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Project
Study of the central neurobiochemical alterations elicited by modulation of the AT4 receptor/insulin-regulated aminopeptidase (IRAP), therapeutic pprospects for cognitive dysfunction and limbic epilepsy (FWOAL440)
1. Aim
The renin-angiotensin system is recognized as the most powerful signalling system for controlling sodium balance, body fluid volumes and arterial blood pressure. The major effector peptide of this system is angiotensin II (Ang II), which causes its hypertensive effects via binding to the AT1 receptor [1]. A new field of research opened when Fisher-Ferraro and co-workers discovered in 1971 an independent angiotensin system in the brain. Later, it was shown that besides Ang II, other fragments are biologically active as well. Ang-(3-8) or Ang IV, has particularly drawn attention since it facilitates memory acquisition and retrieval [2,3] and possesses anticonvulsant properties [4,5] in rodents. This suggests that the Ang IV binding site could become an important drug target for future treatment of brain pathologies such as Alzheimer's dementia and epilepsy.
Since most of Ang IV's physiological effects are already observed at nanomolar concentrations and since Ang IV binds only with low affinity to AT1 and AT2 receptors, it is generally accepted that these are mediated via a specific 'AT4 receptor' [1]. Intriguingly, in 2001, the 'AT4 receptor' was identified as insulin-regulated aminopeptidase (IRAP) or cystinyl aminopeptidase (EC 3.4.11.3), also known as placental leucine aminopeptidase and oxytocinase [6]. IRAP is a type II integral membrane protein homologous to aminopeptidase N (AP-N), and other Zn2+-dependent enzymes of the gluzincin aminopeptidase family [7].
Within this field of central angiotensin research, we have already characterised in vivo the Ang IV-, Ang II- and Ang-(1-7)-mediated effects on striatal dopamine release [8-10] and demonstrated that Ang IV is anticonvulsant against pilocarpine-induced epileptic seizures [5].
In the present project, we aim to further characterize the nature of the binding site for Ang IV and the mechanisms of action behind Ang IV's cognitive enhancing and anticonvulsant effects in order to finally obtain concrete innovative therapies against cognitive decline and limbic epilepsy. To quantify the memory-promoting effects of the ligands of our interest within this project, we already possess a validated set-up of the Morris water maze task and we have currently access to Barnes maze as well as spontaneous alternation tasks. We also possess well-validated chemoconvulsant models for epilepsy, such as the pilocarpine model for limbic epilepsy.
2. Objectives
- We will perform a critical evaluation of the working hypothesis that the IRAP enzyme/AT4 receptor system represents a major cellular recognition and signalling site for Ang IV in the CNS.
- The Ang IV precursor, Ang II, possesses well known AT1-dependent dipsogenic and central pressor effects [1], facilitating effects on memory retention [11] as well as anticonvulsant effects [12]. AT1 receptors were recently shown to contribute also to the Ang IV-mediated central pressor [13] and peripheral vasoconstrictive effects [14]. We will therefore investigate if AT1 and AT2 receptors are involved in the memory enhancing and anticonvulsant effects observed for Ang IV.
- Another objective is to investigate the possible mechanisms of action behind the Ang IV-mediated memory improving and anticonvulsant effects. Several hypotheses need to be tested. First, Ang IV can mediate part of its central effects by inhibiting IRAP's enzymatic activity [6]. We will therefore test if inhibition of IRAP by Ang IV will indeed result in vivo in an increased extracellular concentration of the neuropeptide substrates of IRAP. In addition, selective neuropeptide receptor antagonists will be used to possibly block the Ang IV-mediated effects. Secondly, it is known that alterations in hippocampal neurotransmitter levels play a role in memory acquisition, learning processes and seizure generation. Since there are no in vivo data available we will use in vivo microdialysis to unravel possible effects of Ang IV on hippocampal neurotransmitter release. Finally, IRAP is co-localised with the insulin-dependent glucose transporter GLUT4 in the hippocampus [15] in specific intracellular vesicles. It is known from studies on adipocytes and muscle cells that insulin speeds up the translocation of these vesicles to the cell membrane with significant glucose uptake as a result. We will investigate if Ang IV can alter extracellular hippocampal glucose levels and glucose uptake in brain cells.
