Science Note: Autophagy

Recent research shows that autophagy inhibits protein aggregation and DNA lesions in cells, leading to cellular health and extended lifespan. Here are some of the papers that show some mechanisms and factors that control autophagy for cellular homeostasis.

Autophagy is a cellular process that removes damaged organelles and proteins to maintain cellular homeostasis and function. By reducing the accumulation of toxic aggregates and dysfunctional components, autophagy helps to prevent cellular stress and inflammation, key contributors to aging. Increased autophagic activity is associated with improved proteostasis, stress resistance and increased lifespan in several model organisms. Thus, autophagy is a critical mechanism linking cellular health to healthy aging and longevity.

Point of Interest
- Mesencephalic astrocyte-derived neurotrophic factor 1 (MANF-1 regulates autophagy and lysosomal function via HLH-30/TFEB signaling to promote proteostasis and extend lifespan in C. elegans.

- MANF-1 localizes to lysosomes, modulates autophagic flux and reduces protein aggregation, thereby promoting neuronal survival and stress response.

- MANF is essential for cellular proteostasis, stress response and regulation of ageing through ER-lysosomal localisation and transcriptional signaling.

Point of Interest
- MYTHO, which is conserved across species, promotes lifespan, health and stress resistance by initiating autophagy through the WIPI2-BCAS3 scaffold.

- MYTHO deletion shortens lifespan, reduces oxidative stress resistance and disrupts autophagy, highlighting its role in healthy aging.

- MYTHO is a transcriptionally regulated autophagy initiator essential for stress resistance and longevity in C. elegans and humans.

Point of Interest
- Autophagy processes TOP1cc DNA lesions via lysosomes, enabling DNA repair and genome stability in vertebrates.

- The autophagy receptor TEX264 detects TOP1cc at replication forks and triggers p97-mediated lysosomal delivery through MRE11- and ATR-dependent pathways.

- The conserved role of selective autophagy in DNA repair supports cell survival and is clinically relevant.

Analysis of autophagic flux without transfection

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.

Experimental Data

Experimental Conditions
CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

Procedure
1. HeLa cells were seeded (1.0 x 10⁴ cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37°C in an incubator equilibrated with 95% air and 5% CO₂.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.
  •　MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Control)
  •　Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Starvation)
  •　Amino acid-free medium (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. The supernatant was discarded, bafilomycin A1 working solution (10,000 times dilution, 200 μl), an inhibitor of lysosomal acidification, was added to the well, and the cells were incubated at 37°C for 20 minutes. (Inhibition of autolysosome formation)
5. The stained cells were observed under a confocal fluorescence microscope.

Products in Use
Autophagic Flux Assay Kit

Autophagy is a cellular process that degrades and recycles damaged cellular components and is critical for maintaining cellular health and function. In neurons, autophagy plays an important protective role by removing defective proteins and organelles, preventing the accumulation of toxic aggregates that can lead to neurodegenerative diseases. This process also helps maintain synaptic plasticity and neuronal homeostasis, which are essential for proper neuronal function and brain health. In addition, by regulating the response to neural stress, autophagy contributes to the overall resilience and longevity of neural cells.

Point of Interest
- A microglial population emerges in the cortical regions of aging mice, characterized by a transcriptome indicative of activated autophagy.

- This population is found to be dependent on IL-34, a ligand for CSF1R.  

- Loss of autophagy-dependent microglia leads to neuronal and glial cell death and increased mortality in aged mice exposed to neuroinflammation.  

- Conversely, IL-34-mediated microglial expansion is protective.

Point of Interest
- αSyn aggregates accumulate predominantly in lysosomes, causing them to rupture.

- This rupture seeded the aggregation of endogenous αSyn, initially around damaged lysosomes.  

- Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes, indicating the activation of lysophagy.

- Autophagy-deficient cells treated with αSyn aggregates had an increased number of ruptured lysosomes and enhanced propagation of αSyn aggregates.

Point of Interest
- Hippo kinase MAP4K2 is essential for autophagy and cell survival under energy stress.

- MAP4K2 phosphorylates LC3A at Serine 87 to promote autophagic flux.

- Energy stress induces the formation of the MAP4K2-STRIPAK^STRN4 complex, activating MAP4K2.

- Autophagy mediated by MAP4K2 is critical for the development of head and neck cancer.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

＜NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response＞

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Cell death and the dysfunction of autophagy and lysosomes are closely linked in maintaining cellular health. When autophagy is impaired, cells cannot effectively degrade and recycle damaged components, leading to the accumulation of toxic materials and cellular stress. Lysosomal dysfunction exacerbates this problem, as these organelles are essential for the degradation of autophagosomal contents. The resulting cellular damage and stress can trigger cell death pathways that contribute to various diseases, including neurodegenerative disorders and cancer.

