New Mechanisms of Neurohormonal Regulation under Conditions of Neurodegeneration

It was revealed that a synthetic analogue of hypothalamic neurohormone, proline-rich polypeptide (PRP), possesses a regulatory activity on development of neurochemical, neurohormonal, immunomodulatory and morphofunctional impairments occurring in models of brain neurodegenerative disorders typical for Alzheimer’s disease. These data were obtained in models of aluminum neurotoxicosis and via intracerebroventricular injection of the aggregated form of beta-amyloid peptide. In a model of aluminum neurotoxicosis, it was obviously shown that different doses of PRP produced effect on synthesis of immunocytokines (IL-1, IL-2, and IL-6), prolactin, and insulin-like growth factor-1 (IGF-1), which can be produced in situ in hippocampus and brain cortex directly or indirectly participating in the processes of atypical neuronal amyloid protein formation during neurodegenerative diseases. The regulatory effect of PRP was shown on levels of proand antioxidant metalloproteins of blood and tissues under the oxidative stress typical for aluminum toxicosis that signifies to the influence of PRP on the processes of lipid peroxidation. The antioxidant role of PRP to regulate the intensity of lipid peroxidation processes was quite obviously demonstrated. One of the most significant achievements of the research is detailed characterization of the brain. There was established the ability of PRP to prevent the accumulation of aluminum in the cytoplasm of hippocampus neurons and their penetration through the nuclear membrane. Data concerning the ability of PRP to prevent the development in the neurons of hippocampal complex, magnocellular nuclei of hypothalamus (SO and PV), neuronal cells of motor and limbic parts of cerebral cortex neurodegenerative changes evoked by microinjection of beta amyloid peptide (25-35) are undoubtful. For the first time in the intact animals and in a model of brain amyloid neurodegeneration neurophysiologic studies have revealed the peculiarities of PRP action at the preand post-stimulus impulse current of hippocampus pyramidal neuron during single and frequent excitation of cerebral entorhinal cortex. There were accepted persuasive arguments of functional character proving a developing conception concerning an existence of expressed neuroprotective action of PRP during brain neurodegenerative impairments. Thus, the results of recent research work allowed us to fulfill the present conception about multiform expressions of high biological activity of PRP and convincingly to demonstrate the existence of its central and peripheral immunomodulating action, the ability to regulate the brain free radical processes, the level of lipid peroxidation and as a whole to prevent and correlate the brain neurodegenerative impairments. Address for correspondence: Yerevan State Medical University after M. Heratsi 2 Koryun Street, 0025, Yerevan, Armenia Tel.: (+3741 0) 582412, E-mail: [email protected] H.G. VAHRADYAN, M.I. AGHAJANOV / THE NEW ARMENIAN MEDICAL JOURNAL 7 Introduction One of the fundamental achievements of modern neurochemistry and neuroimmunology is the discovery of a new family of neurohormones, cytokines of hypothalamus, which are synthesized in neurosecretory cells of N. Supraopticus and N. Paraventricularis (NSO, NPV) and are transported by the peptidergic fibers of hypotha lamo-hypophyseal tract into the neurohypophysis [Galoyan A., 1997] . The primary structure of proline-rich polypeptide (PRP) neurohormones, which are transported axonal (and humoral) up to a spinal cord (SC), has been decoded. The investigations have shown that PRP is a product of proteolysis of the neurophysin-vasopressin-associated C-terminal glycoprotein and, along with vasopressin and oxytocin, is transferred from hypotha lamus toward the neurohypophysis by the axonal transport. The discovery of neurosecretion of cells of the hypotha lamic magnocellular nuclei (NPV and NSO), formation of vasopressin and oxytocin in them [Du Vigneaud V., 1954] underlay a new field of research in the modern molecular neuroendocrinology. The next discovery refers to hypotha lamic releasing hormones, the isolation, and study of these peptides [Guillemin R., 1977; Schally A. et al., 1978]. The hormones (LH-RH), (GH-RH), somatostatin