adenosine-kinase and Brain-Ischemia

Adenosine is a modulator of brain function uniquely positioned to integrate excitatory and inhibitory neurotransmission. The past few years brought a wealth of new data fostering our understanding of how the adenosine system is involved in the pathogenesis of neurological diseases. Thus, dysregulation of the adenosine system is implicated in epileptogenesis and cell therapies have been developed to locally augment adenosine in an approach to prevent seizures. While activation of inhibitory adenosine A(1) receptors is beneficial in epilepsy, chronic pain and cerebral ischemia, inhibition of facilitatory A(2A) receptors has profound neuroprotective effects, which are currently exploited in clinical trials in Parkinson's disease. A new era of adenosine-based therapies has begun, with the prospect to cover a wide range of neurological diseases.

Topics: Adenosine; Adenosine Kinase; Alzheimer Disease; Animals; Brain Diseases; Brain Ischemia; Epilepsy; Humans; Huntington Disease; Pain; Parkinson Disease; Schizophrenia; Synaptic Transmission

Adenosine kinase (AK; EC 2.7.1.20) is a key intracellular enzyme regulating intra and extracellular concentrations of adenosine (ADO), an endogenous modulator of intercellular signalling that reduces cell excitability during tissue stress and trauma. The inhibitory effects of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors (GPCR), which regulate membrane cation flux, membrane polarisation and the release of excitatory neurotransmitters. Inhibition of AK potentiates local extracellular ADO levels at cell and tissue sites which are undergoing accelerated ADO release. Thus, AK inhibition represents a mechanism to selectively enhance the endogenous protective actions of ADO during cellular stress while potentially minimising the non-specific effects associated with the systemic administration of ADO receptor agonists. Novel, potent AK inhibitors have recently been synthesised that demonstrate high specificity for this particular enzyme as compared to other ADO metabolic enzymes, transporters and receptors. AK inhibitors have been shown to increase ADO concentrations in various systems in vitro, as well as in an in vivo model of neurotoxicity. In addition, AK inhibitors have demonstrated efficacy in animal models of epilepsy, cerebral ischaemia as well as pain and inflammation, thus suggesting their potential therapeutic utility for these conditions.

Topics: Adenosine Kinase; Animals; Brain Ischemia; Disease Models, Animal; Enzyme Inhibitors; Epilepsy; Humans; Inflammation; Pain; Purinergic P1 Receptor Antagonists; Receptors, Cell Surface

Adenosine kinase (ADK) is the major negative metabolic regulator of the endogenous neuroprotectant and homeostatic bioenergetic network regulator adenosine. We used three independent experimental approaches to determine the role of ADK as a molecular target for predicting the brain's susceptibility to ischemic stroke. First, when subjected to a middle cerebral artery occlusion model of focal cerebral ischemia, transgenic fb-Adk-def mice, which have increased ADK expression in striatum (164%) and reduced ADK expression in cortical forebrain (65%), demonstrate increased striatal infarct volume (126%) but almost complete protection of cortex (27%) compared with wild-type (WT) controls, indicating that cerebral injury levels directly correlate to levels of ADK in the CNS. Second, we demonstrate abrogation of lipopolysaccharide (LPS)-induced ischemic preconditioning in transgenic mice with brain-wide ADK overexpression (Adk-tg), indicating that ADK activity negatively regulates LPS-induced tolerance to stroke. Third, using adeno-associated virus-based vectors that carry Adk-sense or -antisense constructs to overexpress or knockdown ADK in vivo, we demonstrate increased (126%) or decreased (51%) infarct volume, respectively, 4 weeks after injection into the striatum of WT mice. Together, our data define ADK as a possible therapeutic target for modulating the degree of stroke-induced brain injury.

Topics: Adenosine; Adenosine Kinase; Animals; Brain; Brain Ischemia; Cerebral Cortex; Down-Regulation; Gene Deletion; Gene Expression; Infarction, Middle Cerebral Artery; Ischemic Preconditioning; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Up-Regulation

Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5-iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection.