Point of Interest
- Par-4/PAWR activation is critical during ferroptosis, with its depletion blocking and overexpression enhancing this ferroptosis process.

- Upregulation of Par-4 promotes NCOA4-dependent ferritinophagy, a selective autophagy process that degrades ferritin, increasing free iron release, lipid peroxidation, and ROS production.

- Par-4 inhibition blocks ferroptosis-driven tumor suppression, highlighting its potential for cancer therapy.

Point of Interest
- A microglial population emerges in the cortical regions of aging mice, characterized by a transcriptome indicative of activated autophagy.

- This population is found to be dependent on IL-34, a ligand for CSF1R.

- Loss of autophagy-dependent microglia leads to neuronal and glial cell death and increased mortality in aged mice exposed to neuroinflammation.

- Conversely, IL-34-mediated microglial expansion is protective.

Point of Interest
- Autophagy in old hematopoietic stem cells (oHSCs) is a protective response to chronic inflammation, preserving regenerative capacity despite impaired glycolysis.

- Inflammation suppresses glucose uptake and suppresses glycolysis in oHSCs via Socs3 inhibition of AKT/FoxO signaling, but Socs3-mediated autophagy allows metabolic compensation and a shift toward adaptive lipid metabolism.

- Fasting/refeeding restores the glycolytic state and enhances the regenerative potential of oHSCs.

Analysis of autophagic flux without transfection

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.

Experimental Data

Experimental Conditions
CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

Procedure
1. HeLa cells were seeded (1.0 x 10⁴ cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37°C in an incubator equilibrated with 95% air and 5% CO₂.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.
  •　MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Control)
  •　Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Starvation)
  •　Amino acid-free medium (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. The supernatant was discarded, bafilomycin A1 working solution (10,000 times dilution, 200 μl), an inhibitor of lysosomal acidification, was added to the well, and the cells were incubated at 37°C for 20 minutes. (Inhibition of autolysosome formation)
5. The stained cells were observed under a confocal fluorescence microscope.

Products in Use
Autophagic Flux Assay Kit

Cell death and the dysfunction of autophagy and lysosomes are closely linked in maintaining cellular health. When autophagy is impaired, cells cannot effectively degrade and recycle damaged components, leading to the accumulation of toxic materials and cellular stress. Lysosomal dysfunction exacerbates this problem, as these organelles are essential for the degradation of autophagosomal contents. The resulting cellular damage and stress can trigger cell death pathways that contribute to various diseases, including neurodegenerative disorders and cancer.

Point of Interest
- Copper chelators inhibit ferroptosis but do not inhibit other types of cell death, such as apoptosis, necroptosis, and alkaliptosis. 

- On the other hand, exogenous copper directly binds to cysteines of GPX4, leading to ubiquitination and aggregation of GPX4.

- Ubiquitinated and aggregated GPX4 induces autophagy via the Tax1 binding protein and is subsequently degraded.

- Copper enhances ferroptosis and suppresses tumor progression in a mouse model.

Point of Interest
- Scd1, an enzyme that converts saturated fatty acids to monounsaturated fatty acids, is expressed much higher in lipogenic tissues and adipocytes than in other cell types.

- Scd1-deficiency leads to lysosomal dysfunction and ultimately to the accumulation of non-acidic autophagosomes.

- This results in vacuole accumulation and eventual autophagy-dependent cell death. 

- Inhibition of autophagosome formation or supplementation with monounsaturated fatty acids maintains the vitality of Scd1-deficient adipocytes.

Point of Interest
- Glucose starvation causes an increase in autophagic proteins but a decrease in lysosomal protein expression.

- This result leads to dysfunction of lysosomes and accumulation of damaged lysosomes. 

- Lysosomal dysfunction is mainly caused by several enzymes of the glycolytic and NAD pathways, including ALDOA, GAPDH, NAMPT, and PGK1, which do not function effectively under glucose starvation.

- Ferroptosis occurs via iron accumulation due to dysfunction of DMT1, which excretes iron from lysosomes.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Proteostasis refers to the cellular mechanisms that regulate the synthesis, folding, trafficking, and degradation of proteins to maintain overall protein balance and health within the cell. Disruptions in proteostasis, such as misfolded proteins or impaired degradation pathways, can lead to protein aggregates that are toxic to cells, a common feature of neurodegenerative diseases such as Alzheimer's and Parkinson's. Maintaining proteostasis is critical in neurons because their long lifespan and high metabolic activity make them particularly susceptible to the accumulation of protein aggregates. Perturbations in proteostasis can initiate or exacerbate neurodegenerative processes, ultimately contributing to neuronal dysfunction and cell death.

