Chemical and mechanical control of axon fasciculation and defasciculation.

Neural networks are constructed through the development of robust axonal projections from individual neurons, which ultimately establish connections with their targets. In most animals, developing axons assemble in bundles to navigate collectively across various areas within the central nervous system or the periphery, before they separate from these bundles in order to find their specific targets. These processes, called fasciculation and defasciculation respectively, were thought for many years to be controlled chemically: while guidance cues may attract or repulse axonal growth cones, adhesion molecules expressed at the surface of axons mediate their fasciculation. Recently, an additional non-chemical parameter, the mechanical longitudinal tension of axons, turned out to play a role in axon fasciculation and defasciculation, through zippering and unzippering of axon shafts. In this review, we present an integrated view of the currently known chemical and mechanical control of axon:axon dynamic interactions. We highlight the facts that the decision to cross or not to cross another axon depends on a combination of chemical, mechanical and geometrical parameters, and that the decision to fasciculate/defasciculate through zippering/unzippering relies on the balance between axon:axon adhesion and their mechanical tension. Finally, we speculate about possible functional implications of zippering-dependent axon shaft fasciculation, in the collective migration of axons, and in the sorting of subpopulations of axons.

Congenital mirror movements are associated with defective polymerisation of RAD51.

BACKGROUND: Mirror movements are involuntary movements of one hand that mirror intentional movements of the other hand. Congenital mirror movements (CMM) is a rare genetic disorder with autosomal dominant inheritance, in which mirror movements are the main neurological manifestation. CMM is associated with an abnormal decussation of the corticospinal tract, a major motor tract for voluntary movements. RAD51 is known to play a key role in homologous recombination with a critical function in DNA repair. While RAD51 haploinsufficiency was first proposed to explain CMM, other mechanisms could be involved. METHODS: We performed Sanger sequencing of RAD51 in five newly identified CMM families to identify new pathogenic variants. We further investigated the expression of wild-type and mutant RAD51 in the patients' lymphoblasts at mRNA and protein levels. We then characterised the functions of RAD51 altered by non-truncating variants using biochemical approaches. RESULTS: The level of wild-type RAD51 protein was lower in the cells of all patients with CMM compared with their non-carrier relatives. The reduction was less pronounced in asymptomatic carriers. In vitro, mutant RAD51 proteins showed loss-of-function for polymerisation, DNA binding and strand exchange activity. CONCLUSION: Our study demonstrates that RAD51 haploinsufficiency, including loss-of-function of non-truncating variants, results in CMM. The incomplete penetrance likely results from post-transcriptional compensation. Changes in RAD51 levels and/or polymerisation properties could influence guidance of the corticospinal axons during development. Our findings open up new perspectives to understand the role of RAD51 in neurodevelopment.

Alternative polyadenylation produces multiple 3' untranslated regions of odorant receptor mRNAs in mouse olfactory sensory neurons.

BACKGROUND: Odorant receptor genes constitute the largest gene family in mammalian genomes and this family has been extensively studied in several species, but to date far less attention has been paid to the characterization of their mRNA 3' untranslated regions (3'UTRs). Given the increasing importance of UTRs in the understanding of RNA metabolism, and the growing interest in alternative polyadenylation especially in the nervous system, we aimed at identifying the alternative isoforms of odorant receptor mRNAs generated through 3'UTR variation. RESULTS: We implemented a dedicated pipeline using IsoSCM instead of Cufflinks to analyze RNA-Seq data from whole olfactory mucosa of adult mice and obtained an extensive description of the 3'UTR isoforms of odorant receptor mRNAs. To validate our bioinformatics approach, we exhaustively analyzed the 3'UTR isoforms produced from 2 pilot genes, using molecular approaches including northern blot and RNA ligation mediated polyadenylation test. Comparison between datasets further validated the pipeline and confirmed the alternative polyadenylation patterns of odorant receptors. Qualitative and quantitative analyses of the annotated 3' regions demonstrate that 1) Odorant receptor 3'UTRs are longer than previously described in the literature; 2) More than 77% of odorant receptor mRNAs are subject to alternative polyadenylation, hence generating at least 2 detectable 3'UTR isoforms; 3) Splicing events in 3'UTRs are restricted to a limited subset of odorant receptor genes; and 4) Comparison between male and female data shows no sex-specific differences in odorant receptor 3'UTR isoforms. CONCLUSIONS: We demonstrated for the first time that odorant receptor genes are extensively subject to alternative polyadenylation. This ground-breaking change to the landscape of 3'UTR isoforms of Olfr mRNAs opens new avenues for investigating their respective functions, especially during the differentiation of olfactory sensory neurons.

