Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster.

Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. In this paper, we show that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, our data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function.

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.

Impact of a High-Fat Diet on the Metabolomics Profile of 129S6 and C57BL6 Mouse Strains.

Different inbred mouse strains vary substantially in their behavior and metabolic phenotype under physiological and pathological conditions. The purpose of this study was to extend the knowledge of distinct coping strategies under challenging events in two differently adapting mouse strains: C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv). Thus, we aimed to investigate possible similarities and differences in the body weight change, behavior, and several metabolic variables in Bl6 and 129Sv strains in response to high-fat diet (HFD) using the AbsoluteIDQ p180 kit. We found that 9 weeks of HFD induced a significant body weight gain in 129Sv, but not in Bl6 mice. Besides that, 129Sv mice displayed anxiety-like behavior in the open-field test. Metabolite profiling revealed that 129Sv mice had higher levels of circulating branched-chain amino acids, which were even more amplified by HFD. HFD also induced a decrease in glycine, spermidine, and t4-OH-proline levels in 129Sv mice. Although acylcarnitines (ACs) dominated in baseline conditions in 129Sv strain, this strain had a significantly stronger AC-reducing effect of HFD. Moreover, 129Sv mice had higher levels of lipids in baseline conditions, but HFD caused more pronounced alterations in lipid profile in Bl6 mice. Taken together, our results show that the Bl6 line is better adapted to abundant fat intake.

Lipopolysaccharide-Induced Strain-Specific Differences in Neuroinflammation and MHC-I Pathway Regulation in the Brains of Bl6 and 129Sv Mice.

Many studies have demonstrated significant mouse-strain-specific differences in behavior and response to pathogenic and pharmacological agents. This study seeks to characterize possible differences in microglia activation and overall severity of neuroinflammation in two widely used mouse strains, C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv), in response to acute lipopolysaccharide (LPS) administration. Locomotor activity within the open field arena revealed similar 24 h motor activity decline in both strains. Both strains also exhibited significant bodyweight loss due to LPS treatment, although it was more severe in the Bl6 strain. Furthermore, LPS induced a hypothermic response in Bl6 mice, which was not seen in 129Sv. We found that 24 h LPS challenge significantly increased the inflammatory status of microglia in 129Sv mice. On the other hand, we observed that, under physiological conditions, microglia of Bl6 seemed to be in a higher immune-alert state. Gene and protein expression analysis revealed that LPS induces a significantly stronger upregulation of MHC-I-pathway-related components in the brain of Bl6 compared to 129Sv mice. The most striking difference was detected in the olfactory bulb, where we observed significant LPS-induced upregulation of MHC-I pathway components in Bl6 mice, whereas no alterations were observed in 129Sv. We observed significant positive correlations between bodyweight decline and expressions of MHC-I components in the olfactory bulbs of Bl6 mice and the frontal cortex of 129Sv, highlighting different brain regions most affected by LPS in these strains. Our findings suggest that the brains of Bl6 mice exist in a more immunocompetent state compared to 129Sv mice.

Sex-Specific Microglial Activation and SARS-CoV-2 Receptor Expression Induced by Chronic Unpredictable Stress.

The coronavirus disease 2019 (COVID-19) pandemic has generated a lot of stress and anxiety among not only infected patients but also the general population across the globe, which disturbs cerebral immune homeostasis and potentially exacerbates the SARS-CoV-2 virus-induced neuroinflammation, especially among people susceptible to neuropsychiatric disorders. Here, we used a chronic unpredictable mild stress (CUMS) mouse model to study its effects on glia-mediated neuroinflammation and expression of SARS-CoV2 viral receptors. We observed that female mice showed depressive-like behavior after CUMS, whereas male mice showed enhanced anxiety and social withdrawal. Interestingly, CUMS led to increased amounts of total and MHCII+ microglia in the hippocampi of female mice but not male mice. mRNA levels of SARS-CoV-2 viral receptors angiotensin-converting enzyme 2 (Ace2) and basigin (Bsg) were also upregulated in the prefrontal cortices of stressed female mice but not male mice. Similarly, sex-specific changes in SARS-CoV-2 viral receptors FURIN and neuropilin-1 (NRP1) were also observed in monocytes of human caregivers enduring chronic stress. Our findings provided evidence on detrimental effects of chronic stress on the brain and behavior and implied potential sex-dependent susceptibility to SARS-CoV-2 infection after chronic stress.

