The deubiquitinating enzyme USP46 regulates AMPA receptor ubiquitination and trafficking.

Regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) accumulation at synapses is the major molecular mechanism underlying the expression of synaptic plasticity, a process contributing to the execution of higher brain functions including learning and memory, and leading to neurological disorders and neurodegenerative diseases as well (Huganir and Nicoll 2013). The amount of functional AMPARs can be regulated by rapid trafficking of receptors to and from the synaptic surface via vesicle-mediated membrane insertion, internalization, and recycling (Malinow and Malenka 2002; Song and Huganir 2002; Newpher and Ehlers 2008; Wang et al. 2012). In addition to receptor translocation, a change in synaptic receptor levels can result from a change in AMPAR degradation and rate of synthesis, or a rebalance between these two processes (Schwarz et al. 2010; Lin et al. 2011). Ubiquitination on certain proteins has been shown to regulate neuronal development and synaptic plasticity (Speese et al. 2003; Zhao et al. 2003; Yi and Ehlers 2007). Among membrane proteins, including neurotransmitter receptors, ubiquitination serves as a major signal to trigger endocytosis and direct the internalized protein to the lysosome and/or proteasome for degradation (Schwarz et al. 2010; Lin et al. 2011; Lin and Man 2013). Our previous study has shown that AMPARs are subject to Nedd4-mediated ubiquitination, leading to a reduction in cell-surface receptor expression and suppressed synaptic transmission (Lin et al. 2011; Lin and Man 2014). Additionally, a recent study has demonstrated that all AMPAR subunits are subject to ubiquitination, which is regulated by neuronal activity (Widagdo et al. 2015). Ubiquitination is a reversible process mediated via the addition of ubiquitin by E3 ligases and the removal of ubiquitin moieties via deubiquitinating enzymes (DUB) (Nijman et al. 2005; Komander et al. 2009; Reyes-Turcu et al. 2009). In the human genome, there are an estimated 500–600 E3 ligases and less than one hundred DUBs identified thus far, suggesting the specification of DUBs on substrate selection (Komander et al. 2009; Kowalski and Juo 2012). DUBs are categorized into five families based on catalytic domains, including ubiquitin-specific proteases (USPs), ubiquitin c-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephins and JAB1/MPN/MOV34 metalloenzymes (JAMMs) (Komander et al. 2009). Among them, the ubiquitin c-terminal hydrolases, ubiquitin-specific proteases, OTU and Josephin families are Cys proteases and JAMM family members are zinc metallo-proteases. However, in mammalian neurons, the cellular process and molecular components involved in AMPAR deubiquitination remain largely unknown. In this study, we identify USP46 as the DUB specific for AMPARs. We find that USP46 is enriched at the synapse and co-localizes with synaptic marker proteins. Expression of USP46 results in a significant reduction in AMPAR ubiquitination, accompanied by a decreased rate in AMPAR degradation and an increase in AMPAR synaptic accumulation. By contrast, knockdown of USP46 by shRNA leads to elevated AMPAR ubiquitination and a reduction in AMPAR protein amount in neurons. In line with changes in AMPAR synaptic localization, electrophysiological recordings show reduced miniature excitatory postsynaptic currents (mEPSCs) amplitude in neurons transfected with USP46 shRNA, indicating an important role for dynamic regulation in AMPAR ubiquitination in neuronal communication and brain function.