Nxnl2 splicing results in dual functions in neuronal cell survival and maintenance of cell integrity.

The rod-derived cone viability factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration, arises due to the loss of trophic support from RdCVF2. In contrary, the progressive loss of rod visual function of the Nxnl2-/- mouse results from a decrease in outer segment length, mediated by a cell autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2-/- mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.

FMRP regulates postnatal neuronal migration via MAP1B.

The fragile X syndrome (FXS) represents the most prevalent form of inherited intellectual disability and is the first monogenic cause of autism spectrum disorder. FXS results from the absence of the RNA-binding protein FMRP (fragile X messenger ribonucleoprotein). Neuronal migration is an essential step of brain development allowing displacement of neurons from their germinal niches to their final integration site. The precise role of FMRP in neuronal migration remains largely unexplored. Using live imaging of postnatal rostral migratory stream (RMS) neurons in Fmr1-null mice, we observed that the absence of FMRP leads to delayed neuronal migration and altered trajectory, associated with defects of centrosomal movement. RNA-interference-induced knockdown of Fmr1 shows that these migratory defects are cell-autonomous. Notably, the primary Fmrp mRNA target implicated in these migratory defects is microtubule-associated protein 1B (MAP1B). Knocking down MAP1B expression effectively rescued most of the observed migratory defects. Finally, we elucidate the molecular mechanisms at play by demonstrating that the absence of FMRP induces defects in the cage of microtubules surrounding the nucleus of migrating neurons, which is rescued by MAP1B knockdown. Our findings reveal a novel neurodevelopmental role for FMRP in collaboration with MAP1B, jointly orchestrating neuronal migration by influencing the microtubular cytoskeleton.

FMRP regulates tangential neuronal migration via MAP1B.

The Fragile X Syndrome (FXS) represents the most prevalent form of inherited intellectual disability and is the first monogenic cause of Autism Spectrum Disorder. FXS results from the absence of the RNA-binding protein FMRP (Fragile X Messenger Ribonucleoprotein). Neuronal migration is an essential step of brain development allowing displacement of neurons from their germinal niches to their final integration site. The precise role of FMRP in neuronal migration remains largely unexplored. Using live imaging of postnatal Rostral Migratory Stream (RMS) neurons in Fmr1-null mice, we observed that the absence of FMRP leads to delayed neuronal migration and altered trajectory, associated with defects of centrosomal movement. RNA-interference-induced knockdown of Fmr1 shows that these migratory defects are cell-autonomous. Notably, the primary FMRP mRNA target implicated in these migratory defects is MAP1B (Microtubule-Associated Protein 1B). Knocking-down MAP1B expression effectively rescued most of the observed migratory defects. Finally, we elucidate the molecular mechanisms at play by demonstrating that the absence of FMRP induces defects in the cage of microtubules surrounding the nucleus of migrating neurons, which is rescued by MAP1B knockdown. Our findings reveal a novel neurodevelopmental role for FMRP in collaboration with MAP1B, jointly orchestrating neuronal migration by influencing the microtubular cytoskeleton.

Fragile X mental retardation protein regulates new neuron differentiation in the adult olfactory bulb.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein essential for multiple aspects of neuronal mRNA metabolism. Its absence leads to the fragile X syndrome, the most prevalent genetic form of mental retardation. The anatomical landmark of the disease, also present in the Fmr1 knock-out (KO) mice, is the hyperabundance of immature-looking lengthened dendritic spines. We used the well known continuous production of adult-born granule cells (GCs) in the mouse olfactory bulb (OB) to analyze the consequences of Fmrp loss on the differentiation of GCs. Morphological analysis of GCs in the Fmr1 KO mice showed an increase in spine density without a change in spine length. We developed an RNA interference strategy to cell-autonomously mutate Fmr1 in a wild-type OB network. Mutated GCs displayed an increase in spine density and spine length. Detailed analysis of the spines through immunohistochemistry, electron microscopy, and electrophysiology surprisingly showed that, despite these abnormalities, spines receive normal glutamatergic synapses, and thus that mutated adult-born neurons are synaptically integrated into the OB circuitry. Time-course analysis of the spine defects showed that Fmrp cell-autonomously downregulates the level and rate of spine production and limits their overgrowth. Finally, we report that Fmrp does not regulate dendritogenesis in standard conditions but is necessary for activity-dependent dendritic remodeling. Overall, our study of Fmrp in the context of adult neurogenesis has enabled us to carry out a precise dissection of the role of Fmrp in neuronal differentiation and underscores its pleiotropic involvement in both spinogenesis and dendritogenesis.

