Effects of Progesterone and Other Gonadal Hormones on Glutamatergic Circuits in the Retina.

BACKGROUND: Gonadal hormones function in the retina; however, their targets have not yet been identified. Therefore, the present study examined the effects of progesterone and other gonadal hormones on glutamatergic circuits in the retina. METHODS: Extracellular glutamate concentrations, which correspond to the amount of glutamate released, were examined using an enzyme-linked fluorescent assay system. The activity of glutamatergic synapses between bipolar cells and ganglion cells was investigated using a patch clamp technique. Changes in retinal thickness during pregnancy were assessed using optical coherence tomography (OCT) images. RESULTS: Progesterone and pregnenolone sulfate increased extracellular glutamate concentrations, whereas estrogen and testosterone did not. Progesterone increased the activity of glutamatergic synapses between bipolar cells and ganglion cells. A temporal decrease in the thickness of the peripheral retina was observed in the 1st trimester. CONCLUSIONS: Progesterone, but not estrogen or testosterone, activated glutamate release in the mouse retina. Increases in the concentration of progesterone during pregnancy did not induce any detectable change in retinal thickness.

Starburst amacrine cells form gap junctions in the early postnatal stage of the mouse retina.

Although gap junctional coupling in the developing retina is important for the maturation of neuronal networks, its role in the development of individual neurons remains unclear. Therefore, we herein investigated whether gap junctional coupling by starburst amacrine cells (SACs), a key neuron for the formation of direction selectivity, occurs during the developmental stage in the mouse retina. Neurobiotin-injected SACs coupled with many neighboring cells before eye-opening. The majority of tracer-coupled cells were retinal ganglion cells, and tracer coupling was not detected between SACs. The number of tracer-coupled cells significantly decreased after eye-opening and mostly disappeared by postnatal day 28 (P28). Membrane capacitance (Cm), an indicator of the formation of electrical coupling with gap junctions, was larger in SACs before than after eye-opening. The application of meclofenamic acid, a gap junction blocker, reduced the Cm of SACs. Gap junctional coupling by SACs was regulated by dopamine D1 receptors before eye-opening. In contrast, the reduction in gap junctional coupling after eye-opening was not affected by visual experience. At the mRNA level, 4 subtypes of connexins (23, 36, 43, and 45) were detected in SACs before eye-opening. Connexin 43 expression levels significantly decreased after eye-opening. These results indicate that gap junctional coupling by SACs occurs during the developmental period and suggest that the elimination of gap junctions proceeds with the innate system.

Rapid Visualization of Deeply Located Tumors In Vivo by Intravenous Administration of a γ-Glutamyltranspeptidase-Activated Fluorescent Probe.

We previously showed that spraying the fluorescent probe gGlu-HMRG (γ-glutamyl hydroxymethyl rhodamine green) can visualize even tiny tumors on the mesentery and peritoneal wall of tumor-bearing mice. However, during surgery, repeated spraying is necessary to detect tumors located deep within organs. Here, we examine whether deeply located tumors can be stained by intravenous administration of this probe. In mice bearing subcutaneous tumors, intravenous administration of gGlu-HMRG resulted in a rapid and specific increase of fluorescence in the tumor, which was visible to the naked eye within 5 min, and the maximum fluorescence intensity ratio of tumor to normal tissue (T/N = 4.3) was reached at 30 min. In mice bearing lung tumors, the T/N ratio reached approximately 20 at 30 min after administration, and deeply located tumors were clearly visualized. These results suggest that intravenous administration of gGlu-HMRG may be a useful technique in fluorescence-guided surgery of tumors.

P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis.

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high-affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Identification of a novel di-C-glycosyl dihydrochalcone and the thermal stability of polyphenols in model ready-to-drink beverage solutions with Cyclopia subternata extract as functional ingredient.

Heat processing of ready-to-drink beverages is required to ensure a microbiologically safe product, however, this can result in the loss of bioactive compounds responsible for functionality. The objective of this study was to establish the thermal stability of a novel dihydrochalcone, 3',5'-di-ß-d-glucopyranosyl-3-hydroxyphloretin (2), 3',5'-di-ß-d-glucopyranosylphloretin (3) and other Cyclopia subternata phenolic compounds, in model solutions with or without citric acid and ascorbic acid. The solutions were heated at 93, 121 and 135 °C, relevant to pasteurisation, commercial sterilisation and ultra-high temperature (UHT) pasteurisation, respectively. For most compounds, the acids decreased the second order reaction rate constants, up to 27 times. Compound 2 (46.29 ± 0.53 (g/100 g)-1 h-1), and to a lesser extent compound 3 (5.94 ± 0.01 (g/100 g)-1 h-1) were the most thermo-unstable compounds when treated at 135 °C without added acids. Even though differential effects were observed for compounds at different temperatures and formulations, overall, the phenolic compounds were most stable under UHT pasteurisation conditions.

Involvement of the C-terminal domain in cell surface localization and G-protein coupling of mGluR6.

Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gßγ subunits via its intracellular C-terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840-871) was necessary for mGluR6 cell surface localization and G-protein coupling using mGluR6-CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C-terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate-induced G-protein-mediated responses, whereas a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface localization and glutamate-dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.

SENP1 and SENP2 regulate SUMOylation of amyloid precursor protein.

