© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs247841. doi:10.1242/jcs.247841 RESEARCH ARTICLE TrkB deubiquitylation by USP8 regulates receptor levels and BDNF-dependent neuronal differentiation Carlos Martın-Rodŕ ıgueź 1,2,*, Minseok Song3, Begoña Anta1,2, Francisco J. González-Calvo1, Rubén Deogracias1, Deqiang Jing4, Francis S. Lee4 and Juan Carlos Arevalo1,2,*,‡ ABSTRACT (RTK) family, initiate downstream signaling pathways in the Ubiquitylation of receptor tyrosine kinases (RTKs) regulates both the response to neurotrophin binding. Then, they are internalized and levels and functions of these receptors. The neurotrophin receptor localized to different subcellular compartments. Regulation of these TrkB (also known as NTRK2), a RTK, is ubiquitylated upon activation trafficking processes has been known to be coupled to Trk receptor by brain-derived neurotrophic factor (BDNF) binding. Although TrkB signaling specificity and duration. ubiquitylation has been demonstrated, there is a lack of knowledge Upon ligand binding, Trk receptors undergo various post- regarding the precise repertoire of proteins that regulates TrkB translational modifications (PTMs). Most notably, tyrosine ubiquitylation. Here, we provide mechanistic evidence indicating that phosphorylation generates binding sites for proteins that enable the ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF- recruitment of downstream signaling components (Arevalo and Wu, and TrkB-dependent neuronal differentiation. USP8 binds to the 2006). Another relevant PTM for Trk receptors is ubiquitylation, a C-terminus of TrkB using its microtubule-interacting domain (MIT). dynamic and reversible process in which ubiquitin is covalently Immunopurified USP8 deubiquitylates TrkB in vitro,whereas bound to the substrate by E3 ubiquitin ligases and can be knockdown of USP8 results in enhanced ubiquitylation of TrkB upon subsequently removed by deubiquitylases (DUBs). The most BDNF treatment in neurons. As a consequence of USP8 depletion, studied neurotrophin receptor related to ubiquitylation is TrkA, TrkB levels and its activation are reduced. Moreover, USP8 protein which is ubiquitylated by different E3 ubiquitin ligases including regulates the differentiation and correct BDNF-dependent dendritic Nedd4-2 (also known as NEDD4L), TRAF6, c-Cbl, and Cbl-b formation of hippocampal neurons in vitro and in vivo.Weconcludethat (Arevalo et al., 2006; Geetha et al., 2005; Georgieva et al., 2011; USP8 positively regulates the levels and activation of TrkB, modulating Takahashi et al., 2011). In addition, USP8 deubiquitylates TrkA, and BDNF-dependent neuronal differentiation. USP36 binding to Nedd4-2 affects TrkA ubiquitylation (Anta et al., 2016; Ceriani et al., 2015). TrkA ubiquitylation influences receptor This article has an associated First Person interview with the first stability, signaling and subcellular localization playing a pivotal role author of the paper. in the regulation of NGF/TrkA-mediated physiological functions, such as differentiation, survival and nociception (Arevalo et al., 2006; KEY WORDS: TrkB, NTRK2, USP8, BDNF, Neuron, Differentiation, Geetha et al., 2005; Georgieva et al., 2011; Makkerh et al., 2005; Signaling Sanchez-Sanchez and Arevalo, 2017; Yu et al., 2014, 2011). However, very little is known about TrkB and TrkC ubiquitylation. INTRODUCTION It has been reported that c-Cbl E3 ubiquitin ligase can ubiquitylate Neurotrophins are a family of secreted proteins that regulate nervous TrkB (Pandya et al., 2014) and that Ndfip1, an adaptor protein for system development and function. Four members have been HECT E3 ubiquitin ligases, mediates ubiquitylation of TrkB receptor identified in vertebrates: nerve growth factor (NGF), brain-derived and degradation (Murray et al., 2019). In addition, UCH-L1 neurotrophic factor (BDNF), neurotrophin 3 (NT3, also known as deubiquitylates TrkB promoting TrkB-dependent contextual fear NTF3) and neurotrophin 4 (NT4 or NTF4). Each neurotrophin binds conditioning learning and memory (Guo et al., 2017). Further specifically to the tropomyosin receptor kinases (Trks), NGF to research is required to identify new proteins involved in TrkB TrkA (NTRK1), BDNF and NT4 to TrkB (NTRK2), and NT3 to ubiquitylation and deubiquitylation that may regulate BDNF/TrkB- TrkC (NTRK3) (Chao et al., 2006; Huang and Reichardt, 2003). Trk mediated functions. neurotrophin receptors, which belong to the receptor tyrosine kinase USP8 is a DUB directly related with endosomal trafficking of RTKs. It mediates its functions through regulation of components of endosomal sorting complex required for transport (ESCRT)-0 (Row 1Departmento de Biologıá Celular y Patologıa,́ Instituto de Neurociencias de Castilla y León, University of Salamanca, Salamanca 37007, Spain. 