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.

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.

Altered Expression Profile of IgLON Family of Neural Cell Adhesion Molecules in the Dorsolateral Prefrontal Cortex of Schizophrenic Patients.

Neural adhesion proteins are crucial in the development and maintenance of functional neural connectivity. Growing evidence suggests that the IgLON family of neural adhesion molecules LSAMP, NTM, NEGR1, and OPCML are important candidates in forming the susceptibility to schizophrenia (SCZ). IgLON proteins have been shown to be involved in neurite outgrowth, synaptic plasticity and neuronal connectivity, all of which have been shown to be altered in the brains of patients with the diagnosis of schizophrenia. Here we optimized custom 5'-isoform-specific TaqMan gene-expression analysis for the transcripts of human IgLON genes to study the expression of IgLONs in the dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients (n = 36) and control subjects (n = 36). Uniform 5'-region and a single promoter was confirmed for the human NEGR1 gene by in silico analysis. IgLON5, a recently described family member, was also included in the study. We detected significantly elevated levels of the NEGR1 transcript (1.33-fold increase) and the NTM 1b isoform transcript (1.47-fold increase) in the DLPFC of schizophrenia patients compared to healthy controls. Consequent protein analysis performed in male subjects confirmed the increase in NEGR1 protein content both in patients with the paranoid subtype and in patients with other subtypes. In-group analysis of patients revealed that lower expression of certain IgLON transcripts, mostly LSAMP 1a and 1b, could be related with concurrent depressive endophenotype in schizophrenic patients. Additionally, our study cohort provides further evidence that cannabis use may be a relevant risk factor associated with suicidal behaviors in psychotic patients. In conclusion, we provide clinical evidence of increased expression levels of particular IgLON family members in the DLPFC of schizophrenic patients. We propose that alterations in the expression profile of IgLON neural adhesion molecules are associated with brain circuit disorganization in neuropsychiatric disorders, such as schizophrenia. In the light of previously published data, we suggest that increased level of NEGR1 in the frontal cortex may serve as molecular marker for a wider spectrum of psychiatric conditions.

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.

Adaptation to larval crowding in Drosophila ananassae and Drosophila nasuta nasuta: increased larval competitive ability without increased larval feeding rate.

The standard view of adaptation to larval crowding in fruitflies, built on results from 25 years of multiple experimental evolution studies on Drosophila melanogaster, was that enhanced competitive ability evolves primarily through increased larval feeding and foraging rate, and increased larval tolerance to nitrogenous wastes, at the cost of efficiency of food conversion to biomass. These results were at odds from the predictions of classical K-selection theory, notably the expectation that selection at high density should result in the increase of efficiency of conversion of food to biomass, and were better interpreted through the lens of α-selection. We show here that populations of D. ananassae and D. n. nasuta subjected to extreme larval crowding evolve greater competitive ability and pre-adult survivorship at high density, primarily through a combination of reduced larval duration, faster attainment of minimum critical size for pupation, greater time efficiency of food conversion to biomass and increased pupation height, with a relatively small role of increased urea/ammonia tolerance, if at all. This is a very different suite of traits than that seen to evolve under similar selection in D. melanogaster, and seems to be closer to the expectations from the canonical theory of K-selection. We also discuss possible reasons for these differences in results across the three species. Overall, the results reinforce the view that our understanding of the evolution of competitive ability in fruitflies needs to be more nuanced than before, with an appreciation that there may be multiple evolutionary routes through which higher competitive ability can be attained.