Differential roles of Aβ processing in hypoxia-induced axonal damage
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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