Mitochondrial Dynamics in Glial Regulation of Neuroinflammation [Apr. 08, 2025] DOJINDO LABORATORIES

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Mitochondria are increasingly recognized as critical regulators of glial cell function in the central nervous system, influencing both neuroinflammatory responses and neuronal integrity. This Science Note reviews recent advances in our understanding of how mitochondrial activity within glial cells shapes their pathological and protective roles in the inflamed brain and provides insights into potential therapeutic strategies targeting glial mitochondrial metabolism.

Mitochondrial complex I activity in microglia sustains neuroinflammation (Nature, 2025)
In multiple sclerosis, chronic activation of pro-inflammatory microglia leads to sustained CNS inflammation through mitochondrial reverse electron transport at complex I, resulting in increased ROS production. Inhibition of complex I reduces ROS and alleviates neuroinflammation, highlighting its potential as a therapeutic target.

Highlighted technique: To elucidate the relationship between complex I activity and reverse electron transport (RET)-mediated ROS production in microglia, this study used integrated transcriptomic, proteomic and metabolomic analyses to comprehensively assess changes in electron transport chain enzyme expression, metabolic flux and antioxidant responses.

Loss of fatty acid degradation by astrocytic mitochondria triggers neuroinflammation and neurodegeneration (Nature Metabolism, 2023)
Astrocytes are essential support cells for neurons and play a key role in maintaining lipid homeostasis in the brain through mitochondrial fatty acid metabolism. Mitochondrial dysfunction in astrocytes leads to the accumulation of acetyl-CoA, which promotes STAT3 acetylation and triggers a shift towards a reactive astrocyte phenotype associated with neuroinflammation.

Highlighted technique: In this study, astrocyte-specific Tfam knockout mice were generated to model selective mitochondrial OXPHOS deficiency in astrocytes. Using this model, the authors showed that loss of astrocytic mitochondrial OXPHOS leads to cognitive impairment and neuroinflammation.

Microglia rescue neurons from aggregate-induced neuronal dysfunction and death through tunneling nanotubes (Neuron, 2024)
Microglia support neuronal recovery by delivering healthy mitochondria to stressed neurons via tunneling nanotubes (TNTs), reducing oxidative stress and restoring function. In addition, microglia use TNTs to remove toxic aggregates of alpha-synuclein and tau from neurons, helping to suppress neurodegeneration.

Highlighted technique: In this study, neurons and microglia were co-cultured to observe the intercellular transfer of materials such as mitochondria, alpha-synuclein and tau via tunneling nanotubes (TNTs). These transfers were captured using fluorescent labelling and live cell imaging, allowing real-time observation of TNT-mediated dynamics.

Related technique Oxygen Consumption Rate Assay, Total ROS Detection

Previous Science Note

Application Note I
> Induction of Mitophagy in Parkin Expressed HeLa cells

After HeLa cells were washed with HBSS, co-stained with MitoBright ROS Deep Red and mitochondrial membrane potential staining dye (JC-1: code MT09), and the generated mitochondrial ROS and membrane potential were observed simultaneously. As a result, the decrease in mitochondrial membrane potential and the generation of mitochondrial ROS are simultaneously observed.
＜Imaging Conditions＞(Confocal microscopy) JC-1: Green Ex = 488, Em = 490-520 nm, Red: Ex = 561, Em = 560-600 nm MitoBright ROS ：Ex = 633 nm, Em = 640-700 nm Scale bar: 10 μm	＜Examination Conditions＞(Plate Reader)Tecan, Infinite M200 Pro JC-1: Green Ex=480-490 nm, Em=525-545 nm; Red: Ex= 530-540 nm, Em=585-605 nm MitoBright ROS: Ex=545-555 nm, Em = 665-685 nm

Application Note II
> Inhibition of Mitochondrial Electron Transport Chain

Antimycin stimulation of Jurkat cells was used to evaluate the changes in cellular state upon inhibition of the mitochondrial electron transport chain using a variety of indicators.

The results showed that inhibition of the electron transport chain resulted in (1) a decrease in mitochondrial membrane potential and (2) a decrease in OCR. In addition, (3) the NAD⁺/NADH ratio of the entire glycolytic pathway decreased due to increased metabolism of pyruvate to lactate to maintain the glycolytic pathway, (4) GSH depletion due to increased reactive oxygen species (ROS), and (6) increase in the NADP⁺/NADPH ratio due to decreased NADH required for glutathione biosynthesis were observed.

Related Techniques

Target	Kit & Probes
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Mitophagy detection	Mitophagy Detection Kit
Mitochondrial Staining	MitoBright LT Green / Red / Deep Red
Lipid Droplet Staining	Lipi-Blue/ Green/ Red/ Deep Red
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Glycolysis/oxidative phosphorylation assay	Glycolysis/OXPHOS Assay Kit
Cellular senescence detection	SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST