Related Experiment Video
Updated: Nov 1, 2025
Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria
Published on: August 4, 2022
Axotomy Induces Drp1-Dependent Fragmentation of Axonal Mitochondria
Joseph Kedra1, Shen Lin1, Almudena Pacheco1
1Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, United States.
Abstract:
It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dystrophic growth cones due to both intrinsic and extrinsic factors. We sought to investigate the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into the primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for 2 weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDivi-1) blocked fragmentation. Additionally, it was determined that there is increased mitophagy in CST axons following Spinal cord injury (SCI) based on increased colocalization of mitochondria and LC3. In vitro models revealed that mitochondrial divalent ion uptake is necessary for injury-induced mitochondrial fission, as inhibiting the mitochondrial calcium uniporter (MCU) using RU360 prevented injury-induced fission. This phenomenon was also observed in vivo. These studies indicate that following the injury, both in vivo and in vitro, axonal mitochondria undergo increased fission, which may contribute to the lack of regeneration seen in CNS neurons.
Insights
Axonal mitochondria fragment after central nervous system (CNS) injury, impairing neuron repair. Inhibiting mitochondrial fission and calcium uptake promotes healthier mitochondria, potentially aiding CNS axon regeneration.
Area of Science:
- Neuroscience
- Cell Biology
- Regenerative Medicine
Background:
- Central nervous system (CNS) axons typically fail to regenerate after injury due to intrinsic and extrinsic factors.
- The role of axonal mitochondria in the response to CNS injury, particularly spinal cord injury (SCI), remains incompletely understood.
Purpose of the Study:
- To investigate the role of axonal mitochondria in the response of corticospinal tract (CST) axons to injury.
- To determine the impact of mitochondrial dynamics, specifically fission and mitophagy, on CNS axon integrity following injury.
Main Methods:
- Adult rats received viral vector injections (AAV) to label CST axons with fluorescent proteins (mitoDsRed, GFP-LC3).
- Dorsal column lesions were performed to induce SCI, followed by analysis of mitochondrial morphology and mitophagy.
- Pharmacological inhibitors (mDivi-1 for Drp1, RU360 for MCU) were used to modulate mitochondrial fission.
- Both in vivo and in vitro models were employed to study mitochondrial responses to injury.
Main Results:
- Mitochondria in injured CST axons exhibited fragmentation near the injury site, persisting for up to two weeks.
- Inhibition of the GTPase Drp1 using mDivi-1 prevented mitochondrial fragmentation.
- Increased mitophagy, indicated by colocalization of mitochondria and LC3, was observed in CST axons post-SCI.
- Inhibition of the mitochondrial calcium uniporter (MCU) using RU360 blocked injury-induced mitochondrial fission both in vitro and in vivo.
Conclusions:
- Axonal mitochondria undergo increased fission following CNS injury, potentially contributing to the failure of CNS axon regeneration.
- Mitochondrial calcium uptake is necessary for injury-induced mitochondrial fission.
- Targeting mitochondrial dynamics, such as inhibiting fission or calcium uptake, may represent a therapeutic strategy to promote CNS axon repair.
