In vitro and in vivo studies on the role of the IRAP enzyme/AT4 receptor system in learning and memory processes

INTRODUCTION
Ang IV and the IRAP enzyme /AT4 receptor system
The renin-angiotensin system (RAS) is widely recognised as the most powerful signalling system for controlling sodium balance, body fluid volumes and arterial blood pressure. The major RAS peptide is the octapeptide angiotensin II (Ang II). It is formed by enzymatic processing of Ang I by the angiotensin-converting enzyme (ACE) present in plasma as well as in tissues such as brain, kidney and heart. Although Ang II has long been considered to represent the RAS end product, there is now strong evidence that this system comprises additional peptides with particular physiological functions. In this respect, shorter Ang II fragments such as Ang-(1-7), Ang III and Ang IV are formed via the activity of ACE and other peptidases. The hexapeptide Ang IV sparked great interest because of its wide range of physiological effects. Among those, its facilitatory role in memory acquisition and retrieval is of potential therapeutic interest.
Ang IV binds to AT1 and AT2 receptors but only with low affinity. Yet, most of its physiological effects are already observed at nanomolar concentrations and classical non-peptide AT1 and AT2 antagonists do not block these effects. This, together with the discovery of high affinity binding sites for [125I]-Ang IV in the central nervous, vascular and renal systems (1-3) led to the concept of a novel angiotensin receptor subtype: the 'AT4 receptor' (4,5). The pharmacological profile of the AT4 receptor deviates significantly from AT1 and AT2 receptors. Instead, it is activated by Ang IV and by more stable synthetic peptide analogues like Norleucinel-Ang IV (Nlel-Ang IV) (6) and Norleucinal (7). These putative AT4 receptors also constitute cellular targets for hemorphins, a class of endogenous CNS peptides obtained by hydrolysis of the beta chain of hemoglobin (8).
The “AT4 receptors” have recently been identified as the insulin-regulated aminopeptidase (IRAP) enzyme, also known as placental leucine aminopeptidase (P-LAP) and oxytocinase (Otase) (7). IRAP is a type II integral membrane protein homologous to aminopeptidase A (AP-A), aminopeptidase N (AP-N), and other Zn2+-dependent enzymes of the gluzincin aminopeptidase family (9, for review see 10). Its different denominations are related to its independent “discovery” by several research teams as well as to differences in the physiological context in which this enzyme was investigated. In insulin-responsive cells, IRAP co-localises with the insulin-dependent glucose transporter GLUT4 in specific intracellular vesicles (11).
These intriguing findings imply that Ang IV might be a ligand for the putative AT4 receptors as well as a competitive inhibitor of IRAP’s catalytic activity (11). AT4 receptor ligands could thus mediate at least part of their physiological effects by inhibiting IRAP’s enzymatic activity. Moreover, homodimer formation is one of the characteristic features of the membrane-bound M1 metallopeptidase family (12) to which IRAP belongs. As dimers, these enzymes have the potential to convey information across cell membranes in the same way as growth factors and cytokine receptors. In this respect, the structurally related AP-N and dipeptidylpeptidase IV ectoenzymes have already been shown to mediate intracellular signalling (13,14). It is of particular interest that AT4 receptor ligands also inhibit AP-N activity (15). Accordingly, IRAP and AP-N might both act as classical receptors for Ang IV. These new concepts offer a wide range of original opportunities for examining the physiological roles of the “IRAP/AT4” and the AP-N systems as well as the mechanisms of action of Ang IV. This research is of special interest in the field of cognition and it may also contribute to our understanding of pathophysiological conditions such as Alzheimer’s disease.
Role of Ang IV in memory and learning
Initial interest in Ang IV originated from its ability to increase memory recall and learning in passive and conditioned avoidance response studies (16-21). Intracerebroventricular (i.c.v.) administration of the AT4 agonist Nlel-Ang IV facilitated the rate of acquisition to solve a spatial learning task in the circular water maze, an effect that was blocked by the putative “AT4 antagonist” Divalinal-Ang IV (22). This ligand also counteracted scopolamine-induced disruption of spatial learning (23), suggesting an Ang IV-acetylcholine interaction. Electrophysiological and biochemical studies revealed that the cognitive effects of AT4 agonists are mediated via the hippocampus. Ang IV and its analogues significantly enhanced hippocampal long-term potentiation (LTP) in the dentate gyrus and the CA1 field, both in vitro (24) and in vivo (25). Ang IV and LVV-hemorphin-7 facilitated potassium-evoked acetylcholine release from rat hippocampal slices (26) and i.c.v. administration of Ang IV induced c-Fos expression in hippocampal pyramidal cells (27). Ang IV inhibited neurite outgrowth in cultured embryonic chicken sympathetic neurons and may thus play an important role in neuronal development (28). Trophic effects were observed on rat anterior pituitary cells (29). Ang IV restored astrocyte adhesion, growth and morphology, and inhibited apoptosis of hippocampal cells in angiotensinogen knockout (KO) mice (30,31). Autoradiographic studies revealed that AT4 binding sites are prominent in brain structures important to cognitive processing, including hippocampus (32).
Furthermore, widespread epidemiological findings (for review see 33) indicate an intriguing interaction between cardiovascular disorders or its risk factors and pathways influenced in cognitive decline. Massive evidence implicates the cardiovascular risk factor genes, APOEe4 and ACE1 (I/D and I/I genotypes), in the susceptibility to and possibly the pathogenesis of Alzheimer’s disease. Several mechanisms for the involvement of ACE1 polymorphisms and diminished ACE levels in Alzheimer’s disease have been suggested, including malfunctions in the RAS cascade, diminished Ang IV production and inadequate Ang IV-IRAP interactions (33).


WORKING HYPOTHESIS AND AIMS
This innovative project aims to perform a critical evaluation of the working hypotheses that IRAP is indeed the AT4 receptor, and that the IRAP enzyme/AT4 receptor system represents the major cellular recognition and signalling site for Ang IV in the CNS.
In particular, our research will comprise three parts:
(i) The identification and characterization of the IRAP enzyme/AT4 receptor system in neuronal cells in vitro. This will be accomplished by measuring the binding, enzymatic activity and potential signalling mechanisms in cells as well in brain slices and homogenates.
(ii) Investigation of the involvement and the mechanisms of action of the IRAP enzyme/AT4 receptor system in learning and memory processes by measuring the behavioural consequences of i.c.v. and/or intrahippocampal/intracortical application of IRAP/AT4 ligands. These experiments will be carried out in normal rodents and in a further stage of the project in rodent models of Alzheimer’s disease.
(iii) Elucidation of the in vivo physiological relevance of activating and modulating the brain’s IRAP enzyme/AT4 receptor system by measuring both neurotransmitter and neuropeptide release.