Svetlana A. Plesneva

Effect of Hypoxia/Ischemia and Hypoxic Preconditioning/Reperfusion on Expression of Some Amyloid‐Degrading Enzymes

Abstract: Alzheimers disease (AD) is linked to certain common brain pathologies (e.g., ischemia, stroke, and trauma) believed to facilitate its development and progression. One of the logical approaches to this problem is to study the effects of ischemia and hypoxia on the metabolism of amyloid precursor protein, which plays one of the key roles in the pathogenesis of AD. This involves an analysis of (1) proteases, which participate in proteolysis of amyloid precursor protein either by the nonamyloidogenic route (α‐secretase) or the amyloidogenic pathway and lead to formation of toxic β‐amyloid peptides (β‐ and γ‐secretases) and (2) several metallopeptidases that might play a role in degradation of β‐amyloid peptide (Aβ). The study of the effects of prenatal hypoxia and acute hypoxia in adult animals allowed us to conclude that oxygen deprivation results not only in an increase of amyloid precursor protein expression in the brain but also in a decrease in the activity of α‐secretase. In some brain structures involved in AD pathology (the cortex and striatum), we also observed a decrease in the expression of two of the Aβ degrading enzymes, neprilysin and endothelin‐converting enzyme, after hypoxia. A decrease in expression of these metalloproteases was also observed in the model of four‐vessel occlusion ischemia in rats with their restoration to the control levels after reperfusion. Preconditioning to mild hypoxia both in the prenatal period and in adults appeared to have a neuroprotective effect restoring, in particular, the levels of amyloid precursor protein, activity of a‐secretase, and expression of neprilysin and endothelin‐converting enzyme to their control values.

Effect of Sodium Valproate Administration on Brain Neprilysin Expression and Memory in Rats

Alzheimers disease (AD) is accompanied by memory loss due to neuronal cell death caused by toxic amyloid β-peptide (Aβ) aggregates. In the healthy brain, a group of amyloid-degrading enzymes including neprilysin (NEP) maintain Aβ levels at physiologically low concentrations but, with age and under some pathological conditions, expression and activity of these enzymes decline predisposing to late-onset AD. Hence, up-regulation of NEP might be a viable strategy for prevention of Aβ accumulation and development of the disease. As we have recently shown, inhibitors of histone deacetylases, in particular, valproic acid (VA), were capable of up-regulating NEP expression and activity in human neuroblastoma SH-SY5Y cell lines characterised by very low levels of NEP. In the present study, analysing the effect of i.p. injections of VA to rats, we have observed up-regulation of expression and activity of NEP in rat brain structures, in particular, in the hippocampus. This effect was brain region- and age-specific. Administration of VA has also restored NEP activity and memory deficit in adult rats caused by prenatal hypoxia. This suggests that VA and more specific HDAC inhibitors can be considered as potential pharmaceutical agents for up-regulation of NEP activity and improvement of cognitive functions of ageing brain.

Functional defects in adenylyl cyclase signaling mechanisms of insulin and relaxin in skeletal muscles of rat with streptozotocin type 1 diabetes

Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.

A novel view on the mechanisms of action of insulin and other insulin superfamily peptides: involvement of adenylyl cyclase signaling system.

A new signaling mechanism common to mammalian insulin, insulin-like growth factor I, relaxin and mollusc insulin-like peptide, and involving receptor-tyrosine kinase==>G(i) protein (betagamma)==>phosphatidylinositol-3-kinase==>protein kinase Czeta==>adenylyl cyclase==>protein kinase A was discovered in the muscles and some other tissues of vertebrates and invertebrates. The authors data were used to reconsider the problem of participation of the adenylyl cyclase-cAMP system in the regulatory effects of insulin superfamily peptides. A hypothesis has been put forward according to which the adenylyl cyclase signaling mechanism producing cAMP has a triple co-ordinating role in the regulatory action of insulin superfamily peptides on the main cell processes, inducing the mitogenic and antiapoptotic effects and inhibitory influence on some metabolic effects of the peptides. It is suggested that cAMP is a key regulator responsible for choosing the transduction pathway by concerted launching of one (proliferative) program and switching off (suppression) of two others, which lead to cell death and to the predomination of anabolic processes in a cell. The original data obtained give grounds to conclude that the adenylyl cyclase signaling system is a mechanism of signal transduction not only of hormones with serpentine receptors, but also of those with receptors of the tyrosine kinase type (insulin superfamily peptides and some growth factors).

Studies of the molecular mechanisms of action of relaxin on the adenylyl cyclase signaling system using synthetic peptides derived from the LGR7 relaxin receptor.

The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619–629, 619–629-Lys(Palm), and 615–629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619–629-Lys(Palm) and 615–629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385–394 of the αs subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with Gs protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the Gs protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C-terminal segment of the αs subunit of the G protein.

