Employing Autophagy to Eliminate Disease Origins [Jun. 3, 2025] DOJINDO LABORATORIES

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Autophagy is increasingly recognized as a key regulator in disease pathogenesis, influencing the mechanisms of protein aggregation, secretion, and cellular clearance. This Science Note introduces three studies highlighting how autophagy suppression by PHGDH, the secretion mechanism of the antioxidant protein PARK7, and p97/VCP-dependent aggrephagy contribute to the progression of neurodegeneration and cancer.

Transcriptional regulation by PHGDH drives amyloid pathology in Alzheimer’s disease (Cell, 2025)
Summary: Alzheimer’s disease can develop in individuals without disease-causing genetic mutations and typically presents as late-onset (LOAD). This study shows that phosphoglycerate dehydrogenase (PHGDH) ,a recognised LOAD biomarker, promotes amyloid pathology by suppressing autophagy in astrocytes through a transcriptional role independent of its enzymatic activity.

Highlighted technique: This study demonstrated that PHGDH increases the expression of IKKα and HMGB1, which are involved in the suppression of autophagy via the mTOR pathway, at both the mRNA and protein levels in brain organoid astrocytes. Impaired autophagy was also confirmed by a reduction in autophagosomes using fluorescent small-molecule probes.

Related technique Autophagic Flux Detection, Apoptosis Detection

Unconventional secretion of PARK7 requires lysosomal delivery via chaperone-mediated autophagy and specialized SNARE complex (PNAS, 2025)
Summary: An unconventional autophagy pathway involving chaperone-mediated lysosomal translocation mediates the oxidative stress–induced secretion of PARK7/DJ-1, a protein with antioxidant and mitochondrial functions whose dysfunction is linked to neurodegeneration and cancer. This study offers mechanistic insight to guide therapeutic strategies targeting PARK7 secretion.

Highlighted technique: In this study, autophagy was monitored by expressing LC3 fused with either GFP or mRFP in cells. While both GFP and mRFP fluorescence are visible in autophagosomes, only mRFP remains detectable in autolysosomes due to the quenching of GFP in the acidic environment.

Related technique Autophagic Flux Detection Lysosome function

p97/VCP is required for piecemeal autophagy of aggresomes (Nature Communications, 2025)
Summary: Aggresomes, which form near the centrosome when protein quality control fails, are cleared through aggrephagy via a mechanism requiring the disaggregase p97/VCP and the cargo receptor TAX1BP1. This study shows that p97/VCP-mediated disassembly is essential for proteostasis and may offer a therapeutic target in neurodegenerative diseases.

Highlighted technique: In this study, retinoic acid-differentiated SH-SY5Y cells, which mimic neuron-like properties and are commonly used to study aggresomes, were utilized as a disease-relevant model. Autophagy was assessed by immunofluorescence staining of LC3, revealing that p97/VCP inhibition blocked aggresome clearance.

Related technique Autophagic Flux Detection Mitophagy Detection

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Related Techniques (click to open/close)

Target	Kit & Probes
First-time autophagy research	Autophagic Flux Assay Kit
Autophagy detection	DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Mitophagy detection	Mitophagy Detection Kit
Lysosomal function	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
ROS Detection	ROS Assay Kit -Highly Sensitive DCFH-DA- and ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST

Application Note I (click to open/close)
> NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response

We 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.

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. a l., EMBO J., 40(8), e105268 (2021)

Application Note II (click to open/close)
> Simultaneous 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 Deep Red - 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
- Lysosomal Acidic pH Detection Kit
(combination kit of LysoPrime Green / pHLys Red)
- MitoBright ROS Deep Red - Mitochondrial Superoxide Detection