Proteomics Analysis of CA1 Region of the Hippocampus in Pre-, Progression and Pathological Stages in a Mouse Model of the Alzheimer's Disease.

BACKGROUND: CA1 subregion of the hippocampal formation is one of the primarily affected structures in AD, yet not much is known about proteome alterations in the extracellular milieu of this region. OBJECTIVE: In this study, we aimed to identify the protein expression alterations throughout the pre-pathological, progression and pathological stages of AD mouse model. METHODS: The CA1 region perfusates were collected by in-vivo intracerebral push-pull perfusion from transgenic 5XFAD mice and their non-transgenic littermates at 3, 6 and 12 wereßmonths of age. Morris water maze test and immunohistochemistry staining of A performed to determine the stages of the disease in this mouse model. The protein expression differences were analyzed by label-free shotgun proteomics analysis. RESULTS: A total of 251, 213 and 238 proteins were identified in samples obtained from CA1 regions of mice at 3, 6 and 12 months of age, respectively. Of these, 68, 41 and 33 proteins showed statistical significance. Pathway analysis based on the unique and common proteins within the groups revealed that several pathways are dysregulated during different stages of AD. The alterations in glucose and lipid metabolisms respectively in pre-pathologic and progression stages of the disease, lead to imbalances in ROS production via diminished SOD level and impairment of neuronal integrity. CONCLUSION: We conclude that CA1 region-specific proteomic analysis of hippocampal degeneration may be useful in identifying the earliest as well as progressional changes that are associated with Alzheimer's disease.

Early Stage Alterations in CA1 Extracellular Region Proteins Indicate Dysregulation of IL6 and Iron Homeostasis in the 5XFAD Alzheimer's Disease Mouse Model.

In recent years, an increasing number of research papers revealed that the compositional and volumetric alterations in the extracellular matrix are the consequences of aging and may be related to Alzheimer's disease (AD). In this study, we aimed to demonstrate the alterations in hippocampal extracellular fluid proteins in vivo using the 5XFAD mouse model. Samples were obtained from hippocampi of 5XFAD mice (n = 6) and their non-transgenic littermates by intracerebral push-pull perfusion technique at 3 months of age, representing the pre-pathological stage of the AD. Proteins in the hippocampal perfusates were analyzed by Ultra Performance Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UPLC-ESI-qTOF-MS/MS). 178 proteins were identified and 19 proteins of them were found to be statistically significantly altered (p≤0.05, fold change ≥40%, unique peptide count ≥3) in the hippocampal CA1 extracellular fluid of the 5XFAD mouse model. Ingenuity pathway analysis of the protein expression results identified IL6 as an upstream regulator. The upregulation of IL6 was validated by immunohistochemical staining of the hippocampus and cortex of the 5XFAD mice prior to Aß plaque formation. Furthermore, the iron level in the hippocampus was measured by inductively coupled plasma-mass spectrometry as IL6 is mentioned in several studies to take part in iron homeostasis and inflammation and found to be increased in 5XFAD mice hippocampus. Alterations in extracellular matrix proteins in addition to increasing amount of hippocampal IL6 and iron in the early stages of AD may reveal inflammation-mediated iron dyshomeostasis in the early stages of neurodegeneration.

The effect of Gly-Gln [ß-endorphin30-31] on morphine-evoked serotonin and GABA efflux in the nucleus accumbens of conscious rats.

Glycyl-L-glutamine (Gly-Gln; ß-endorphin30-31) is an endogenous dipeptide synthesized through the post-translational processing of ß-endorphin1-31. Central Gly-Gln administration inhibits the rewarding properties of morphine and attenuates morphine tolerance, dependence and withdrawal although it does not interfere with morphine analgesia. In an earlier study, we found that Gly-Gln inhibits morphine-induced dopamine efflux in the nucleus accumbens (NAc), consistent with its ability to inhibit morphine reward. To further investigate the mechanism responsible for its central effects we tested whether i.c.v. Gly-Gln administration influences the rise in extracellular serotonin and GABA concentrations evoked by morphine in the NAc. Conscious rats were treated with Gly-Gln (100nmol/5µl) or saline i.c.v. followed, 2min later, by morphine (2.5mg/kg) or saline i.p. and extracellular serotonin and GABA concentrations were analyzed by microdialysis and HPLC. Morphine administration increased extracellular serotonin and GABA concentrations significantly within 20min, as shown previously. Unexpectedly, Gly-Gln also increased extracellular serotonin concentrations significantly in control animals. Combined treatment with Gly-Gln+morphine also elevated extracellular serotonin concentrations although the magnitude of the response did not differ significantly from the effect of Gly-Gln or morphine, given alone suggesting that Gly-Gln suppressed morphine induced serotonin efflux. Gly-Gln abolished the morphine-induced rise in extracellular GABA concentrations but had no effect on extracellular GABA when given alone to otherwise untreated animals. These data show that Gly-Gln stimulates NAc serotonin efflux and, together with earlier studies, support the hypothesis that Gly-Gln inhibits the rewarding effects of morphine by modulating morphine induced dopamine, GABA and serotonin efflux in the NAc.

