DOI: 10.1126/science.1074549, 425 (2002);298 Science et al.Jia LuoSubthalamic GAD Gene Therapy in a Parkinson's Disease Rat Model This copy is for your personal, non-commercial use only. clicking here.colleagues, clients, or customers by , you can order high-quality copies for yourIf you wish to distribute this article to others here.following the guidelines can be obtained byPermission to republish or repurpose articles or portions of articles ): September 3, 2014 www.sciencemag.org (this information is current as ofThe following resources related to this article are available online at http://www.sciencemag.org/content/298/5592/425.full.htmlversion of this article at: including high-resolution figures, can be found in the onlineUpdated information and services, http://www.sciencemag.org/content/suppl/2002/10/10/298.5592.425.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/298/5592/425.full.html#relatedfound at:can berelated to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/298/5592/425.full.html#ref-list-1, 4 of which can be accessed free:cites 28 articlesThis article 118 article(s) on the ISI Web of Sciencecited by This article has been http://www.sciencemag.org/content/298/5592/425.full.html#related-urls18 articles hosted by HighWire Press; see:cited by This article has been http://www.sciencemag.org/cgi/collection/medicineMedicine, Diseasessubject collections:This article appears in the following registered trademark of AAAS. is aScience2002 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience on September 3, 2014www.sciencemag.orgDownloaded from on September 3, 2014www.sciencemag.orgDownloaded from on September 3, 2014www.sciencemag.orgDownloaded from on September 3, 2014www.sciencemag.orgDownloaded from on September 3, 2014www.sciencemag.orgDownloaded from on September 3, 2014www.sciencemag.orgDownloaded fromSubthalamic GAD Gene Therapyin a Parkinson’s Disease RatModelJia Luo,1,2Michael G. Kaplitt,3Helen L. Fitzsimons,1*David S. Zuzga,2Yuhong Liu,2Michael L. Oshinsky,2Matthew J. During1,2†The motor abnormalities of Parkinson’s disease (PD) are caused by alterationsin basal ganglia network activity, including disinhibition of the subthalamicnucleus (STN), and excessive activity of the major output nuclei. Using adeno-associated viral vector–mediated somatic cell gene transfer, we expressedglutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of theneurotransmitter GABA, in excitatory glutamatergic neurons of the STN in rats.The transduced neurons, when driven by electrical stimulation, produced mixedinhibitory responses associated with GABA release. This phenotypic shift re-sulted in strong neuroprotection of nigral dopamine neurons and rescue of theparkinsonian behavioral phenotype. This strategy suggests that there is plas-ticity between excitatory and inhibitory neurotransmission in the mammalianbrain that could be exploited for therapeutic benefit.Degeneration of specific groups of cells char-acterizes many neurological disorders. In PD,neurons of the substantia nigra pars compacta(SNc) are particularly vulnerable, leading tomarked depletion of dopamine in the primaryprojection region, the striatum. As a result,the major inhibitory-output nuclei of the bas-al ganglia, the substantia nigra pars reticulata(SNr) and internal segment of the globuspallidus (GPi), are driven by a disinhibitedand thereby overactive subthalamic nucleus(1–4 ) whose projection axons end in asym-metric, excitatory synapses on target neuronsin the SNr (5). Marked improvement of themotor symptoms of PD occurs following ei-ther STN ablation (6, 7 ), electrical inhibitionwith high-frequency stimulation (8, 9), orpharmacological silencing by local lidocaineor muscimol infusion (10). Here, we describea genetic approach to test the hypothesis thatthe glutamatergic neurons of the STN can beinduced to express GAD, and thereby changefrom an excitatory nucleus to a predominant-ly inhibitory system that releases GABA at itsterminal region in the substantia nigra (SN),leading to suppression of firing activity ofthese SN neurons. Moreover, we show thatsuch an intervention also results in neuropro-tection with resistance to 6-hydroxydopamine(6-OHDA)–induced degeneration of dopami-nergic neurons.We used recombinant adeno-associatedvirus (rAAV) to transduce neurons in theSTN (11). This vector not only provides forhighly efficient and stable gene transfer (12,13), but also results in minimal inflammatoryand immunological responses (14 ). GABA,the brain’s major inhibitory transmitter, canbe generated by two isoforms of GAD,GAD65 and GAD67 (supporting text online).We generated rAAV vectors (11) containingboth GAD65 and GAD67 cDNAs using thecytomegalovirus enhancer/chicken -actin(CBA) promoter (15) and a woodchuck hep-atitis virus postregulatory element (WPRE)(16 ) to further enhance expression (fig. S1A).Mouse neural progenitor C17.2 cells wereinfected with both the GAD65 and GAD67vectors with functional expression of thetransgene confirmed by immunocytochemis-try with antibodies specific to each GADisoform and GABA (11) (fig. S1, B to J).GABA release was quantified by high-perfor-mance liquid chromatography (11, 17 ) (fig.S1K).Adult male rats were stereotactically in-jected into the left STN with GAD65,GAD67, or control GFP (green fluorescentprotein) vectors. Four to 5 months after sur-gery, expression of the transgenes was deter-mined by immunofluorescence (11). Robustexpression confined to the STN was obtainedfor all transgenes (Fig. 1, A to I) with anuclear halo in the GAD65-transduced neu-rons consistent with membrane-bound en-zyme (Fig. 1F), whereas both GAD67 (Fig.1I) and GFP (Fig. 1C) filled the cell somacompletely. The STN neurons send a majorprojection to the SNr, with efferents also to1Functional Genomics and Translational NeuroscienceLaboratory, Department of Molecular Medicine andPathology, University of Auckland, Auckland, NewZealand.2CNS Gene Therapy Center, Jefferson Med-ical College, Philadelphia, PA 19107, USA.3Center forStereotactic and Functional Neurosurgery, Depart-ment of
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