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Asparagine Endopeptidase (Δ Secretase), an Enzyme Research Article: New Research | Disorders of the Nervous System Asparagine endopeptidase (δ secretase), an enzyme implicated in Alzheimer’s disease pathology, is an inhibitor of axon regeneration in peripheral nerves https://doi.org/10.1523/ENEURO.0155-20.2020 Cite as: eNeuro 2020; 10.1523/ENEURO.0155-20.2020 Received: 24 April 2020 Revised: 20 November 2020 Accepted: 23 November 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 English et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Asparagine endopeptidase (δ secretase), an enzyme implicated in Alzheimer’s disease pathology, 2 is an inhibitor of axon regeneration in peripheral nerves. Abbreviated title: δ secretase and nerve regeneration List of Author Names and Affiliations Arthur W. English Department of Cell Biology Emory University School of Medicine Atlanta, GA 30322 Xia Liu Department of Pathology and Laboratory Medicine Emory University School of Medicine Atlanta, GA 30322 Olivia C. Mistretta Department of Cell Biology Emory University School of Medicine Atlanta, GA 30322 Patricia J. Ward Department of Cell Biology Emory University School of Medicine Atlanta, GA 30322 Keqiang Ye Department of Pathology and Laboratory Medicine Emory University School of Medicine Atlanta, GA 30322 Author Contributions AWE: Designed research, Analyzed data, Wrote the paper; XL: Performed research, Analyzed data; OCM: Performed research, Analyzed data; PJW: Designed research, Performed research, Analyzed data; KY: Designed research, Analyzed data. Correspondence should be addressed to: Arthur W. English, Ph.D. Department of Cell Biology Emory University School of Medicine Whitehead Research Building 615 Michael Street, Room 405P Atlanta, GA 30322 V – 404-727-6250 F- 404-727-6256 [email protected] Number of figures: 6 Number of Tables: 0 Number of words for Abstract: 173 Number of Multimedia: 0 Number of words for Significance: 105 Number of words for Introduction: 631 Number of words for Discussion: 1197 1 3 Acknowledgements: Special thanks are due to Supriya Wariyar, Robin Isaacson, and 4 Alden Brown for their technical assistance. 5 Conflict of Interest: The authors declare no conflict of interest interests. Funding Sources: This work was supported by NIH grant NS105982 from the USPHS. 6 English, A.W. et al 7 Abstract 8 Asparagine endopeptidase (AEP) is a lysosomal protease implicated in the pathology of 9 Alzheimer’s disease (AD). It is known to cleave the axonal microtubule associated protein, Tau, 10 and amyloid precursor protein (APP), both of which might impede axon regeneration following 11 peripheral nerve injury. Active AEP, AEP-cleaved fragments of Tau (Tau N368), and APP (APP 12 N585) were found in injured peripheral nerves. In AEP null mice, elongation of regenerating 13 axons after sciatic nerve transection and repair was increased relative to wild type (WT) 14 controls. Compound muscle action potentials (M responses) were restored in reinnervated 15 muscles twice as fast after injury in AEP knockout mice as WT controls. Neurite elongation in 16 cultures of adult dorsal root ganglion neurons derived from AEP knockout mice was increased 17 significantly relative to cultures from wild type controls. In AEP knockout mice exposed to one 18 hour of 20 Hz electrical stimulation at the time of nerve injury, no further enhancement of axon 19 regeneration was observed. These findings support inhibition of AEP as a therapeutic target to 20 enhance axon regeneration after peripheral nerve injury. 21 Significance Statement 22 Axons in injured peripheral nerves can regenerate, but recovery from such injuries is poor. 23 Asparagine endopeptidase (AEP) is an enzyme linked to the major pathological features of 24 Alzheimer’s disease. We show here that it is highly expressed in injured peripheral nerves and 25 both reduces the microtubule stabilizing effect of Tau on axons and degrades amyloid precursor 26 protein in a manner that hinders any axon pathfinding role. Removing AEP by gene knockout 27 enhanced the elongation of regenerating axons in cut nerves and promoted an accelerated 28 restoration of neuromuscular function. Inhibition of AEP is thus a target for development of novel 29 strategies to treat peripheral nerve injuries. 30 Introduction 3 English, A.W. et al 31 Even though only about 10% of more than 200,000 new traumatic peripheral nerve 32 injuries that occur in the US each year ever recover function (Frostick et al., 1998; Scholz et al., 33 2009), there is currently no widely used non-surgical treatment available. Enhancing axon 34 regeneration has emerged as a goal for the development of new treatments following peripheral 35 nerve injury (Höke and Brushart, 2010). 