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.

The Primary Cilium and Neuronal Migration.

The primary cilium (PC) is a microtubule-based tiny sensory organelle emanating from the centrosome and protruding from the surface of most eukaryotic cells, including neurons. The extremely severe phenotypes of ciliopathies have suggested their paramount importance for multiple developmental events, including brain formation. Neuronal migration is an essential step of neural development, with all neurons traveling from their site of birth to their site of integration. Neurons perform a unique type of cellular migration called cyclic saltatory migration, where their soma periodically jumps along with the stereotyped movement of their centrosome. We will review here how the role of the PC on cell motility was first described in non-neuronal cells as a guide pointing to the direction of migration. We will see then how these findings are extended to neuronal migration. In neurons, the PC appears to regulate the rhythm of cyclic saltatory neuronal migration in multiple systems. Finally, we will review recent findings starting to elucidate how extracellular cues sensed by the PC could be intracellularly transduced to regulate the machinery of neuronal migration. The PC of migrating neurons was unexpectedly discovered to display a rhythmic extracellular emergence during each cycle of migration, with this transient exposure to the external environment associated with periodic transduction of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. The PC in migrating neurons thus uniquely appears as a beat maker, regulating the tempo of cyclic saltatory migration.

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.

Role of Adult-Born Versus Preexisting Neurons Born at P0 in Olfactory Perception in a Complex Olfactory Environment in Mice.

Olfactory perceptual learning is defined as an improvement in the discrimination of perceptually close odorants after passive exposure to these odorants. In mice, simple olfactory perceptual learning involving the discrimination of two odorants depends on an increased number of adult-born neurons in the olfactory bulb, which refines the bulbar output. However, the olfactory environment is complex, raising the question of the adjustment of the bulbar network to multiple discrimination challenges. Perceptual learning of 1 to 6 pairs of similar odorants led to discrimination of all learned odor pairs. Increasing complexity did not increase adult-born neuron survival but enhanced the number of adult-born neurons responding to learned odorants and their spine density. Moreover, only complex learning induced morphological changes in neurons of the granule cell layer born during the first day of life (P0). Selective optogenetic inactivation of either population confirmed functional involvement of adult-born neurons regardless of the enrichment complexity, while preexisting neurons were required for complex discrimination only.

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.

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.

Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway.

Hypothalamic tanycytes are specialized glial cells lining the third ventricle. They are recently identified as adult stem and/or progenitor cells, able to self-renew and give rise to new neurons postnatally. However, the long-term neurogenic potential of tanycytes and the pathways regulating lifelong cell replacement in the adult hypothalamus are largely unexplored. Using inducible nestin-CreER(T2) for conditional mutagenesis, we performed lineage tracing of adult hypothalamic stem and/or progenitor cells (HySC) and demonstrated that new neurons continue to be born throughout adult life. This neurogenesis was targeted to numerous hypothalamic nuclei and produced different types of neurons in the dorsal periventricular regions. Some adult-born neurons integrated the median eminence and arcuate nucleus during aging and produced growth hormone releasing hormone. We showed that adult hypothalamic neurogenesis was tightly controlled by insulin-like growth factors (IGF). Knockout of IGF-1 receptor from hypothalamic stem and/or progenitor cells increased neuronal production and enhanced α-tanycyte self-renewal, preserving this stem cell-like population from age-related attrition. Our data indicate that adult hypothalamus retains the capacity of cell renewal, and thus, a substantial degree of structural plasticity throughout lifespan.

Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

BACKGROUND: In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. METHODS: Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. RESULTS: Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. CONCLUSIONS: Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause.

A role for dendritic translation of CaMKIIα mRNA in olfactory plasticity.

Local protein synthesis in dendrites contributes to the synaptic modifications underlying learning and memory. The mRNA encoding the α subunit of the calcium/calmodulin dependent Kinase II (CaMKIIα) is dendritically localized and locally translated. A role for CaMKIIα local translation in hippocampus-dependent memory has been demonstrated in mice with disrupted CaMKIIα dendritic translation, through deletion of CaMKIIα 3'UTR. We studied the dendritic localization and local translation of CaMKIIα in the mouse olfactory bulb (OB), the first relay of the olfactory pathway, which exhibits a high level of plasticity in response to olfactory experience. CaMKIIα is expressed by granule cells (GCs) of the OB. Through in situ hybridization and synaptosome preparation, we show that CaMKIIα mRNA is transported in GC dendrites, synaptically localized and might be locally translated at GC synapses. Increases in the synaptic localization of CaMKIIα mRNA and protein in response to brief exposure to new odors demonstrate that they are activity-dependent processes. The activity-induced dendritic transport of CaMKIIα mRNA can be inhibited by an NMDA receptor antagonist and mimicked by an NMDA receptor agonist. Finally, in mice devoid of CaMKIIα 3'UTR, the dendritic localization of CaMKIIα mRNA is disrupted in the OB and olfactory associative learning is severely impaired. Our studies thus reveal a new functional modality for CaMKIIα local translation, as an essential determinant of olfactory plasticity.

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.

Regulation of survival in adult hippocampal and glioblastoma stem cell lineages by the homeodomain-only protein HOP.