Evidence for the existence of pyrimidinergic transmission in rat brain.

The uridine nucleotides uridine-5'-triphosphate (UTP) and uridine-5'-diphosphate (UDP) have previously been identified in media from cultured cells. However, no study to date has demonstrated their presence in brain extracellular fluid (ECF) obtained in vivo. Using a novel method, we now show that UTP and UDP, as well as uridine, are detectable in dialysates of striatal ECF obtained from freely-moving rats. Intraperitoneal (i.p.) administration of uridine or exposure of striatum to depolarizing concentrations of potassium chloride increases extracellular uridine, UTP and UDP, while tetrodotoxin (TTX) decreases their ECF levels. Uridine administration also enhances cholinergic neurotransmission which is accompanied by enhanced brain levels of diacylglycerol (DAG) and inositol trisphosphate (IP3) and blocked by suramin, but not by PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) or MRS2578 suggesting a possible mediation of P2Y2 receptors activated by UTP. These observations suggest that uridine, UTP and UDP may function as pyrimidinergic neurotransmitters, and that enhancement of such neurotransmission underlies pharmacologic effects of exogenous uridine on the brain.

The mediation of the central histaminergic system in the pressor effect of intracerebroventricularly injected melittin, a phospholipase A2 activator, in normotensive rats.

Melittin is a polypeptide component of bee venom that leads to an increase in arachidonic acid release and subsequently in prostaglandin synthesis by activating phospholipase A(2). Recently we demonstrated that centrally or peripherally administrated melittin caused pressor effect and central thromboxane A(2) (TXA(2)) and cholinergic system mediated these effects of melittin. Also centrally injected histamine leads to pressor and bradycardic response by activating central histamine receptors in normotensive rats and central cholinergic system involved the effects of histamine. The present study demonstrates an involvement of the central histaminergic system in melittin-induced cardiovascular effect in normotensive rats. Experiments were carried out in male Sprague Dawley rats. Intracerebroventricularly (i.c.v.) injected melittin (0.5, 1 and 2 nmol) caused dose- and time-dependent increases in mean arterial pressure (MAP) and decrease in heart rate (HR) as we reported previously. Moreover, H(2) receptor antagonist ranitidine (50 nmol; i.c.v.) almost completely and H(3)/H(4) receptor antagonist thioperamide (50 nmol; i.c.v.) partly blocked melittin-evoked cardiovascular effects, whereas H(1) receptor blocker chlorpheniramine (50 nmol; i.c.v.) had no effect. Also centrally injected melittin was accompanied by 28% increase in extracellular histamine concentration in the posterior hypothalamus, as shown in microdialysis studies. In conclusion, results show that centrally administered melittin causes pressor and bradycardic response in conscious rats. Moreover, according to our findings, there is an involvement of the central histaminergic system in melittin-induced cardiovascular effects.

Investigation of serum amino acid and serum amyloid A concentrations in chickens with amyloid arthropathy.