and others were revealed. The role of the analogue of releasing hormones (LH-RH, etc.) in treatment of cancers of various organs has been established [Schally A. et al., 2001]. Further development of mentioned directions has an important value for understanding brain functions and organization of neurohormonal functions as a whole. This is the basis for creation of new endogenic neurohormonal preparations for treatment of nervous, endocrine, immune, and other diseases. The detailed study of signal molecules of the brain neuroendocrine immune system revealed the occurrence of peptides synthesized in hypothalamus, many of which are produced in the hypotha lamus neurosecretory cells. Their primary structure was established [Galoyan A., 1997]. In particular, in the hypotha lamus neurosecretory cells there was revealed an immunophiline consisting of 107 amino acids and participating in the mechanisms of interleukin biosynthesis (in particular IL-2). The biosynthesis of interleukins IL-lα, IL-1β, IL-2, IL-6, TNF, etc. in the hypotha lamus neurosecretory cells and their output in neurohypophysis was simultaneously established [Galoyan A., 1997; Zilfyan A.V., Soghbatyan L.T., 2000]. During the last decade, in laboratory of Professor A. Galoyan there was discovered a new family of PRPs including 4 peptides, each of which possesses specific properties referring to various functions of an organism. PRP as having a wide spectrum of biological activity is the most investigated one; in our work it is marked as PRP [Galoyan A. et al., 2004a;b; Vahradyan H. et al., 2004]. The wide spectrum of PRP biological properties is considered in the aspect of their metabolic activity, antibacterial and antiviral action, myelogenesis, lymphopoiesis, immunomodulatory action, and neuroprotective as well as neurohormonal properties [Aghajanov M. et al., 2000; Galoyan A., 2000; Gladkevich A. et al., 2007]. Numerous data specify the polyfunctionality of PRP and the involvement of mentioned neuropeptides in many biologically significant processes in norm and in case of pathology [Aghajanov M. et al., 2002; Shakhlamov V. et al., 2002; Galoyan А. et al., 2008]. Thus, it is established that PRP participates in the expression mechanisms of interleukins (TNF, IL-1, IL-6) in fibroblasts, macrophages, and astrocytes. The investigations performed by A. Galoyan on PRPmediated inhibition of pro-apoptotic caspase-3 and -9, as well as activation of caspase-2 and -6 [Galoyan A. et al., 2000b], were the beginning of studies on universal protective properties of PRP in the neurodegene rative processes. PRP, being immunomodulator and stimulator of immunocompetent cells activates the formation of IL-1, IL-6 and TNF-α in astrocytes [Galoyan A., 1997], exerts the neuroprotective action in case of intoxications evoked by snake poisons, hemisection of a spinal cord [Galoyan A. et al., 2000a; Ð.¶.ì2Ðð2 ̧Ú2Ü, Ø.Æ. 2Ô2æ2Üàì / Üàð Ð2ÚÎ2Î2Ü ́ÄÞÎ2Î2Ü Ð2Ü ̧oê 8 Sarkisyan J. et al., 2003], shows the antibacterial influence in vivo and antiviral action in vitro [Aprikyan V., Galoyan A., 2003]. Finally, PRP is the NGF-like compound stimulating the secretion of those compounds from the neuroglial elements [Chekhonin V. et al., 2001]. The problem of modeling neurodegenerative lesions of CNS, to which the Alzheimer's disease (AD) also pertains, is closely connected to extremely actual problem to study both pathogenesis and treatment of this most severe human disease [Vahradyan H., Vahradyan V., 2003] Data available on AD is based on results of postmortem investigations/studies, therefore they provide information only about the finishing, occasionally terminal stages of disease, thus bringing forth a problem of working-out the correct AD experimental models to the utmost approximated to the used pathology in a number of actual problems of neuroscience. Reproducing an adequate model assumes a discovery of new priority directions in studies of the cellular-and-molecular bases of its pathogenesis and purposeful development of ways for prophylaxis and therapy. The purpose of research was to study the protective and corrective actions of synthetic analogue of hypothalamic neurohormone, PRP, on mechanisms peculiar to development of neurochemical, neurohormonal, immunomodulatory and morphofunctional disorders arising in case of aluminum