Genetic deletion of the striatum-enriched phosphodiesterase PDE10A: Evidence for altered striatal function
Introduction
The cyclic nucleotides cAMP and cGMP are second messengers mediating intracellular signal transduction across the phylogenetic spectrum. A key element in regulation of these signaling pathways is metabolic inactivation of cyclic nucleotides by cyclic nucleotide phosphodiesterases (PDEs) (for review, Soderling and Beavo, 2000). In mammals, PDEs comprise a superfamily of enzymes divided into 11 families that are differentially localized throughout the organism. The ability to selectively regulate cyclic nucleotide signaling through pharmacological manipulation of these enzymes offers unique therapeutic opportunities.
The newly described PDE10A is particularly interesting in this regard due to its relatively specific expression in the brain. PDE10A was first identified by homology screening of EST databases and homologues of the mouse, rat and human enzymes have been characterized (Fujishige et al., 1999, Loughney et al., 1999, Soderling et al., 1999). PDE10A is an 89 kDA protein capable of hydrolyzing both cAMP and cGMP. Initial northern and dot blot studies demonstrated PDE10A expression within the brain and in particular, the striatum (Fujishige et al., 1999, Loughney et al., 1999, Soderling et al., 1999). Subsequent localization studies using in situ hybridization and immunohistochemistry indicate PDE10A mRNA and protein are highly expressed in the medium spiny projection neurons of the striatum (Seeger et al., 2003). High levels of PDE10A protein are found in the cell bodies and dendritic fields of these neurons as well as in the regions to which these neurons project.
The medium spiny neurons of the striatum function as the principal input site to the basal ganglia (Wilson, 1998). The basal ganglia are a series of interconnected subcortical nuclei that integrate widespread cortical input with dopaminergic signaling to plan, facilitate, and execute relevant motor and cognitive patterns while suppressing unwanted or irrelevant patterns (for review, see Graybiel, 2000). Striatal dysfunction is implicated in a number of CNS disorders including Parkinson's disease, Huntington's disease, schizophrenia and obsessive-compulsive disorders. There is considerable evidence that the cAMP (Girault and Greengard, 2004) and cGMP (West and Grace, 2004) signaling pathways play significant roles in the regulation of medium spiny neuron excitability. Thus, the pattern of localization of PDE10A within these neurons suggests that changes in the activity of this enzyme may affect striatal function and PDE10A inhibitors may provide therapeutic benefit in the treatment of diseases associated with this brain region.
Until recently, speculation around the functional role of PDE10A in the central nervous system has been based solely on its localization within the brain. We have now produced a mouse in which the gene for PDE10A is constitutively disrupted and in the present studies use these mice to explore the physiological function of PDE10A. Specifically, PDE10A knockout (PDE10A−/−) mice were characterized across a broad spectrum of well-established behavioral tests including an assessment of behavioral responsiveness to dopamine releasing agents and NMDA receptor antagonists. The results suggest that PDE10A normally functions to inhibit striatal output by reducing medium spiny neuron excitability.
Section snippets
Gene targeting of PDE10A
A 2.2 kb murine PDE10A genomic fragment was used as a probe to identify PDE10A genomic clones from a murine DBA/1LacJ genomic phage library (Stratagene, La Jolla CA). The PDE10A targeting vector was created by cloning 2.0 kb of 5′ homology and 5.1 kb of 3′ homology in pJNS2 (PGK-Neo/TK vector). The targeting vector was designed to replace 3.9 kb of the PDE10A genomic locus, containing bps 1499–1861 of murine PDE10A mRNA (AF110507), with the PGK-Neo cassette. DBA/1LacJ ES cells were electroporated
Targeted disruption of the PDE10A Gene
The targeting vector designed to generate PDE10A knockout mice replaced approximately 3.9 kb of the PDE10A genomic locus with the PGK-neo cassette. This genomic region included three exons corresponding to bases 1499–1861 of the coding region (AF110507) (Fig. 1). Correctly targeted ES cells were identified by Southern hybridization analysis (Fig. 2A). Homozygous PDE10A−/− mice were genotyped by a two-primer approach, as described in Section 2. As expected, no band was amplified from a PCR primer
Discussion
Disruption of the PDE10A gene in mice did not lead to any gross alteration of brain development and/or brain function. PDE10A−/− mice appeared healthy and generally behaved normally. Although a truncated PDE10A immunoreactive protein was present in the striatum of PDE10A−/− mice, the mutant protein was found to lack catalytic activity. In addition, immunoprecipitated PDE activity was significantly decreased in striatal homogenates from PDE10A−/− mice consistent with the loss of significant
Acknowledgements
Portions of this work have been presented at the Society for Neuroscience Meeting, 2003 and 2004 and the International Congress on Schizophrenia Research, 2003 and 2005. The authors thank Kimberly Crimin for expert advice on the statistical analysis of the Morris Water Maze data. The authors would also like to thank Marsha Roach and Linyan He for their ES cell culture contributions and Linda Loverro, Ed Glynn, Benjamin Capers, Henry Anderson, and Steve Linskens for their part in the colony
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