Mechanism Analysis of Mitochondrial Autophagy in Cerebral Stroke

Mitochondria play key roles in a variety of cellular processes, including ATP production, Ca²⁺ homeostasis, reactive oxygen species (ROS) generation, and apoptosis. Mitophagy is a mechanism of specific autophagic elimination of mitochondria. Many stimuli can activate mitophagy to alleviate cellular damage caused by oxidative stress. Abnormal mitophagy is closely related to the occurrence, development, and pathological mechanism of ischemic stroke. Enhanced mitophagy has been shown to potentially improve ROS accumulation in cerebral ischemic stroke. In summary, mitophagy and the activation of various signaling pathways are closely related to the pathophysiology of ischemic stroke. Targeting these signals may improve the pathological changes and symptoms of ischemic stroke and help discover new treatments for stroke.

Fig. 1. An overview of mitophagy in ischemic neurons. (Li et al., 2023)

Our Services

At Ace Therapeutics, we are committed to providing comprehensive mechanism analysis services for mitophagy in stroke. Our team of experienced researchers and state-of-the-art facilities enable us to study the molecular mechanisms, pathophysiological roles, and possible signal transduction pathways of mitophagy regulation in ischemic brain injury. We aim to help clients develop the rational design of novel stroke therapeutic interventions by targeting mitophagy.

We can study the signaling events of multiple mitophagy receptors and their physiological significance in in vitro and in vivo models of ischemic stroke.

• PTEN induced putative kinase 1 (PINK1)/Parkin signaling pathway
• BNIP3 (B Lymphoma-2 gene/adenovirus E1B interacting protein 3) and NIX (BNIP3L) signaling pathway
• FUN14 domain-containing 1 (FUNDC1) signaling pathway

In addition, our experts help clients identify mitophagy targets derived from different neuronal cells as potential therapeutic targets for ischemic stroke, including but not limited to:

Our Mitophagy Detection Methods

We offer a variety of biochemical and cell biology methods for monitoring mitophagy in animal models of stroke, ensuring you with accurate and reliable data.

• Fluorescence Microscopy

Our fluorescence microscopy platform is widely used to observe mitochondria-related proteins or structures. We used labeling of mitochondria with MitoTracker dye to visualize mitochondria in living neural cells. For fixed cells, we label mitochondria using antibodies that stain mitochondrial proteins which allow monitoring of mitochondria-independent mitochondrial membrane potential. Furthermore, we used colocalization of Lysotracker or lysosome-associated membrane proteins (LAMP-1 and LAMP-2) to monitor the mitophagy process.

• Immunoblotting of Mitochondrial Proteins

We offer western blotting analysis of multiple mitochondrial proteins to quantitatively assess the mitophagy process, such as TOM20 (an extramitochondrial protein), TIM23 (an intramitochondrial protein), cytochrome C oxidase subunit II (COXII) (an intramitochondrial protein).

• Mitochondrial DNA (mtDNA) Quantification

Our approach quantifies mitochondrial DNA (mtDNA) relative to nuclear DNA (nDNA). By measuring changes in the ratio between mtDNA and nDNA, we can assess the occurrence of mitophagy. Real-time PCR uses nDNA as the standard to amplify mtDNA, and determines relative quantification by analyzing the difference in Ct values between mtDNA and nDNA.

• Ultrastructural Assessment

We use electron microscopy to directly observe autophagosomes containing engulfed mitochondria, which provides direct images of autophagosomes engulfing mitochondria.

• Targeted mKeima Assay

We use the mitochondria-targeted mKeima to detect and analyze mitophagy. By measuring the ratio of fluorescence at different excitation wavelengths, we can differentiate between intact (green) and degraded (red) mitochondria. This method provides sensitive and quantitative visualization of mitophagy.

Ace Therapeutics provides comprehensive analysis services to elucidate the role of mitophagy in ischemic stroke. Through advanced methodologies and partnerships, we help our clients develop new perspectives, therapeutic strategies, and potential therapeutic targets for stroke. If you are interested in our services, please do not hesitate to contact us!

1. Li, J., et al. (2023). Targeting neuronal mitophagy in ischemic stroke: an update. Burns & Trauma, 11, tkad018.