Award Number: W81XWH-04-1-0881 TITLE: Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson’s Disease

submission form Targeting Adenosine A2 A Receptors in Parkinson’s Disease and Other CNS Disorders May 17 19, 2006  Boston  USA  presented by  MassGeneral Institute for Neurodegenerative Disease  1 AAV-Cre/loxP conditional KO of adenosine A2A receptors in striatal neurons Augusta Pisanu*, Miguel Sena-Esteves & Michael A. Schwarzschild Department of Neurology, Massachusetts General Hospital, Charlestown, MA USA; *Presenting author (apisanu@partners.org.) Gene knockout (KO) approaches to receptor function complement traditional pharmacological methods by complete specificity and inactivation. However, standard KO strategies globally eliminate the targeted receptor, and thus their use for investigating the role of receptors in the adult brain can be confounded by developmental or systemic phenotypes. The transgenic Cre/loxP conditional KO system can achieve partial control over the timing and distribution of receptor inactivation using a specific promoter to direct cre expression, e.g., to study the effects of the adenosine A2A receptor (A2AR) in postnatal forebrain. To achieve an even higher degree of precision in eliminating the A2AR from discrete regions on one side of the brain, and in pursuit of a better understanding of A2AR involvement in neurodegeneration, we adopted an AAV-Cre/loxP system. Adeno-associated virus (AAV)-Cre vectors were stereotactically infused into homozygous floxed A2AR adult mice, resulting in a conditional KO of the A2AR at the site and time of infection. Working with several serotypes of AAV-Cre and AAV-GFP, injected into the striatum of floxed A2AR mice, we demonstrated an infection and GFP expression largely restricted to the targeted striatum for AAV1 serotype (Fig. A) but not AAV1/8 (which produced a widespread infection extending into the overlying cortex). Focusing on AAV1 serotype we characterized the titer-dependence and time-course of neuronal Cre expression and A2AR loss, visualized by IHC (as in Fig. B). Cre expression was detectable 8 days post-infusion of AAV vectors but the loss of A2AR was not evident until the 16 day, reaching a maximum extent at the 32 day post-injection. Figure: (A) Fluorescence of rostralcaudal coronal sections stained with a Hoechst 33258 one month after AAV1-GFP infusion (arrow). (B) Striatal Cre expression and the coincident elimination of striatal A2AR in a floxed A2AR mouse one month after unilateral intrastriatal injection of AAV1-Cre (arrow). The AAV-Cre/loxP conditional KO system provides a precise tool with which to explore the neurobiology of adenosine receptors and their pathophysiology in models of CNS disease. Funded by DoD W81XWH-04-1-0881. To the best of my knowledge and judgment I, the presenting author, report that: [Please check one. ]  The authors have no financial conflict of interest in the presentation of this work.  The authors may have a financial conflict(s) of interest as I have described on the next page. ‡ Significant financial conflict of interest is explained under the abstract submission policy (rule #11) and scientific communication guildelines (section 1.6) of the Society for Neuroscience. A B