neurotoxicosis and neurodegenerative changes, induced by the β-amyloid peptide (the AD-model). The following tasks were formulated according to the aim of the study: • under conditions of aluminum neurotoxicosis to investigate immunomodulatory action of various concentrations of PRP – its ability to influence the production of immunocytokines (IL-1 IL-2, IL-6) and insulin-like growth factor (IGF-1) in various formations of a brain (an integral brain, hippocampus and cerebral cortex), thymus, liver and blood serum of experimental animals; • in a model of the aluminum neurotoxicosis to study the role of neuroendocrine mechanisms and, in particular, that of hypophyseal prolactin in realization of immunomodulating action of optimal and low PRP doses on synthesis of various immunocytokines in an integral brain, hippocampus, thymus, liver and blood serum; • to characterize integrally the ability of PRP to prevent and correct various manifestations of the oxidative stress developing in case of the acute and chronic aluminum toxicosis as disorders in the system of blood proand antioxidant metalloproteins, including a level of cytochromes: b5, ∑b558 I-IV of the serum, ∑b558 of erythrocyte membranes, the content and O2-producing ability of suprol, activity of Cu, Zn-SOD, catalase, ceruloplasmin and transferrin; • to investigate the influence of PRP on the shifts of tissue level of metalloproteins as the enzymes of antiradical protection of a cell, Cu, Zn-SOD, catalase, cytochrome c and content of malone dialdehyde (MDA) in an integral brain and hippocampus, developing in case of the acute and chronic aluminum neurotoxicosis; • to study the ability of PRP to prevent the ultrastructural neurodegenerative lesions of different structures of the brain, developing in neurons of hippocampal complex, of motor and limbic zones of cortex and cells of magnocellular nuclei of hypothalamus (supraoptical and paraventricular) in case of aluminum neurotoxicosis and intracerebroventricular introduction of the aggregated form of β-amyloid peptide (25-35); • to study the preventive and corrective PRP action on preand post-stimulus characteristics of a pulse stream flow of the pyramidal neuron in СА2 of hippocampus under conditions of a single and frequency irritation of the entorhinal cortex of a brain of the same hemisphere in norm and in model of neurodegenerative lesion induced by introduction of β-amyloid peptide into the lateral ventricles of the brain. Material and Methods The experimental models. According to the tasks put forward at the various stages, the investigations were carried out in different variants of AD experimental modeling. The research was carried out in white male mice H.G. VAHRADYAN, M.I. AGHAJANOV / THE NEW ARMENIAN MEDICAL JOURNAL 9 (weight = 30-40 g) and pubertal inbred white rats (weight = 180-200 g). • The acute aluminum neurotoxicosis was caused by subcutaneous injection of 0.2 ml 10% solution of aluminum chloride to white rats. Likewise, the control animals were administered 0.9% solution of NaCl. Animals were decapitated under the mild ethereal anesthesia. Blood and other organs (brain, hippocampus, and liver) were drawn and placed in various containers on a cold and processed simultaneously. In case of double introduction of aluminum chloride there was revealed a rather low mortality of animals that formed the basis for using the specified dose for AD-modeling in rats. An experimental model of AD in mice was obtained by the single subcutaneous introduction of 0.2 ml of 3% aluminum chloride. The animals were divided into the following three groups: Control Group of intact animals, Experimental Group I with the single injection of АlСl3, Experimental Group II– at the 4th day after introduction of АlСl3 animals were exposed to intraperitoneal (i/p) administration of PRP at a dose level of 15 and 1.5 μg/40 g of animal weight. The animals were sacrificed on the 8th or 12th day after injection of АlСl3 (mice and rats, accordingly). • The chronic aluminum neurotoxicosis in white rats was induced by oral administration (with drinking water) of the 10% solution of aluminum chloride to animals within one and three months; • The model of amyloidal brain damage. With the purpose to induce the amyloid damage of brain and study the neurotoxic properties of βamyloid peptide