IF du Neurocentre

76 publications

* equal contribution
Les IF indiqués ont été collectés par le Web of Sciences en Juin 2021

27/04/2021 | Sci Rep   IF 4.4
Early loss of Scribble affects cortical development, interhemispheric connectivity and psychomotor activity.
Ezan J, Moreau MM, Mamo TM, Shimbo M, Decroo M, Richter M, Peyroutou R, Rachel R, Tissir F, de Anda FC, Sans N, Montcouquiol M

Neurodevelopmental disorders arise from combined defects in processes including cell proliferation, differentiation, migration and commissure formation. The evolutionarily conserved tumor-suppressor protein Scribble (Scrib) serves as a nexus to transduce signals for the establishment of apicobasal and planar cell polarity during these processes. Human SCRIB gene mutations are associated with neural tube defects and this gene is located in the minimal critical region deleted in the rare Verheij syndrome. In this study, we generated brain-specific conditional cKO mouse mutants and assessed the impact of the Scrib deletion on brain morphogenesis and behavior. We showed that embryonic deletion of Scrib in the telencephalon leads to cortical thickness reduction (microcephaly) and partial corpus callosum and hippocampal commissure agenesis. We correlated these phenotypes with a disruption in various developmental mechanisms of corticogenesis including neurogenesis, neuronal migration and axonal connectivity. Finally, we show that Scrib cKO mice have psychomotor deficits such as locomotor activity impairment and memory alterations. Altogether, our results show that Scrib is essential for early brain development due to its role in several developmental cellular mechanisms that could underlie some of the deficits observed in complex neurodevelopmental pathologies.

03/10/2020 | Aging Cell   IF 7.2
Age-related impairment of declarative memory: linking memorization of temporal associations to GluN2B redistribution in dorsal CA1.
Al Abed AS, Sellami A, Potier M, Ducourneau EG, Gerbeaud-Lassau P, Brayda-Bruno L, Lamothe V, Sans N, Desmedt A, Vanhoutte P, Bennetau-Pelissero C, Trifilieff P, Marighetto A

GluN2B subunits of NMDA receptors have been proposed as a target for treating age-related memory decline. They are indeed considered as crucial for hippocampal synaptic plasticity and hippocampus-dependent memory formation, which are both altered in aging. Because a synaptic enrichment in GluN2B is associated with hippocampal LTP in vitro, a similar mechanism is expected to occur during memory formation. We show instead that a reduction of GluN2B synaptic localization induced by a single-session learning in dorsal CA1 apical dendrites is predictive of efficient memorization of a temporal association. Furthermore, synaptic accumulation of GluN2B, rather than insufficient synaptic localization of these subunits, is causally involved in the age-related impairment of memory. These challenging data identify extra-synaptic redistribution of GluN2B-containing NMDAR induced by learning as a molecular signature of memory formation and indicate that modulating GluN2B synaptic localization might represent a useful therapeutic strategy in cognitive aging.

09/06/2020 | Cell Rep   IF 8.1
Vangl2 in the Dentate Network Modulates Pattern Separation and Pattern Completion.
Robert BJA, Moreau MM, Dos Santos Carvalho S, Barthet G, Racca C, Bhouri M, Quiedeville A, Garret M, Atchama B, Al Abed AS, Guette C, Henderson DJ, Desmedt A, Mulle C, Marighetto A, Montcouquiol M, Sans N

The organization of spatial information, including pattern completion and pattern separation processes, relies on the hippocampal circuits, yet the molecular and cellular mechanisms underlying these two processes are elusive. Here, we find that loss of Vangl2, a core PCP gene, results in opposite effects on pattern completion and pattern separation processes. Mechanistically, we show that Vangl2 loss maintains young postmitotic granule cells in an immature state, providing increased cellular input for pattern separation. The genetic ablation of Vangl2 disrupts granule cell morpho-functional maturation and further prevents CaMKII and GluA1 phosphorylation, disrupting the stabilization of AMPA receptors. As a functional consequence, LTP at lateral perforant path-GC synapses is impaired, leading to defects in pattern completion behavior. In conclusion, we show that Vangl2 exerts a bimodal regulation on young and mature GCs, and its disruption leads to an imbalance in hippocampus-dependent pattern completion and separation processes.