The renin-angiotensin system is recognized as the most powerful signalling system for controlling sodium balance, body fluid volumes and arterial blood pressure. The major effector peptide of this system is angiotensin II (Ang II), which causes its hypertensive effects via binding to the AT1 receptor [1]. A new field of research opened when Fisher-Ferraro and co-workers discovered in 1971 an independent angiotensin system in the brain. Later, it was shown that besides Ang II, other fragments are biologically active as well. Ang-(3-8) or Ang IV, has particularly drawn attention since it facilitates memory acquisition and retrieval [2,3] and possesses anticonvulsant properties [4,5] in rodents. This suggests that the Ang IV binding site could become an important drug target for future treatment of brain pathologies such as Alzheimer's dementia and epilepsy.
Since most of Ang IV's physiological effects are already observed at nanomolar concentrations and since Ang IV binds only with low affinity to AT1 and AT2 receptors, it is generally accepted that these are mediated via a specific 'AT4 receptor' [1]. Intriguingly, in 2001, the 'AT4 receptor' was identified as insulin-regulated aminopeptidase (IRAP) or cystinyl aminopeptidase (EC 3.4.11.3), also known as placental leucine aminopeptidase and oxytocinase [6]. IRAP is a type II integral membrane protein homologous to aminopeptidase N (AP-N), and other Zn2+-dependent enzymes of the gluzincin aminopeptidase family [7].
Within this field of central angiotensin research, we have already characterised in vivo the Ang IV-, Ang II- and Ang-(1-7)-mediated effects on striatal dopamine release [8-10] and demonstrated that Ang IV is anticonvulsant against pilocarpine-induced epileptic seizures [5].
In the present project, we aim to further characterize the nature of the binding site for Ang IV and the mechanisms of action behind Ang IV's cognitive enhancing and anticonvulsant effects in order to finally obtain concrete innovative therapies against cognitive decline and limbic epilepsy. To quantify the memory-promoting effects of the ligands of our interest within this project, we already possess a validated set-up of the Morris water maze task and we have currently access to Barnes maze as well as spontaneous alternation tasks. We also possess well-validated chemoconvulsant models for epilepsy, such as the pilocarpine model for limbic epilepsy.
2. Objectives
- We will perform a critical evaluation of the working hypothesis that the IRAP enzyme/AT4 receptor system represents a major cellular recognition and signalling site for Ang IV in the CNS.
- The Ang IV precursor, Ang II, possesses well known AT1-dependent dipsogenic and central pressor effects [1], facilitating effects on memory retention [11] as well as anticonvulsant effects [12]. AT1 receptors were recently shown to contribute also to the Ang IV-mediated central pressor [13] and peripheral vasoconstrictive effects [14]. We will therefore investigate if AT1 and AT2 receptors are involved in the memory enhancing and anticonvulsant effects observed for Ang IV.
- Another objective is to investigate the possible mechanisms of action behind the Ang IV-mediated memory improving and anticonvulsant effects. Several hypotheses need to be tested. First, Ang IV can mediate part of its central effects by inhibiting IRAP's enzymatic activity [6]. We will therefore test if inhibition of IRAP by Ang IV will indeed result in vivo in an increased extracellular concentration of the neuropeptide substrates of IRAP. In addition, selective neuropeptide receptor antagonists will be used to possibly block the Ang IV-mediated effects. Secondly, it is known that alterations in hippocampal neurotransmitter levels play a role in memory acquisition, learning processes and seizure generation. Since there are no in vivo data available we will use in vivo microdialysis to unravel possible effects of Ang IV on hippocampal neurotransmitter release. Finally, IRAP is co-localised with the insulin-dependent glucose transporter GLUT4 in the hippocampus [15] in specific intracellular vesicles. It is known from studies on adipocytes and muscle cells that insulin speeds up the translocation of these vesicles to the cell membrane with significant glucose uptake as a result. We will investigate if Ang IV can alter extracellular hippocampal glucose levels and glucose uptake in brain cells.
Date:1 Jan 2008 → 31 Dec 2011
Keywords:epilepsy, pharmacology
Disciplines:Basic sciences