fragment – βА (25-35) in processes of neurodegeneration, 5 series of the investigation were carried out in male rats with body weight of 250-300 g under the nembutal narcosis: 1) intact animals; 2) animals injected by the sterile distilled water intracerebroventricularly (icv) into the lateral ventricles of brain from both sides on the coordinates of stereotaxic atlas [Paxinos G., Watson C., 2007] АР-1 mm, L±l.5 mm, DV+3.5 mm with the speed of 1 mcl/min and being kept before the experiment within 45 days; 3) animals injected icv by 30 nmol of βА (25-35), aggregated according to T. Maurice and A. Privat [Maurice T., Privat A., 1998] as l mg/ml in sterile distilled water at t=37°C within 4 days into the lateral ventricles of brain from both sides with the rate of 1 mcl/min and being kept before the experiment within 45 days; 4) animals injected icv by aggregated βА (25-35) in a combination with the single intra-abdominal (i/a) injection of 15 μg of PRP, 24 hours prior to introduction of βА; 5) animals icv injected aggregated βА (25-35) in a combination with single intra-peritoneal (i/p) injection of 15 mсg of PRP on the 6th day after introduction of βА. The neuromorphological investigations. The isolated rat brain was fixed during 1-3 days in 10% formalin at 4°С and then washed out overnight in physiological solution containing 15% of saccharose for preserving the structures. The brain was put in paraplast and frontal sections with the thickness of 10 microns were obtained. The frontal frozen sections (10 microns) and free-floating microtome sections (50 microns) from the various parts of the brain were washed out in 10% and 5% solution of saccharose and then in a physiological solution. With the purpose of revealing the nervous structures, sections were stained with toluidine blue by Rokhman’s method. After staining within 15 minutes in 0.1%-solution of toluidine blue prepared in acetate buffer with рН=4.8, the sections were washed out in abundant amount of buffer, then incubated within 10 minutes in 1% solution of potassium ferrocyanide prepared in distilled water. The material was dehydrated in three portions of 70° alcohol and in two portions of xylol, and then put into the canadian balm; in sections stained with toluidine blue, the estimation of neurodegeneration was carried out. Synthesis and identification of a new hypothalamic PRP, consisting of 15-amino acid residues, as well as β-amyloid neurоpeptide (25-35) was synthesized in the laboratory of Prof. Galoyan by the use of F-moc amino acid residue. The molecular weight and purity degree of peptide were determined with the help of high performance Ð.¶.ì2Ðð2 ̧Ú2Ü, Ø.Æ. 2Ô2æ2Üàì / Üàð Ð2ÚÎ2Î2Ü ́ÄÞÎ2Î2Ü Ð2Ü ̧oê 10 liquid chromatography (HPLC) and the mass spectrometry analysis (Matrix-assis ted laser desorption/ionization – MALDI). The preparation was dissolved in the NaCl solution, sterilized by filtration (the size of pores equal to 0.22 microns) with the subsequent spectrophotometric determination of its content in 1 ml solution. Determination of proand antioxidant metalloproteins. White pubertal rats were used in order to study the neuroprotective influences of PRP on content of proand antioxidant action metalloproteins. Animals were divided into four groups: Control Group (i/p injection of 0.2 ml of physiological solution on the 1st and 3rd days of the experiment); Experimental Group I (s/c introduction of 0.2 ml of 10% so lution of А1С13 on the 1st and 3rd days of experiment); the Experimental Group II (i/p introduction of 20 μg of PRP on the 6th day after А1С13 injection); Experimental Group III (i/p introduction of 20 μg of PRP one hour prior to an injection of aluminum chloride). In the following series of experiment the dosedependent effect of PRP on the content of the mentioned metalloproteins was investigated. The animals were sacrificed on the 12th day of the experiment. Blood metalloproteins of prooxidant (cytochrome В5 from the hemolysate, В558I and В558II from blood serum, B558III and B558IV from the erythrocyte membranes and suprol from the blood serum) and antioxidant action (Cu, Zn-SOD and catalase from hemolysate, ceruloplasmin (CP) and transferrin (TF) from the blood serum) were simultaneously obtained according to the method suggested by M. Simonyan [Simonyan M., 1988]. After separation of