Topics: Adenosine; Adenosine Kinase; Animals; Brain Ischemia; Cell Death; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Functional Laterality; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroprotective Agents; Promoter Regions, Genetic; Pyramidal Cells; Receptor, Adenosine A1; Tubercidin; Ubiquitin

Protective mechanisms of the brain may reduce the extent of injury after focal cerebral ischemia. Here, we explored in a mouse model of focal cerebral ischemia potential synergistic neuroprotective effects of two mediators of neuroprotection: (i) neuronal or glial precursor cells and (ii) the inhibitory neuromodulator adenosine. Embryonic stem (ES) cells, engineered to release adenosine by biallelic disruption of the adenosine kinase gene, and respective wild-type cells were induced to differentiate into either neural or glial precursor cells and were injected into the striatum of mice 1 week before middle cerebral artery occlusion. All stem cell-derived graft recipients were characterized by a significant reduction in infarct volume, an effect that was augmented by the release of adenosine. Neuroprotection was strongest in adenosine-releasing glial precursor cell recipients, which were characterized by an 85% reduction of the infarct area. Graft-mediated neuroprotection correlated with a significant improvement of general and focal neurologic scores. Histologic analysis before and after ischemia revealed clusters of implanted cells within the striatum of all treated mice. We conclude that ES cell derived adenosine-releasing brain implants provide neuroprotection by synergism of endogenous precursor cell-mediated effects and paracrine adenosine release.

Topics: Adenosine; Adenosine Kinase; Alleles; Animals; Brain Ischemia; Cerebral Infarction; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nervous System Diseases; Neurons; Stem Cell Transplantation

In the brain, the levels of adenosine increase up to 100-fold during cerebral ischernia; however, the roles of specific cell types, enzymatic pathways and membrane transport processes in regulating intra- and extracellular concentrations of adenosine are poorly characterized. Rat primary cortical neurons and astrocytes were incubated with [(3)H]adenine for 30 min to radiolabel intracellular ATP. Cells were then treated with buffer, glucose deprivation (GD), oxygen-glucose deprivation (OGD), 100 micro M sodium cyanide (NaCN) or 500 micro M iodoacetate (IAA) for 1 h to stimulate the metabolism of ATP and cellular release of [(3)H]purines. The nucleoside transport inhibitor dipyridamole (DPR) (10 micro M), the adenosine kinase inhibitor iodotubercidin (ITU) (1 micro M), the adenosine deaminase inhibitor EHNA (1 micro M) and the purine nucleoside phosphorylase inhibitor BCX-34 (10 micro M) were tested to investigate the contribution of specific enzymes and transporters in the metabolism and release of purines from each cell type. Our results indicate that (a). under basal conditions astrocytes released significantly more [(3)H]adenine nucleotides and [(3)H]adenosine than neurons, (b). OGD, NaCN and IAA conditions produced significant increases in [(3)H]adenosine release from neurons but not astrocytes, and (c) DPR blocked [(3)H]inosine release from both astrocytes and neurons but only blocked [(3)H]adenosine release from neurons. These data suggest that, in these experimental conditions, adenosine was formed by an intracellular pathway in neurons and then released via a nucleoside transporter. In contrast, adenine nucleotide release and extracellular metabolism to adenosine appeared to predominate in astrocytes.

Topics: Adenine Nucleotides; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine Triphosphate; Animals; Antimetabolites; Astrocytes; Brain Ischemia; Cells, Cultured; Cerebral Cortex; Chromatography, Thin Layer; Enzyme Inhibitors; Extracellular Space; Glucose; Hypoxanthine; Hypoxia-Ischemia, Brain; Inosine; Iodoacetates; Neurons; Purines; Rats; Sodium Cyanide

5-Iodotubercidin is a potent inhibitor of the enzyme adenosine kinase. It has a recognized ability to enhance interstitial fluid levels of the cerebroprotective purine, adenosine, in the hypoxic brain, and an anticonvulsant action, which is thought to be a consequence of its ability to increase extracellular adenosine levels. 5-Iodotubercidin (1 mg/kg, i.p.) was therefore tested for its ability to reduce cerebral ischemic injury in a gerbil model. Unanesthetized gerbils were subjected to a 5 min period of bilateral carotid artery occlusion and then maintained in an environmental chamber at 30 degrees C for 5 hr to counteract the hypothermia-inducing action of 5-iodotubercidin. As estimated from the extent of the increases in locomotor activity and the magnitude of hippocampal CAI layer pyramidal cell loss, 5-iodotubercidin (1 mg/kg) failed to have a cerebroprotective effect against ischemic injury.

Topics: Adenosine Kinase; Animals; Body Temperature; Brain Ischemia; Gerbillinae; Hippocampus; Male; Motor Activity; Tubercidin