Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila.

Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.

The emerging roles of microRNAs in the pathogenesis of frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) spectrum disorders.

Increasing evidence suggests that frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) share some clinical, pathological, and molecular features as part of a common neurodegenerative spectrum disorder. In recent years, enormous progress has been made in identifying both pathological proteins and genetic mutations associated with FTD-ALS. However, the molecular pathogenic mechanisms of disease onset and progression remain largely unknown. Recent studies have uncovered unexpected links between FTD-ALS and multiple aspects of RNA metabolism, setting the stage for further understanding of the disorder. Here, the authors will focus on microRNAs and review the emerging roles of these small RNAs in several aspects of FTD-ALS pathogenesis.

Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons.

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.

Brief and long maternal separation in C57Bl6J mice: behavioral consequences for the dam and the offspring.

Introduction: Animal models, especially rodents, have become instrumental to experimentally investigate the effects of an adverse post-natal environment on the developing brain. For this purpose, maternal separation (MS) paradigms have been widely used in the last decades. Nonetheless, how MS affects maternal behavior and, ultimately, the offspring depend on multiple variables. Methods: To gain further insights into the consequences of MS, we decided to thoroughly measure and compare the effects of short (15 min, 3 times/day) vs. long (3 h, 1 time/day) separation on multiple maternally-associated behaviors and across the entire post-natal period. Results: Compared to unhandled control litters, our results confirmed previous studies and indicated that SMS enhanced the time and variety of maternal care whereas LMS resulted in poor caregiving. We also showed that SMS-accrued caregiving persisted during the whole post-natal period. In contrast, LMS effects on maternal behavior were restricted to the early life (P2-P10). Finally, we also analyzed the behavioral consequences of these different rearing social environments on the offspring. We found that MS has profound effects in social tasks. We showed that affiliative touch, a type of prosocial behavior that provides comfort to others, is particularly sensitive to the modification of maternal caregiving. Discussion: Our results provide further support to the contention that interactions during the early post-natal period critically contribute to emotional processing and brain co-construction.

Optimized miR-124 reporters uncover differences in miR-124 expression among neuronal populations in vitro.

Introduction: Although intensively studied in the last decades, how microRNAs (miRNAs) are expressed across different cell types in the brain remains largely unknown. Materials: To address this issue, we sought to develop optimized fluorescence reporters that could be expressed in precise cellular subsets and used to accurately quantify miR contents in vivo. Results: Focusing on miR-124, we tested different reporter designs whose efficiency was confirmed in different in vitro settings including cell lines and primary neuronal cultures from different brain structures. Unlike previous reporters, we provide experimental evidence that our optimized designs can faithfully translate miR levels in vitro. Discussion: Tools developed here would enable assessing miRNA expression at the single cell resolution and are expected to significantly contribute to future miRNA research in vivo.

Region-specific microRNA alterations in marmosets carrying SLC6A4 polymorphisms are associated with anxiety-like behavior.

BACKGROUND: Psychiatric diseases such as depression and anxiety are multifactorial conditions, highly prevalent in western societies. Human studies have identified a number of high-risk genetic variants for these diseases. Among them, polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4) have attracted much attention. However, due to the paucity of experimental models, molecular alterations induced by these genetic variants and how they correlate to behavioral deficits have not been examined. In this regard, marmosets have emerged as a powerful model in translational neuroscience to investigate molecular underpinnings of complex behaviors. METHODS: Here, we took advantage of naturally occurring genetic polymorphisms in marmoset SLC6A4 gene that have been linked to anxiety-like behaviors. Using FACS-sorting, we profiled microRNA contents in different brain regions of genotyped and behaviorally-phenotyped marmosets. FINDINGS: We revealed that marmosets bearing different SLC6A4 variants exhibit distinct microRNAs signatures in a region of the prefrontal cortex whose activity has been consistently altered in patients with depression/anxiety. We also identified Deleted in Colorectal Cancer (DCC), a gene previously linked to these diseases, as a downstream target of the differently expressed microRNAs. Significantly, we showed that levels of both microRNAs and DCC in this region were highly correlated to anxiety-like behaviors. INTERPRETATION: Our findings establish links between genetic variants, molecular modifications in specific cortical regions and complex behavioral responses, providing new insights into gene-behavior relationships underlying human psychopathology. FUNDING: This work was supported by France National Agency, NRJ Foundation, Celphedia and Fondation de France as well as the Wellcome Trust.

