New Mitophagy Pathways: Next Therapy Targets? [Sep. 23, 2025] DOJINDO LABORATORIES

Defects in mitophagy through the autophagy–lysosome pathway, particularly in the PINK1/Parkin system, are closely linked to neurodegenerative disease. Recent studies reveal additional mechanisms, including a mitophagic stress response that relieves autophagy inhibition and a PINK1–SMAD3 transcriptional loop, highlighting new therapeutic targets in mitochondrial quality control.

1. Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy (Nature Communications, 2025)
Summary: Damaged mitochondria are usually cleared by the Pink1/Parkin pathway, but this study identifies a parallel Mitophagic Stress Response (MitoSR) triggered by mitochondrial damage in neurons. MitoSR degrades autophagy inhibitors (MTMR5, MTMR2, Rubicon) via the ubiquitin–proteasome system, thereby enhancing autophagosome formation, lysosome fusion, and overall mitochondrial quality control.

Highlighted technique: The study used fluorescent pH probes to monitor lysosomal acidity under mitochondrial stress. With this approach, the authors showed that reducing Rubicon, a protein that suppresses autophagy, restored lysosomal activity and enabled more efficient clearance of damaged mitochondria.

Related techniques Lysosomal Function Analysis (as used in this article), Autophagosome Detection (as used in this article)

2. A positive feedback loop between SMAD3 and PINK1 in regulation of mitophagy (Cell Discovery, 2025)
Summary: PINK1 is a protein that protects cells by helping remove damaged mitochondria, and SMAD3 is a transcription factor that controls gene activity. This study reveals, for the first time, that PINK1 and SMAD3 activate each other in a unique self-amplifying loop independent of the usual TGFβ pathway, uncovering a powerful new mechanism that strengthens mitochondrial quality control and offers fresh insight into diseases like Parkinson’s.

Highlighted technique: To evaluate mitophagy, researchers used the Mito-Keima assay, which tracks mitochondria entering acidic lysosomes as a measure of degradation. In HeLa cells expressing YFP-Parkin and mito-Keima, SMAD3 knockdown combined with Oligomycin/Antimycin A treatment enabled flow cytometric quantification of this process.

Related techniques Mitophagy Detection, Autophagic Flux Assay Kit

Previous Science Note

Related Techniques (click to open/close)

Target	Kits & Probes
Mitophagy detection	Mitophagy Detection Kit
First-time autophagy research	Autophagic Flux Assay Kit
Autophagy detection	DAPGreen, DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Lysosomal function Analysis	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
ROS Detection	ROS Assay Kit -Highly Sensitive DCFH-DA- and ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-
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)
> Accurate Autophagy Imaging with Lysosome Staining

We performed fluorescence imaging under amino acid starvation in HeLa cells stained with the autophagosome detection probe DAPRed (Code: D677) and LysoPrime Green (Code: L261). The fluorescence signal of LysoPrime Green increased, and lysosomal localization was confirmed over time. These results indicate that the co-localization rate with DAPRed fluorescence is sufficient, enabling accurate autophagy analysis.

Application Note II (click to open/close)
> Induction of Mitophagy in Parkin Expressed HeLa cells

CCCP(carbonyl cyanide m-chlorophenyl hydrazone) has been added to normal and Parkin expressed cells. The strong fluorescence was not observed in normal HeLa cells(A)(B). On the other hand, the trong fluorescence was shown in Parkin expressed cells in 18 hours after additon of CCCP(C). Some of the puncta are co-localized with the autophagy marker(GFP-LC3). In addition, suppressed fluorescence of Mtphagy dye was observed when autophagy inhibitor, bafilomycin was added to Parking expressed cells(D). Because lysosomal pH was increased by the additon of bafilomycin.

Previous Science Note