If overexpression of ADK is sufficient

If overexpression of ADK is sufficient to induce spontaneous seizures, then reduction of ADK in engineered mice should prevent epileptogenesis. The generation of mice with moderately reduced levels of ADK in brain rather than complete ADK deficiency seems to be essential, since several lines of evidence suggest that only small changes in ADK levels are permissible: (i) the homozygous disruption of ADK is lethal (Boison et al., 2002b); (ii) ADK is highly conserved in evolution (Spychala et al., 1996), suggesting that mutations are not easily tolerated; (iii) no mutations of human ADK are known in man (OMIM, Online Mendelian Inheritance in Man, Victor A. McKusick et al., Johns Hopkins University) indicating that most mutations are lethal; (iv) excessive levels of ADK lead to severe deficits in A1R activation and to lethal status epilepticus or cell death after otherwise non-lethal triggers (Fedele et al., 2006, Kochanek et al., 2006, Li et al., 2008, Pignataro et al., 2007b); (v) inadequate levels of ADK in brain are expected to lead to a rise in adenosine to unacceptably high levels, with likely lethal consequences, e.g. central Deferoxamine mesylate receptor and perinatal mortality induced by elevated adenosine (Montandon et al., 2006). Therefore, a new mouse line (fb-Adk-def) was generated with a moderate reduction of ADK (62% of normal) restricted to the forebrain (Li et al., 2008). This was achieved by breeding Adk-tg mice, which contain a loxP-flanked ubiquitously expressed Adk transgene, with Emx1-Cre-tg3 mice (Iwasato et al., 2004), yielding a forebrain selective excision of the transgene in most cells (except GABAergic interneurons). As expected, fb-Adk-def mice were resistant to KA-induced SE and subsequent neuronal cell loss (Li et al., 2008). However, when intraamygdaloid injection of KA was paired with the A1 receptor antagonist DPCPX, wild-type like SE and corresponding CA3 selective neuronal cell loss (i.e. trigger for subsequent epileptogenesis) could be restored. However, despite the presence of the epileptogenesis triggering acute injury DPCPX-treated fb-Adk-def mice were resistant to subsequent epileptogenesis. Neither astrogliosis, nor upregulation of ADK, nor spontaneous seizures were found three weeks after CA3 injury (Li et al., 2007). These studies demonstrate that epileptogenesis was prevented by reduced levels of ADK.
Adenosine-releasing stem cell-derived brain implants prevent epileptogenesis As outlined above, seizure susceptibility is increased under conditions of elevated ADK and reduced adenosine (either in the context of astrogliosis or via transgenic ADK). Conversely, reconstitution of the adenosine system should suppress seizures in established epilepsy but should also prevent epileptogenesis. Ample evidence has been provided for the seizure suppressive potential of adenosine-releasing cell transplants (Boison, 2007a, Boison, 2007b). In these studies, kindled seizures in the rat could be suppressed by intraventricular implants of encapsulated fibroblasts, myoblasts or stem cells engineered to release adenosine based on disruption of the Adk gene (Boison et al., 2002a, Güttinger et al., 2005a, Güttinger et al., 2005b, Huber et al., 2001). These studies demonstrated that the focal paracrine release of adenosine is sufficient to suppress seizures (Güttinger et al., 2005a), that implant efficacy is not compromised by receptor desensitization (Güttinger et al., 2005b), and that focal delivery of adenosine in contrast to systemic activation of A1Rs or systemic inhibition of ADK is not accompanied by sedative side effects (Güttinger et al., 2005b). These studies also demonstrated that rather small doses of adenosine in the nanomolar range, when delivered locally, are sufficient to suppress seizures (Huber et al., 2001). However, cell encapsulation limited the long-term viability of this first generation of adenosine-releasing therapeutic cells (Boison, 2007a).