The 2018 Mark A. Smith Prize Winner

CONGRATULATIONS TO

for his paper Synaptic activity induces input-specific rearrangements in a targeted

synaptic protein interaction network

Jonathan D. Lautz

The 2017 Mark A. Smith Prize Winner

for his paper An isogenic blood-brain barrier model comprising brain endothelial cells,

astrocytes, and neurons derived from human induced pluripotent stem cells

Scott Glen CanfieldCONGRATULATIONS TO

19 - RM000526

Find out more about the Prize and past Awardees

onlinelibrary.wiley.com/page/ journal/14714159/homepage/ mark_a_smith_prize.htm

MORE ABOUT THE PRIZEThe Mark A. Smith Prize recognizes the contribution of an outstanding young scientist to an exceptional research paper published in the Journal of Neurochemistry.

The Editors’ Award, instigated in 2009, was renamed Mark A. Smith prize to pay tribute to Mark’s long service to the Journal as a Handling Editor and Deputy Chief Editor. From 2019 onwards, any first author less than 8 years post PhD will qualify.

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The Mark A. Smith Prize 2015 / 2016 AwardeesThe deubiquitinating enzyme USP46 regulates AMPA receptor ubiquitination and traffickingYuda Huo, Natasha Khatri, Qingming Hou, James Gilbert, Guan Wang, Heng-Ye ManDepartment of Biology, Boston University, Boston, Massachusetts, USADepartment of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts, USAEmail: hman@bu.edu

Abstract

Background

Hypothesis

Summary

References

Regulation of AMPAR accumulation at synapse 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 neurodegenera-tive disease as well ( Huganir & 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, internaliza-tion and recycling (Malinow & Malenka, 2002; Newpher & Ehlers, 2008; Song & Huganir 2002; Wang, Gilbert, & Man, 2012). In addition to re-ceptor translocation, a change in synaptic receptor level can result from a change in AMPAR degradation and synthesis rate, or a rebalance between these two processes (A. Lin et al., 2011; Schwarz, Hall, & Patrick, 2010).Ubiquitination on certain proteins has been shown to regulate neuronal development and synaptic plasticity (Speese, Trotta, Rodesch, Aravamu-dan, & Broadie, 2003; Yi & Ehler, 2007; Zhao, Hedge, & Marin, 2003). Among membrane proteins including neurotransmitter receptors, ubi-quitination serves as a major signal to trigger endocytosis and direct the internalized protein to lysosome and/or proteasome for degradation (A. Lin et al., 2011; A. W. Lin & Man, 2013; Schwarz et al., 2010). Our previous study has shown that AMPARs are subject to Nedd4-media-ted ubiquitination, leading to a reduction in cell-surface receptor ex-pression and suppressed synaptic transmission (A. Lin et al., 2011; A. Lin & 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 rever-sible process mediated via the addition of ubiquitin by E3 ligases and the removal of ubiquitin meidties via deubiquitinating enzymes (DUB) (Nijman et al. 2005; Komander et al. 2009; Reyes-Turcu et al. 2009). However, the deubiquitination of AMPARs remains largely unknown.

AMPAR trafficking plays a crucial role for the expression of synaptic plasticity. Our previous work has shown that AMPARs are subject to protein ubiquitination leading to receptor internalization and degra-dation. However, whether ubiquitination is the final stage of AMPAR turnover remains unclear. We hypothesize that the ubiquitin chain on AMPARs could be removed by deubiquitinase (DUB) to counterba-lance receptor endocytosis and bypass the proteasomal degradation.

Huganir R. L. and Nicoll R. A. (2013) AMPARs and synaptic plasticity:the last 25 years. Neuron 80, 704–717. doi: 10.1016/j.neuron.2013.10.025.Komander D., Clague M. J. and Urbe S. (2009) Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550-563. doi: 10.1155/2012/892749.Lin A., Hou Q., Jarzylo L., Amato S., Gilbert J., Shang F. and Man H. Y. (2011) Nedd4-mediated AMPA recep-tor ubiquitination regulates receptor turnover and trafficking. J. Neurochem. 119, 27-39. doi:10.1111/j.1471-4159.2011.07221.x.Lin A. W. and Man H. Y. (2013) Ubiquitination of neurotransmitter receptors and postsynaptic scaffolding prote-ins. Neural. Plast. 2013, 432057. doi: 10.1155/2013/432057.Lin A. and Man H. Y. (2014) Endocytic adaptor epidermal growth factor receptor substrate 15(Eps15) is involved in the trafficking of ubiquitinated alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors. J. Biol. Chem. 289. 24652-24664. doi: 10.1074/jbc.M114.582114.Malinow R. and Malenka R. C. (2002) AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103-126. doi: 10.1146/annurev. Neuro. 25.112701.142758.Newpher T. M. and Ehlers M. D. (2008) Glutamate receptor dynamics in dendritic microdomains. Neuron. 58, 472-497. doi: 10.1016/j.neuron.2008.04.030.Nijman S. M., Luna-Vargas M. P., Velds A., Brummelkamp T.R., Dirac A. M., Sixma T. K. and Bernards R. (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773-786. doi: 10.1016/j.cell.2005.11.007.Schwarz L. A., Hall B. J. and Patrick G. N. (2010) Activity-dependent ubiquitination of GluA1 mediates a dis-tinct AMPA receptor endocytosis and sorting pathway. J. Neurosci. 30, 16718-16729. doi: 10.1523/JNEUROS-CI.3686-10.2010.Song I. and Huganir R. L. (2002) Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 25, 578-588.Speese S. D., Trotta N., Rodesch C. K., Aravamudan B. and Broadie K. (2003) The ubiquitin proteasome system actutely regulates presynaptic protein turnover and synaptic efficacy. Curr. Biol. 13, 899-910.Wang G., Gilbert J. and Man H. Y. (2012) AMPA receptor trafficking in homeostatic synaptic plasticity: functio-nal molecules and signaling cascades. Neural Plast. 2012, 825464. doi: 10.1155/2012/825364.Yi J. J. and Ehlers M. D. (2007) Emerging roles for ubiquitin and protein degradation in neuronal function. Phar-macol. Rev. 59, 14-39. doi: 10.1124/pr.59.1.4.Zhao Y., Hegde A. N. and Martin K. C. (2003) The ubiquitin proteasome system fucntions as an inhibitory cons-traint on synaptic strengthening. Curr. Biol. 13, 887-898.Reyes-Turcu F. E., Ventii K. H. and Wikinson K. D. (2009) Regulation and cellular roles of ubiquitin-specific deu-

Alpha-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) are the primary mediators for inter-neuronal communication and play a crucial role in higher brain functions including learning and me-mory. Our previous work demonstrated that AMPARs are subject to ubiquitination by the E3 ligase Nedd4, re-sulting in EPS15-mediated receptor internalization and Ubiquitin (Ub)-proteasome pathway (UPP)-dependent degradation. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. However, deubiquitination of mammalian AMPARs and the responsible deubiquitinating enzymes (DUBs) remain elusive. In this study, we identify a DUB, USP46, which specifically removes ubiquitination modification from AMPARs. USP46 is enriched at the synapse and co-localizes with sy-naptic marker proteins. In heterologous cells and neurons, expression of USP46 results in a significant reduction in AMPAR ubiquitination, accompanied with a reduced rate in AMPAR degradation and an increase in AM-PAR synaptic accumulation. By contrast, knockdown of USP46 by RNAi leads to elevated AMPAR ubiquitination and a reduction in AMPAR protein amount in neurons. Consistently, mEPSC recordings show reduced synaptic strength in neurons expressing USP46-selective RNAi. These results demonstrate USP46-mediated regulation of AMPAR ubiquitination and turnover, which may play an important role in synaptic plasticity and brain function.

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FIGURE 1. UPS46 synaptic distribution and developme.ntal expression.

FIGURE 3. USP46 deubiquitinates GluA1 in vivo and in vitro.

FIGURE 4. Knock down of USP46 enhances AMPAR ubiquitination.

A and B. Double staining of USP46 (Red) and postsynaptic protein PSD-95 (Green) or GluA1 (Green) in cultured DIV14 hippocampal neu-rons. The selected region was enlarged for cla-rity (right). Arrowheads indicate co-localized puncta. Scale bar represents 20 μm. C. USP46 and other proteins including Nedd4, PSD-95 and GluA1 are enriched in the synaptosomal P2 fraction purified from adult rat hippocam-pal tissue. In contrast, Akt is mainly distribut-ed in the solubilized fraction. The same amount of protein (3 μg) was loaded for each lane. D. USP46 expression in different brain regions. USP46 is relatively higher in prefrontal cortex (PFC). The AMPAR E3 ligase Nedd4 and tubu-lin was also probed. E. USP46 expression time course was measured from cultured cortical neurons at varied days in vitro (DIV) after pla-ting. USP46 amounts peaked in the 2nd and 3rd weeks. GluA1 and tubulin were also probed.

A and B. HEK cells were co-transfected with HA-ubiquitin (HA-Ubi), GFP-GluA1 together with either USP46 or USP12. GFP-GluA1 iso-lated by immunoprecipitation was probed for HA-ubiquitin. Ubiquitin signals (HA-Ubi) were reduced in cells expressing USP46, but not USP12. The same western blot was re-pro-bed with GluA1 antibody to confirm the im-munoprecipitation. Tubulin from cell lysates was probed to indicate equal amount of lysa-tes (N = 3 independent experiments). Bar gra-phes represent Mean ± SEM, *p