07/01/2020 | eLife   IF 7.1
Vangl2 acts at the interface between actin and N-cadherin to modulate mammalian neuronal outgrowth.
Dos-Santos Carvalho S, Moreau MM, Hien YE, Garcia M, Aubailly N, Henderson DJ, Studer V, Sans N, Thoumine O, Montcouquiol M

Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of Vangl2 decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.

09/12/2019 | J Neurosci Methods   IF 2.8
Alpha technology: A powerful tool to detect mouse brain intracellular signaling events.
Zanese M*, Tomaselli G*, Roullot-Lacarriere V, Moreau M, Bellocchio L, Grel A, Marsicano G, Sans N, Vallee M, Revest JM

BACKGROUND: Phosphorylation by protein kinases is a fundamental molecular process involved in the regulation of signaling activities in living organisms. Understanding this complex network of phosphorylation, especially phosphoproteins, is a necessary step for grasping the basis of cellular pathophysiology. Studying brain intracellular signaling is a particularly complex task due to the heterogeneous complex nature of the brain tissue, which consists of many embedded structures. NEW METHOD: Overcoming this degree of complexity requires a technology with a high throughput and economical in the amount of biological material used, so that a large number of signaling pathways may be analyzed in a large number of samples. We have turned to Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology. COMPARISON WITH EXISTING METHOD: Western blot is certainly the most commonly used method to measure the phosphorylation state of proteins. Even though Western blot is an accurate and reliable method for analyzing modifications of proteins, it is a time-consuming and large amounts of samples are required. Those two parameters are critical when the goal of the research is to comprehend multi-signaling proteic events so as to analyze several targets from small brain areas. RESULT: Here we demonstrate that Alpha technology is particularly suitable for studying brain signaling pathways by allowing rapid, sensitive, reproducible and semi-quantitative detection of phosphoproteins from individual mouse brain tissue homogenates and from cell fractionation and synaptosomal preparations of mouse hippocampus. CONCLUSION: Alpha technology represents a major experimental step forward in unraveling the brain phosphoprotein-related molecular mechanisms involved in brain-related disorders.

07/01/2019 | Cold Spring Harb Perspect Med   IF 5.6
Development and Patterning of the Cochlea: From Convergent Extension to Planar Polarity.
Montcouquiol M, Kelley MW

Within the mammalian cochlea, sensory hair cells and supporting cells are aligned in curvilinear rows that extend along the length of the tonotopic axis. In addition, all of the cells within the epithelium are uniformly polarized across the orthogonal neural-abneural axis. Finally, each hair cell is intrinsically polarized as revealed by the presence of an asymmetrically shaped and apically localized stereociliary bundle. It has been known for some time that many of the developmental processes that regulate these patterning events are mediated, to some extent, by the core planar cell polarity (PCP) pathway. This article will review more recent work demonstrating how components of the PCP pathway interact with cytoskeletal motor proteins to regulate cochlear outgrowth. Finally, a signaling pathway originally identified for its role in asymmetric cell divisions has recently been shown to mediate several aspects of intrinsic hair cell polarity, including kinocilia migration, bundle shape, and elongation.