serum and erythrocytes and washing-up of the latter from various corpuscles and plasma with the use of physiological solution, cleaned erythrocytes were hemolyzed in water. The erythrocyte membranes were separated by centrifugation at рН=5.6 and the membrane proteins were solubilized by the 0.5% non-ionic detergent (a triton X-100 and nonidet Р-40, Germany). After the dialysis of hemolysate, the serum and fractions of membrane proteins were dialyzed against water and after centrifugation the supernatants were analyzed by the ionexchange chromatography on cellulose of KA-52 and DЕ-52 (Whatman, England) and on Sephadex DЕАЕ A-50 (Pharmacia, Sweden). The enriched protein fractions were purified on biogels Р-100 and Р-150 (Reanal, Hungary). In procedure of obtaining and cleaning metalloproteins the centrifuges K-24, K-70, spectrophotometer "Specord M-40" (Germany) and glass columns with filters (2×20 cm and 2×80 cm) were used. Superoxide dismutase (SOD) activity and superoxide-producing activity of suprol were determined in blood serum and rat tissues by nitrotetrazolium method [Nishikimi М. et al., 1972] by determination of inhibition percentage or increase (in case of suprol) in formation of formazan at the rate calculated per 1 ml of blood serum or 1 g tissue, correspondingly. The catalase activity of fractions obtained from homogenates of brain tissues, hippocampus, liver and erythrocyte membranes was determined by the spectral method or permanganatometry with calculation of the amount of hydrogen peroxide cleavage in presence of the certain amount of fractions for 1 minute at 20°С. The specific activities were determined by calculation per 1 ml of serum (for suprol) and 1 ml of erythrocytes (for SOD and catalase). The ferroxidase activity of ceruloplasmin in blood serum of rats was determined by the spectral method [McGahan М. et al., 1989]. The values of density of characteristic maximum optical absorptions were used as a quantitative index of blood metalloproteins. The amount of transferrin was defined by a density value of the maximum optical absorption at 470 nm. The isolation of metalloproteins of antioxidant action (Cu, Zn-COD and catalase) from brain was carried out by M.A. Simonyan's method [Simonyan M., 1988], with some modification. The tissues of brain, hippocampus and liver in the certain ratio were homogenated in 0.04 M potassium phosphate buffer at рН=7.4. A part of homogenates was left for MDA by the method of H.G. VAHRADYAN, M.I. AGHAJANOV / THE NEW ARMENIAN MEDICAL JOURNAL 11 Yu. Vladimirov and A. Archakov [Vladimirov Yu., Archakov A., 1972]. Further, the homogenates were homogenized in acetone. The acetonic powders were mixed with 0.04 M potassium phosphate buffer and incubated for one hour. The insoluble residue was removed by centrifugation, and supernatant was dialyzed against water. The dialyzed supernatants of these mixes were subjected to ion-exchange chromatography on cellulose DЕ-52. SOD and catalase from columns were eluted by the 0.03 M and 0.1 M potassium phosphate buffer, correspondingly. The neuroimmunological investigations. In order to study the PRP-immunomodulating influence on the content of cytokines (IL-1, IL-2, IL-6), prolactin and insulin-like growth factor-1 (IGF-1), the blood serum and supernatants prepared from the internal organs (an integral brain tissue, hippocampus, thymus, and liver) of mice were subjected to immune-enzyme analysis (ELISA). For this purpose, the animals were decapitated and an integral brain, liver, and thymus were taken. After cleaning the brain from dura mater and vessels, as well as washing off blood by the cooled isotonic NaCl-solution the material was exposed separately on ice. The cortex of cerebral hemispheres and hippocampus were kept in liquid nitrogen freeze until the research was performed. Then the tissues were weighed and homogenated in the separation medium of 0.9%-solution of NaCl (1:10) in homogenizer "Potter S", with the speed 1500 rot/min within 1 minute. The homogenates were centrifuged at 3000 rot/min during 10 minutes at 4°С, and then the supernatants were selected. The cytokines were determined by means of appropriate kits (DRG International Inc., USA) and their amount expressed in pg/ml. The content of prolactin was determined using corresponding kits (MicrowellTM FSH EIA, USA). The