Point of Interest
- Chronic inhibition of γ-secretase leads to a decrease in lysosomal Ca²⁺ and ultimately to resulting in endolysosomal collapse.

- APP(amyloid precursor protein) C-terminal fragments (APP-CTFs) localize to late endosome/lysosome-ER contacts and an excess of APP-CTFs reduces lysosomal Ca²⁺ replenishment from the ER.

- Balanced levels of APP-CTF are important for lysosomal homeostasis.

Point of Interest
- Loss of autophagy in neurons leads to a metabolic defect and cell death.

- Dysfunctional autophagy causes NAD depletion due to hyperactivation of NAD-consuming enzymes.
- NAD depletion leads to cell death via mitochondrial depolarization in neurons.

- Increasing intracellular NAD levels rescues the viability of autophagy-deficient neurons.

Point of Interest
- Drosophila ortholog of APP, Appl, plays a role in controlling multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling, and proteostasis in the aging brain.

- Appl regulated autophagy through TGFβ signaling in mouse genetic, human iPSC and in vivo tauopathy models.

- The role of APP in controlling age-dependent proteostasis is conserved and associated with Alzheimer's disease.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Autophagy is a cellular process that involves the degradation and recycling of cellular components through the formation of autophagosomes, which subsequently fuse with lysosomes for content degradation. In the context of cancer, autophagy plays a dual role; it can suppress tumor initiation by eliminating damaged organelles and proteins, but can also promote tumor survival and growth under metabolic stress by providing nutrients through the recycling of cellular components.

Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion
Click here for the original article: Ronghui Yan, et. al., Nature Immunology, 2024.

Point of Interest
- Carnosine synthase, CARNS2, promotes carnosine synthesis under hypoxia.

- Carnosine controls lysosomal subcellular distribution, acidification, and activity by maintaining intracellular pH homeostasis.

- By maintaining lysosomal activity, carnosine facilitates NFX1 degradation, which triggers galectin-9 and T-cell-mediated immune escape and tumorigenesis.

Point of Interest

-Chronic selective autophagy suppresses NF-κB signaling in MCD-type Diffuse large B cell lymphoma (DLBCL).

-MCD-type DLBCL develops genetic and epigenetic alterations that attenuate selective autophagy.

-Disruption of selective autophagy leads to the accumulation of ubiquitinated MYD^88L265P.

-BTK and mTOR inhibitors interfere with the My-T-BCR pathway by enhancing the autophagic degradation of MYD^88L265P.

Point of Interest

-An in vivo CRISPR screen identifies p47 as a suppressor of HER2+ breast cancer metastasis.

-Depletion of p47 reduces NEMO endosomal trafficking and enhances NF-κB signaling.

-Ablation of p47 impairs lysosomal repair and autophagic flux, leading to increased metastasis.

-Lower expression of p47 correlates with increased metastasis in human breast cancer.

Analysis of autophagic flux without transfection

Detection Principle

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.

Experimental Data

Experimental Conditions
CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

Procedure
1. HeLa cells were seeded (1.0 x 10⁴ cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37°C in an incubator equilibrated with 95% air and 5% CO₂.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.
  •　MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Control)
  •　Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Starvation)
  •　Amino acid-free medium (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. The supernatant was discarded, bafilomycin A1 working solution (10,000 times dilution, 200 μl), an inhibitor of lysosomal acidification, was added to the well, and the cells were incubated at 37°C for 20 minutes. (Inhibition of autolysosome formation)
5. The stained cells were observed under a confocal fluorescence microscope.

Products in Use
Autophagic Flux Assay Kit

Autophagy plays a critical role in neurodegeneration and aging by maintaining cellular health through the degradation and recycling of damaged cellular components. In the context of neurodegeneration, such as Alzheimer's, Parkinson's and Huntington's diseases, impaired autophagy contributes to the accumulation of toxic protein aggregates that exacerbate neuronal damage and dysfunction. Conversely, enhancing autophagy has been shown to mitigate these deleterious effects and improve neuronal survival, suggesting a protective role in aging and neurodegenerative diseases. Research continues to explore autophagy modulators as potential therapeutic agents to slow the progression of neurodegenerative diseases and promote healthier aging.

The aging mouse CNS is protected by an autophagy-dependent microglia population promoted by IL-34
Click here for the original article: Rasmus Berglund, et. al., Nature Communications, 2024.

