Mitochondrial swelling and microtubule depolymerization are associated with energy depletion in axon degeneration

doi:10.1016/j.neuroscience.2013.02.033

Neuroscience

Volume 238, 15 May 2013, Pages 258-269

https://doi.org/10.1016/j.neuroscience.2013.02.033 Get rights and content

Abstract

Although mitochondrial dysfunction is intimately related to axonal degeneration following nerve injury, the molecular mechanisms of mitochondrial swelling and its mechanistic relation to axonal degeneration are largely unknown. Previous studies have demonstrated that axonal degeneration in the injured peripheral nerves shows two morphologically distinct phases: (1) A latency period (∼24 h), in which the morphology of axonal cytoskeletons seems unchanged, followed by (2) an execution period (36–48 h), which shows a catastrophic granular degeneration of most axonal structures in rodent axons. In the present study, we found that, in the sciatic nerve axotomy model, energy failure and microtubule depolymerization occurred during the latency period whereas mitochondrial swelling and neurofilament degradation started in the execution period. The energy repletion with NAD or an NAD/pyruvate mixture inhibited microtubule depolymerization, mitochondrial swelling and axonal degeneration in transected sciatic nerve axons. In addition, microtubule perturbing agents enhanced axonal degeneration and mitochondrial swelling. Extracellular calcium chelation did not affect energy failure, microtubule depolymerization or mitochondrial swelling, but it did prevent neurofilament degradation. These findings suggest that an early disturbance in energy dynamics regardless of mitochondrial swelling might be a key trigger for the initiation of axonal degeneration and that extracellular calcium influx is a late effector for neurofilament degradation.

Highlights

► We examined the mechanism of mitochondrial swelling and axonal degeneration. ► Energy failure occurred prior to mitochondrial swelling and axonal degeneration. ► Energy repletion prevented mitochondrial swelling and microtubule depolymerization. ► Perturbing microtubule dynamics enhanced axonal degeneration and mitochondrial swelling. ► Calcium was not related to mitochondrial swelling and microtubule depolymerization.

Introduction

After traumatic injury of axons, the degeneration of the distal axons is performed by several enzymatic processes that are distinct from cell death signaling pathways (Ikegami and Koike, 2003, Coleman and Freeman, 2010). The importance of the axonal influx of extracellular calcium has received attention as a molecular mechanism of axonal degeneration (Glass et al., 1994, George et al., 1995, Stirling and Stys, 2010). It was recently reported that the ubiquitin–proteasome pathway is implicated in regulating microtubule fragmentation in the early phase of axonal degeneration (Zhai et al., 2003, Wakatsuki et al., 2011). The discovery of the slow Wallerian degeneration (Wlds) gene sheds new light on the molecular mechanism of axonal degeneration (Coleman and Freeman, 2010). Wlds protein is a fusion protein composed of the amino-terminal 70 amino acids of a ubiquitinating enzyme and the full-length of nicotinamide mononucleotide adenylyltransferase (Nmnat), that produces NAD. The overexpression of Nmnat also delayed axonal degeneration after nerve injury (Sasaki et al., 2009, Sasaki and Milbrandt, 2010). Furthermore, NAD levels decreased with ATP levels in the transected axons of cultured neurons, and repletion of NAD or pyruvate could rescue these axons from degeneration by preventing the decrease of ATP (Araki et al., 2004, Wang et al., 2005). This suggests that local energy status might be an important factor regulating axonal stability and degeneration.

Because axonal mitochondria are essential for energy supply in axons, a failure in axonal energy metabolism may be caused by impaired axonal transport of mitochondria and/or mitochondrial dysfunction following nerve injury (Court and Coleman, 2012). Previous studies have demonstrated mitochondrial swelling in degenerated axons after nerve injury (Vial, 1958, Barrientos et al., 2011). Since swollen mitochondria were dysfunctional in many cases (Mazzeo et al., 2009), a mechanism related to mitochondrial swelling seems intimately related to axonal degeneration. Recently, an active involvement of the activation of mitochondrial permeability transition pore (mPTP) in mitochondrial swelling and axonal degeneration has been reported (Okonkwo and Povlishock, 1999, Barrientos et al., 2011). However, the molecular mechanisms of mitochondrial swelling and its mechanistic relation to axonal degeneration are still unclear. In the present study, we examined how factors of energy status and extracellular calcium influx affect microtubule depolymerization, mitochondrial swelling and axonal degeneration using biochemical, immunocytochemical and ultrastructural analyses.

Section snippets

Sciatic nerve axotomy

Sciatic nerve axotomy of adult mice (C57BL/6) was performed as previously described (Shin et al., 2010, Jung et al., 2011). Wlds mice were originally obtained from Harlan (England) (Sasaki et al., 2009). All procedures were performed according to the protocols approved by the Dong-A University Committee on animal research, which follows the guide for animal experiments established by the Korean Academy of Medical Sciences. Briefly, sciatic nerves were sectioned 5 mm proximal to the tibioperoneal

Temporal changes in mitochondrial morphology in degenerating axons in vivo

In cross sections of uninjured sciatic nerves, most axonal mitochondria showed electron-dense round structures that rarely reveal mitochondrial cristae or double membranes (Fig. 1A). In longitudinal sections, rod-shaped mitochondria are frequently found in axons, and we measured the length index of mitochondria (length/diameter). The mean length index and diameter of mitochondria in normal axons were 6.92 and 0.21 μm, respectively (Fig. 1B, C). At 24 h after axotomy, the mean diameter of axonal

Discussion

It has been reported that mitochondrial dysfunction is pivotal for axonal degeneration in many neurodegenerative diseases (Morfini et al., 2009, Court and Coleman, 2012). Because dysfunctional mitochondria swell in many pathological conditions, mitochondrial swelling may be a causal event for energy failure and for subsequent axonal degeneration. Consistent with findings, Barrientos et al. (2011) recently showed an involvement of mPTP in the mitochondrial swelling and axonal degeneration.

Acknowledgements

This research was supported by Mid-career Research Program and Science Research Center through NRF grant funded by the Ministry of Education, Science and Technology of Republic of Korea (2012-0000900, 2012-0005295), and by grants from Ministry of Health, Labour and Welfare of Japan. The authors declare no competing financial interests.

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^†: These authors contributed equally to this article.

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