BACKGROUND: Increased proteolytic cleavage of serum amyloid A (SAA) may potentially contribute to the development of AA amyloid deposition. OBJECTIVE: To study the possible relationship between amyloid artropathy and expression of SAA and some serum amino acids. ANIMALS AND METHODS: Values of 15 serum amino acids and SAA were investigated in chickens with experimentally induced amyloid arthropathy. Thirty-four, 5-week-old chicks were allocated into two groups: one group was injected intra-articularly with 0.25 mL complete Freund's adjuvant at the left tibio-metatarsal joint to induce amyloid arthropathy, whereas the other group served as control. All pullets were necropsied 13 weeks after injection. Collected tissue samples were examined histopathologically. Blood samples were collected and SAA concentrations were measured with enzyme-linked immunosorbent assay. High-performance liquid chromatography was used to assess the amino acid concentrations in serum. RESULTS: Amyloid accumulation in joints occurred only in the experimental group (89%). SAA concentrations of 166 ± 17 and 423 ± 39 (SD) ng/mL were found in the control and experimental groups, respectively (p < 0.001). In the experimental group, an increase was observed in all examined amino acid concentrations except for citrulline. The most significant (p < 0.001) increases were noticed in serine (from 159 ± 15 to 360 ± 29 µmol/L), glycine (from 151 ± 20 to 279 ± 16 µmol/L), isoleucine (from 48 ± 2 to 80 ± 6 µmol/L), and phenylalanine (from 49 ± 2 to 90 ± 3 µmol/L). CONCLUSION: The results of this study suggest that there is a positive correlation between some serum amino acid values, especially serine, glycine, isoleucine, and phenylalanine, and the high concentrations of SAA in chickens with amyloid arthropathy.

Serum choline levels in patients with stable angina and acute coronary syndromes: relation to the severity of coronary artery disease.

OBJECTIVE: We sought to investigate whether serum choline levels are increased across the spectrum of coronary artery disease (CAD) manifestations and correlate with the severity of coronary stenosis. METHODS: A total of 36 patients with acute coronary syndrome (ACS) [22 patients with non-ST-segment elevation ACS and 14 patients with ST-segment elevation acute myocardial infarction (STEMI)], 22 patients with stable angina pectoris (SAP), and 18 controls were recruited for the study. In ACS patients, serum choline levels were measured on admission, and at 24 and 48 h thereafter, using high-performance liquid chromatography. The severity of CAD was assessed using the Gensini score. RESULTS: Serum choline levels on admission were significantly higher in the entire group of patients with ACS than in controls. The highest level of choline was observed in the STEMI group, followed by the SAP, and the non-ST-segment elevation ACS groups. Serum choline levels decreased gradually in patients with STEMI over the 48-h period. Serum choline levels on admission, and at 24 or 48 h thereafter, did not correlate with the presence of CAD neither in patients with ACS (P=0.78, 0.98 and 0.98, respectively) nor in those with SAP (P=0.92). CONCLUSION: Our results suggest that serum choline levels are increased in ACS patients. However, there was no clear correlation between levels of choline and the severity and extent of CAD in this patient group.

Vitamin a deficiency in patients with common variable immunodeficiency.

Vitamin A, a naturally occuring antioxidant micronutrient, has immunomodulating effect in patients with immunodeficiency, including an influence on cytokine production and lymphocyte growth and functions. Vitamin A deficiency is associated with a shift from type 2 cytokines to predominantly type 1 cytokines. The aims of this study were to determine Vitamin A status in Common variable immunodeficiency (CVID) patients and the relationship between Vitamin A status and cytokines production. Serum Vitamin A, neopterin, TNF-alpha, IL-2, IL-4, and IL-10 levels were determined in 19 CVID patients and 15 healthy children. Effects of 9-cis retinal, Vitamin A derivative, on cytokines (TNF-alpha, IL-2, IL-4 and IL-10) production in lymphocytes were tested in vitro condition using lymphocyte cultures obtained from CVID patients and healthy children.Serum Vitamin A level in CVDI patients was, 21.1+/- 1.5 microg/dL, significantly (p < 0.001) lower than the value, 35.7+/- 1.8 microg/dL, observed in healthy children. Serum neopterin level in the patients was, 9.8+/- 2.9 nmol/L, higher (p < 0.05) than the value, 3.9+/- 0.7 nmol/L, observed in control group. Common variable immunodeficiency patients, serum IL-4 level was significantly (p < 0.05) lower than the value observed for healthy children. Serum TNF-alpha, IL-2 and IL-10 levels were similar in the patients and healthy children. Vitamin A derivative, 9-cis retinal, increased TNF-alpha and IL-4 production in cultured mononuclear cells obtained from control and CVID patients. Vitamin A derivative, also, increased IL-2 and Il-4 production in cultured mononuclear cells obtained from CVID patients. These results show that CVID patients have low serum Vitamin A levels and high serum neopterin levels. A supplementation with Vitamin A may have role in downregulation of inflammatory responses in CVID patients.