Disturbed Prefrontal Cortex Activity in the Absence of Schizophrenia-Like Behavioral Dysfunction in Arc/Arg3.1 Deficient Mice.

Arc/Arg3.1, an activity regulated immediate early gene, is essential for learning and memory, synaptic plasticity, and maturation of neural networks. It has also been implicated in several neurodevelopmental disorders, including schizophrenia. Here, we used male and female constitutive and conditional Arc/Arg3.1 knock-out (KO) mice to investigate the causal relationship between Arc/Arg3.1 deletion and schizophrenia-linked neurophysiological and behavioral phenotypes. Using in vivo local field potential recordings, we observed dampened oscillatory activity in the prefrontal cortex (PFC) of the KO and early conditional KO (early-cKO) mice, in which Arc/Arg3.1 was deleted perinatally. Whole-cell patch-clamp recordings from neurons in PFC slices revealed altered synaptic properties and reduced network gain in the KO mice as possible mechanisms underlying the oscillation deficits. In contrast, we measured normal oscillatory activity in the PFC of late conditional KO (late-cKO) mice, in which Arc/Arg3.1 was deleted during late postnatal development. Our data show that constitutive Arc/Arg3.1 KO mice exhibit no deficit in social engagement, working memory, sensorimotor gating, native locomotor activity, and dopaminergic innervation. Moreover, adolescent social isolation, an environmental stressor, failed to induce deficits in sociability or sensorimotor gating in adult KO mice. Thus, genetic removal of Arc/Arg3.1 per se does not cause schizophrenia-like behavior. Prenatal or perinatal deletion of Arc/Arg3.1 alters cortical network activity, however, without overtly disrupting the balance of excitation and inhibition in the brain and not promoting schizophrenia. Misregulation of Arc/Arg3.1 rather than deletion could potentially tip this balance and thereby promote emergence of schizophrenia and other neuropsychiatric disorders.SIGNIFICANCE STATEMENT The activity-regulated and memory-linked gene Arc/Arg3.1 has been implicated in the pathogenesis of schizophrenia, but direct evidence and a mechanistic link are still missing. The current study asks whether loss of Arc/Arg3.1 can affect brain circuitry and cause schizophrenia-like symptoms in mice. The findings demonstrate that genetic deletion of Arc/Arg3.1 before puberty alters synaptic function and prefrontal cortex activity. Although brain networks are disturbed, genetic deletion of Arc/Arg3.1 does not cause schizophrenia-like behavior, even when combined with an environmental insult. It remains to be seen whether misregulation of Arc/Arg3.1 might critically imbalance brain networks and lead to emergence of schizophrenia.

Arc/Arg3.1 mediates a critical period for spatial learning and hippocampal networks.

During early postnatal development, sensory regions of the brain undergo periods of heightened plasticity which sculpt neural networks and lay the foundation for adult sensory perception. Such critical periods were also postulated for learning and memory but remain elusive and poorly understood. Here, we present evidence that the activity-regulated and memory-linked gene Arc/Arg3.1 is transiently up-regulated in the hippocampus during the first postnatal month. Conditional removal of Arc/Arg3.1 during this period permanently alters hippocampal oscillations and diminishes spatial learning capacity throughout adulthood. In contrast, post developmental removal of Arc/Arg3.1 leaves learning and network activity patterns intact. Long-term memory storage continues to rely on Arc/Arg3.1 expression throughout life. These results demonstrate that Arc/Arg3.1 mediates a critical period for spatial learning, during which Arc/Arg3.1 fosters maturation of hippocampal network activity necessary for future learning and memory storage.

Transcriptional ERRgamma2-mediated activation is regulated by sentrin-specific proteases.

Modification with SUMOs (small ubiquitin-related modifiers) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The ERRgamma [oestrogen receptor-related receptor gamma; ERR3 or NR3B3 (nuclear receptor subfamily 3, group B, gene3)] is a constitutively active orphan nuclear receptor. A PDSM, (phosphorylation-dependent sumoylation motif) is located in the close vicinity of the N-terminally located ERRgamma2-specific AF-1 (activation function-1). Its function can be replaced by an NDSM (negatively charged amino acid-dependent sumoylation motif). A mutational analysis reveals that ERRgamma2 activity is modulated through sumoylation of a lysine residue at position 40, which in turn is regulated by phosphorylation. Phosphorylation at the +5 position relative to the sumoylation target is directly visualized by a high-resolution EMSA (electrophoretic mobility-shift assay). Sumoylation represses the activity of ERRgamma both with and without forced expression of the PGC-1beta (peroxisome-proliferator-activated receptor gamma co-activator-1beta). Fusion proteins of a heterologous DNA-binding domain with the ERRgamma2 N-terminus demonstrate the function of the PDSM as the RF-1 (repression function-1) for the neighbouring AF-1. De-repression is achieved by co-expression of sentrin/SENP (sentrin-specific protease) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor.

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37.

The neuropeptide head activator (HA) is a mitogen for mammalian cell lines of neuronal or neuroendocrine origin. HA signalling is mediated by a G-protein-coupled receptor (GPCR). Orphan GPCRs with homology to peptide receptors were screened for HA interaction. Electrophysiological recordings in frog oocytes and in mammalian cell lines as well as Ca(2+) mobilisation assays revealed nanomolar affinities of HA to GPR37. HA signal transduction through GPR37 was mediated by an inhibitory G protein and required Ca(2+) influx through a channel of the transient receptor potential (TRP) family. It also required activation of Ca(2+)-dependent calmodulin kinase and phosphoinositide 3-kinase. Respective inhibitors blocked HA signalling and HA-induced mitosis in GPR37-expressing cells. HA treatment resulted in internalisation of GPR37. Overexpression of GPR37 led to aggregate formation, retention of the receptor in the cytoplasm and low survival rates of transfected cells, confirming the notion that misfolded GPR37 contributes to cell death, as observed in Parkinson's disease.

Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.

By searching the human and mouse genomic databases we found two G-protein-coupled receptors, GPR139 and GPR142, with characteristic motifs of the rhodopsin family of receptors. The gene for GPR139 maps to chromosome 7F1 of mouse and 16p12.3 of human and that for GPR142 to 11E2 of mouse and 17q25.1 of human. We isolated GPR139 from a cDNA library of adult mouse brain and GPR142 from a cDNA library of brains from 15-day-old mouse embryos. GPR139 mRNA was predominantly expressed in specific areas of human and mouse brains, whereas GPR142 mRNA showed a more ubiquitous expression both in the brain and in various peripheral glands and organs. A 50% identity and a 67% homology at the amino-acid level between the two receptors and only 20-25% identity with other G-protein-coupled receptors established them as a new subbranch within the phylogenetic tree and hints at a common or similar ligand(s). Preliminary results suggest that the cognate ligand is present in brain extracts and is, most likely, a small peptide. GPR139 signal transduction in Chinese hamster ovary cells requires coupling to an inhibitory G-protein and is mediated by phospholipase C. Dimer formation may be necessary for proper function.

The orphan G-protein-coupled receptor GPR19 is expressed predominantly in neuronal cells during mouse embryogenesis.

G-protein-coupled receptors (GPCRs) are characterized by seven transmembrane domains and constitute the largest and structurally best-conserved family of signaling molecules. They are present in a diversity of organs and tissues and are involved in virtually all physiological processes. Here we report the expression of GPR19, an orphan GPCR, during mouse embryonic development and in the adult brain. Transcripts of GPR19 were detected early in embryonic development and were prominent in tissues of neuroectodermal origin. With ongoing differentiation, the localization of GPR19 transcripts became restricted to distinct regions of the developing brain, and the overall signal intensity declined in parallel. In the adult mouse, GPR19 showed high levels of transcription in several regions of the brain, including the olfactory bulb, the hippocampus, hypothalamic nuclei, and the cerebellum, and in testis. Lower levels of GPR19 expression were detected in heart, liver, and kidney. These data suggest that, amongst several other functions in the adult organism, GPR19 probably exerts its most characteristic effects during the early development of the nervous system.

Characterization of calmodulin binding to the orphan nuclear receptor Errgamma.

The estrogen receptor-related receptor gamma (ERRgamma/ ERR3/NR3B3), a member of the nuclear receptor superfamily, activates transcription in the absence of ligands. In order to identify ligand-independent mechanisms of activation, we tested whether calmodulin (CaM), a key regulator of numerous cellular processes and a predominant intracellular receptor for Ca2+-signals, interacts with ERRgamma. In vitro pull-down experiments with calmodulin-Sepharose demonstrated a Ca2+-dependent interaction with cellularly expressed ERRgamma. As shown by truncation analysis, the CaM binding site is highly unusual in that it is composed of two discontinuous elements. Moreover, by surface plasmon resonance (SPR) biosensor technology, we detected a direct interaction of immobilized bacterially expressed ERR-gamma fusion protein with Ca2+-calmodulin. This is best described by a model which assumes a conformational change of the initially formed complex to a more stable form. Whereas in vitro DNA binding was calmodulin-independent, transient transfection analysis revealed a Ca2+-influx-dependent ERRgamma-mediated transcriptional activation of a luciferase reporter gene. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of ERRgamma.

PGC-1 and PERC, coactivators of the estrogen receptor-related receptor gamma.

The mouse nuclear receptor ERRgamma (estrogen receptor-related receptor gamma) is highly expressed in heart, skeletal muscle, kidney, and brain, as well as in the developing nervous system. We found that the expression of the coactivators PGC-1 (PGC-1alpha) and PERC (PGC-1beta) in mammalian cells augmented potently the transcriptional activation by ERRgamma. The constitutive activation function 2 (AF-2) of the orphan receptor was important for the synergistic enhancement. Functional receptor truncation analysis revealed an additional amino-terminal activation function, specific for the ERRgamma2 isoform and PGC-1. In vitro experiments showed a direct interaction of ERRgamma with both coactivators. Our findings suggest distinct regulatory functions for PGC-1 and PERC as tissue-specific coactivators for ERRgamma.

Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding.

The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is an orphan member of the nuclear receptor superfamily closely related to the estrogen receptors. To explore the DNA binding characteristics, the protein-DNA interaction was studied in electrophoretic mobility shift assays (EMSAs). In vitro translated ERRgamma binds as a homodimer to direct repeats (DR) without spacing of the nuclear receptor half-site 5'-AGGTCA-3' (DR-0), to extended half-sites, and to the inverted estrogen response element. Using ERRgamma deletion constructs, binding was found to be dependent on the presence of sequences in the ligand binding domain (LBD). A far-Western analysis revealed that ERRgamma forms dimers even in the absence of DNA. Two elements, located in the hinge region and in the LBD, respectively, are necessary for DNA-independent dimerization. DNA binding of bacterial expressed ERRgamma requires additional factors present in the serum and in cellular extracts. Fusion proteins of the germ cell nuclear factor (GCNF/NR6A1) with ERRgamma showed that the characteristic feature to be stimulated by additional factors can be transferred to a heterologous protein. The stimulating activity was further characterized and its target sequence narrowed down to a small element in the hinge region.