Ruani N. Fernando

The angiotensin IV/AT4 receptor.

Abstract.The angiotensin AT4 receptor was originally defined as the specific, high-affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT4 receptor. Central administration of Ang IV, its analogues or LVV-hemorphin 7 markedly enhance learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The AT4 receptor has a broad distribution and is found in a range of tissues, including the adrenal gland, kidney, lung and heart. In the kidney Ang IV increases renal cortical blood flow and decreases Na+ transport in isolated renal proximal tubules. The AT4 receptor has recently been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). IRAP is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and is predominantly found in GLUT4 vesicles in insulin-responsive cells. Three hypotheses for the memory-potentiating effects of the AT4 receptor/IRAP ligands, Ang IV and LVV-hemorphin 7, are proposed: (i) acting as potent inhibitors of IRAP, they may prolong the action of endogenous promnestic peptides; (ii) they may modulate glucose uptake by modulating trafficking of GLUT4; (iii) IRAP may act as a receptor, transducing the signal initiated by ligand binding to its C-terminal domain to the intracellular domain that interacts with several cytoplasmic proteins.

Loss of system x(c)- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility.

System xc− exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system xc− (xCT−/−) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system xc− does not induce oxidative stress in vivo. Young xCT−/− mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations in xCT−/− mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system xc− inhibitors lowered extracellular glutamate, whereas the system xc− activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system xc− may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. These novel findings sustain that system xc− is an important source of extracellular glutamate in the hippocampus. System xc− is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

Identification and characterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase

Approximately one‐quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effec tive cognitive‐enhancing agents. Insulin‐regulated ami nopeptidase (IRAP) is potentially an innovative tar get for the development of cognitive enhancers, as its peptide inhibitors exhibit memory‐enhancing effects in both normal and memory‐impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small‐molecule inhibitors of IRAP. Structure‐based computational development of an initial “hit” resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (Ki 20‐700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 μl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibi tors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cogni tive enhancers. Notably, this study also provides unequiv ocal proof of principal that inhibition of IRAP results in memory enhancement.— Albiston, A. L., Morton, C. J., Ng, H. L., Pham, V., Yeatman, H. R., Ye, S., Ruani, N., Fernando, R. N., De Bundel, D., Ascher, D. B., Men delsohn, F. A. O., Parker, M. W., Chai, S. Y. Identification and characterization of a new cognitive enhancer based on inhibition of insulin‐regulated aminopeptidase. FASEB J. 22, 4209–4217 (2008)

The insulin-regulated aminopeptidase IRAP is colocalised with GLUT4 in the mouse hippocampus - Potential role in modulation of glucose uptake in neurones?

It is proposed that insulin‐regulated aminopeptidase (IRAP) is the site of action of two peptides, angiotensin IV and LVV‐hemorphin 7, which have facilitatory effects on learning and memory. In fat and muscles, IRAP codistributes with the insulin‐responsive glucose transporter GLUT4 in specialised vesicles, where it plays a role in the tethering and/or trafficking of these vesicles. This study investigated whether an analogous system exists in two functionally distinct regions of the brain, the hippocampus and the cerebellum. In the hippocampus, IRAP was found in the pyramidal neurones where it exhibited a high degree of colocalisation with GLUT4. Consistent with the role of GLUT4 in insulin‐responsive tissues, the glucose transporter was thought to be responsible for facilitating glucose uptake into these pyramidal neurones in response to potassium‐induced depolarisation or cAMP activation as the glucose influx was sensitive to indinavir treatment. Angiotensin IV and LVV‐hemorphin 7 enhanced this activity‐dependent glucose uptake in hippocampal slices. In contrast, in the cerebellum, where the distribution of IRAP was dissociated from GLUT4, the effect of the peptides on glucose uptake was absent. We propose that the modulation of glucose uptake by angiotensin IV and LVV‐hemorphin 7 is region‐specific and is critically dependent on a high degree of colocalisation between IRAP and GLUT4. These findings also confirm a role for IRAP and GLUT4 in activity‐dependent glucose uptake in hippocampal neurones.