The nuclear receptor ROR(alpha) exerts a bi-directional regulation of IL-6 in resting and reactive astrocytes.

Astrocytes and one of their products, IL-6, not only support neurons but also mediate inflammation in the brain. Retinoid-related orphan receptor-alpha (RORalpha) transcription factor has related roles, being neuro-protective and, in peripheral tissues, anti-inflammatory. We examined the relation of ROR(alpha) to astrocytes and IL-6 using normal and ROR(alpha) loss-of-function mutant mice. We have shown ROR(alpha) expression in astrocytes and its up-regulation by pro-inflammatory cytokines. We have also demonstrated that ROR(alpha) directly trans-activates the Il-6 gene. We suggest that this direct control is necessary to maintain IL-6 basal level in the brain and may be a link between the neuro-supportive roles of ROR(alpha), IL-6, and astrocytes. Furthermore, after inflammatory stimulation, the absence of ROR(alpha) results in excessive IL-6 up-regulation, indicating that ROR(alpha) exerts an indirect repression probably via the inhibition of the NF-kappaB signaling. Thus, our findings indicate that ROR(alpha) is a pluripotent molecular player in constitutive and adaptive astrocyte physiology.

Roadbumps at the Crossroads of Integrating Behavioral and In Vitro Approaches for Neurotoxicity Assessment.

With the appreciation that behavior represents the integration and complexity of the nervous system, neurobehavioral phenotyping and assessment has seen a renaissance over the last couple of decades, resulting in a robust database on rodent performance within various testing paradigms, possible associations with human disorders, and therapeutic interventions. The interchange of data across behavior and other test modalities and multiple model systems has advanced our understanding of fundamental biology and mechanisms associated with normal functions and alterations in the nervous system. While there is a demonstrated value and power of neurobehavioral assessments for examining alterations due to genetic manipulations, maternal factors, early development environment, the applied use of behavior to assess environmental neurotoxicity continues to come under question as to whether behavior represents a sensitive endpoint for assessment. Why is rodent behavior a sensitive tool to the neuroscientist and yet, not when used in pre-clinical or chemical neurotoxicity studies? Applying new paradigms and evidence on the biological basis of behavior to neurobehavioral testing requires expertise and refinement of how such experiments are conducted to minimize variability and maximize information. This review presents relevant issues of methods used to conduct such test, sources of variability, experimental design, data analysis, interpretation, and reporting. It presents beneficial and critical limitations as they translate to the in vivo environment and considers the need to integrate across disciplines for the best value. It proposes that a refinement of behavioral assessments and understanding of subtle pronounced differences will facilitate the integration of data obtained across multiple approaches and to address issues of translation.

The transcription factor Engrailed-2 guides retinal axons.

Engrailed-2 (En-2), a homeodomain transcription factor, is expressed in a caudal-to-rostral gradient in the developing midbrain, where it has an instructive role in patterning the optic tectum--the target of topographic retinal input. In addition to its well-known role in regulating gene expression through its DNA-binding domain, En-2 may also have a role in cell-cell communication, as suggested by the presence of other domains involved in nuclear export, secretion and internalization. Consistent with this possibility, here we report that an external gradient of En-2 protein strongly repels growth cones of Xenopus axons originating from the temporal retina and, conversely, attracts nasal axons. Fluorescently tagged En-2 accumulates inside growth cones within minutes of exposure, and a mutant form of the protein that cannot enter cells fails to elicit axon turning. Once internalized, En-2 stimulates the rapid phosphorylation of proteins involved in translation initiation and triggers the local synthesis of new proteins. Furthermore, the turning responses of both nasal and temporal growth cones in the presence of En-2 are blocked by inhibitors of protein synthesis. The differential guidance of nasal and temporal axons reported here suggests that En-2 may participate directly in topographic map formation in the vertebrate visual system.

Loss of floor plate Netrin-1 impairs midline crossing of corticospinal axons and leads to mirror movements.

