Science Note: Mitochondria

Recent research shows that mitochondria are critical for T cell function, regulating metabolic pathways essential for immune responses. Here are some of the papers that show mechanisms and strategies, such as mitochondrial transfer or genetic modification, to improve mitochondrial health and reinvigorate T cell activity against tumours.

Mitochondria play a critical role in T cell function, providing energy and regulating metabolic pathways essential for immune responses. In cancer, the tumour microenvironment induces mitochondrial dysfunction in T cells, leading to exhaustion and reduced anti-tumour efficacy. Strategies to improve mitochondrial health, such as mitochondrial transfer or genetic modification, have shown promise in reinvigorating T cell activity against tumours. Targeting mitochondrial pathways in T cells offers a novel approach to improving cancer immunotherapies and overcoming tumour-induced immunosuppression.

Intercellular nanotube-mediated mitochondrial transfer enhances T cell metabolic fitness and antitumor efficacy
Click here for the original article: Jeremy G. Baldwin, et. al., Cell, 2024.

PGE₂ inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function
Click here for the original article: Matteo Morotti, et. al., Nature, 2024.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy
Click here for the original article: Jack D. Chan, et. al., Nature, 2024.

Point of Interest

- Bone marrow stromal cells transfer mitochondria to CD8+ T cells via nanotubes, requiring Talin 2 for optimal transfer.

- Mitochondria-boosted T cells exhibit improved respiration, tumor infiltration, and reduced exhaustion, enhancing antitumor responses.

- Intercellular mitochondrial transfer offers a new organelle-based therapy, advancing next-generation cell therapies for cancer treatment.

Point of Interest

- Prostaglandin E2 (PGE2) impairs IL-2 sensing in CD8+ tumour-infiltrating lymphocytes (TILs) by downregulating IL-2 receptor components, causing oxidative stress and cell death.

- Blocking PGE2 signalling during TIL expansion restores IL-2 responsiveness, increasing TIL proliferation and improving tumour control in adoptive cell therapy.

- Targeting PGE2 pathways enhances IL-2-driven T cell expansion, offering new strategies to improve cancer immunotherapy outcomes.

Point of Interest

- CAR T-cell therapy is less effective in solid tumours due to the immunosuppressive microenvironment that causes T-cell dysfunction.

- Overexpression of FOXO1 enhances the stem-like phenotype, mitochondrial fitness and persistence of CAR T cells, thereby improving antitumour efficacy.

- Engineering FOXO1 in CAR T cells offers a promising strategy to increase efficacy against solid tumours in cancer therapy.

Recent research on cancer progression shows that mtDNA mutations affect cellular metabolism and promote tumor growth. Here are some of the studies showing how mtDNA mutations drive metabolic changes and have implications for cancer therapy.

Mitochondrial DNA (mtDNA) plays a key role in cancer progression by influencing cellular metabolism and oxidative phosphorylation. Mutations in mtDNA can lead to metabolic shifts, such as the Warburg effect, that promote tumor growth and alter the tumor microenvironment. These mtDNA alterations can increase the production of reactive oxygen species (ROS), which drive pro-tumor signaling pathways such as TGFβ/Smad. In addition, mtDNA mutations and their transfer via extracellular vesicles (EVs) can affect tumor-stroma communication and potentially serve as biomarkers and therapeutic targets in cancer treatment.

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma
Click here for the original article: Mahnoor Mahmood, et. al., Nature Cancer, 2024.

Mitochondrial genome transfer drives metabolic reprogramming in adjacent colonic epithelial cells promoting TGFβ1-mediated tumor progression
Click here for the original article: Bingjie Guan, et. al., Nature Communications, 2024.

Plasma cell–derived mtDAMPs activate the macrophage STING pathway, promoting myeloma progression
Click here for the original article: Aisha Jibril, et. al., Blood, 2024.

Point of Interest

- DNA mutations in tumors promote metabolic shifts and anti-tumor immune responses, sensitizing them to checkpoint blockade therapies.

- Engineered mtDNA mutations in melanoma reshaped the tumor microenvironment, reducing neutrophils and enhancing immune responses in mice and humans.

- Tumors with >50% mtDNA mutations showed a 2.5-fold improved response to checkpoint blockade, highlighting the therapeutic potential of mtDNA.

Point of Interest

- Tumor-derived EV-mtDNA enhances mitochondrial respiration, ROS production and TGFβ1 expression, promoting colon cancer progression.

