Depression-Associated Negr1 Gene-Deficiency Induces Alterations in the Monoaminergic Neurotransmission Enhancing Time-Dependent Sensitization to Amphetamine in Male Mice.

In GWAS studies, the neural adhesion molecule encoding the neuronal growth regulator 1 (NEGR1) gene has been consistently linked with both depression and obesity. Although the linkage between NEGR1 and depression is the strongest, evidence also suggests the involvement of NEGR1 in a wide spectrum of psychiatric conditions. Here we show the expression of NEGR1 both in tyrosine- and tryptophan hydroxylase-positive cells. Negr1-/- mice show a time-dependent increase in behavioral sensitization to amphetamine associated with increased dopamine release in both the dorsal and ventral striatum. Upregulation of transcripts encoding dopamine and serotonin transporters and higher levels of several monoamines and their metabolites was evident in distinct brain areas of Negr1-/- mice. Chronic (23 days) escitalopram-induced reduction of serotonin and dopamine turnover is enhanced in Negr1-/- mice, and escitalopram rescued reduced weight of hippocampi in Negr1-/- mice. The current study is the first to show alterations in the brain monoaminergic systems in Negr1-deficient mice, suggesting that monoaminergic neural circuits contribute to both depressive and obesity-related phenotypes linked to the human NEGR1 gene.

High-Fat Diet Induces Pre-Diabetes and Distinct Sex-Specific Metabolic Alterations in Negr1-Deficient Mice.

In the large GWAS studies, NEGR1 gene has been one of the most significant gene loci for body mass phenotype. The purpose of the current study was to clarify the role of NEGR1 in the maintenance of systemic metabolism, including glucose homeostasis, by using both male and female Negr1-/- mice receiving a standard or high fat diet (HFD). We found that 6 weeks of HFD leads to higher levels of blood glucose in Negr1-/- mice. In the glucose tolerance test, HFD induced phenotype difference only in male mice; Negr1-/- male mice displayed altered glucose tolerance, accompanied with upregulation of circulatory branched-chain amino acids (BCAA). The general metabolomic profile indicates that Negr1-/- mice are biased towards glyconeogenesis, fatty acid synthesis, and higher protein catabolism, all of which are amplified by HFD. Negr1 deficiency appears to induce alterations in the efficiency of energy storage; reduced food intake could be an attempt to compensate for the metabolic challenge present in the Negr1-/- males, particularly during the HFD exposure. Our results suggest that the presence of functional Negr1 allows male mice to consume more HFD and prevents the development of glucose intolerance, liver steatosis, and excessive weight gain.

Alternative Promoter Use Governs the Expression of IgLON Cell Adhesion Molecules in Histogenetic Fields of the Embryonic Mouse Brain.

The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.

Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system.

Limbic system associated membrane protein (Lsamp) is a neural adhesion protein which has been recently found to be differentially expressed between serotonergic neuron subtypes. We have previously shown elevated serotonin (5-HT) turnover rate in Lsamp-deficient mice. The purpose of the current study was to elucidate the role of Lsamp in serotonergic neurotransmission. Chronic (18 days) administration of serotonin reuptake inhibitor (SSRI) escitalopram (10 mg/kg) significantly increased general activity in wild-type mice in the open field and protected exploration in Lsamp-/- mice in the elevated-plus maze. An important psychopathology-related endophenotype, elevated 5-HT turnover in the brain of Lsamp-deficient mice, was reproduced in the saline group. Escitalopram restored the elevated 5-HT turnover of Lsamp-deficient mice to a level comparable with their wild-type littermates, suggesting that high 5-HT turnover in mutants is mediated by the increased activity of serotonin transporter (SERT protein encoded by Slc6a4 gene). The baseline level of Slc6a4 transcript was not changed in Lsamp-deficient mice, however, our immunohistochemical analysis showed partial co-expression of Lsamp with both SERT and Tph2 proteins in raphe. Overactivity of SERT in Lsamp-/- mice is further supported by significant elevation of Maoa transcript and increase of DOPAC, another Mao A product, specifically in the raphe. Again, elevation of DOPAC was reduced to the level of wild-type by chronic SSRI treatment. The activity of Lsamp gene promoters varied in 5-HT producing nuclei: both Lsamp 1a and 1b promoters were active in the dorsal raphe; most of the expression in the median raphe was from 1b promoter, whereas Lsamp 1a promoter was almost exclusively active in the caudal subgroup of raphe nuclei. We suggest that Lsamp may have an impact on the integrity of serotonergic synapses, which is possibly the neurochemical basis of the anxiety- and sociability-related phenotype in Lsamp-deficient mice.

Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders.

Neuronal growth regulator 1 (NEGR1) belongs to the immunoglobulin (IgLON) superfamily of cell adhesion molecules involved in cortical layering. Recent functional and genomic studies implicate the role of NEGR1 in a wide spectrum of psychiatric disorders, such as major depression, schizophrenia and autism. Here, we investigated the impact of Negr1 deficiency on brain morphology, neuronal properties and social behavior of mice. In situ hybridization shows Negr1 expression in the brain nuclei which are central modulators of cortical-subcortical connectivity such as the island of Calleja and the reticular nucleus of thalamus. Brain morphological analysis revealed neuroanatomical abnormalities in Negr1-/- mice, including enlargement of ventricles and decrease in the volume of the whole brain, corpus callosum, globus pallidus and hippocampus. Furthermore, decreased number of parvalbumin-positive inhibitory interneurons was evident in Negr1-/- hippocampi. Behaviorally, Negr1-/- mice displayed hyperactivity in social interactions and impairments in social hierarchy. Finally, Negr1 deficiency resulted in disrupted neurite sprouting during neuritogenesis. Our results provide evidence that NEGR1 is required for balancing the ratio of excitatory/inhibitory neurons and proper formation of brain structures, which is prerequisite for adaptive behavioral profiles. Therefore, Negr1-/- mice have a high potential to provide new insights into the neural mechanisms of neuropsychiatric disorders.

The combined impact of IgLON family proteins Lsamp and Neurotrimin on developing neurons and behavioral profiles in mouse.

Cell surface neural adhesion proteins are critical components in the complex orchestration of cell proliferation, apoptosis, and neuritogenesis essential for proper brain construction and behavior. We focused on the impact of two plasticity-associated IgLON family neural adhesion molecules, Neurotrimin (Ntm) and Limbic system associated membrane protein (Lsamp), on mouse behavior and its underlying neural development. Phenotyping neurons derived from the hippocampi of Lsamp-/-, Ntm-/- and Lsamp-/-Ntm-/- mice was performed in parallel with behavioral testing. While the anatomy of mutant brains revealed no gross changes, the Ntm-/- hippocampal neurons exhibited premature sprouting of neurites and manifested accelerated neurite elongation and branching. We propose that Ntm exerts an inhibitory impact on neurite outgrowth, whereas Lsamp appears to be an enhancer of the said process as premature neuritogenesis in Ntm-/- neurons is apparent only in the presence of Lsamp. We also show interplay between Lsamp and Ntm in regulating tissue homeostasis: the impact of Ntm on cellular proliferation was dependent on Lsamp, and Lsamp appeared to be a positive regulator of apoptosis in the presence of Ntm. Behavioral phenotyping indicated test-specific interactions between Lsamp and Ntm. The phenotypes of single mutant lines, such as reduced swimming speed in Morris water maze and increased activity in the elevated plus maze, were magnified in Lsamp-/-Ntm-/- mice. Altogether, evidence both from behavioral experiments and cultured hippocampal cells show combined and differential interactions between Ntm and Lsamp in the formation of hippocampal circuits and behavioral profiles. We demonstrate that mutual interactions between IgLON molecules regulate the initiation of neurite sprouting at very early ages, and even cell-autonomously, independent of their regulation of cell-cell adhesion.

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of ß-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Promoter-Specific Expression and Genomic Structure of IgLON Family Genes in Mouse.