In humans, execution of unimanual movements requires lateralized activation of the primary motor cortex, which then transmits the motor command to the contralateral hand through the crossed corticospinal tract (CST). Mutations in NTN1 alter motor control lateralization, leading to congenital mirror movements. To address the role of midline Netrin-1 on CST development and subsequent motor control, we analyze the morphological and functional consequences of floor plate Netrin-1 depletion in conditional knockout mice. We show that depletion of floor plate Netrin-1 in the brainstem critically disrupts CST midline crossing, whereas the other commissural systems are preserved. The only associated defect is an abnormal entry of CST axons within the inferior olive. Alteration of CST midline crossing results in functional ipsilateral projections and is associated with abnormal symmetric movements. Our study reveals the role of Netrin-1 in CST development and describes a mouse model recapitulating the characteristics of human congenital mirror movements.

Evidence for developmentally regulated local translation of odorant receptor mRNAs in the axons of olfactory sensory neurons.

Odorant receptor mRNAs are transported within axons of olfactory sensory neurons that project into the olfactory bulb. Odorant receptor proteins have been identified along the distal part of these axons, which raises the possibility of their local synthesis in axons. We took advantage of the anatomical separation between the olfactory mucosa (which contains the sensory neuron cell bodies) and the bulb (which contains sensory axons but no sensory neuron cell bodies) to address this issue using a quantitative biochemical approach. Combining a method that separates polysome-associated mRNAs from untranslated mRNAs with a reverse transcription-quantitative PCR approach, we demonstrate that significant amounts of odorant receptor mRNAs are associated with polysomes in the sensory axons of the adult mouse bulb. We thus provide the first evidence for local synthesis of odorant receptor proteins in these axons. Interestingly, the rate of odorant receptor mRNA translation in axons is significantly greater during periods when the proportion of immature axons is higher (i.e., at postnatal day 4 or on regeneration after chemical lesion in adults). In contrast, the olfactory marker protein mRNA, which is restricted to mature axons, is translated at a low and constant level. Overall, we demonstrate that translation levels of odorant receptor mRNAs in axons are developmentally regulated, and positively correlated to the stage of axonal growth into the bulb. Given the established function of odorant receptors in the axonal wiring of sensory projections, we propose that this regulated axonal translation may play a role in the development and maintenance of the glomerular array.

Primary cilium-dependent cAMP/PKA signaling at the centrosome regulates neuronal migration.

The primary cilium (PC) is a small centrosome-assembled organelle, protruding from the surface of most eukaryotic cells. It plays a key role in cell migration, but the underlying mechanisms are unknown. Here, we show that the PC regulates neuronal migration via cyclic adenosine 3'-5' monosphosphate (cAMP) production activating centrosomal protein kinase A (PKA). Biosensor live imaging revealed a periodic cAMP hotspot at the centrosome of embryonic, postnatal, and adult migrating neurons. Genetic ablation of the PC, or knockdown of ciliary adenylate cyclase 3, caused hotspot disappearance and migratory defects, with defective centrosome dynamics and altered nucleokinesis. Delocalization of PKA from the centrosome phenocopied the migratory defects. Our results show that the PC and centrosome form a single cAMP signaling unit dynamically regulating migration, further highlighting the centrosome as a signaling hub.

Specific interaction between Sam68 and neuronal mRNAs: implication for the activity-dependent biosynthesis of elongation factor eEF1A.

In cultured hippocampal neurons and in adult brain, the splicing regulatory protein Sam68 is partially relocated to the somatodendritic domain and associates with dendritic polysomes. Transfer to the dendrites is activity-dependent. We have investigated the repertoire of neuronal mRNAs to which Sam68 binds in vivo. By using coimmunoprecipitation and microarray screening techniques, Sam68 was found to associate with a number of plasticity-related mRNA species, including Eef1a1, an activity-responsive mRNA coding for translation elongation factor eEF1A. In cortical neuronal cultures, translation of the Eef1a1 mRNA was strongly induced by neuronal depolarisation and correlated with enhanced association of Sam68 with polysomal mRNAs. The possible function of Sam68 in Eef1a1 mRNA utilization was studied by expressing a dominant-negative, cytoplasmic Sam68 mutant (GFP-Sam68DeltaC) in cultured hippocampal neurons. The level of eEF1A was lower in neurons expressing GFP-Sam68DeltaC than in control neurons, supporting the proposal that endogenous Sam68 may contribute to the translational efficiency of the Eef1a1 mRNA. These findings are discussed in the light of the complex, potentially crucial regulation of eEF1A biosynthesis during long-term synaptic change.

Preparation and Manipulation of Olfactory Epithelium Explant Cultures for Measurement of the Mechanical Tension of Individual Axons Using the Biomembrane Force Probe.