Distribution and cellular localization of insulin-regulated aminopeptidase in the rat central nervous system

Central infusions of angiotensin IV enhance spatial learning, memory retention and retrieval, neurotransmitter release, and long‐term potentiation via interaction with a specific, high‐affinity binding site. This site was recently purified and identified as the insulin‐regulated aminopeptidase (IRAP). This enzyme was previously characterized as the marker protein of specialized insulin‐responsive vesicles containing GLUT4 in muscle and adipose tissue. The present study provides the first comprehensive description of IRAP distribution in the adult rat brain. By using immunohistochemistry, IRAP was found to be highly expressed in selected olfactory regions, in septal and hypothalamic nuclei, throughout the hippocampal formation and cerebral cortex, and in motor and motor associated nuclei. IRAP was expressed exclusively in neurons in these regions. At the cellular level, IRAP was localized within cell bodies, excluding the nucleus, in a punctate vesicular pattern of expression. IRAP‐positive immunoreactivity was also found in some proximal processes but was not detected in synaptic nerve terminals. The neurochemical composition of IRAP‐containing neurons was further characterized by dual‐label immunohistochemistry. IRAP was expressed in cholinergic cell bodies of the medial septum, a source of cholinergic projections to the hippocampus and cerebral cortex. The distribution of IRAP in motor and motor‐associated nuclei; the colocalization of the enzyme with potential in vivo substrates, oxytocin and vasopressin in the hypothalamus; and the colocalization with GLUT4 in selected nuclei all suggest diverse physiological roles for IRAP in the rat central nervous system. J. Comp. Neurol. 487:372–390, 2005.

Identification and development of specific inhibitors for insulin-regulated aminopeptidase as a new class of cognitive enhancers

Two structurally distinct peptides, angiotensin IV and LVV‐haemorphin 7, both competitive high‐affinity inhibitors of insulin‐regulated aminopeptidase (IRAP), were found to enhance aversion‐associated and spatial memory in normal rats and to improve performance in a number of memory tasks in rat deficits models. These findings provide compelling support for the development of specific, high‐affinity inhibitors of the enzyme as new cognitive enhancing agents. Different classes of IRAP inhibitors have been developed including peptidomimetics and small molecular weight compounds identified through in silico screening with a homology model of the catalytic domain of IRAP. The proof of principal that inhibition of IRAP activity results in facilitation of memory has been obtained by the demonstration that the small‐molecule IRAP inhibitors also exhibit memory‐enhancing properties.

Gene knockout of insulin-regulated aminopeptidase: Loss of the specific binding site for angiotensin IV and age-related deficit in spatial memory

The AT(4) ligands, angiotensin IV and LVV-hemorphin 7, elicit robust effects on facilitating memory by binding to a specific site in the brain historically termed the angiotensin AT(4) receptor. The identification of the AT(4) receptor as insulin-regulated aminopeptidase (IRAP) is controversial, with other proteins speculated to be the target(s) of these peptides. In this study we have utilized IRAP knockout mice to investigate IRAP in the brain. We demonstrate that the high-affinity binding site for angiotensin IV is absent in IRAP knockout mice brain sections in parallel with the loss of IRAP immunostaining, providing irrefutable proof that IRAP is the specific high-affinity binding site for AT(4) ligands. However, our characterization of the behavioural phenotype of the IRAP knockout mice revealed a totally unexpected finding. In contrast to the acute effects of IRAP inhibitors in enhancing memory, deletion of the IRAP gene resulted in mice with an accelerated, age-related decline in spatial memory that was only detected in the Y maze paradigm. Moreover, no alterations in behaviour of the IRAP knockout mice were observed that could assist in elucidating the endogenous substrate(s). Our results highlight the importance of analysing the behavioural phenotype of knockout mice across different ages and in distinct memory paradigms.

Sub-cellular localization of insulin-regulated membrane aminopeptidase, IRAP to vesicles in neurons.