amount of prolactin was expressed in ng/ml. The radioisotope method for determination of insulin-like growth factor-1 (IGF-1). IGF-1 in a cerebral cortex and blood serum of mice was determined with the help of the radioisotope counter "Gamma" (Russia–Ukraine) by means of IGF-1 I125 kit (Amersham Biotech., USA). The radioisotope activity was expressed in pulses per minute. The determined indices were inversely proportional to IGF-1 concentration, i.e. the high activity corresponded to low concentrations of a hormone, and vice versa. Electronic microscopy studies in case of aluminum neurotoxicosis were carried out by Prof. A. Shakhlamov and Prof. A. Galoyan at the Institute of Human Morphology of the Russian Academy of Medical Science (Moscow). The investigations were carried out in 30 inbred male rats weighing 120 g arranging the following series: the I series: 3 rats (control’ 1) – intact, without injection; the II series: 2 rats (control’ 2), with daily subcutaneous injection of 0.2 ml 10%solution of АlСl3 within 3 days; the III series: 5 rats preliminary administered 0.5 ml of PRP-solution containing 0.1 mg of PRP, next day the same animals were injected 0.2 ml 10%-АlСl3, on the 3rd day again 0.2 ml of 10%-АlСl3, and on the 4th day 0.2. ml 10%-АlСl3 as well. The animals were sacrificed under the ethereal anesthesia, slices of tissues from hippocampus were fixed by 2.5%-solution on a cold glutaraldehyde on the cocadilate buffer (рН=7.3). After double wash-up within 24 hours the tissue slices were fixed by 1%-solution of osmium tetroxide on cocadilate solution (рН =7.3) during 2 hours, and it was washed out again by the buffer, and then dipped in alcohols of increasing concentrations. The embedding of tissue slices was carried out in an epon-araldite. Ultra-thin slices were studied using electron microscope JEH-100СХ with the accelerating voltage 80 kV. Results 1. Corrective effect of PRP on the content of interleukins, prolactin and insulin-like growth factor-1 in the rat tissues at acute aluminum neurotoxicosis. Recently it was established that IL-1, IL-6 and TNF can be the provoking factors, which provide the synthesis of precursor of an amyloid protein in neuronal structures of the brain [Shigematsu K., McGreer P., 1992; Muller T. et al., 1998; Wen Т. Ð.¶.ì2Ðð2 ̧Ú2Ü, Ø.Æ. 2Ô2æ2Üàì / Üàð Ð2ÚÎ2Î2Ü ́ÄÞÎ2Î2Ü Ð2Ü ̧oê 12 et al., 1998; Zalsman S. et al., 1998]. It was also revealed that the synthesis of immunocytokines in neuronal structures under conditions of physiological activity of an organism is rather precisely balanced due to secretion of growth hormones by an adenohypophysis referring also to prolactin and insulin-like growth factor-1 (IGF-1). As indicated by results of the performed immune-enzyme analysis, after administration of А1С13 the trace amounts of IL-1 are determined in the blood serum of mice (Figure 2), while IL-1 was not revealed in control group animals. After the single PRP injection the IL-1 level in blood of mice with aluminum neurotoxicosis raised appreciably (more than twice). In supernatant prepared from the thymus homogenate of the Experimental Group I mice (with aluminum neurotoxicosis), there were determined only the trace amounts of mentioned cytokine, while the PRP injection was accompanied by the tendency directed toward normalization of IL-1 content. However, the indices of IL-1 in animals of the Experimental Group II were 1.3 times lower than the similar parameters in intact animals. In liver of animals with the aluminum toxicosis the contents of IL-1 decreased, while the PRP injection resulted in observable increasing of its level. IL-1 was revealed in trace amounts in brain homogenates of control mice (Figure 1). In the brain supernatant the non-significant amount of IL-1 was determined in case of aluminum chloride injection. Under administration of PRP the level of IL-1 in a brain tissue lowered almost twice in comparison with the Experimental Group I. IL-1 was not revealed in hippocampus of animals from either control or experimental groups. The content of IL-2 in blood serum, liver, and brain of mice with aluminum neurotoxicosis sharply decreased (more than two-fold). The administration of 15 μg PRP was accompanied by a various degree of IL-2-normalization. In thymus of mice of Experimental Group I, in Figure 1. The content