Cholinergic interneuron inhibition potentiates corticostriatal transmission in direct medium spiny neurons and rescues motor learning in parkinsonism.

Striatal cholinergic interneurons (CINs) respond to salient or reward prediction-related stimuli after conditioning with brief pauses in their activity, implicating them in learning and action selection. This pause is lost in animal models of Parkinson's disease. How this signal regulates the striatal network remains an open question. Here, we examine the impact of CIN firing inhibition on glutamatergic transmission between the cortex and the medium spiny neurons expressing dopamine D1 receptor (D1 MSNs). Brief interruption of CIN activity has no effect in control conditions, whereas it increases glutamatergic responses in D1 MSNs after dopamine denervation. This potentiation depends upon M4 muscarinic receptor and protein kinase A. Decreasing CIN firing by optogenetics/chemogenetics in vivo partially rescues long-term potentiation in MSNs and motor learning deficits in parkinsonian mice. Our findings demonstrate that the control exerted by CINs on corticostriatal transmission and striatal-dependent motor-skill learning depends on the integrity of dopaminergic inputs.

Hepatocyte growth factor-Met signaling is required for Runx1 extinction and peptidergic differentiation in primary nociceptive neurons.

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

MicroRNA-9: functional evolution of a conserved small regulatory RNA.

The functional significance of microRNA-9 (miR-9) during evolution is evidenced by its conservation at the nucleotide level from flies to humans but not its diverse expression patterns. Recent studies in several model systems reveal that miR-9 can regulate neurogenesis through its actions in neural or non-neural cell lineages. In vertebrates, miR-9 exerts diverse cell-autonomous effects on the proliferation, migration, and differentiation of neural progenitor cells by modulating different mRNA targets. In some developmental contexts, miR-9 suppresses apoptosis and is misregulated in several types of cancer cells, influencing proliferation or metastasis formation. Moreover, downregulation of miR-9 in postmitotic neurons is also implicated in some neurodegenerative diseases. Thus, miR-9 is emerging as an important regulator in development and disease through its ability to modulate different targets in a manner dependent on the developmental stage and the cellular context.

Fibroblast growth factor-2 overexpression in transplanted neural progenitors promotes perivascular cluster formation with a neurogenic potential.

Stem/progenitor cell-based therapies hold promises for repairing the damaged nervous system. However, the efficiency of these approaches for neuronal replacement remains very limited. A major challenge is to develop pretransplant cell manipulations that may promote the survival, engraftment, and differentiation of transplanted cells. Here, we investigated whether overexpression of fibroblast growth factor-2 (FGF-2) in grafted neural progenitors could improve their integration in the host tissue. We show that FGF-2-transduced progenitors grafted in the early postnatal rat cortex have the distinct tendency to associate with the vasculature and establish multiple proliferative clusters in the perivascular environment. The contact with vessels appears to be critical for maintaining progenitor cells in an undifferentiated and proliferative phenotype in the intact cortex. Strikingly, perivascular clusters of FGF-2 expressing cells seem to supply immature neurons in an ischemic environment. Our data provide evidence that engineering neural progenitors to overexpress FGF-2 may be a suitable strategy to improve the integration of grafted neural progenitor cells with the host vasculature thereby generating neurovascular clusters with a neurogenic potential for brain repair.

Social Isolation and Enrichment Induce Unique miRNA Signatures in the Prefrontal Cortex and Behavioral Changes in Mice.

An extensive body of evidence supports the notion that exposure to an enriched/impoverished environment alters brain functions via epigenetic changes. However, how specific modifications of social environment modulate brain functions remains poorly understood. To address this issue, we investigate the molecular and behavioral consequences of briefly manipulating social settings in young and middle-aged wild-type mice. We observe that, modifications of the social context, only affect the performance in socially related tasks. Social enrichment increases sociability whereas isolation leads to the opposite effect. Our work also pointed out specific miRNA signatures associated to each social environment. These miRNA alterations are reversible and found selectively in the medial prefrontal cortex. Finally, we show that miRNA modifications linked to social enrichment or isolation might target rather different intracellular pathways. Together, these observations suggest that the prefrontal cortex may be a key brain area integrating social information via the modification of precise miRNA networks.

GPR56/ADGRG1 is associated with response to antidepressant treatment.

