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.
Highlights
Spinal cord injury (SCI) is a severe medical problem with high mortality and long-term morbidity (Eckert and Martin, 2017) for which there is no effective treatment
To assess axonal mitochondrial morphology following spinal cord injury, the mitochondria of corticospinal tract (CST) neurons of adult Sprague–Dawley rats were labeled with mitochondrially targeted DsRed 4 weeks before performing dorsal column lesions at the C4-C5 level
To determine if the mitochondrial fragmentation along CST axons observed after spinal cord injury correlates with increased mitophagy, we examined the association of mitochondria with the autophagocytic marker LC3 (Maday, 2016; Maday and Holzbaur, 2016)
Summary
Spinal cord injury (SCI) is a severe medical problem with high mortality and long-term morbidity (Eckert and Martin, 2017) for which there is no effective treatment. Following injury, severed axons often undergo dieback/retraction from the site of injury and fail to exhibit subsequent regeneration up to and beyond the injury site. Injury Induced Mitochondria Fragmentation within the CNS form retraction bulbs at the cut axon tip as opposed to new growth cones (Hill et al, 2001; Hill, 2017). Regenerative failure is due to cell intrinsic (Fawcett and Verhaagen, 2018) and extrinsic factors (Fitch and Silver, 2008). For functional regeneration and repair of injured circuitry to occur, it will be necessary to manipulate both intrinsic and extrinsic factors. We focus on elucidating the intrinsic factors that impair axon regeneration, focusing on the mitochondrial response to axonal injury in the severed axon
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