2Institute of et al., 2006; Mizuno et al., 2006; Berlin et al., 2010b; Zhang et al., Biomedical Research of Salamanca, 37007 Salamanca, Spain. 3Department of Life 2014) and of ESCRT-III (Row et al., 2007; Crespo-Yanez et al., Sciences, Yeungnam University, Gyeongsan, Gyeongbuk 38541, Korea. 2018). Importantly, USP8 controls the stability of numerous 4Department of Psychiatry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA. proteins including epidermal growth factor receptor (Mizuno *Present address: Department of Biochemistry and Molecular Biology, Faculty of et al., 2005, 2006; Row et al., 2007), hepatocyte growth factor Pharmacy, Complutense University of Madrid, Madrid, Spain. receptor (Row et al., 2006; Niendorf et al., 2007), ErbB3 (Wu et al., ‡Author for correspondence ([email protected]) 2004), ENaC (Zhou et al., 2013), CXCR4 (Berlin et al., 2010a), KCA3.1 (Balut et al., 2011), connexin 43 (Sun et al., 2018) and C.M.-R., 0000-0002-9621-2133; M.S., 0000-0001-9561-8505; R.D., 0000-0002- SHANK3 (Kerrisk Campbell and Sheng, 2018). In addition to this 9279-7867; J.C.A., 0000-0003-1994-3095 canonical function, recent studies suggest that USP8 plays an Handling Editor: Giampietro Schiavo important role in cell cycle progression, apoptosis and genomic Received 18 April 2020; Accepted 13 November 2020 integrity (for a review see Dufner and Knobeloch, 2019). The Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs247841. doi:10.1242/jcs.247841 relevance of USP8 functions comes from studies with knockout To assess whether these in vitro results can be extrapolated to what (KO) mice, which are early embryonically lethal (Niendorf et al., happens in neurons, we evaluated whether the ubiquitylation of TrkB 2007) and its involvement in Cushing’s syndrome (CS) (Ma et al., in response to BDNF is affected by USP8 in cultured cortical neurons. 2015; Reincke et al., 2015). Although numerous substrates have We employed a shRNA strategy to knock down the expression of been identified for USP8, new ones still need to be identified to USP8 using different lentiviruses expressing shRNAs against USP8 explain all its mechanistically known functions. (Fig. S2). At 6 days after transduction, neurons were stimulated with Here, we present evidence that USP8 and TrkB interact using its BDNF for 15 min, lysed and TrkB was immunoprecipitated. We found MIT and C-terminal domain, respectively. USP8 deubiquitylates that TrkB ubiquitylation was enhanced when USP8 expression was TrkB regulating the receptor levels and activation impacting on reduced with two different shRNAs (Fig. 2C,D). Quantification of downstream signaling pathways dependent on BDNF/TrkB. these experiments resulted in a significant increase in TrkB Moreover, USP8 depletion impairs differentiation of cultured ubiquitylation (Fig. 2E). Together, these results indicate that USP8 neurons and BDNF-dependent dendritic formation of hippocampal deubiquitylates TrkB in vitro and in cortical neurons. neurons in vitro and in vivo. Our study identifies USP8 as a key regulator for TrkB ubiquitylation in the CNS. USP8 protein regulates degradation and TrkB receptor levels USP8 regulates the trafficking and stability of different RTKs, RESULTS altering their ubiquitylation (Berlin et al., 2010b; Niendorf et al., USP8 interacts with TrkB receptors 2007). To investigate whether USP8 controls TrkB levels, we In a previous screen looking for deubiquitinases for TrkA, we examined the degradation rate of TrkB neurotrophin receptors. reported that USP8 altered TrkA activation (Anta et al., 2016) and, Surface proteins from cortical neurons infected with control or independently, another group reported that USP8 acts as a DUB for USP8 shRNA-expressing lentiviruses were labeled using standard TrkA (Ceriani et al., 2015). To address whether USP8 interacts biotinylation assays followed by stimulations with BDNF (25 ng/ with other Trk neurotrophin receptors, co-immunoprecipitation ml) for 60 and 120 min. Degradation of surface-labeled TrkB was experiments were performed in HEK293 transfected cells. An analyzed by immunoblot analysis (Fig. 3A). Interestingly, we interaction with all Trk neurotrophin receptors was observed but, observed a transiently enhanced TrkB degradation upon USP8 strikingly, a strong association was detected with TrkB (Fig. 1A), but knockdown at 60 min compared to that in control neurons (Fig. 3B). not with the p75 neurotrophin receptor (NGFR) (Fig. S1). To The pattern of activated TrkB (phosphorylated; denoted pTrk) determine whether TrkB and USP8 interact endogenously, we carried resembles the one of the total TrkB. These data suggest that USP8