Autophagy : Reagent Selection Guide

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.