Amyloid ß, a key molecule in the pathogenesis of Alzheimer's disease (AD), is produced from amyloid precursor protein (APP) by the cleavage of secretases. APP is SUMOylated near the cleavage site of ß-secretase. SUMOylation of APP reduces amyloid ß production, but its regulatory system is still unclear. SUMOylation, a modification at a lysine residue of a target protein, is mediated by activating, conjugating, and ligating enzymes and is reversed by a family of sentrin/SUMO-specific proteases (SENPs). Here, we found that both SENP1 and SENP2 induced de-SUMOylation of APP. Using quantitative PCR, we also found that expression of SENP1 but not SENP2 increased in an age-dependent manner only in female mice. The results of immunoblot analyses showed that the protein expression was consistent with the PCR results. Females, compared to males, have a higher incidence of AD in humans and show more aggressive amyloid pathology in AD mouse models. Our results provide a clue to understanding the role of SUMOylation in the sex difference in AD pathogenesis.

Alteration of global protein SUMOylation in neurons and astrocytes in response to Alzheimer's disease-associated insults.

SUMOylation, a post-translational modification of lysine residues by small ubiquitin-like modifier (SUMO) proteins, has been implicated in the pathogenesis of neurodegenerative disorders including Alzheimer's disease (AD), and in neuron- and astrocyte-specific physiological functions. Global SUMOylation is increased in the AD mouse brain in the pre-plaque-forming stage but returns to wild-type levels in the plaque-bearing stage. To clarify the reason for the transient change in SUMOylation, we analyzed the alteration of global SUMOylation induced by AD-associated cytotoxic stimuli in neurons and astrocytes individually. In neurons, amyloid ß42 oligomers induced some but not significant increase in levels of SUMO1-modified proteins. Both hydrogen peroxide and glutamate significantly reduced SUMO1-modified protein levels. These changes were more prominent in neurons than in astrocytes. The opposite effect of Aß and oxidative/excitotoxic stimuli on SUMO1 modification may cause the pathological stage-associated change in the level of SUMO-modified proteins in the AD mouse brain.

Humanin ameliorates diazepam-induced memory deficit in mice.

Humanin (HN) is an endogenous 24-residue peptide. A highly potent HN derivative, S14G-HN, which has a substitution of serine 14 to glycine, reduced amyloid burden and suppressed cognitive impairment in a mouse model of Alzheimer's disease. S14G-HN also suppressed amnesia induced by a muscarinic receptor antagonist in rodents. To understand the effects of HN on brain function, we tested the effect of S14G-HN on diazepam (DZP)-induced memory impairment and anxiety in mice using the object recognition test and zero-maze test, respectively. Intraperitoneal injection of S14G-HN reversed the DZP-induced memory deficit, whereas no significant change was observed in behavioral markers of anxiety. S14G-HN had no effect on locomotor activity in either test, indicating that S14G-HN did not affect physical functioning or motivation. These results suggest that HN preferentially influences cognitive function but not emotional function in the central nervous system.

L-Fucose-containing arabinogalactan-protein in radish leaves.

The carbohydrate moieties of arabinogalactan-proteins (AGPs) have ß-(1 → 3)-galactan backbones to which side chains of (1 → 6)-linked ß-Gal residues are attached through O-6. Some of these side chains are further substituted with other sugars. We investigated the structure of L-Fuc-containing oligosaccharides released from the carbohydrate moieties of a radish leaf AGP by digestion with α-L-arabinofuranosidase, followed by exo-ß-(1 → 3)-galactanase. We detected a series of neutral ß-(1 → 6)-galactooligosaccharides branching variously at O-3 of the Gal residues, together with corresponding acidic derivatives terminating in 4-O-methyl-GlcA (4-Me-GlcA) or GlcA at the non-reducing terminals. In neutral oligosaccharides with degree of polymerization (dp) mainly higher than 10, L-Fuc groups were attached through L-Ara residues as the sequence, α-L-Fucp-(1 → 2)-α-L-Araf-(1 →. This sequence was verified by isolation of the pentasaccharide α-L-Fuc-(1 → 2)-α-L-Araf-(1 → 3)-ß-Gal-(1 → 6)-ß-Gal-(1 → 6)-Gal upon digestion of the higher oligosaccharides with endo-ß-(1 → 6)-galactanase. By contrast, in lower polymerized (predominantly dp 4) acidic oligosaccharides, L-Fuc groups were attached directly at the non-reducing terminals through α-(1 → 2)-linkages, resulting in the release of the tetrasaccharides, α-L-Fucp-(1 → 2)-ß-GlcA-(1 → 6)-ß-Gal-(1 → 6)-Gal and α-L-Fucp-(1 → 2)-ß-4-Me-GlcA-(1 → 6)-ß-Gal-(1 → 6)-Gal. In long acidic oligosaccharides with dp mainly higher than 13, L-Fuc groups localized on branches were attached to the uronic acids directly and/or L-Ara residues as in the neutral oligosaccharides.

Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) approximately 7-12 microm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.

Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.

Killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor expressed on subsets of natural killer (NK) cells and T cells, for which no endogenous ligands are known. Here, we show that KLRG1 binds three of the classical cadherins (E-, N-, and R-), which are ubiquitously expressed in vertebrates and mediate cell-cell adhesion by homotypic or heterotypic interactions. By expression cloning using the mouse KLRG1 tetramer as a probe, we identified human E-cadherin as a xenogeneic ligand. We also identified a syngeneic interaction between mouse KLRG1 and mouse E-cadherin. Furthermore, we show that KLRG1 binds N- and R-cadherins. Finally, we demonstrate that E-cadherin binding of KLRG1 prevents the lysis of E-cadherin-expressing targets by KLRG1+ NK cells. These results suggest that KLRG1 ligation by E-, N-, or R-cadherins may regulate the cytotoxicity of killer cells to prevent damage to tissues expressing the cadherins.