Glyphosate-induced changes in the expression of galanin and GALR1, GALR2 and GALR3 receptors in the porcine small intestine wall.

Glyphosate is the active ingredient of glyphosate-based herbicides and the most commonly used pesticide in the world. The goal of the present study was to verify whether low doses of glyphosate (equivalent to the environmental exposure) evoke changes in galanin expression in intramural neurons in the small intestine in pigs and to quantitatively determine changes in the level of galanin receptor encoding mRNA (GALR1, GALR2, GALR3) in the small intestine wall. The experiment was conducted on 15 sexually immature gilts divided into three study groups: control (C)-animals receiving empty gelatin capsules; experimental 1 (G1)-animals receiving a low dose of glyphosate (0.05 mg/kg b.w./day); experimental 2 (G2)-animals receiving a higher dose of glyphosate (0.5 mg/kg b.w./day) orally in gelatine capsules for 28 days. Glyphosate ingestion led to an increase in the number of GAL-like immunoreactive intramural neurons in the porcine small intestine. The results of RT-PCR showed a significant increase in the expression of mRNA, which encodes the GAL-receptors in the ileum, a decreased expression in the duodenum and no significant changes in the jejunum. Additionally, intoxication with glyphosate increased the expression of SOD2-encoding mRNA in the duodenum and decreased it in the jejunum and ileum, but it did not affect SOD1 expression. The results suggest that it may be a consequence of the cytotoxic and/or neurotoxic properties of glyphosate and/or its ability to induce oxidative stress.

Development of a high-throughput screening-compatible cell-based functional assay to identify small molecule probes of the galanin 3 receptor (GalR3).

The galanin 3 receptor (GalR3) belongs to the large G protein-coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening-compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.

GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu.

Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin-mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ-derived NSCs and the involvement of apoptosis (Bcl-2, cleaved caspase-3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP), spliced X-box binding protein 1 (XBP1), c-Jun N-terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24 h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3-mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.

Amino substituted analogs of 1-phenyl-3-phenylimino-2-indolones with potent galanin Gal3 receptor binding affinity and improved solubility.

A series of amino analogs of 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (1) were synthesized to improve aqueous solubility, while retaining high affinity for the human galanin Gal3 receptor. A very potent analog (9e, 1,3-dihydro-1-[3-(2-pyrrolidinylethoxy)phenyl]-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one, Ki=5 nM) shows good selectivity and solubility of 48 microg/mL at pH 7.4.