Angiotensin IV and LVV‐hemorphin 7 promote robust enhancing effects on learning and memory. These peptides are also competitive inhibitors of the insulin‐regulated membrane aminopeptidase, suggesting that the biological actions of these peptides may result from inhibition of IRAP activity. However, the normal function of IRAP in the brain is yet to be determined. The present study investigated the sub‐cellular distribution of IRAP in four neuronal cell lines and in the mouse brain. Using sub‐cellular fractionation, IRAP was found to be enriched in low density microsomes, while lower levels of IRAP were also present in high density microsomes, plasma membrane and mitochondrial fractions. Dual‐label immunohistochemistry confirmed the presence of IRAP in vesicles co‐localized with the vesicular maker VAMP2, in the trans Golgi network co‐localized with TGN 38 and in endosomes co‐localized with EEA1. Finally using electron microscopy, IRAP specific immunoreactivity was predominantly associated with large 100–200 nm vesicles in hippocampal neurons. The location, appearance and size of these vesicles are consistent with neurosecretory vesicles. IRAP precipitate was also detected in intracellular structures including the rough endoplasmic reticulum, Golgi stack and mitochondrial membranes. The sub‐cellular localization of IRAP in neurons demonstrated in the present study bears striking parallels with distribution of IRAP in insulin responsive cells, where the enzyme plays a role in insulin‐regulated glucose uptake. Therefore, we propose that the function of IRAP in neurons may be similar to that in insulin responsive cells.

Distinct distribution of GLUT4 and insulin regulated aminopeptidase in the mouse kidney.

The physiological importance of the insulin responsive glucose transporter GLUT4 in adipocytes and muscle in maintaining glucose homeostasis is well established. A key protein associated with this process is the aminopeptidase IRAP which co-localizes with GLUT4 in specialized vesicles, where it plays a tethering role. In this study, we investigated the distribution of both GLUT4 and IRAP in the kidney to gain insights into the potential roles of these proteins in this organ. Both IRAP and GLUT4 immunostaining was observed in the epithelial cells of the proximal and distal tubules and thick ascending limbs in the cortex, but very little overlap between GLUT4 and IRAP immunoreactivity was observed. GLUT4 staining was consistent with a vesicular localization, whereas IRAP staining was predominantly on the luminal surface. In the principal cells of the inner medulla collecting duct (IMCD), IRAP immunoreactivity was detected throughout the cell, with limited overlap with the vasopressin responsive water channel aquaporin-2 (AQP-2). AQP-2 levels were observed to be two-fold higher in IRAP knockout mice. Based on our results, we propose that GLUT4 plays a role in shunting glucose across epithelial cells. In the kidney cortex, IRAP, in concert with other peptidases, may be important in the generation of free amino acids for uptake, whereas in the principal cells of the inner medulla IRAP may play a localized role in the regulation of vasopressin bioactivity.

Insulin-regulated aminopeptidase

Although insulin-regulated aminopeptidase (IRAP) was first described as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter, GLUT4, the protein has subsequently been shown to be identical to oxytocinase or the AT4 receptor. In insulin responsive tissues, IRAP is almost exclusively co-localized with GLUT4, being retained in intracellular compartments in the basal state or translocating with GLUT4 to the plasma membrane under insulin stimulation. The function of IRAP in these tissues has not been elucidated — the protein is thought to be involved in the tethering of GLUT4 vesicles to intracellular compartments. In the placenta, IRAP was isolated as the enzyme that degrades oxytocin and is therefore thought to play a role in the prevention of premature labour and maintenance of an adequate blood flow to the uterus. Potentially the most exciting physiological role attributed to IRAP is the involvement of the enzyme in memory processing. AT4 ligands, upon binding to the catalytic site of IRAP, enhance spatial learning, facilitate memory retention and retrieval and reverse amnesia. We postulate that the AT4 ligands act via either one of these mechanisms: (1) The peptides bind to the catalytic site of IRAP and inhibit its enzymatic activity thereby prolonging the half-life of its neuropeptide substrates with memory-enhancing properties or (2) Upon binding to IRAP, the AT4 ligands regulate the level of GLUT4 expressed at the cell surface resulting in an increase in glucose uptake into neurones.