Autophagy : Reagent Selection Guide

Science Note

[May. 14, 2024]                                                                                                                                                                                                                       Previous Science Note
Disruption of proteostasis leading to neurodegeneration 

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.

Accumulation of APP C-terminal fragments causes endolysosomal dysfunction through the dysregulation of late endosome to lysosome-ER contact sites
Click here for the original article: Marine Bretou, et. al., Developmental Cell, 2024.
NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency
Click here for the original article: Congxin Sun​, et. al., Cell Reports, 2023.
Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging
Click here for the original article: Nature Communications, et. al., Cell Reports, 2023.

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.

Related Techniques
     Lysosomal function Acidic pH Detection Kit-Green/Red and Green/Deep Red
     First-time autophagy research Autophagic Flux Assay Kit NEW
     Autophagy detection DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
     Mitophagy detection dye Mitophagy Detection Kit and Mtphagy Dye
     Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.
     Glycolysis/Oxidative phosphorylation Assay Extracellular OCR Plate Assay Kit, Glycolysis/OXPHOS Assay Kit
     NAD(H) and NADP(H) redox couples assay NAD/NADH and NADP/NADPH Assay Kit 

Related Applications

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.



Autophagic Pathway and Reagent Selection Guide

Autophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles. In this process, an isolation membrane composed of double membrane appear in cytosol, expands gradually, enfold with the aggregated proteins and damaged organelles, and close to form autophagosomes. The autophagosomes are fused with lysosomes to form autolysosomes in which are acidic environment. The contents in autolysosomes are decomposed by digestive enzymes in lysosomes. Since this cellular function is said to be related to aging as well as neurodegenerative diseases such as Parkinson’s disease, a simple autophagy detection method is being required.



  Small Fluorescent Molecules Fluorescent Protein
Autophagosome -
Autolysosome -

Autophagic Flux Assay Kit
This kit contains autophagosome and autolysosome detection dye (DAPRed), autolysosome detection dye (DALGreen), and lysosomal acidification inhibitor (bafilomycin A1). The Autophagic Flux Assay Kit allows the accurate evaluation of autophagic flux by monitoring autophagosome formation, lysosome fusion, and digestion of contents.

Time-lapse imaging of autophagy with DALGreen

For more details, click application data

H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, “Small fluorescent molecules for monitoring autophagic flux“, FEBS Lett.2018, 592, (4), 559–567.

Some data mentioned on this page is provided by the reference above.

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