Point of Interest
 - A microglial population emerges in the cortical regions of aging mice, characterized by a transcriptome indicative of activated autophagy.

- This population is found to be dependent on IL-34, a ligand for CSF1R.

- Loss of autophagy-dependent microglia leads to neuronal and glial cell death and increased mortality in aged mice exposed to neuroinflammation.

- Conversely, IL-34-mediated microglial expansion is protective.

Point of Interest
 - Mitophagy is either increased or unchanged in older mice compared to younger ones.

- There is a marked upregulation of the type I interferon response in the retinas of older mice.

- This response correlates with increased levels of cytosolic mtDNA and activation of the cGAS/STING pathway.

- In older mice, induction of mitophagy with urolithin A attenuates cGAS/STING activation and ameliorates the deterioration of neurological function.

Point of Interest
 - SKA2 inhibits SA-dependent IL-1β release by counteracting FKBP5 function.

- Hippocampal Ska2 knockdown in male mice hyperactivates secretory autophagy (SA), leading to neuroinflammation and subsequent neurodegeneration.

- Hyperactivation of SA increases IL-1β release, contributing to an inflammatory feed-forward vicious cycle including NLRP3 inflammasome activation and Gasdermin D-mediated neurotoxicity.

- Results from protein analyses of postmortem human brains show that SA is hyperactivated in Alzheimer's disease.

Analysis of Lysosomal Mass and pH Exchange 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

Scientists have revealed that Scd1, which is induced by exposure to cool temperatures, and monounsaturated lipids are crucial for the survival of adipocytes. Inhibition of Scd1 leads to autophagy and cell death, while supplementation with monounsaturated fatty acids can prevent this.

Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes
Click here for the original article: Hiroyuki Mori, et. al., Molecular Metabolism, 2024.

Point of Interest
 - Inhibition of Scd1 results in autophagy-dependent cell death.

 - Blockade of autophagy or supplementation with monounsaturated fatty acids rescues Scd1-deficient adipocytes from death.

 - Reduced bone marrow adipocyte volume and number in mice deficient for Scd1 specifically in bone marrow adipocytes.

 - Scd1 deficiency causes cell death characterized by the accumulation of autophagosomes due to lysosomal dysfunction.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

＜NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response＞

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Autophagy is a cellular process that degrades and recycles damaged organelles, misfolded proteins, and other cellular debris, and is critical for maintaining cellular homeostasis. Dysregulated autophagy is often observed in the context of neurodegeneration, where either excessive or insufficient autophagic activity can contribute to the accumulation of toxic proteins and organelle dysfunction that are hallmarks of neurodegenerative diseases. Enhancing autophagy has been shown to alleviate symptoms and pathology in models of diseases such as Alzheimer's, Parkinson's, and Huntington's, suggesting a protective role against neurodegeneration. Conversely, excessive autophagy can lead to cell death, suggesting that a delicate balance is required for neuronal health and highlighting the complexity of targeting autophagy as a therapeutic strategy for neurodegenerative diseases.

Point of Interest
- Inhibiting microglial autophagy leads to the disengagement of microglia from amyloid plaques, exacerbating neuropathology in mice with Alzheimer's Disease (AD).

- An autophagy deficiency fosters the development of senescent microglia.

- Pharmacological removal of autophagy-deficient senescent microglia can alleviate neuropathology in mice with AD.

Point of Interest
- Activated microglia mediate non-cell-autonomous inhibition of neuronal autophagy.

- CCL-5, CCL-4, and CCL-3 derived from microglia activate the neuronal CCR5 receptor, leading to the inhibition of neuronal autophagy.

- Levels of CCR5, CCL-3, CCL-4, and CCL-5 are elevated in the brains of mice with Huntington’s disease and tauopathy.

- Inhibiting CCR5, either pharmacologically or genetically, ameliorates neurodegeneration in mice.

Point of Interest
- The translocator protein (TSPO) is pivotal for respiratory metabolism and energy supply in microglia.

- Hexokinase-2 (HK) affects glycolytic metabolism and phagocytosis through its interaction with mitochondria.

- Mitochondrial HK influences glycolysis and inflammation, and its displacement improves phagocytosis in TSPO-deficient microglia.

- Alzheimer’s beta-amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in Alzheimer’s disease.

