Forebrain Deletion of aGDI in Adult Mice Worsens the Pre-Synaptic Deficit at Cortico-Lateral Amygdala Synaptic Connections Veronica Bianchi1., Fre´ de´ ric Gambino2,3., Luca Muzio4, Daniela Toniolo5, Yann Humeau2,6, Patrizia 1 D’Adamo * 1 Dulbecco Telethon Institute at Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy, 2 Centre National de la Recherche Scientifique UPR3212, CNRS, University of Strasbourg, Strasbourg, France, 3 De´partement des Neurosciences Fondamentales, CMU, Gene`ve, Suisse, 4 Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy, 5 Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy and Institute of Molecular Genetics-CNR, Pavia, Italy, 6 Institut Interdiciplinaire de Neuroscience Centre National de la Recherche Scientifique UMR5297, University of Bordeaux, Bordeaux, France Abstract The GDI1 gene encodes aGDI, which retrieves inactive GDP-bound RAB from membranes to form a cytosolic pool awaiting vesicular release. Mutations in GDI1 are responsible for X-linked Intellectual Disability. Characterization of the Gdi1-null mice has revealed alterations in the total number and distribution of hippocampal and cortical synaptic vesicles, hippocampal short-term synaptic plasticity and specific short-term memory deficits in adult mice, which are possibly caused by alterations of different synaptic vesicle recycling pathways controlled by several RAB GTPases. However, interpretation of these studies is complicated by the complete ablation of Gdi1 in all cells in the brain throughout development. In this study, we generated conditionally gene-targeted mice in which the knockout of Gdi1 is restricted to the forebrain, hippocampus, cortex and amygdala and occurs only during postnatal development. Adult mutant mice reproduce the short-term memory deficit previously reported in Gdi1-null mice. Surprisingly, the delayed ablation of Gdi1 worsens the pre-synaptic phenotype at cortico-amygdala synaptic connections compared to Gdi1-null mice. These results suggest a pivotal role of aGDI via specific RAB GTPases acting specifically in forebrain regions at the pre-synaptic sites involved in memory formation. Citation: Bianchi V, Gambino F, Muzio L, Toniolo D, Humeau Y, et al. (2012) Forebrain Deletion of aGDI in Adult Mice Worsens the Pre-Synaptic Deficit at Cortico- Lateral Amygdala Synaptic Connections. PLoS ONE 7(1): e29763. doi:10.1371/journal.pone.0029763 Editor: Judith Homberg, Radboud University, The Netherlands Received August 31, 2011; Accepted December 5, 2011; Published January 23, 2012 Copyright: 2012 Bianchi 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 grants from Telethon, Italy [TCP04015 to PD]; by Centre Nationale de la Recherche Scientifique and the Agence Nationale de la Recherche contract 06-003-Neuro-01 to YH, and by the European Neuroscience Institutes Network (Project Contract Nu LSHM-CT-2005-019063), and Sanofi and the Re´gion Aquitaine to YH. 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: p.dadamo@hsr.it . These authors contributed equally to this work. Introduction Human intellectual disability (ID), also referred to as Mental Retardation (MR), is a common human neurodevelopment disorder with onset early in postnatal life. This condition affects approximately 2–3% of the human population [1]. ID is classified based on intelligence quotient (IQ); the presence of other clinical features distinguishes syndromic ID (S-ID) from non-syndromic ID (NS-ID). Over the past 15 years, mutations in , 40 genes have been associated with NS-ID and , 80% of these are found on the X-chromosome. The identification and functional characterization of these genes have greatly enlarged our understanding of human cognition and intellect. Because NS-ID is characterized clinically only by intellectual impairment, the genes underlying this clinical condition are likely to be involved in learning and memory formation. Additionally, the functional study of these genes might help to understand neural development and provide treatment strategies for NS-ID. The GDI1 gene encodes aGDI, a protein that controls the cycling of RAB GTPase [2]. RAB GTPases are proteins involved in the control of intracellular traffic; they act as molecular switches between active GTP-bound and inactive GDP-bound conforma- tions. The role of aGDI is to bind the RAB in its GDP-bound state from membranes and to maintain a cytosolic pool of GDP- associated RAB. The discovery of mutations in the GDI1 [3] and RAB39B [4] genes in ID patients supports the importance of the intracellular trafficking mediated by the aGDI-RAB GTPase pathway in the development of cognitive function. We have previously demonstrated that the lack of aGDI in Gdi1-null mice impaired hippocampal dependent forms of short- term memory, namely, the working and associative fear-related memory formation, and greatly reduced male aggression, thereby modifying social interaction [5]. We also showed that the absence of aGDI in the brain leads to alterations in the total number of hippocampal and cortical synaptic vesicles (SV) during synaptic differentiation at postnatal days (PND) 10 and 30. In adult mice at PND 90, the distribution of SV was altered at differentiated neuronal terminals, and the reserve pool (RP) appeared to be specifically affected [6]; this likely resulted in slow recovery after SV depletion and a short-term memory deficit in Gdi1-null mice. Thus, the complete ablation of Gdi1 leads to a complex phenotype that is most likely the result of alterations in the temporal and spatial functioning of aGDI and some of the associated RABs. Our previous results suggest that these PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29763