Differential Effects of UCHL1 Modulation on Alpha- Synuclein in PD-Like Models of Alpha-Synucleinopathy Anna E. Cartier1,5, Kiren Ubhi1, Brian Spencer1, Ruben A. Vazquez-Roque3, Kori Ann Kosberg1, Lawrence Fourgeaud4, Priya Kanayson 1 , Christina Patrick1, Edward Rockenstein1, Gentry N. Patrick5, Eliezer Masliah1,2* 1 Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Pathology, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 3 Departamento de Fisiolog´ıa, Escuela Nacional de Ciencias Biolo´ gicas, Instituto Politecnico Nacional, Mexico City, Me´xico, 4 Molecular Neurobiology Laboratory, The Salk Institute of Biological Studies, La Jolla, California, United States of America, 5 Division of Biological Sciences, Section of Neurobiology, University of California San Diego, La Jolla, California, United States of America Abstract Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH- L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/ aggregated a-syn species in the PD brain. Citation: Cartier AE, Ubhi K, Spencer B, Vazquez-Roque RA, Kosberg KA, et al. (2012) Differential Effects of UCHL1 Modulation on Alpha-Synuclein in PD-Like Models of Alpha-Synucleinopathy. PLoS ONE 7(4): e34713. doi:10.1371/journal.pone.0034713 Editor: Philipp J. Kahle, Hertie Institute for Clinical Brain Research and German Center for Neurodegenerative Diseases, Germany Received October 19, 2011; Accepted March 5, 2012; Published April 13, 2012 Copyright: 2012 Cartier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by National Institutes of Heath grants AG18840, AG5131, AG022074, AG03197, AG010435. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: emasliah@ucsd.edu Introduction Parkinson’s disease (PD) is a neurodegenerative disorder that affects 1–2% of the population over age 60 [1,2]. PD is characterized by progressive accumulation of a-synuclein (a-syn) in cortical and subcortical regions leading to neuronal degener- ation culminating in motor dysfunctions and dementia [3,4]. To date several genes including that encoding a-syn have been conclusively linked to PD [5]. However, the link to ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), remains controversial. UCH-L1 is a multi-functional protein and is highly abundant in the brain [6]. UCH-L1, through its hydrolase activity, is responsible for generating free monomeric ubiquitin from precursor poly-ubiquitin chains [6] and can also associate with mono-ubiquitin to inhibit its degradation and, therefore, stabilize and maintain ubiquitin levels [7]. UCH-L1 also acts as a ligase in vitro, and such ligase activity has been demonstrated towards a-syn [8]. The implied involvement of UCH-L1 in PD is based on the discovery of a I93M mutation in the UCH-L1 gene reported in a German family with autosomal dominant PD [9]. The UCH- L1I93M mutant displays significant decreased hydrolase activity in vitro, implying that loss of UCH-L1 function may contribute to a decrease in the availability of free ubiquitin, and an impaired clearance of proteins by the Ubiquitin Proteasome System (UPS). Furthermore, transgenic mice that over express the I93M mutant, show behavioral and pathological deficits characteristic of Parkinsonism [10]. A previous report demonstrated that UCH- L1 is localized to synaptic vesicles, and co-immunoprecipitates with a-syn from rat brains [8]. a-syn is enriched in pre-synaptic terminals, and is potentially involved in synaptic plasticity and neurotransmitter release [11]. Moreover, a-syn is a major component of pathogenic insoluble proteinaceous deposits known as Lewy bodies (LBs) commonly found in PD brains [12]. In vitro, UCH-L1 was found to possess a dimerization-dependent ligase activity responsible for K63-linked polyubiquitination of a-syn [8], which may potentially block its degradation and lead to its accumulation and aggregation within neurons. Interestingly, a polymorphism in the UCH-L1 gene (S18Y), was found to be associated with a lower risk of developing PD, has a significantly reduced ligase activity leading to reduced levels of ubiquitinated a- syn [8,13,14]. However, more recent studies have failed to find significant association between S18Y polymorphism and reduced PD risk [15,16]. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34713