- EV-mtDNA from cancer cells activates ROS-driven RelA translocation in neighboring cells, regulating TGFβ1 and promoting tumor growth.

- EV-mtDNA serves as a potential biomarker and therapeutic target for paracrine metabolic communication in colon cancer.

Point of Interest

- Elevated cell-free mtDNA in multiple myeloma activates macrophages via the STING pathway and promotes disease progression in the bone marrow.

- Multiple myeloma (MM) cell-derived mtDAMPs induce chemokine upregulation in bone marrow macrophages, contributing to MM cell retention and tumor growth.

- Inhibition of STING signaling or chemokine response reduces multiple myeloma tumor burden and induces MM cell egress from the bone marrow.

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.

• - 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

Mitochondria are essential for maintaining lysosomal and autophagic homeostasis by providing the ATP required for lysosomal acidification and enzymatic function. They interact with lysosomes through signaling pathways and physical contacts, and coordinate the degradation and recycling of cellular components via autophagy. Efficient mitochondrial function supports the autophagic process by ensuring the removal of damaged organelles and proteins, thereby preventing cellular stress. Disruptions in mitochondrial activity can impair lysosomal function and autophagy, leading to the accumulation of cellular debris and compromised cellular homeostasis.

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.

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity
Click here for the original article: Yuki Takakura, et. al., Nature Communications, 2024.

Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis
Click here for the original article: Yura Do, et. al., Bioscience Reports, 2024.

Point of Interest

-Mitofissin is a mitochondrial fission factor located in the mitochondrial intermembrane space that is required for receptor-mediated mitophagy.

-The mechanisms and roles of membrane fission by Atg44 differ from those of the known mitochondrial fission factors, the dynamin-related proteins Dnm1.

-Mitofissin-deficient cells cannot be enwrapped by the autophagosome precursor, the phagophore, due to the lack of mitochondrial fission. 

-Mitofissin binds directly to lipid membranes and induces lipid membrane fragility to facilitate membrane fission required for mitophagy.

Point of Interest

-Thymic epithelial cells (TECs) may use stress-independent autophagy to degrade self-protein for generation of self-antigen peptides.

-The mitochondrial protein C15ORF48 induced by inflammatory stimulation reduces mitochondrial membrane potential, decreases intracellular ATP levels, activates AMPK, and ultimately induces autophagy.

-Autophagy dependent on C15ORF48 increases intracellular glutathione levels and promotes cell survival by reducing oxidative stress. 

-C15orf48 deficient mice show a reduction in stress-independent autophagy, but not in typical starvation-induced autophagy.

-C15orf48 deficient mice develop autoimmunity, and furthermore, engraftment of C15orf48-deficient thymus into nude mice results in autoimmunity.

Point of Interest

-Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in iron uptake into erythrocyte mitochondria..

-Cardiomyocyte-specific Abcb10 knockout mice show progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities.

-In addition, the mice exhibit increased Hif1α expression, decreased NAD synthase expression, reduced NAD+ levels and lysosomal dysfunction. 

-Impairment of Abcb10 leads to the accumulation of iron and lipid peroxides in lysosomes, resulting in ferroptosis and contributing to the development of chronic heart failure.

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 - Mitochondrial Superoxide Detection

Co-staining of Lysosome and Mitophagy

We performed fluorescence imaging by stimulating mitophagy induction in SHSY-5Y cells stained with Mitophagy Detection(Code: MD01) and LysoPrime Green or existing products. The fluorescence signal of LysoPrime Green did not decrease and the lysosomal localization over time was confirmed. This means that the co-localization rate of the fluorescent spots of the Mtphagy Dye is higher than that of the existing product, and thus more accurate mitophagy analysis can be performed.

LysoPrime Green: Ex= 488 nm, Em= 500-570 nm
Mtphagy Dye: Ex= 561 nm, Em= 560-650 nm

Products in Use
   - LysoPrime Green
   - Mitophagy Detection Kit and Mtphagy Dye

Mitochondrial activity and transfer are increasingly recognized for their roles in cancer progression and therapy. Tumor cells can enhance their survival and resistance to therapy by acquiring mitochondria from surrounding stromal cells through a process known as mitochondrial transfer. This transfer not only helps cancer cells meet their increased energy and metabolic demands but also aids in evading apoptosis induced by therapeutic agents. As a result, targeting the mechanisms of mitochondrial transfer and disrupting these interactions is being explored as a novel therapeutic strategy to enhance the effectiveness of cancer treatments and reduce resistance.

Mitochondrial metabolism sustains CD8⁺ T cell migration for an efficient infiltration into solid tumors
Click here for the original article: Luca Simula, et. al., Nature Communications, 2024.

Cancer cells reprogram to metastatic state through the acquisition of platelet mitochondria
Click here for the original article: Wenkan Zhang, et. al., Cell Reports, 2023.

Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells
Click here for the original article: Sinforosa Gagliardi, et. al., Cell Commun Signal., 2024.

Point of Interest

- Mitochondrial oxidation of glucose and glutamine, but not fatty acids, is required for interstitial motility of CD8⁺ T cells.

- For T cells to migrate, both ATP and ROS produced by mitochondria are required.

- Increasing mitochondrial activity improves intratumoral migration of CD8⁺ T cells and recruitment of CAR T cells to tumor islets.

Point of Interest

- Cancer cells acquire platelet mitochondria via the PINK1/Parkin-Mfn2 pathway to be reprogrammed to a metastatic state.

- Transferred platelet mitochondria promoted glycolytic metabolism and increased glutathione peroxidase expression.

- GSH eliminates ROS and increase, which leads to increase GSSG levels and ultimately to enhance lung metastasis of osteosarcoma in the presence of platelet mitochondria.

Point of Interest

- Cisplatin chemoresistant cells are characterized by impaired late endocytic trafficking and increased secretion of RAB7-positive mitochondria-derived vesicles (MDVs).

- MDVs can be secreted by cisplatin chemoresistant cells and deliver cisplatin outside the cells.

- MDVs purified from chemoresistant cells induce chemoresistance in recipient cells via a RAB7-modulated process.

- The MDVs localize to mitochondria and slow down mitochondrial activity, leading to mitochondrial dysfunction, lysosomal deficit, and ultimately increased MDVs.

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.

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

Mitochondrial metabolism involves the biochemical processes within mitochondria that convert nutrients into energy and building blocks necessary for cell function, primarily through the citric acid cycle and oxidative phosphorylation. This energy production produces adenosine triphosphate (ATP), the cell's primary energy currency. Oxidative stress occurs when there's an imbalance between the production of reactive oxygen species (ROS) in the mitochondria and the cell's ability to detoxify these harmful byproducts or repair the resulting damage. Over time, excessive oxidative stress can lead to cellular damage that contributes to aging and several diseases, including neurodegenerative disorders and cancer. 

Autoregulatory control of mitochondrial glutathione homeostasis
Click here for the original article: Yuyang Liu, et. al., Science, 2023.

ApoE enhances mitochondrial metabolism via microRNA-142a/146a-regulated circuits that suppress hematopoiesis and inflammation in hyperlipidemia
Click here for the original article: Tuan Anh Phu et. al., Cell Reports, 2023.

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity
Click here for the original article: Yuki Takakura et. al., Nature Communications, 2024.

Point of Interest
- The mitochondrial glutathione (GSH) transporter, SLC25A39, undergoes rapid degradation by the mitochondrial protease AFG3L2.

- Depletion of GSH dissociates AFG3L2 from SLC25A39, which triggers enhancement in the uptake of GSH by mitochondria.

- This regulatory mechanism is dependent on a putative iron-sulfur cluster within SLC25A39, linking the control of mitochondrial iron homeostasis to GSH import.

Point of Interest
- Apolipoprotein E (ApoE) elevates the levels of miR-146a in myeloid cells and hematopoietic stem and progenitor cells, leading to decreased glucose absorption and glycolysis.

- By diminishing miR-142a levels, ApoE enhances fatty acid oxidation, thereby improving mitochondrial metabolism.

- ApoE plays a critical role in regulating immune cell metabolism, influencing hematopoiesis and inflammation in conditions of hyperlipidemia.

Point of Interest
- The mitochondrial protein C15ORF48 reduces mitochondrial membrane potential and intracellular ATP levels, resulting in the activation of Unc-51-like kinase 1.

- C15ORF48-dependent induction of autophagy upregulates intracellular glutathione levels and promotes cell survival by reducing oxidative stress.

- Mice deficient in C15orf48 show a reduction in stress-independent autophagy in thymic epithelial cells (TECs).

- C15orf48-/- mice develop autoimmunity, suggesting that stress-independent autophagy in TECs is critical for thymic self-tolerance.

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.

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