BACKGROUND: Homeodomain proteins play critical roles in shaping the development of the embryonic central nervous system in mammals. After birth, neurogenic activities are relegated to stem cell niches, which include the subgranular layer of the dentate gyrus of the hippocampus. Here, we have analyzed the function of HOP (Homeodomain only protein) in this stem cell niche and in human glioblastomas. RESULTS: We find that HOP is strongly expressed by radial astrocytes of the dentate gyrus in mice, which are stem cells that give rise to hippocampal granular neurons throughout adulthood. Deletion or down-regulation of HOP results in a decrease of apoptosis of these stem cells without changes in proliferation, and in an increase in the number of newly formed granule neurons. We also find that human glioblastomas largely lack HOP expression and that reintroduction of HOP function in glioma cells cultured as gliomaspheres leads to enhanced apoptosis in a subset of cases. In these cells, HOP function decreases clonogenicity. CONCLUSION: These data suggest that HOP participates in the regulation of the adult mouse hippocampal stem cell niche by negatively affecting cell survival. In addition, HOP may work as a tumor suppressor in a subset of glioblastomas. HOP function thus appears to be critical in the adult brain in a region of continued plasticity, and its deregulation may contribute to disease.

Dopamine depletion impairs precursor cell proliferation in Parkinson disease.

Cerebral dopamine depletion is the hallmark of Parkinson disease. Because dopamine modulates ontogenetic neurogenesis, depletion of dopamine might affect neural precursors in the subependymal zone and subgranular zone of the adult brain. Here we provide ultrastructural evidence showing that highly proliferative precursors in the adult subependymal zone express dopamine receptors and receive dopaminergic afferents. Experimental depletion of dopamine in rodents decreases precursor cell proliferation in both the subependymal zone and the subgranular zone. Proliferation is restored completely by a selective agonist of D2-like (D2L) receptors. Experiments with neural precursors from the adult subependymal zone grown as neurosphere cultures confirm that activation of D2L receptors directly increases the proliferation of these precursors. Consistently, the numbers of proliferating cells in the subependymal zone and neural precursor cells in the subgranular zone and olfactory bulb are reduced in postmortem brains of individuals with Parkinson disease. These observations suggest that the generation of neural precursor cells is impaired in Parkinson disease as a consequence of dopaminergic denervation.

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.

EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.

Neural stem cells in the subventricular zone (SVZ) continue to generate new neurons in the adult brain. SVZ cells exposed to EGF in culture grow to form neurospheres that are multipotent and self-renewing. We show here that the majority of these EGF-responsive cells are not derived from relatively quiescent stem cells in vivo, but from the highly mitotic, Dlx2(+), transit-amplifying C cells. When exposed to EGF, C cells downregulate Dlx2, arrest neuronal production, and become highly proliferative and invasive. Killing Dlx2(+) cells dramatically reduces the in vivo response to EGF and neurosphere formation in vitro. Furthermore, purified C cells are 53-fold enriched for neurosphere generation. We conclude that transit-amplifying cells retain stem cell competence under the influence of growth factors.

Lack of the cell-cycle inhibitor p27Kip1 results in selective increase of transit-amplifying cells for adult neurogenesis.

The subventricular zone (SVZ) is the largest germinal layer in the adult mammalian brain and comprises stem cells, transit-amplifying progenitors, and committed neuroblasts. Although the SVZ contains the highest concentration of dividing cells in the adult brain, the intracellular mechanisms controlling their proliferation have not been elucidated. We show here that loss of the cyclin-dependent kinase inhibitor p27Kip1 has very specific effects on a population of CNS progenitors responsible for adult neurogenesis. Using bromodeoxyuridine and [(3)H]thymidine incorporation to label cells in S phase and cell-specific markers and electron microscopy to identify distinct cell types, we compared the SVZ structure and proliferation characteristics of wild-type and p27Kip1-null mice. Loss of p27Kip1 had no effect on the number of stem cells but selectively increased the number of the transit-amplifying progenitors concomitantly with a reduction in the number of neuroblasts. We conclude that cell-cycle regulation of SVZ adult progenitors is remarkably cell-type specific, with p27Kip1 being a key regulator of the cell division of the transit-amplifying progenitors.

Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents.

The lateral walls of the forebrain lateral ventricles are the richest source of stem cells in the adult mammalian brain. These stem cells give rise to new olfactory neurons that are renewed throughout life. The neurons originate in the subventricular zone (SVZ), migrate within the rostral extension (RE) of the SVZ along the rostral migratory stream (RMS) within tube-like structures formed of glial cells, to eventually reach the olfactory bulb (OB). We demonstrate that, contrary to the current view, multipotential (neuronal-astroglial-oligodendroglial) precursors with stem cell features can be isolated not only from the SVZ but also from the entire RE, including the distal portion within the OB. Specifically, these stem cells do not derive from the migratory neuroblasts coming from the SVZ. Interestingly, stem cells isolated from the proximal RE generate significantly more oligodendrocytes, and those from the distal RE proliferate significantly more slowly than stem cells derived from the SVZ and other RE regions. These findings demonstrate that stem cells are not confined to the forebrain periventricular region and indicate that stem cells endowed with different functional characteristics occur at different levels of the SVZ-RE pathway.