Science Note: Mitochondria

Mitochondrial stress responses are a set of cellular reactions triggered when mitochondria, the energy-producing organelles in cells, encounter various forms of stress. This can include damage from reactive oxygen species (ROS), fluctuations in nutrient levels, or other environmental stressors. In response to such stress, mitochondria can undergo changes in their dynamics, such as fission and fusion, to maintain cellular homeostasis and energy production. Additionally, cells activate protective pathways, like the mitochondrial unfolded protein response (UPRmt), to repair or degrade damaged mitochondrial proteins and mitigate the potentially harmful effects of mitochondrial dysfunction. 

Mitochondrial fission drives neuronal metabolic burden to promote stress susceptibility in male mice
Click here for the original article: Wan-Ting Dong, et. al., Nature Metabolism, 2023.

Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis
Click here for the original article: Yi Fu, et. al., Molecular Cell, 2023.

Glial-derived mitochondrial signals affect neuronal proteostasis and aging
Click here for the original article: Raz Bar-Ziv et. al., Science Advances, 2023.

Point of Interest
- Inhibition of Drp1 in neurons ameliorates stress-related depressive-like behavior.

- Enhancing Drp1-dependent mitochondrial fission promotes stress susceptibility.

- Increased stress susceptibility is alleviated by increasing mitochondrial ATP production.

Point of Interest
- Mitochondrial double-strand breaks (mtDSBs) lead to significant mitochondrial defects and impede protein import.

- mtDSBs initiate an integrated stress response (ISR).

- Inhibition of the ISR exacerbates mitochondrial defects and slows recovery of mitochondrial DNA.

- ATAD3A is a potential signaling molecule from damaged genomes to the inner membrane of mitochondria.

Point of Interest
- The mitochondrial unfolded protein response (UPRmt) activation in four astrocyte-like glial cells can alleviate protein aggregation in neurons.

- Glial cells use small clear vesicles (SCVs) to signal the mitochondrial proteotoxic stress to neurons.

- Neurons relay the signal to the periphery using dense-core vesicles.

In recent years, the discovery of several novel mitochondrial stress responses has attracted much attention. A mitochondrial iron-responsive pathway was discovered that protects cells from iron deficiency. In other breakthroughs, a new anticancer target is identified and the nucleus-to-mitochondria ROS-sensing pathway is implicated in resistance to platinum-based treatments for ovarian cancer. Research also uncovers the role of the protein in promoting mitochondrial fission, essential for effective mitophagy.
Dojindo has a number of unique probes that can be used to evaluate different mitochondrial stress responses: Mitochondrial Fe²⁺ detection MitoFerro-Green, Mitochondrial ROS Detection MitoBright ROS - Mitochondrial Superoxide Detection, and so on. 

A mitochondrial iron-responsive pathway regulated by DELE1
Click here for the original article: Yusuke Sekine, et. al., Molecular Cell, 2023.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway
Click here for the original article: Junbing Zhang, et. al., Cell, 2023.

The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy
Click here for the original article: Tomoyuki Fukuda , et. al., Molecular Cell, 2023.

Point of Interest
- In a steady state, DELE1 is swiftly degraded by the matrix-resident LONP1 post-import.
- When iron is deficient, DELE1 import is halted, leading to its stabilization on mitochondria.
- The stabilized full-length form of DELE1 activates HRI on the surface of mitochondria.
- The DELE1-HRI-ISR pathway serves to protect erythroid cells from cell death induced by iron deficiency.

Point of Interest
- The integration of chemical proteomics and CRISPRi screens identifies ROS-target proteins.
- Nuclear H₂O₂ oxidizes C408 within the autoinhibitory domain of CHK1, thereby leading to its activation.
- CHK1 regulates mitochondrial translation by suppressing the mtDNA-binding protein, SSBP1.
-  SSBP1 promotes resistance to platinum-based agents and nuclear H₂O₂ in cases of ovarian cancer.

Point of Interest
- Mitofissin is a mitochondrial fission factor located within the inner mitochondrial space.
- The yeast Mitofissin, known as Atg44, promotes mitochondrial fission, which is essential for mitophagy.
- Atg44 directly induces membrane fragility, facilitating membrane fission.
- The mechanisms and roles of membrane fission performed by Atg44 differ from those of Dnm1.

Scientists have unveiled that hypoxia transforms regular tubular mitochondria into larger structures called megamitochondria through inter-mitochondrial contact and fusion. During hypoxia, mitochondria-lysosome interaction is enhanced, and some lysosomes get engulfed by megamitochondria, which escalates mitochondrial ROS production.  
We offer lysosomal mass/pH detecting Dyes: Lysosomal Acidic pH Detection kit-Green/Red, Green/Deep Red, and mitochondrial superoxide detecting dye: MitoBright ROS - Mitochondrial Superoxide Detection (These products are promotional now! Click here for Summer Promotion 2023).

Hypoxia-reprogramed megamitochondrion contacts and engulfs lysosome to mediate mitochondrial self-digestion    

Click here for the original article: Tianshu Hao, et. al., Nature Communications (2023)

Point of Interest
- Hypoxia transforms regular tubular mitochondria into larger structures through inter-mitochondrial contact and fusion.  
- Mitochondria-lysosome interaction is enhanced during hypoxia, and some lysosomes get engulfed by larger mitochondria.
- The STX17-SNAP29-VAMP7 complex aids this process and contributes to a self-digestion mode of mitochondrial degradation.  This self-digestion mode escalates mitochondrial ROS production.

NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron–Sulfur Cluster Proteins    Naiara Santana-Coodina, et. al., Cancer Discovery (2022)

Point of Interest
- Ferritinophagy, the process by which iron is released from storage for use in cells, is found to be upregulated in pancreatic ductal adenocarcinoma (PDAC), promoting tumor growth.
- It's shown to support the synthesis of iron–sulfur cluster proteins, thereby maintaining mitochondrial stability.
- Blocking the ferritinophagy adapter NCOA4 delays tumor growth and extends survival, although compensatory iron-gathering pathways may develop.

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

The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging    Chin-Ling Wang, et. al., Cell Metabolism (2023)

Point of Interest
- Elevated mitochondrial protein folding stress in aging neural stem cells
- SIRT7 safeguards neural stem cells by reducing mitochondrial protein folding stress
- SIRT7 overexpression enhances neurogenesis and cognitive function in aged mice

Metabolic shift to glycolysis in senescenct cells

	NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.                        *S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471
Products in Use    ① DNA Damage Detection Kit - γH2AX    ② Cellular Senescence Detection Kit - SPiDER-βGal    ③ NAD/NADH Assay Kit-WST    ④ JC-1 MitoMP Detection Kit    ⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

2. Simultaneously detection of Lysosomal and Mitochondrial Dysfunction

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

Products in Use
   - LysoPrime Green
   - pHLys Red
   - MitoBright ROS - Mitochondrial Superoxide Detection

Related Product
   - Lysosomal Acidic pH Detection Kit

TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit    Francisco J. Roca, et. al., Cell (2019)

Point of Interest
- TNF triggers mitochondrial ROS production in mycobacterium-infected macrophages, causing necrosis.  
- This process involves mitochondrial ROS activating lysosomal enzymes, leading to BAX activation and subsequent ER activation causing Ca²⁺ influx into mitochondria.  
- Targeting this mitochondrial Ca²⁺ overload can help prevent detrimental macrophage necrosis in TB.

1. Simultaneously detection of Lysosomal and Mitochondrial Dysfunction

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with mitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

Products in Use
   - LysoPrime Green
   - pHLys Red
   - MitoBright ROS - Mitochondrial Superoxide Detection

Related Product
   - Lysosomal Acidic pH Detection Kit

2. Monitoring ROS in Macrophage Phagocytosis

Dead cells (2&3) phagocytosed by Cell1 resulted in increased ROS(green).

ROS detection reagent allowed for reliable analysis of the role of ROS in phagocytosis. Its high intracellular residence and low background noise made it possible to perform long-term analysis of ROS production in the cell. This information can provide important insights into the mechanisms of phagocytosis and contribute to the development of treatments for diseases associated with macrophage dysfunction.