Autolysosome
(DALGreen)

＜Cellular Landscape: autophagy-related pathway＞

Link to each product (red at left figure)

Autophagy Detection
 Autophagic Flux Assay Kit - First-choice product
 DAPRed - Autophagosome detection
 DAPGreen - Autophagosome detection
 DALGreen - Autolysosome detection

Mitophagy Detection
 Mitophagy Detection Kit and Mtphagy Dye

Mitochondrial Membrane Potential Detection
 JC-1 / MT-1

Lysosomal Analysis
 Lysosomal Acidic pH Detection Kit
                   Green/Red, Green/Deep Red

Endocytosis Detection
 ECGreen-Endocytosis Detection
 AcidSensor Labeling Kit

Exosome
 Exosome Isolation Kit
 Exosome Membrane Labeling Kit
                          Green / Red / Deep Red

• - The nematode Caenorhabditis elegans nuclear envelope anchor protein ANC-1 and its m ammlian ortholog nesprin-2 are cleared out by autophagy and restrict the nucleolar size, a biomarker of aging.
• - Nucleolar degradation at the most proximal oocyte by ANC-1 and key autophagic components relates to a germline immortality.
• - Perturbation of this clearance pathway causes tumor-like structures in C. elegans, and genetic ablation of nesprin-2 causes ovarian carcinomas in mice.

• - Nutrient starvation triggers changes in metabolism that are coordinated across the cell and its organelles.
• - Jang et al. studied how endosomal signaling lipid turnover through MTM1 reshapes the endoplasmic reticulum to control mitochondrial morphology and oxidative metabolism.
• - A lipid-controlled organellar relay transmits nutrient-triggered changes in endosomal signaling lipid levels to mitochondria to enable metabolic rewiring.

• - Chaperone-mediated autophagy (CMA) contributes to the rhythmic removal of clock machinery proteins and the circadian remodeling.
• - Disruption of this autophagic pathway in vivo leads to fragmented circadian patterns, resembling those in sleep disorders and aging.
• - Conversely, loss of the circadian clock abolishes the rhythmicity of CMA, leading to pronounced changes in the CMA-dependent cellular proteome. 

Autophagy is a cellular process that degrades and recycles damaged cellular components and is critical for maintaining cellular health and function. In neurons, autophagy plays an important protective role by removing defective proteins and organelles, preventing the accumulation of toxic aggregates that can lead to neurodegenerative diseases. This process also helps maintain synaptic plasticity and neuronal homeostasis, which are essential for proper neuronal function and brain health. In addition, by regulating the response to neural stress, autophagy contributes to the overall resilience and longevity of neural cells.

Point of Interest
- A microglial population emerges in the cortical regions of aging mice, characterized by a transcriptome indicative of activated autophagy.

- This population is found to be dependent on IL-34, a ligand for CSF1R.  

- Loss of autophagy-dependent microglia leads to neuronal and glial cell death and increased mortality in aged mice exposed to neuroinflammation.  

- Conversely, IL-34-mediated microglial expansion is protective.

Point of Interest
- αSyn aggregates accumulate predominantly in lysosomes, causing them to rupture.

- This rupture seeded the aggregation of endogenous αSyn, initially around damaged lysosomes.  

- Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes, indicating the activation of lysophagy.

- Autophagy-deficient cells treated with αSyn aggregates had an increased number of ruptured lysosomes and enhanced propagation of αSyn aggregates.

Point of Interest
- Hippo kinase MAP4K2 is essential for autophagy and cell survival under energy stress.

- MAP4K2 phosphorylates LC3A at Serine 87 to promote autophagic flux.

- Energy stress induces the formation of the MAP4K2-STRIPAK^STRN4 complex, activating MAP4K2.

- Autophagy mediated by MAP4K2 is critical for the development of head and neck cancer.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

＜NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response＞

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Scientists have discovered that protein aggregates and components of the autophagy machinery accumulate in developmental radial glia-like neural stem cells (NSCs) as they enter quiescence. They also uncover an important role of autophagy in the transition of developmental NSCs to their quiescent adult form by modulating autophagy activity in vivo.

Autophagy drives the conversion of developmental neural stem cells to the adult quiescent state
Click here for the original article: Isabel Calatayud-Baselga, et. al., Nature Communications, 2023.

Point of Interest
- Protein aggregates and autophagy components accumulate in radial glia-like NSCs during quiescence.
- Blocking autophagy hinders the establishment and maintenance of this quiescent state.
- Enhancing autophagy via AMPK/ULK1 activation promotes NSC quiescence independent of BMP signaling.
- Disruption of autophagy in radial glia-like NSCs affects initial and sustained quiescence during NSC niche formation in the hippocampal dentate gyrus.

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

＜Impact on Lysosomal Acidification and pH Detection＞

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

＜NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response＞

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.