It remains unclear why many patients with depression do not respond to antidepressant treatment. In three cohorts of individuals with depression and treated with serotonin-norepinephrine reuptake inhibitor (N = 424) we show that responders, but not non-responders, display an increase of GPR56 mRNA in the blood. In a small group of subjects we also show that GPR56 is downregulated in the PFC of individuals with depression that died by suicide. In mice, we show that chronic stress-induced Gpr56 downregulation in the blood and prefrontal cortex (PFC), which is accompanied by depression-like behavior, and can be reversed by antidepressant treatment. Gpr56 knockdown in mouse PFC is associated with depressive-like behaviors, executive dysfunction and poor response to antidepressant treatment. GPR56 peptide agonists have antidepressant-like effects and upregulated AKT/GSK3/EIF4 pathways. Our findings uncover a potential role of GPR56 in antidepressant response.

Deficits in Social Behavior Precede Cognitive Decline in Middle-Aged Mice.

An extensive literature details deterioration of multiple brain functions, especially memory and learning, during aging in humans and in rodents. In contrast, the decline of social functions is less well understood. It is presently not clear whether age-dependent deficits observed in social behavior mainly reflect the disruption of social networks activity or are simply secondary to a more general impairment of cognitive and executive functions in older individuals. To address this issue, we carried out a battery of behavioral tasks exploring different brain functions in young (3 months) and middle-aged wild-type mice (9 months). Consistent with previous reports, our results show no obvious differences between these two groups in most of the domains investigated including learning and memory. Surprisingly, in social tasks, middle-aged animals showed significantly reduced levels of interactions when exposed to a new juvenile mouse. In the absence of overt cognitive decline, our findings suggest that social impairments may precede the disruption of other brain functions and argue for a selective vulnerability of social circuits during aging.

Adverse effects of methylene blue on the central nervous system.

BACKGROUND: An increasing number of clinical observations suggest adverse neurologic outcome after methylene blue (MB) infusion in the setting of parathyroid surgery. Hence, the aim of the current study was to investigate the potentially neurotoxic effects of MB using a combination of in vivo and in vitro experimental approaches. METHODS: Isoflurane-anesthetized adult rats were used to evaluate the impact of a single bolus intravascular administration of MB on systemic hemodynamic responses and on the minimum alveolar concentration (MAC) of isoflurane using the tail clamp test. In vivo, MB-induced cell death was evaluated 24 h after MB administration using Fluoro-Jade B staining and activated caspase-3 immunohistochemistry. In vitro, neurotoxic effects of MB were examined in hippocampal slice cultures by measuring excitatory field potentials as well as propidium iodide incorporation after MB exposure. The impact of MB on dendritic arbor was evaluated in differentiated single cell neuronal cultures. RESULTS: Bolus injections of MB significantly reduced isoflurane MAC and initiated widespread neuronal apoptosis. Electrophysiologic recordings in hippocampal slices revealed a rapid suppression of evoked excitatory field potentials by MB, and this was associated with a dose-dependent effect of this drug on cell death. Dose-response experiments in single cell neuronal cultures revealed that a 2-h-long exposure to MB at non-cell-death-inducing concentrations could still induce significant retraction of dendritic arbor. CONCLUSIONS: These results suggest that MB exerts neurotoxic effects on the central nervous system and raise questions regarding the safety of using this drug at high doses during parathyroid gland surgery.

Expression of FGF-2 in neural progenitor cells enhances their potential for cellular brain repair in the rodent cortex.

Strategies to enhance the capacity of grafted stem/progenitors cells to generate multipotential, proliferative and migrating pools of cells in the postnatal brain could be crucial for structural repair after brain damage. We investigated whether the over-expression of basic fibroblast growth factor 2 (FGF-2) in neural progenitor cells (NPCs) could provide a robust source of migrating NPCs for tissue repair in the rat cerebral cortex. Using live imaging we provide direct evidence that FGF-2 over-expression significantly enhances the migratory capacity of grafted NPCs in complex 3D structures, such as cortical slices. Furthermore, we show that the migratory as well as proliferative properties of FGF-2 over-expressing NPCs are maintained after in vivo transplantation. Importantly, after transplantation into a neonatal ischaemic cortex, FGF-2 over-expressing NPCs efficiently invade the injured cortex and generate an increased pool of immature neurons available for brain repair. Differentiation of progenitor cells into immature neurons was correlated with a gradual down-regulation of the FGF-2 transgene. These results reveal an important role for FGF-2 in regulating NPCs functions when interacting with the host tissue and offer a potential strategy to generate a robust source of migrating and immature progenitors for repairing a neonatal ischaemic cortex.

