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Neuronal Preconditioning Requires the Mitophagic Activity of C-Terminus of HSC70-Interacting Protein

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Neuronal Preconditioning Requires the Mitophagic Activity of C-Terminus of HSC70-Interacting Protein This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular Neuronal Preconditioning Requires the Mitophagic Activity of C-terminus of HSC70-Interacting Protein Britney N. Lizamaa,b, Amy M. Palubinskya,b, Vineeth A. Raveendranc, Annah M. Moored, Joel D. Federspiele, Simona G. Codreanue, Daniel C. Lieblere and BethAnn McLaughlinb,f,g aNeuroscience Graduate Group bVanderbilt Brain Institute cVanderbilt International Summer Research Academy dVanderbilt Interdisciplinary Graduate Program eDepartments of Biochemistry fNeurology gPharmacology, Vanderbilt University Medical Center, 465 21st Ave S MRB III, Nashville, TN, 37240 DOI: 10.1523/JNEUROSCI.0699-18.2018 Received: 13 March 2018 Revised: 8 June 2018 Accepted: 13 June 2018 Published: 22 June 2018 Author contributions: B.N.L., A.M.P., and B.M. designed research; B.N.L., A.M.P., V.R., A.M., J.F., S.G.C., and D.C.L. performed research; B.N.L., A.M.P., J.F., S.G.C., and B.M. analyzed data; B.N.L. wrote the first draft of the paper; B.N.L., A.M.P., V.R., A.M., J.F., S.G.C., D.C.L., and B.M. edited the paper; B.N.L. and B.M. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. The authors thank Dr. Cam Patterson for providing the original CHIP knockout animals; Ms. Sharon Klein, Ms. Arulita Gupta, and Ms. Dominique Szymkiewicz for technical assistance and ImageJ expertise; Dr. Joshua Fessel and Dr. Alice Soragni for electron microscopy expertise; Dr. Christopher E. Wright and Mr. Jeff Duryea for imaging assistance; and Ms. Ama J. Winland for cell culture maintenance. We also thank the Vanderbilt Cell Imaging Shared Resource for transmission electron microscopy preparation and imaging. This work was supported by the Walter and Suzanne Scott Foundation funding of the J.B. Marshall Laboratory (B.M, A.M.P., B.N.L.), the Dan Marino Foundation (B.M., A.M.P.), NIH grants NS050396 (B.M.) and RO1ES022936 (B.M., D.C.L.), a Vanderbilt Brain Institute Scholarship (B.N.L., A.M.P.), a VISRA Program Scholarship (V.R.), and predoctoral fellowships from the AHA 15PRE25100000 (A.M.P.), 14PRE2003500007 (B.N.L.). The authors declare no conflict of interest. Corresponding author: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0699-18.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors ͳ Neuronal Preconditioning Requires the Mitophagic Activity of C-terminus of HSC70- ʹ Interacting Protein ͵ Ͷ Britney N. Lizamaa,b, Amy M. Palubinskya,b, Vineeth A. Raveendranc, Annah M. Moored, Joel D. Federspiele, ͷ Simona G. Codreanue, Daniel C. Lieblere, & BethAnn McLaughlin*b,f,g ͸ ͹ aNeuroscience Graduate Group, bVanderbilt Brain Institute, cVanderbilt International Summer Research Academy, ͺ dVanderbilt Interdisciplinary Graduate Program, Departments of eBiochemistry, fNeurology and gPharmacology, ͻ Vanderbilt University Medical Center, 465 21st Ave S MRB III, Nashville, TN, 37240 *Corresponding author: ͳͲ [email protected] ͳͳ ͳʹ Abstract: ͳ͵ C-terminus of HSC70-interacting protein (CHIP, STUB1) is a ubiquitously expressed cytosolic E3-ubiquitin ͳͶ ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction prior to premature death. This ͳͷ phenotype is more consistent with animal models in which master regulators of autophagy are affected rather ͳ͸ than with the mild phenotype of classic E3-ubiquitin ligase mutants. The cellular and biochemical events that ͳ͹ contribute to neurodegeneration and premature aging in CHIP KO models remain poorly understood. ͳͺ Electron and fluorescent microscopy demonstrates that CHIP deficiency is associated with greater numbers of ͳͻ mitochondria, but these organelles are swollen and misshapen. Acute bioenergetic stress triggers CHIP ʹͲ induction and re-localization to mitochondria where it plays a role in the removal of damaged organelles. This ʹͳ mitochondrial clearance is required for protection following low-level bioenergetic stress in neurons. CHIP ʹʹ expression overlaps with stabilization of the redox stress sensor PTEN-inducible kinase 1 (PINK1) and is ʹ͵ associated with increased LC3-mediated mitophagy. Introducing human promoter-driven vectors with ʹͶ mutations in either the E3 ligase or TPR domains of CHIP in primary neurons derived from CHIP-null animals ʹͷ enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests the increase in ʹ͸ mitochondrial CHIP is likely due to diminished clearance of these CHIP-tagged organelles. Proteomic analysis ʹ͹ of WT and CHIP KO mouse brains (4 male, 4 female per genotype) reveals proteins essential for maintaining ʹͺ energetic, redox and mitochondrial homeostasis undergo significant genotype-dependent expression changes. ʹͻ Together these data support the use of CHIP deficient animals as a predictive model of age-related ͵Ͳ degeneration with selective neuronal proteotoxicity and mitochondrial failure. ͵ͳ ͵ʹ Significance Statement: Mitochondria are recognized as central determinants of neuronal function and ͵͵ survival. We demonstrate that C-terminus of HSC70-Interacting Protein (CHIP) is critical for neuronal ͵Ͷ responses to stress. CHIP upregulation and localization to mitochondria is required for mitochondrial ͵ͷ autophagy (mitophagy). Unlike other disease-associated E3 ligases such as Parkin and Mahogunin, CHIP ͵͸ controls homeostatic and stress-induced removal of mitochondria. While CHIP deletion results in greater ͵͹ numbers of mitochondria, these organelles have distorted inner membranes without clear cristae. Neuronal ͵ͺ cultures derived from animals lacking CHIP are more vulnerable to acute injuries, and transient loss of CHIP ͵ͻ renders neurons incapable of mounting a protective response following low-level stress. Together these data ͶͲ suggest that CHIP is an essential regulator of mitochondrial number, cell signaling and survival. Ͷͳ Ͷʹ Ͷ͵ Introduction Ͷͺ Garza, 2012). HSP70 binding partners alter the ͶͶ The HSP70 complex is capable of blocking Ͷͻ localization, activity and expression of the Ͷͷ neurodegeneration triggered by a host of genetic ͷͲ chaperone complex itself and its client proteins. E3 Ͷ͸ mutations, physiological events and environmental ͷͳ ligases, such as Parkin and C-terminus of HSC70- Ͷ͹ stressors (Franklin et al., 2005; Gestwicki and ͷʹ Interacting Protein (CHIP), regulate HSP70- ͳ ͷ͵ mediated protein degradation (Ballinger et al., ͳͲͲ In this work, we sought to understand the ͷͶ 1999; Jiang et al., 2001). CHIP is a 303 amino acid ͳͲͳ underpinnings of the devastating phenotype ͷͷ cytosolic protein that contains an N-terminal ͳͲʹ caused by loss of CHIP by evaluating its role in cell ͷ͸ tetratricopeptide repeat (TPR) domain regulating ͳͲ͵ signaling using neuronal models of acute ͷ͹ HSP/HSC70-docking, a helix-loop-helix domain, ͳͲͶ pathophysiological stress. Using a combination of ͷͺ and a C-terminal Ubox domain that is necessary for ͳͲͷ cellular imaging, proteomics, biochemistry, ͷͻ binding to E2-conjugating enzymes (Ballinger et al., ͳͲ͸ molecular targeting and ultrastructural assays, we ͸Ͳ 1999; Jiang et al., 2001; Xu et al., 2008). CHIP is ͳͲ͹ determined that CHIP deficiency results in radical ͸ͳ unique among E3 ligases in that its expression is ͳͲͺ mitochondrial reorganization not seen in other E3 ͸ʹ induced by acute bioenergetic stress and in post- ͳͲͻ ligase deficient animals. These data reinforce a ͸͵ mortem samples from patients with stroke ͳͳͲ model in which CHIP functions are more in ͸Ͷ (Stankowski et al., 2011; Lizama et al., 2017). CHIP ͳͳͳ keeping with a role as an essential autophagy ͸ͷ is also unique in that it forms asymmetric ͳͳʹ regulator than a standard E3 ligase. Taken together, ͸͸ homodimers, which have not been observed in ͳͳ͵ our data supports the use of these animals as a ͸͹ other E3 ligase proteins and are only formed in ͳͳͶ powerful and robust model of neurological ͸ͺ higher vertebrates, suggesting it may perform ͳͳͷ dysfunction induced by mitochondrial failure and ͸ͻ distinctive roles in cell biology (Zhang et al., 2005; ͳͳ͸ secondary proteotoxicity. ͹Ͳ Ye et al., 2017). ͳͳ͹ ͹ͳ Further evidence of the uniqueness of CHIP can ͳͳͺ Experimental Methods ͹ʹ be found in studies demonstrating that mice ͳͳͻ Materials and Reagents ͹͵ deficient in CHIP have pervasive dysfunction far ͳʹͲ Tissue culture: Fetal bovine serum (SH30070.03) ͹Ͷ more robust than would be predicted based on a ͳʹͳ was obtained from Hyclone. Dulbecco’s modified ͹ͷ primary role as a conventional E3 ligase. CHIP ͳʹʹ Eagle’s medium (DMEM, 11995) with high glucose, ͹͸ insufficiency results in decreased life expectancy ͳʹ͵ minimum essential medium (MEM, 51200), ͹͹ and profound lipid oxidation, as well as poor ͳʹͶ Neurobasal medium, B27 supplement (17504044), ͹ͺ performance on behavioral assessments of motor ͳʹͷ N2 supplement (17502048), 0.25% Trypsin–EDTA, ͹ͻ function and anxiety (McLaughlin et al., 2012; ͳʹ͸ trypan blue stain 0.4%, and penicillin-streptomycin ͺͲ Palubinsky et al., 2015). Notably, CHIP knockout ͳʹ͹ (15140-122) were purchased from Invitrogen. ͺͳ (KO) animals are unable to complete even simple ͳʹͺ LipoJet In Vitro Transfection Kit (Ver. II) was ͺʹ behavioral assessments given their moribund ͳʹͻ purchased from SignaGen. ͺ͵ nature. ͳ͵Ͳ Western blotting: XT-MOPS running buffer, ͺͶ Mutations in CHIP have been identified in ͳ͵ͳ Tris-Glycine transfer buffer, Criterion Bis-Tris gels, ͺͷ patients with an early-onset, recessive form of ͳ͵ʹ Laemmli buffer, and precision plus protein all blue ͺ͸ spinocerebellar ataxia, in which mutations in major ͳ͵͵ standards were purchased from Bio-Rad ͺ͹ structural domains confer loss of CHIP function ͳ͵Ͷ Laboratories. Membrane-blocking solution was ͺͺ (Heimdal et al., 2014; Shi et al., 2014; Synofzik et al., ͳ͵ͷ from Zymed. Hybond P polyvinylidene difluoride ͺͻ 2014; Bettencourt et al., 2015). Patients exhibit some ͳ͵͸ membranes were acquired from GE Healthcare.
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