IgLON family is composed of five genes: Lsamp, Ntm, Opcml, Negr1, and Iglon5; encoding for five highly homologous neural adhesion proteins that regulate neurite outgrowth and synapse formation. In the current study we performed in silico analysis revealing that Ntm and Opcml display similar genomic structure as previously reported for Lsamp, characterized by two alternative promotors 1a and 1b. Negr1 and Iglon5 transcripts have uniform 5' region, suggesting single promoter. Iglon5, the recently characterized family member, shares high level of conservation and structural qualities characteristic to IgLON family such as N-terminal signal peptide, three Ig domains, and GPI anchor binding site. By using custom 5'-isoform-specific TaqMan gene-expression assay, we demonstrated heterogeneous expression of IgLON transcripts in different areas of mouse brain and several-fold lower expression in selected tissues outside central nervous system. As an example, the expression of IgLON transcripts in urogenital and reproductive system is in line with repeated reports of urogenital tumors accompanied by mutations in IgLON genes. Considering the high levels of intra-family homology shared by IgLONs, we investigated potential compensatory effects at the level of IgLON isoforms in the brains of mice deficient of one or two family members. We found that the lack of IgLONs is not compensated by a systematic quantitative increase of the other family members. On the contrary, the expression of Ntm 1a transcript and NEGR1 protein was significantly reduced in the frontal cortex of Lsamp-deficient mice suggesting that the expression patterns within IgLON family are balanced coherently. The actions of individual IgLONs, however, can be antagonistic as demonstrated by differential expression of Syp in deletion mutants of IgLONs. In conclusion, we show that the genomic twin-promoter structure has impact on both anatomical distribution and intra-family interactions of IgLON family members. Remarkable variety in the activity levels of 1a and 1b promoters both in the brain and in other tissues, suggests complex functional regulation of IgLONs by alternative signal peptides driven by 1a and 1b promoters.

Gene expression patterns and environmental enrichment-induced effects in the hippocampi of mice suggest importance of Lsamp in plasticity.

Limbic system associated membrane protein (Lsamp) gene is involved in behavioral adaptation in social and anxiogenic environments and has been associated with a broad spectrum of psychiatric diseases. Here we studied the activity of alternative promoters of Lsamp gene in mice in three rearing conditions (standard housing, environmental enrichment and social isolation) and in two different genetic backgrounds (129S6/SvEv and C57BL/6). Isolation had no effect on the expression levels of Lsamp. Environmental enrichment elevated the expression levels of Lsamp 1b transcript specifically in the hippocampus in B6 mice, and the same tendency existed across both mouse lines and both transcripts. Furthermore, we showed that the density of cells exhibiting 1b promoter activity is remarkably higher in the subgranular zone of the dentate gyrus in the hippocampal formation which is a specific area of enrichment-induced neurogenesis in adult rodents. On the contrary to 1b, 1a promoter is selectively active in the pyramidal and granule cell layers. We provide evidence that Lsamp modulates enrichment-induced activation of Bdnf as the enrichment-induced elevation of Bdnf in the hippocampus is significantly diminished in Lsamp-deficient mice; furthermore, a significant correlation was found between the expression levels of Lsamp and Bdnf transcripts in the hippocampus and frontal cortex. Significant strain differences in Lsamp expression were detected in the hippocampus, frontal cortex and thalamus that could be related to the different behavioral phenotype of B6 and 129Sv mice. Our data provides further evidence that LSAMP is implicated in the hippocampal connectivity and plasticity thereby modulating adaptability in changing environments.

Input coding for neuro-electronic hybrid systems.

Liquid State Machines have been proposed as a framework to explore the computational properties of neuro-electronic hybrid systems (Maass et al., 2002). Here the neuronal culture implements a recurrent network and is followed by an array of linear discriminants implemented using perceptrons in electronics/software. Thus in this framework, it is desired that the outputs of the neuronal network, corresponding to different inputs, be linearly separable. Previous studies have demonstrated this by either using only a small set of input stimulus patterns to the culture (Hafizovic et al., 2007), large number of input electrodes (Dockendorf et al., 2009) or by using complex schemes to post-process the outputs of the neuronal culture prior to linear discriminance (Ortman et al., 2011). In this study we explore ways to temporally encode inputs into stimulus patterns using a small set of electrodes such that the neuronal culture's output can be directly decoded by simple linear discriminants based on perceptrons. We demonstrate that network can detect the timing and order of firing of inputs on multiple electrodes. Based on this, we demonstrate that the neuronal culture can be used as a kernel to transform inputs which are not linearly separable in a low dimensional space, into outputs in a high dimension where they are linearly separable. Thus simple linear discriminants can now be directly connected to outputs of the neuronal culture and allow for implementation of any function for such a hybrid system.

Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival.

Delaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.