36 Following injury to a peripheral nerve, regenerative sprouts in the proximal segment lead 37 to formation of regenerating axons that pass into a regeneration pathway. This process of 38 elongation, which is heavily influenced by axon guidance molecules (Dun et al., 2019; Dun and 39 Parkinson, 2020), involves distal addition of cytoskeletal elements, such as microtubules 40 (Bamburg et al., 1986). Microtubules are polymers of α and β tubulin monomers and they can 41 change dynamically between polymerization and de-polymerization states (Conde and Cáceres, 42 2009). The microtubule-associated protein, Tau, binds to microtubules and promotes their 43 stabilization in growing axons (Black et al., 1996; Drubin, 1986). Asparagine endopeptidase 44 (AEP, also known as δ-secretase or legumain) is a lysosomal cysteine protease that cleaves on 45 the C terminal side of asparagine residues. It cleaves Tau at N255 and N368, thereby removing 46 its microtubule-binding domain completely. AEP cleaves Tau in vitro and in human Alzheimer’s 47 disease (AD) brains, abolishing its microtubule stabilizing function, inducing Tau aggregation, 48 and triggering neurodegeneration (Zhang et al., 2014). 49 Another AEP substrate implicated in AD, amyloid precursor protein (APP), is often 50 associated with axon pathfinding (Farah, 2012). It is found in growth cones (Sosa et al., 2013), 51 and contains a receptor for the Slit family of glycoprotein axon guidance molecules on its 52 extracellular domain (Wang et al., 2017). It also has been suggested to play a role as a co- 53 receptor with several other axon guidance partners (Southam et al., 2019). Most of the 54 ectodomain of APP can be cleaved by β-site amyloid precursor protein cleaving enzyme 1 55 (BACE1 or β-secretase), removing its Slit binding site. After peripheral nerve crush, increasing 4 English, A.W. et al 56 BACE1 activity inhibits regeneration (Tallon et al., 2017) while in mice null for BACE1, 57 regeneration is enhanced (Farah et al., 2011). Subsequent to BACE1 cleavage of APP, the C- 58 terminal fragment of APP can be cleaved by Ɣ-secretase to form amyloid-β protein (Aβ). When 59 applied to cultures of cortical neurons, Aβ stimulated growth cone collapse and neurite 60 retraction (Kuboyama, 2018). In the brain, AEP cleaves APP at N585, removing its Slit binding 61 site but also shortening the long extracellular domain of APP, thereby reducing steric hindrance 62 to BACE1 activity and enhancing Aβ generation (Zhang et al., 2015). Thus, increased AEP 63 activity after PNI could inhibit axon regeneration both by interfering with the guidance role of 64 APP and via increased production of Aβ. 65 It is not known whether enzymatically active AEP is found in injured nerves, but if it is, it 66 could impair axon regeneration by its actions on both Tau and APP. If removed or inhibited, 67 AEP could result in enhanced axon regeneration. One goal of this study was to investigate the 68 activity of AEP in a widely used model of peripheral nerve injury and to evaluate axon 69 regeneration in this model in mice in which AEP is knocked out. 70 Brief electrical stimulation (ES) has been used to enhance axon regeneration after 71 peripheral nerve injury (PNI). Its success depends on an increase in signaling by brain derived 72 neurotrophic factor (BDNF) and its TrkB receptor in the regenerating axons (Gordon and 73 English, 2016). Because BDNF-TrkB signaling can drive an Akt-mediated block of AEP activity, 74 both in vitro and in mouse brains (Wang et al., 2018), we hypothesized that the effectiveness of 75 ES in promoting axon regeneration after PNI is the result of AEP inhibition. Thus, a second goal 76 of this study was to evaluate whether ES would enhance axon regeneration in mice null for 77 AEP. 78 5 English, A.W. et al 79 Materials and Methods 80 Experimental Design and Statistical Analysis –When comparing parametric data from 81 more than two groups, a one-way ANOVA was used. If the omnibus result of that analysis was 82 significant (p<0.05), groups were compared in a post-hoc manner using Tukey’s honest 83 significant difference test. For non-parametric analyses (distributions of axon profile lengths), 84 comparisons were made in a pairwise manner using a Kolmogorov-Smirnov two sample test. 85 For time-series data, comparisons were made using multiple linear regression analysis. 86 Significance of all statistical comparisons was set at p<0.05. All statistical comparisons were 87 performed using GraphPad-Prism. 88 Animals – All mice used in both in vivo experiments and as a source of cells for in vitro 89 studies were on a mixed C57BL6-129/Ola background.
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