Effects of prenatal hypoxia on expression of amyloid precursor protein and metallopeptidases in the rat brain

In the present study we have investigated the effect of prenatal hypoxia on expression of amyloid precursor protein (APP) and some metallopeptidases, which regulate β-amyloid peptide (Aβ) levels (neprilysin (NEP) and endothelin-converting enzyme (ECE-1)) in the cortex of rats during different periods of postnatal development. We have found that the level of APP in the sensorimotor cortex (SMC) of rats, analysed by Western blotting, increases from days 1 to 5 of postnatal development and then steadily decreases with age, with the most dramatic decline in the period from day 180 to 600. In the cortex of rats subjected to prenatal hypoxia on day 13.5 of embryogenesis, the postnatal levels of APP were higher than in the control. Secretion of the soluble form of APP (sAPP) by α-secretase was found to be the most active on day 30 of postnatal development and there was a significant decrease in the production of sAPP after prenatal hypoxia. NEP was found to be expressed in the cortex of rats only at the early stages of postnatal development and it was barely detectable in adult rats. The decline of NEP levels during ageing might contribute to accumulation of Aβ in later life in humans. Prenatal hypoxia resulted in a significant decrease of NEP expression on day 10, but its level was recovered when animals were preconditioned to mild hypoxia. A similar phenomenon was observed when the expression of ECE-1 was analysed. Overall, prenatal hypoxia leads to significant changes in the levels of APP and expression of metallopeptidases involved in amyloid metabolism during all postnatal life and preconditioning to hypoxia appeared to be neuroprotective.

Decrease in functional activity of G-proteins hormone-sensitive adenylate cyclase signaling system, during experimental type II diabetes mellitus.

The development of experimental type II diabetes mellitus in rats was accompanied by dysfunction of inhibitory and stimulatory heterotrimeric G-proteins, components of hormone-sensitive adenylate cyclase signal system. The function of inhibitory G-proteins decreased most significantly under these conditions, which is seen from weakened regulatory effects of somatostatin (in the myocardium) and bromocriptine (in the brain striatum) realized via inhibitory G-proteins in diabetic rats compared to controls. These hormones produce less pronounced inhibitory effect on forskolin-induced activation of adenylate cyclase. In the myocardium of diabetic rats, the stimulatory effects of isoproterenol and relaxin on adenylate cyclase realized via stimulatory G-proteins were decreased to a lesser extent. In the striatum of diabetic rats the stimulatory effect of serotonin and relaxin did not differ from the control. Therefore, dysfunction of stimulatory G-proteins during type II diabetes mellitus is characterized by tissue specificity. Synthetic peptides corresponding to functionally important regions in α-subunits of G-proteins and relaxin receptor LGR7 less effectively inhibited hormone signal transduction via the adenylate cyclase system in rats with type II diabetes. These changes reflect abnormal coupling between receptors and G-proteins in tissues of diabetic rats.

Effects of biogenic amines and glucagon on the adenylate cyclase system in molluscan, holothurian and ascidian muscle membranes

Abstract The in vitro effects of biogenic amines and glucagon on the adenylate cyclase activity of muscle membranes of molluscs ( Tonicella marmorea, Mytilus edulis, Mizuhopecten yessoensis, Spisula sachalinesis, Littorina littorea, Lymnaea stagnalis, Planorbarius corneus ), holothurian ( Cucumaria japonica ) and ascidian ( Halocyntia aurantium ) were studied. Glucagon, β-adrenergic agonists and serotonin have a stimulating effect on the sarcolemmal adenylate cyclase activity potentiated by guanine nucleotides. The serotonin antagonist, cyproheptadine, and β-antagonists blocked the effect of hormones. Serotonin receptor (5-HTR) and β-adrenoreceptor (β-AR) in the sarcolemma of Anodonta cygnea, Mizuhopecten yessoensis, Spisula sachalinesis, Cucumaria japonica and Halocyntia aurantium were identified. A negative regulation of 5-HTR and β-AR affinity by guanine nucleotides was revealed, which provides evidence of the functional coupling of the receptors with GTP-binding protein. The results of the comparative study of the hormone-sensitive adenylate cyclase system suggest a certain similarity in the structural and functional organization of the system in invertebrates and vertebrates.

Molecular Mechanisms of Modified Sensitivity of the Adenylate Cyclase Signaling System to Biogenic Amines during Streptozotocin-Induced Diabetes

We demonstrated changes in the sensitivity of the adenylate cyclase signaling system to biogenic amines (adrenoceptor agonists and serotonin) underwent a change in skeletal muscles of rats with 30-day streptozotocin-induced diabetes. Isoproterenol had a less significant stimulatory effect on adenylate cyclase in diabetic rats. Hormonal signals via Gi proteins were suppressed in animals with diabetes, which determined a greater stimulatory effect of norepinephrine and serotonin on adenylate cyclase. Hormones less significantly increased guanosine triphosphate-binding activity of G proteins in diabetic rats, which reflects the impairment of their functional coupling with receptors.

Role of phosphatidylinositol-3-kinase and protein kinase Cζ in the adenylate cyclase signal mechanism of action of relaxin in muscle tissues of rats and mollusks

We showed that phosphatidylinositol-3-kinase and protein kinase Cζ are involved in the adenylate cyclase signal mechanism of relaxin action. A selective inhibitor of phosphatidylinositol-3-kinase wortmannin blocked the stimulatory effect of relaxin on adenylate cyclase in rat skeletal muscles and Anodonta cygnea smooth muscles. Antibodies against protein kinase Cζ abolished the relaxin-induced stimulation of adenylate cyclase in rat muscles, but not in mollusk muscles. Our results indicate that phosphatidylinositol-3-kinase and protein kinase Cζ play a role in the adenylate cyclase signal mechanism of relaxin action.