membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. MT-1 is extremely photostable and more sensitive than JC-1 and can provide equivalent detection sensitivity to TMRE. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Diffusion of the probes occurs in a damaged mitochondria that has the decreased membrane potential.
Fixed cell staining – – –
Live-cell staining Yes Yes Yes
Fixation after live-cell staining – Yes –
Staining time 30-60 min 30 min 30-60 min
Ex / Em Monomer: 514 / 529
J-aggregation: 585/590		 530-560 / 570-640 550 / 575
Product code MT09 MT13 –

Mitochondria staining

Mitochondria staining
Reagent MitoBright LT series MitoBright IM Red MitoTracker series Rhodamine 123
Principle Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential and covalently binds to proteins and other biomolecules. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dye that accumulates in intact mitochondria due to the membrane potential.
Fixed cell staining – – – –
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining Yes Yes – –
Staining time 30 min 30 min 15-45 min > 15 min
Ex / Em 493 / 508, 547 / 563, 643 / 663 548 / 566 490 / 516 ~
644 / 665		 507 / 529
Product code MT10, MT11,MT12 MT15 – R233

Metal Ion Detection

   Iron ion (Fe2+) Calcium ion (Ca2+)
Reagent Mito-FerroGreen Rhod2-AM
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with ferrous ion in mitochondria to emit green fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with calcium ion in mitochondria to emit red fluorescence.
Fixed cell staining – –
Live-cell staining Yes Yes
Fixation after live-cell staining – –
Staining time 30 min 30-60 min
Ex / Em 505 / 535 553 / 576
Product code M489 R002 
Application Products
Mitophagy Detection Mitophagy Detection Kit
Mitochondrial Phospholipid Peroxidase Detection MitoPeDPP
Mitochondrial Ferrous Ion Detection Mito-FerroGreen
Mitochondrial Superoxide MitoBright ROS - Mitochondrial Superoxide Detection
Mitochondrial Singlet Oxygen Detection Si-DMA for Mitochondrial Singlet Oxygen Imaging
Mitochondrial Membrane Potential JC-1 MitoMP Detection Kit
MT-1 MitoMP Detection Kit
Mitochondria Staining MitoBright LT Green
MitoBright LT Red
MitoBright LT Deep Red
MitoBright IM Red for Immunostaining

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