Polysialic acid-neural cell adhesion molecule in brain plasticity: from synapses to integration of new neurons.

Isoforms of the neuronal cell adhesion molecule (NCAM) carrying the linear homopolymer of alpha 2,8-linked sialic acid (polysialic acid, PSA) have emerged as particularly attractive candidates for promoting plasticity in the nervous system. The large negatively charged PSA chain of NCAM is postulated to be a spacer that reduces adhesion forces between cells allowing dynamic changes in membrane contacts. Accumulating evidence also suggests that PSA-NCAM-mediated interactions lead to activation of intracellular signaling cascades that are fundamental to the biological functions of the molecule. An important role of PSA-NCAM appears to be during development, when its expression level is high and where it contributes to the regulation of cell shape, growth or migration. However, PSA-NCAM does persist in adult brain structures such as the hippocampus that display a high degree of plasticity where it is involved in activity-induced synaptic plasticity. Recent advances in the field of PSA-NCAM research have not only consolidated the importance of this molecule in plasticity processes but also suggest a role for PSA-NCAM in the regulation of higher cognitive functions and psychiatric disorders. In this review, we discuss the role and mode of actions of PSA-NCAM in structural plasticity as well as its potential link to cognitive processes.

GABA regulates dendritic growth by stabilizing lamellipodia in newly generated interneurons of the olfactory bulb.

The initial formation and growth of dendrites is a critical step leading to the integration of newly generated neurons into postnatal functional networks. However, the cellular mechanisms and extracellular signals regulating this process remain mostly unknown. By directly observing newborn neurons derived from the subventricular zone in culture as well as in olfactory bulb slices, we show that ambient GABA acting through GABA(A) receptors is essential for the temporal stability of lamellipodial protrusions in dendritic growth cones but did not interfere with filopodia dynamics. Furthermore, we provide direct evidence that ambient GABA is required for the proper initiation and elongation of dendrites by promoting the rapid stabilization of new dendritic segments after their extension. The effects of GABA on the initial formation of dendrites depend on depolarization and Ca2+ influx and are associated with a higher stability of microtubules. Together, our results indicate that ambient GABA is a key regulator of dendritic initiation in postnatally generated olfactory interneurons and offer a mechanism by which this neurotransmitter drives early dendritic growth.

Low concentrations of ketamine initiate dendritic atrophy of differentiated GABAergic neurons in culture.

Administration of subanesthetic concentrations of ketamine, a noncompetitive antagonist of the N-methyl-d-aspartate (NMDA) type of glutamate receptors, is a widely accepted therapeutic modality in perioperative and chronic pain management. Although extensive clinical use has demonstrated its safety, recent human histopathological observations as well as laboratory data suggest that ketamine can exert adverse effects on central nervous system neurons. To further investigate this issue, the present study was designed to evaluate the effects of ketamine on the survival and dendritic arbor architecture of differentiated gamma-aminobutyric acidergic (GABAergic) interneurons in vitro. We show that short-term exposure of cultures to ketamine at concentrations of > or =20 microg/ml leads to a significant cell loss of differentiated cells and that non-cell death-inducing concentrations of ketamine (10 microg/ml) can still initiate long-term alterations of dendritic arbor in differentiated neurons, including dendritic retraction and branching point elimination. Most importantly, we also demonstrate that chronic (>24 h) administration of ketamine at concentrations as low as 0.01 microg/ml can interfere with the maintenance of dendritic arbor architecture. These results raise the possibility that chronic exposure to low, subanesthetic concentrations of ketamine, while not affecting cell survival, could still impair neuronal morphology and thus might lead to dysfunctions of neural networks.

Effects of ketamine on the developing central nervous system.

Ketamine is a widely used drug in pediatric anesthesia practice, acting primarily through the blockade of the N-methyl-D-aspartate (NMDA) type of glutamate receptors. A growing body of laboratory evidence, accumulated during the past few years, suggests that this drug could have potential adverse effects on the developing central nervous system. The goal of this short review is to give a brief synopsis of experimental work indicating ketamine-induced developmental neurotoxicity as well as to discuss potential limitations concerning extrapolation of these studies to clinical practice.