In this paper, we describe a protocol allowing measurement of the mechanical tension of individual axons grown ex vivo from neural tissue explants. This protocol was developed with primary cultures of olfactory epithelium explants from embryonic (E13.5) mice. It includes a detailed description of explant dissection and culture, as well as the main steps of the procedure for axon tension measurement using the previously established Biomembrane Force Probe.

Axon tension regulates fasciculation/defasciculation through the control of axon shaft zippering.

While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behavior that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and mechanical tension in the axons, and provide the first quantification of the force of axon-axon adhesion. Furthermore, we introduce a biophysical model of the zippering dynamics, and we quantitatively relate the individual zipper properties to global characteristics of the developing axon network. Our study uncovers a new role of mechanical tension in neural development: the regulation of axon fasciculation.

BFPTool: a software tool for analysis of Biomembrane Force Probe experiments.

BACKGROUND: The Biomembrane Force Probe is an approachable experimental technique commonly used for single-molecule force spectroscopy and experiments on biological interfaces. The technique operates in the range of forces from 0.1 pN to 1000 pN. Experiments are typically repeated many times, conditions are often not optimal, the captured video can be unstable and lose focus; this makes efficient analysis challenging, while out-of-the-box non-proprietary solutions are not freely available. RESULTS: This dedicated tool was developed to integrate and simplify the image processing and analysis of videomicroscopy recordings from BFP experiments. A novel processing feature, allowing the tracking of the pipette, was incorporated to address a limitation of preceding methods. Emphasis was placed on versatility and comprehensible user interface implemented in a graphical form. CONCLUSIONS: An integrated analytical tool was implemented to provide a faster, simpler and more convenient way to process and analyse BFP experiments.

CaMKIIα Expression Defines Two Functionally Distinct Populations of Granule Cells Involved in Different Types of Odor Behavior.

Granule cells (GCs) in the olfactory bulb (OB) play an important role in odor information processing. Although they have been classified into various neurochemical subtypes, the functional roles of these subtypes remain unknown. We used in vivo two-photon Ca2+ imaging combined with cell-type-specific identification of GCs in the mouse OB to examine whether functionally distinct GC subtypes exist in the bulbar network. We showed that half of GCs express Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα+) and that these neurons are preferentially activated by olfactory stimulation. The higher activity of CaMKIIα+ neurons is due to the weaker inhibitory input that they receive compared to their CaMKIIα-immunonegative (CaMKIIα-) counterparts. In line with these functional data, immunohistochemical analyses showed that 75%-90% of GCs expressing the immediate early gene cFos are CaMKIIα+ in naive animals and in mice that have been exposed to a novel odor and go/no-go operant conditioning, or that have been subjected to long-term associative memory and spontaneous habituation/dishabituation odor discrimination tasks. On the other hand, a perceptual learning task resulted in increased activation of CaMKIIα- cells. Pharmacogenetic inhibition of CaMKIIα+ GCs revealed that this subtype is involved in habituation/dishabituation and go/no-go odor discrimination, but not in perceptual learning. In contrast, pharmacogenetic inhibition of GCs in a subtype-independent manner affected perceptual learning. Our results indicate that functionally distinct populations of GCs exist in the OB and that they play distinct roles during different odor tasks.

Non cell-autonomous role of DCC in the guidance of the corticospinal tract at the midline.

DCC, a NETRIN-1 receptor, is considered as a cell-autonomous regulator for midline guidance of many commissural populations in the central nervous system. The corticospinal tract (CST), the principal motor pathway for voluntary movements, crosses the anatomic midline at the pyramidal decussation. CST fails to cross the midline in Kanga mice expressing a truncated DCC protein. Humans with heterozygous DCC mutations have congenital mirror movements (CMM). As CMM has been associated, in some cases, with malformations of the pyramidal decussation, DCC might also be involved in this process in human. Here, we investigated the role of DCC in CST midline crossing both in human and mice. First, we demonstrate by multimodal approaches, that patients with CMM due to DCC mutations have an increased proportion of ipsilateral CST projections. Second, we show that in contrast to Kanga mice, the anatomy of the CST is not altered in mice with a deletion of DCC in the CST. Altogether, these results indicate that DCC controls CST midline crossing in both humans and mice, and that this process is non cell-autonomous in mice. Our data unravel a new level of complexity in the role of DCC in CST guidance at the midline.

New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes.

Cytoplasmic FMRP interacting protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene), whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC), thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models - fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction.