Senescence Resarch and Mitochondria DOJINDO LABORATORIES

Topics

Cellular Senescence and Mitochondria at a Glance
Key Literature on Mitochondrial Function and Aging
Accumulation of Lipid Peroxides and Their Connection to Cellular Senescence and Mitochondria
Mitochondrial and Lysosomal, and Iron Regulation of Senescence
Related Technical information

Cellular Senescence and Mitochondria at a Glance

In aged cells, due to mitochondrial dysfunction, ATP is primarily generated through the anaerobic glycolysis pathway, leading to an increase in lactate production²⁾. DNA damage is one of the causes of mitochondrial dysfunction in cellular aging. The accumulation of DNA damage activates DNA repair mechanisms and increases NAD+ consumption. The decrease in NAD+ levels reduces SIRT1 activity, an important factor in maintaining mitochondrial function, leading to impaired mitochondrial function (inhibition of electron transfer → ATP production / reduction of NAD+ levels)^1),3).

Reference:

1. J. Wu, Z. Jin, H. Zheng and L. Yan, “Sources and implications of NADH/NAD+redox imbalance in diabetes and its complications”, Diabetes Metab. Syndr. Obes., 2016, 9, 145

2. Z. Feng, R. W. Hanson, N. A. Berger and A. Trubitsyn, “Reprogramming of energy metabolism as a driver of aging”, Oncotarget., 2016, 7(13), 15410.

3. S. Imai and L. Guarente, “NAD+ and sirtuins in aging and disease”, Trends in Cell Biology, 2014, 24(8), 464.

Oxidative stress & accelerated aging:

① SA-β-gal

Impairment of mitochondrial function:

③ Mitochondrial membrane potential

④ Oxygen consumption rate (OCR)

⑤ ADP/ATP ratio

Upregulation of glycolysis pathway and glutamine metabolism

⑥ Glucose consumption

⑦ Lactate production

⑧ Glutamine consumption

Reduction in antioxidant capacity：

⑨ NADPH/NADP⁺ ratio

DNA repair mechanisms：

⑩ NAD⁺/NADH Ratio

Key Literature on Mitophagy and Aging

	
			Title
			Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila

			Schmid, E. et al., Nature Aging, 2022, 2, 494-507
		
			Key Points
			1. Aging leads to a decline in mitophagy in the brain of Drosophila, while mitochondrial mass increases.
2. Inducing BNIP3 in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aging brain.
3. Neuronal induction of BNIP3-mediated mitophagy extends the organism's lifespan and healthspan.
4. BNIP3-mediated mitophagy in the nervous system improves muscle and gut homeostasis in aging flies.

			Title
			Mitochondrial contribution to lipofuscin formation

			König, J. et al., Redox Biol, 2017, 11, 673-681

			Key Points
			1. Impaired mitophagy in aged cells leads to increased mitochondrial mass and superoxide formation. Additionally, inhibiting mitochondrial fission also increases lipofuscin formation.
2. Downregulation of Lon protease is associated with increased lipofuscin formation, while the application of mitochondrial-targeted antioxidant mitoTEMPO prevents the accumulation of these protein aggregates.

			Title
			Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy

			Bektas, A. et al., Aging, 2019, 11(21), 9234-9263
		
			Key Points
			1. Aging is associated with persistent mitochondrial dysfunction in CD4+ T lymphocytes, and defects in mitophagy turnover may trigger chronic inflammation and lead to impaired immune defense in the elderly.
2. These references and insights highlight the connection between mitochondrial dysfunction, impaired mitophagy, and aging, supporting the critical role of mitochondrial quality control in cellular aging and age-related diseases.

Accumulation of Lipid Peroxides and Their Connection to Cellular Senescence and Mitochondria

Lipotoxicity is caused by intracellular lipid accumulation and is indicative of mitochondrial disfunction. Lipotoxicity accelerates the degenerative process of cellular senescence, influencing cancer development.

① Lipid Droplet Detection

② Mitochondria Dynamics

③ Senescence Marker Detection

④ Monitoring Mitochondrial Membrane Potential

⑤ Mitophagy Detection/Lysosome Detection

⑥ Lipid Peroxide Detection

⑦ Glucose Uptake Detection

⑧ Total ROS Detection/Mitochondrial Superoxide Detection

⑨ Cell Cycle Analysis

References

1. Clara, C. al., “Mitochondria: Are they causal players in cellular senescence?”, Biochimica et Biophysica Acta – Bioenergetics, 2015, 1847(11), 1373-1379.

2. Huizhen, Z. et al., “Lipidomics reveals carnitine palmitoyltransferase 1C protects cancer cells from lipotoxicity and senescence”, Journal of Pharmaceutical Analysis, 2020.

3. Xiaojuan, H. et al., “Astrocyte Senescence and Alzheimer’s Disease: A Review”, Front. Aging Neurosci., 2020.

4. Borén, J. et al., “Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation”, Cell Death Differ, 2012, 19(9), 1561-1570.

5. Na, L. et al., “Aging and stress induced β cell senescence and its implication in diabetes development”, Aging (Albany NY), 2019, 11(21), 9947–9959.

Mitochondrial and Lysosomal, and Iron Regulation of Senescence

Senescence is a cellular process that results in the cessation of cell division, often serving as a protective mechanism against the proliferation of damaged cells, including potential cancer cells. This process is intricately regulated by numerous factors including, but not limited to, tumor suppressor genes, DNA damage response (DDR) pathways, and various signaling molecules. In addition, the senescence-associated secretory phenotype (SASP), consisting of cytokines, growth factors, and proteases, is regulated by NF-κB and other transcription factors that influence the tissue microenvironment and impact aging and disease processes.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence
Click here for the original article: Mengying Cui, et. al., PNAS, 2023.

Point of Interest
- HKDC1, a protein involved in glycolysis, is a direct target of the transcription factor TFEB and is essential for maintaining both mitochondrial and lysosomal function.

- This activity helps avert cellular senescence, playing a vital role in maintaining cellular homeostasis.

- Beyond its role in glycolysis, HKDC1 contributes to mitophagy and lysosomal repair processes independently.

- The absence of HKDC1 may result in cellular senescence and the buildup of damaged organelles

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype
Click here for the original article: Mate Maus, et. al., Nature, 2023.

Point of Interest
- Vascular and hemolytic injury trigger iron accumulation, which causes senescence and promotes fibrosis.

- Senescent cells persistently accumulate iron, even after the increase in extracellular iron has subsided.

- Cells exposed to various types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes.
- The high levels of labile iron fuel the generation of reactive oxygen species and the SASP.

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging
Click here for the original article: Monami Ogura, et. al., EMBP Reports, 2023.

Point of Interest
- Microautophagy in the repair of damaged lysosomes prevents aging.

- STK38 and GABARAPs are key regulators of this process.

- STK38 is required for lysosomal recruitment of VPS4 and GABARAPs are involved in ESCRT assembly.

- Depletion of these regulators leads to accelerated cellular senescence and shortened lifespan.

Related Technique in This Topic

Cellular senescence detection

SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.

First-time autophagy research

Autophagic Flux Assay Kit

Autophagy detection

DAPGreen / DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)

Lysosomal function

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

Ferrous ion (Fe²⁺) detection

FerroOrange(intracellular), Mito-FerroGreen(mitochondria)

Mitochondrial superoxide detection

MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Oxygen consumption rate assay

Extracellular OCR Plate Assay Kit

Related Applications

Analysis of Lysosomal Mass and pH change in Senescence-induced Cells

Purpose: To investigate changes in lysosomal mass and pH in A549 cells induced to senescence by treatment with Doxorubicin (DOX).

Methods: Senescence-associated acidic β-galactosidase (SA-βGal) activity was detected using Cellular Senescence Detection Kit - SPiDER-βGal. Lysosomal mass was detected using LysoPrime Deep Red, and pH was detected using pHLys Red. Fluorescence imaging was used to observe changes in lysosomal mass and pH in senescent cells compared to non-senescent cells. The normalized fluorescence intensity of lysosomal mass and pH was also measured by a plate reader.

Results: Our findings indicate that senescence induced by DOX resulted in an increase in lysosomal mass and acidification of pH compared to non-senescent cells. The obtained results are consistent with previous reports* that demonstrated enhanced lysosomal activity in senescent cells induced by the CDK4/6 inhibitor, palbociclib. The fluorescence imaging and plate reader data both support these findings.

* Miguel Rovira, et. al., Aging Cell (2022)

＜Experimental Conditions for Microscopy＞
SA-βGal(Green):Ex = 488 nm, Em = 490 – 550 nm
Lysosomal pH (Red):Ex = 561 nm, Em = 560 – 620 nm
Lysosomal mass (Deep Red):Ex = 633 nm, Em = 640 – 700 nm

＜Experimental Conditions for Plate Reader＞
SA-βGal: Ex = 525 – 535 nm, Em = 550 – 570 nm
Lysosomal pH: Ex = 555 – 565 nm, Em = 590 – 610 nm
Lysosomal mass: Ex = 645 – 655 nm, Em = 690 – 710 nm

<Products in Use>
- Cellular Senescence Detection Kit
- Lysosomal pH and mass detection Kit
> More about Lysosomal Function Analysis