Netrin-G/NGL complexes encode functional synaptic diversification.

Synaptic cell adhesion molecules are increasingly gaining attention for conferring specific properties to individual synapses. Netrin-G1 and netrin-G2 are trans-synaptic adhesion molecules that distribute on distinct axons, and their presence restricts the expression of their cognate receptors, NGL1 and NGL2, respectively, to specific subdendritic segments of target neurons. However, the neural circuits and functional roles of netrin-G isoform complexes remain unclear. Here, we use netrin-G-KO and NGL-KO mice to reveal that netrin-G1/NGL1 and netrin-G2/NGL2 interactions specify excitatory synapses in independent hippocampal pathways. In the hippocampal CA1 area, netrin-G1/NGL1 and netrin-G2/NGL2 were expressed in the temporoammonic and Schaffer collateral pathways, respectively. The lack of presynaptic netrin-Gs led to the dispersion of NGLs from postsynaptic membranes. In accord, netrin-G mutant synapses displayed opposing phenotypes in long-term and short-term plasticity through discrete biochemical pathways. The plasticity phenotypes in netrin-G-KOs were phenocopied in NGL-KOs, with a corresponding loss of netrin-Gs from presynaptic membranes. Our findings show that netrin-G/NGL interactions differentially control synaptic plasticity in distinct circuits via retrograde signaling mechanisms and explain how synaptic inputs are diversified to control neuronal activity.

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

BACKGROUND: Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. METHODS AND FINDINGS: Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. CONCLUSIONS: FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants. Please see later in the article for the Editors' Summary.

Diversification of behavior and postsynaptic properties by netrin-G presynaptic adhesion family proteins.

BACKGROUND: Vertebrate-specific neuronal genes are expected to play a critical role in the diversification and evolution of higher brain functions. Among them, the glycosylphosphatidylinositol (GPI)-anchored netrin-G subfamily members in the UNC6/netrin family are unique in their differential expression patterns in many neuronal circuits, and differential binding ability to their cognate homologous post-synaptic receptors. RESULTS: To gain insight into the roles of these genes in higher brain functions, we performed comprehensive behavioral batteries using netrin-G knockout mice. We found that two netrin-G paralogs that recently diverged in evolution, netrin-G1 and netrin-G2 (gene symbols: Ntng1 and Ntng2, respectively), were responsible for complementary behavioral functions. Netrin-G2, but not netrin-G1, encoded demanding sensorimotor functions. Both paralogs were responsible for complex vertebrate-specific cognitive functions and fine-scale regulation of basic adaptive behaviors conserved between invertebrates and vertebrates, such as spatial reference and working memory, attention, impulsivity and anxiety etc. Remarkably, netrin-G1 and netrin-G2 encoded a genetic "division of labor" in behavioral regulation, selectively mediating different tasks or even different details of the same task. At the cellular level, netrin-G1 and netrin-G2 differentially regulated the sub-synaptic localization of their cognate receptors and differentiated the properties of postsynaptic scaffold proteins in complementary neural pathways. CONCLUSIONS: Pre-synaptic netrin-G1 and netrin-G2 diversify the complexity of vertebrate behaviors and differentially regulate post-synaptic properties. Our findings constitute the first genetic analysis of the behavioral and synaptic diversification roles of a vertebrate GPI protein and presynaptic adhesion molecule family.

A family-based association study and gene expression analyses of netrin-G1 and -G2 genes in schizophrenia.

BACKGROUND: The netrin-G1 (NTNG1) and -G2 (NTNG2) genes, recently cloned from mouse, play a role in the formation and/or maintenance of glutamatergic neural circuitry. Accumulating evidence strongly suggests that disturbances of neuronal development and the N-methyl-d-aspartate receptor-mediated signaling system might represent a potential pathophysiology in schizophrenia. We therefore set out to examine the genetic contribution of human NTNG1 and NTNG2 to schizophrenia. METHODS: Twenty-one single nucleotide polymorphisms (SNPs) from NTNG1 and 10 SNPs from NTNG2 were analyzed in 124 schizophrenic pedigrees. All genotypes were determined with the TaqMan assay. The expression levels of NTNG1 and NTNG2 were examined in the frontal (Brodmann's Area [BA]11 and BA46) and temporal (BA22) cortices from schizophrenic and control postmortem brains. The isoform-specific expression of NTNG1 splice variants was assessed in these samples. RESULTS: Specific haplotypes encompassing alternatively spliced exons of NTNG1 were associated with schizophrenia, and concordantly, messenger ribonucleic acid isoform expression was significantly different between schizophrenic and control brains. An association between NTNG2 and schizophrenia was also observed with SNPs and haplotypes that clustered in the 5' region of the gene. CONCLUSIONS: The NTNG1 and NTNG2 genes might be relevant to the pathophysiology of schizophrenia.

Complementary expression and neurite outgrowth activity of netrin-G subfamily members.

Classical members of the UNC6/netrin family are secreted proteins which play a role as long-range cues for directing growth cones. We here identified in mice a novel member netrin-G2 which constitute a subfamily with netrin-G1 among the UNC6/netrin family. Both of these netrin-Gs are characterized by glycosyl phosphatidyl-inositol linkage onto cells, molecular variants presumably generated by alternative splicing and lack of any appreciable affinity to receptors for classical netrins. These genes are preferentially expressed in the central nervous system with complementary distribution in most brain areas, that is netrin-G1 in the dorsal thalamus, olfactory bulb and inferior colliculus, and netrin-G2 in the cerebral cortex, habenular nucleus and superior colliculus. Consistently, immunohistochemical analysis revealed that netrin-G1 molecules are present on thalamocortical but not corticothalamic axons. Thalamic and neocortical neurons extended long neurites on immobilized recombinant netrin-G1 or netrin-G2 in vitro. Immobilized anti-netrin-G1 antibodies altered shapes of cultured thalamic neurons. We propose that netrin-Gs provide short-range cues for axonal and/or dendritic behavior through bi-directional signaling.

Laminar organization of the developing lateral olfactory tract revealed by differential expression of cell recognition molecules.

The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immunostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5-bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the superficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons.

Human netrin-G1 isoforms show evidence of differential expression.

The recently identified netrins-G1 and -G2 form a distinct subgroup within the UNC-6/netrin gene family of axon guidance molecules. In this study, we determined the size and structure of the exon/intron layout of the human netrin-G1 (NTNG1) and -G2 (NTNG2) genes. Northern analysis of both genes showed limited nonneuronal but wide brain expression, particularly for NTNG2. Reverse transcriptase PCR detected nine alternatively spliced isoforms including four novel variants of NTNG1 from adult brain. A semiquantitative assay established that major expression was restricted to isoforms G1c, G1d, G1a, and G1e in the brain and to G1c in the kidney. There is also evidence of developmental regulation of these isoforms between fetal and adult brain. In conclusion, NTNG1 may use alternative splicing to diversify its function in a developmentally and tissue-specific manner.