Elsevier

Neurobiology of Disease

Volume 77, May 2015, Pages 94-105
Neurobiology of Disease

Differential roles of Aβ processing in hypoxia-induced axonal damage

https://doi.org/10.1016/j.nbd.2015.02.027Get rights and content

Highlights

  • Hypoxia induces loss of structural integrity and transport capacity in RGC axons.

  • Aβ mediates hypoxia-induced structural compromise of RGC axons.

  • Aβ blockade does not restore active axonal transport capacity during hypoxia.

  • Hypoxia-induced compromise of axonal structure but not transport depends on Aβ.

  • Aβ inhibition could provide clinical benefit for aspects of axonal degeneration.

Abstract

Axonopathy is a common and early phase in neurodegenerative and traumatic CNS diseases. Recent work suggests that amyloid β (Aβ) produced from amyloid precursor protein (APP) may be a critical downstream mediator of CNS axonopathy in CNS diseases, particularly those associated with hypoxia. We critically tested this hypothesis in an adult retinal explant system that preserves the three-dimensional organization of the retina while permitting direct imaging of two cardinal features of early-stage axonopathy: axonal structural integrity and axonal transport capacity. Using this system, we found via pharmacological inhibition and genetic deletion of APP that production of Aβ is a necessary step in structural compromise of retinal ganglion cell (RGC) axons induced by the disease-relevant stressor hypoxia. However, identical blockade of Aβ production was not sufficient to protect axons from associated hypoxia-induced reduction in axonal transport. Thus, Aβ mediates distinct facets of hypoxia-induced axonopathy and may represent a functionally selective pharmacological target for therapies directed against early-stage axonopathy in CNS diseases.

Introduction

Axonopathy, encompassing compromise of both axonal structure and function, is an early consequence of stress across a broad range of central neurons and neuropathological conditions (Coleman, 2005). While clearly associated with physical trauma to the nervous system (Rotshenker, 2011), axonopathy has also been increasingly appreciated as an early event in neurodegeneration in neurological disorders including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and glaucoma (Coleman, 2005, Morfini et al., 2009). Common features of compromised axonal structure include the development of axonal varicosities, accumulation of organelles, and loss of synaptic contacts, whereas deficits in transport capacity is a core consequence of impaired axonal function. However, the pathways mediating such structural and functional compromise, and whether these pathways are intersecting or distinct, remain under investigation.

A growing body of evidence has implicated the production of amyloid beta (Aβ) from amyloid precursor protein (APP) as a common pathway associated with axonopathy. While Aβ has been studied most extensively in relation to AD (Hardy & Selkoe, 2002), aberrant processing through this cascade has now also been reported in axonopathic diseases such as glaucoma (Yoneda et al., 2005), multiple sclerosis (Ferguson et al., 1997), ALS (Calingasan et al., 2005), epilepsy (Borges et al., 2003), stroke (Ohgami et al., 1992), HIV-dementia (Raja et al., 1997), Creutzfeld–Jakob disease (Liberski & Budka, 1999), and traumatic brain and optic nerve injury (Olsson et al., 2004, Reichard et al., 2004). Aβ itself impairs axonal structure and function in a variety of experimental paradigms (Kasa et al., 2000, Pike et al., 1992, Stokin et al., 2005), and blockade of the enzymes necessary for Aβ production from APP (BACE1 and the γ-secretase complex (Hardy and Selkoe, 2002, Scheuner et al., 1996, Vassar et al., 1999) protects central axons from diverse stressors (Farah et al., 2011, Nikolaev et al., 2009, Yoon et al., 2006, Jurynczyk et al., 2005). Together, these data imply a broad role for Aβ as an effector of axonopathy across CNS degenerative diseases.