  > for detail experimental notes are available at Nikon web site.

Products in Use
   - ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-

Related Product
  - ROS Assay Kit -Highly Sensitive DCFH-DA-

Loss of fatty acid degradation by astrocytic mitochondria triggers neuroinflammation and neurodegeneration
Yashi Mi, et. al., Nature Metabolism (2023)

Point of Interest
- This study demonstrates that astrocytic OxPhos is crucial for breaking down fatty acids and maintaining lipid balance in the brain.
- When fatty acid levels overwhelm astrocytic OxPhos, this leads to lipid droplet buildup and neurodegeneration with similarities to Alzheimer's disease, including cognitive decline and demyelination.
- This process occurs as high acetyl-CoA levels trigger astrocyte reactivity through the activation of STAT3. 

1. Monitoring ROS in Macrophage Phagocytosis

Dead cells (2&3) phagocytosed by Cell1 resulted in increased ROS(green).

ROS detection reagent allowed for reliable analysis of the role of ROS in phagocytosis. Its high intracellular residence and low background noise made it possible to perform long-term analysis of ROS production in the cell. This information can provide important insights into the mechanisms of phagocytosis and contribute to the development of treatments for diseases associated with macrophage dysfunction.

  > for detail experimental notes are available at Nikon web site.

Products in Use
   - ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-

Related Product
  - ROS Assay Kit -Highly Sensitive DCFH-DA-

2. Mitochondrial Superoxide Detection in Senescent Cells

Background fluorescence caused by lipofuscin can be minimized by using a better fluorescent probe, as tested in TIG-1 cells. 

Lipofuscin accumulates in senescent cells, causing increased background fluorescence during observation. To minimize the effects of endogenous fluorescence from lipofuscin and other substances, a better fluorescent probe was tested in TIG-1 cells. Company T's product exhibited endogenous fluorescence, while MitoBright ROS Deep Red showed less background fluorescence. Researchers should compare sensitivity, wavelength, and channels and select the appropriate fluorescent probe to minimize endogenous fluorescence for accurate cellular senescence research.

Products in Use
  - MitoBright ROS - Mitochondrial Superoxide Detection

Mitohormesis reprogrammes macrophage metabolism to enforce tolerance
Greg A. Timblin, et. al., nature metabolism (2021)

   Point of Interest
   - Mitochondrial stress response, mitohormesis, occurs as macrophages transition from an LPS-responsive to LPS-tolerant state, with impaired pro-inflammatory gene transcription.
   - Hydroxyoestrogen-triggered mitohormesis also suppresses mitochondrial oxidative metabolism and acetyl-CoA production enforcing an LPS-tolerant state.
   - Mitochondrial ROS and mitochondrial reactive electrophilic species are TLR-dependent signaling molecules that activate mitohormesis as a negative feedback mechanism to control inflammation through tolerance.

• - Brown adipocytes eliminate damaged mitochondrial parts through EVs
• - Thermogenic stimuli increase the release of mitochondrial EVs
• - EVs exert a negative autocrine action on brown adipocyte thermogenesis
• - bMACs actively take up mitochondrial EVs ensuring optimal brown adipose tissue thermogenesis

• - Flunarizine (FNZ), a drug whose chronic use causes parkinsonism, led to parkinsonism-like motor dysfunction in mice
• - FNZ induced mitochondrial dysfunction and decreased mitochondrial mass specifically in the brain
• - Mitochondria were engulfed by lysosomes independent of mitophagy, followed by VAMP2- and STX4-dependent exocytosis
• - The mitochondria-free cells generated FNZ-dependent method could survive for nearly 1 month

• - Aging leads to a decline in mitophagy in the Drosophila brain with a concomitant increase in mitochondrial content
• - Induction of BNIP3 in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aged brain
• - Neuronal induction of BNIP3-mediated mitophagy increases organismal longevity and healthspan
• - BNIP3-mediated mitophagy in the nervous system improves muscle and intestinal homeostasis in aged flies