Angiogenin in Parkinson Disease Models: Role of Akt Phosphorylation and Evaluation of AAV-Mediated Angiogenin Expression in MPTP Treated Mice Trent U. Steidinger 1 , Sunny R. Slone 2 , Huiping Ding 2 , David G. Standaert 2 , Talene A. Yacoubian 2 * 1 Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 2 Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America Abstract The angiogenic factor, angiogenin, has been recently linked to both Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). We have recently shown that endogenous angiogenin levels are dramatically reduced in an alpha-synuclein mouse model of PD and that exogenous angiogenin protects against cell loss in neurotoxin-based cellular models of PD. Here, we extend our studies to examine whether activation of the prosurvival Akt pathway is required for angiogenin’s neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+), as observed in ALS models, and to test the effect of virally-mediated overexpression of angiogenin in an in vivo PD model. Using a dominant negative Akt construct, we demonstrate that inhibition of the Akt pathway does not reduce the protective effect of angiogenin against MPP+ toxicity in the dopaminergic SH-SY5Y cell line. Furthermore, an ALS-associated mutant of angiogenin, K40I, which fails to induce Akt phosphorylation, was similar to wildtype angiogenin in protection against MPP+. These results confirm previous work showing neuroprotective effects of angiogenin against MPP+, and indicate that Akt is not required for this protective effect. We also investigated whether adeno-associated viral serotype 2 (AAV2)-mediated overexpression of angiogenin protects against dopaminergic neuron loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. We found that angiogenin overexpression using this approach does not reduce the MPTP-induced degeneration of dopaminergic cells in the substantia nigra, nor limit the depletion of dopamine and its metabolites in the striatum. Together, these findings extend the evidence for protective effects of angiogenin in vitro, but also suggest that further study of in vivo models is required to translate these effects into meaningful therapies. Citation: Steidinger TU, Slone SR, Ding H, Standaert DG, Yacoubian TA (2013) Angiogenin in Parkinson Disease Models: Role of Akt Phosphorylation and Evaluation of AAV-Mediated Angiogenin Expression in MPTP Treated Mice. PLoS ONE 8(2): e56092. doi:10.1371/journal.pone.0056092 Editor: Malu ´ G. Tansey, Emory University, United States of America Received October 10, 2012; Accepted January 8, 2013; Published February 7, 2013 Copyright: ß 2013 Steidinger 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 supported by a National Institutes of Health - National Institute of Neurological Disorders and Stroke K08 (NS060948), by the American Parkinson Disease Association, and by the Parkinson’s Association of Alabama. 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: tyacoub@uab.edu Introduction The potent angiogenic factor, angiogenin, has recently been associated with neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD) [1,2]. Angiogenin was first linked to ALS through genetic studies that revealed the association of certain angiogenin mutations with both sporadic and familial forms of ALS [1,2,3,4,5,6,7,8,9]. Wildtype angiogenin has been shown to reduce motoneuron cell death in response to hypoxia, serum deprivation, ER stress, and excitotox- icity, while mutant forms of angiogenin associated with ALS fail to reduce toxicity in these models [10,11,12]. Angiogenin treatment delays motor dysfunction and motor neuron loss, and prolongs survival in the superoxide dismutase 1 (SOD1) mouse model of ALS [10]. More recently, angiogenin has been linked to PD. Our lab previously demonstrated a robust down-regulation of angio- genin expression in transgenic mice overexpressing human alpha- synuclein, a mouse model of PD [13,14]. We also demonstrated that exogenous angiogenin reduced toxicity by rotenone and 1- methyl-4-phenylpyridine (MPP+) in neuroblastoma cell lines [14]. Just recently two genetic screens showed several angiogenin variants to be associated with PD [2,15]. How angiogenin may promote cell survival in neurodegener- ation is not well understood. Angiogenin has been identified to induce several signaling pathways associated with survival and cellular maintenance [10,16,17,18]. In endothelial and smooth muscle cells, angiogenin is linked to the induction of several pathways including stress-associated protein kinase/c-Jun N- terminal kinase (SAPK/JNK), phospholipase C (PLC), extracellu- lar signal-related kinase 1/2 (ERK1/2), and Akt [19,20,21,22]. Angiogenin is also known to localize to the nucleus where it induces rRNA translation and pro-survival protein expression [17,23]. Activation of the PI3K/Akt pathway has been linked to angiogenin’s neuroprotective effects in ALS models. In motoneu- rons and in the SOD1 ALS mouse model, angiogenin induced phosphorylation of Akt, whose activation was required for angiogenin’s neuroprotective effect. In contrast, the ALS-associ- ated mutant K40I failed to induce Akt phosphorylation and failed to be protective in motoneurons [10]. We previously demonstrated angiogenin to induce Akt phosphorylation in the SH-SY5Y PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e56092