Such a role for Aβ would have particular relevance in the setting of CNS hypoxia. Hypoxia has long been known to induce both structural and functional axonopathy (Def Webster and Ames, 1965, Ochs and Ranish, 1970), and the enzymatic pathway necessary for Aβ production is sensitive to tissue oxygen status (Peers et al., 2009). CNS hypoxia in fact predisposes central neurons to degeneration in well-studied diseases like Alzheimer's and optic neuropathy (Grimm and Willmann, 2012, Zlokovic, 2011), and such hypoxia-induced stress mechanisms as hypoxia-inducible factor-mediated pathways (Koh & Powis, 2012), heat-shock-factor-mediated pathways (Baird et al., 2006, Shen et al., 2005), and the unfolded protein response (Kim et al., 2008) are now understood to be central to the protein homeostasis defense mechanisms triggered in a wide range of neurodegenerative and neuroinflammatory conditions in the CNS (Powers et al., 2009, Mehta et al., 2009). Thus, elucidating hypoxia-activated axonopathic mechanisms likely to intersect with those in CNS disease remains an important goal.

Therefore, we sought here to test critically the hypothesis that Aβ is a critical downstream effector of hypoxic stress on central axons, focusing on the axons of retinal ganglion cells (RGCs), the long projection neurons of the eye that resemble other projection neurons of the brain and spinal cord in terms of function, connectivity, and susceptibility to neurodegenerative conditions (Dowling, 2012). Using hypoxic stress to induce early-stage axonopathy in these mature CNS neurons within their native tissue environment in explanted retinas, we found that Aβ generated from APP is indeed both sufficient and necessary for the structural degeneration of RGC axons in response to hypoxic stress, and that blockade of either BACE1 or γ-secretase activity can provide quantitative structural protection to stressed axons. Surprisingly, whereas blockade of Aβ production maintained the structural integrity of RGC axons, it was not sufficient to restore impaired active transport capacity. Moreover, we found that active transport could still be maintained even through frank distortions in axonal structure induced by Aβ short of overt breakdown of the axon. Thus, our work supports the potential benefit of anti-amyloidogenic therapies in treating neurodegenerative conditions of the eye but suggests that such maintenance of structural integrity is required but not sufficient for full protection of RGC axons against hypoxic stress-associated dysfunction.

Section snippets

Retinal explant cultures

All experiments were done in explant cultures from adult (> 3 months) rats or mice. Briefly, eyes were enucleated from CD Sprague–Dawley rats (Charles River, Wilmington, MA) or C57Bl6 or APP-deficient mice (Jackson Labs, Bar Harbor, ME) immediately following sacrifice in accordance with NIH guidelines and under Duke IACUC approval and oversight. A circumferential cut was made 1 mm posterior to the limbus, then the retina was gently coaxed away from the posterior sclera to permit separation of the

Hypoxia impairs axonal structure and net retrograde transport in explanted RGCs

The explanted retina offers an accessible experimental context where mature, three-dimensional native tissue architecture is preserved. Acute whole retinal explant cultures have been used for a wide range of physiological and mechanistic studies (Koriyama et al., 2011, Patzke et al., 2010, Donovan and Dyer, 2006), and in general intact tissue models preserve intercellular and local neural circuit interactions to a much greater degree that dissociated cultures and cell lines (Cho et al., 2007,

Discussion

Together, our results indicate that APP-dependent processes mediate specific and separable components of early-stage axonal damage caused by hypoxic stress: whereas Aβ generated from APP is both necessary and sufficient for structural compromise of RGC axons induced by hypoxic stress, Aβ generation is not obligatory for hypoxia-induced impairment of net retrograde axonal transport. Our findings thus support the existence of parallel axonopathic pathways (Vohra et al., 2010) that selectively

Conflict of interest

The authors declare that no conflict of interest exists.

Acknowledgments

We are much indebted to Drs. Peter Reinhart, Julia Cho, Warren Hirst, Steven Braithwaite, and Robert Martone for their valuable input and advice, including providing all of the BACE and γ-secretase inhibitors used in this study; to D. He for assistance and data in the APP processing studies; and to Drs. David Calkins, Rebecca Sappington, Stuart McKinnon, Pate Skene, Dona Chikaraishi, and Francesca Cordeiro for their guidance and support throughout this work, which was supported in part by a

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