Subthalamic GAD Gene Therapy in a Parkinson s Disease Rat Model Jia Luo et al Science 298 425 2002 DOI 10 1126 science 1074549 This copy is for your personal non commercial use only If you wish to distribute this article to others you can order high quality copies for your colleagues clients or customers by clicking here The following resources related to this article are available online at www sciencemag org this information is current as of September 3 2014 Updated information and services including high resolution figures can be found in the online version of this article at http www sciencemag org content 298 5592 425 full html Supporting Online Material can be found at http www sciencemag org content suppl 2002 10 10 298 5592 425 DC1 html A list of selected additional articles on the Science Web sites related to this article can be found at http www sciencemag org content 298 5592 425 full html related This article cites 28 articles 4 of which can be accessed free http www sciencemag org content 298 5592 425 full html ref list 1 This article has been cited by 118 article s on the ISI Web of Science This article has been cited by 18 articles hosted by HighWire Press see http www sciencemag org content 298 5592 425 full html related urls This article appears in the following subject collections Medicine Diseases http www sciencemag org cgi collection medicine Science print ISSN 0036 8075 online ISSN 1095 9203 is published weekly except the last week in December by the American Association for the Advancement of Science 1200 New York Avenue NW Washington DC 20005 Copyright 2002 by the American Association for the Advancement of Science all rights reserved The title Science is a registered trademark of AAAS Downloaded from www sciencemag org on September 3 2014 Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here REPORTS Subthalamic GAD Gene Therapy in a Parkinson s Disease Rat Model Jia Luo 1 2 Michael G Kaplitt 3 Helen L Fitzsimons 1 David S Zuzga 2 Yuhong Liu 2 Michael L Oshinsky 2 Matthew J During1 2 The motor abnormalities of Parkinson s disease PD are caused by alterations in basal ganglia network activity including disinhibition of the subthalamic nucleus STN and excessive activity of the major output nuclei Using adenoassociated viral vector mediated somatic cell gene transfer we expressed glutamic acid decarboxylase GAD the enzyme that catalyzes synthesis of the neurotransmitter GABA in excitatory glutamatergic neurons of the STN in rats The transduced neurons when driven by electrical stimulation produced mixed inhibitory responses associated with GABA release This phenotypic shift resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype This strategy suggests that there is plasticity between excitatory and inhibitory neurotransmission in the mammalian brain that could be exploited for therapeutic bene t Degeneration of specific groups of cells characterizes many neurological disorders In PD neurons of the substantia nigra pars compacta SNc are particularly vulnerable leading to marked depletion of dopamine in the primary projection region the striatum As a result the major inhibitory output nuclei of the basal ganglia the substantia nigra pars reticulata SNr and internal segment of the globus pallidus GPi are driven by a disinhibited and thereby overactive subthalamic nucleus 1 4 whose projection axons end in asymmetric excitatory synapses on target neurons in the SNr 5 Marked improvement of the motor symptoms of PD occurs following either STN ablation 6 7 electrical inhibition with high frequency stimulation 8 9 or pharmacological silencing by local lidocaine or muscimol infusion 10 Here we describe a genetic approach to test the hypothesis that the glutamatergic neurons of the STN can be induced to express GAD and thereby change from an excitatory nucleus to a predominantly inhibitory system that releases GABA at its terminal region in the substantia nigra SN leading to suppression of firing activity of these SN neurons Moreover we show that such an intervention also results in neuropro tection with resistance to 6 hydroxydopamine 6 OHDA induced degeneration of dopaminergic neurons We used recombinant adeno associated virus rAAV to transduce neurons in the STN 11 This vector not only provides for highly efficient and stable gene transfer 12 13 but also results in minimal inflammatory and immunological responses 14 GABA the brain s major inhibitory transmitter can be generated by two isoforms of GAD GAD65 and GAD67 supporting text online We generated rAAV vectors 11 containing both GAD65 and GAD67 cDNAs using the cytomegalovirus enhancer chicken actin CBA promoter 15 and a woodchuck hepatitis virus postregulatory element WPRE 16 to further enhance expression fig S1A Mouse neural progenitor C17 2 cells were infected with both the GAD65 and GAD67 vectors with functional expression of the transgene confirmed by immunocytochemistry with antibodies specific to each GAD isoform and GABA 11 fig S1 B to J GABA release was quantified by high performance liquid chromatography 11 17 fig S1K Adult male rats were stereotactically injected into the left STN with GAD65 GAD67 or control GFP green fluorescent protein vectors Four to 5 months after surgery expression of the transgenes was determined by immunofluorescence 11 Robust expression confined to the STN was obtained for all transgenes Fig 1 A to I with a nuclear halo in the GAD65 transduced neurons consistent with membrane bound enzyme Fig 1F whereas both GAD67 Fig 1I and GFP Fig 1C filled the cell soma completely The STN neurons send a major projection to the SNr with efferents also to 1 Functional Genomics and Translational Neuroscience Laboratory Department of Molecular Medicine and Pathology University of Auckland Auckland New Zealand 2CNS Gene Therapy Center Jefferson Medical College Philadelphia PA 19107 USA 3Center for Stereotactic and Functional Neurosurgery Department of Neurological Surgery Weill Medical College of Cornell University New York NY 10021 USA Present address Neurologix Inc Delaware Biotechnology Institute 15 Innovation Way Newark DE 19711 USA To whom correspondence should be addressed Email m during auckland ac nz Fig 1 rAAV mediated transgene expression in the STN AAV vectors were injected into the left STN A D G Low power ipsilateral left STN bar 100 m B E H Contralateral C F I
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