21/06/2018 | cell physiol biochem   IF 5.5
Galphai Proteins are Indispensable for Hearing.
Beer-Hammer S, Lee SC, Mauriac SA, Leiss V, Groh IAM, Novakovic A, Piekorz RP, Bucher K, Chen C, Ni K, Singer W, Harasztosi C, Schimmang T, Zimmermann U, Pfeffer K, Birnbaumer L, Forge A, Montcouquiol M, Knipper M, Nurnberg B, Ruttiger L

BACKGROUND/AIMS: From invertebrates to mammals, Galphai proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Galphai3-deficiency in pre-hearing murine cochleae pointed to a role of Galphai3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ('hair') bundle, a requirement for the progression of mature hearing. We found that the lack of Galphai3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. METHODS: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Galphai proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Galphai isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. RESULTS: Here we report that lack of Galphai3 but not of the ubiquitously expressed Galphai2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Galphai2 or Galphai3 had no impact. In contrast, double-deficiency for Galphai2 and Galphai3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Galphai3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Galphai3 is selectively involved in generation of neural gain during auditory processing. CONCLUSION: We propose a so far unrecognized complexity of isoform-specific and overlapping Galphai protein functions particular during final differentiation processes.

25/05/2018 | Nat Commun   IF 12.4
Author Correction: Defective Gpsm2/Galphai3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome.
Mauriac SA, Hien YE, Bird JE, Carvalho SD, Peyroutou R, Lee SC, Moreau MM, Blanc JM, Gezer A, Medina C, Thoumine O, Beer-Hammer S, Friedman TB, Ruttiger L, Forge A, Nurnberg B, Sans N, Montcouquiol M

This corrects the article DOI: 10.1038/ncomms14907.

03/06/2017 | Neuroscience   IF 3.3
The embryonic development of hindbrain respiratory networks is unaffected by mutation of the planar polarity protein Scribble.
Chevalier M, Cardoit L, Moreau M, Sans N, Montcouquiol M, Simmers J, Thoby-Brisson M

The central command for breathing arises mainly from two interconnected rhythmogenic hindbrain networks, the parafacial respiratory group (pFRG or epF at embryonic stages) and the preBotzinger complex (preBotC), which are comprised of a limited number of neurons located in confined regions of the ventral medulla. In rodents, both networks become active toward the end of gestation but little is known about the signaling pathways involved in their anatomical and functional establishment during embryogenesis. During embryonic development, epF and preBotC neurons migrate from their territories of origin to their final positions in ventral brainstem areas. Planar Cell Polarity (PCP) signaling, including the molecule Scrib, is known to control the developmental migration of several hindbrain neuronal groups. Accordingly, a homozygous mutation of Scrib leads to severe disruption of hindbrain anatomy and function. Here, we aimed to determine whether Scrib is also involved in the prenatal development of the hindbrain nuclei controlling breathing. We combined immunostaining, calcium imaging and electrophysiological recordings of neuronal activity in isolated in vitro preparations. In the Scrib mutant, despite severe neural tube defects, epF and preBotC neurons settled at their expected hindbrain positions. Furthermore, both networks remained capable of generating rhythmically organized, respiratory-related activities and exhibited normal sensitivity to pharmacological agents known to modify respiratory circuit function. Thus Scrib is not required for the proper migration of epF and preBotC neurons during mouse embryogenesis. Our findings thus further illustrate the robustness and specificity of the developmental processes involved in the establishment of hindbrain respiratory circuits.

07/04/2017 | Nat Commun   IF 12.1
Defective Gpsm2/Galphai3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome.
Mauriac SA, Hien YE, Bird JE, Carvalho SD, Peyroutou R, Lee SC, Moreau MM, Blanc JM, Geyser A, Medina C, Thoumine O, Beer-Hammer S, Friedman TB, Ruttiger L, Forge A, Nurnberg B*, Sans N*, Montcouquiol M*

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Galphai3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an approximately 200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Galphai3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Galphai3 in the regulation of actin dynamics in epithelial and neuronal tissues.

27/03/2017 | Development   IF 5.8
Wnts contribute to neuromuscular junction formation through distinct signaling pathways.
Messeant J, Ezan J, Delers P, Glebov K, Marchiol C, Lager F, Renault G, Tissir F, Montcouquiol M, Sans N, Legay C, Strochlic L

Understanding the developmental steps shaping the formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers, is critical. Wnt morphogens are key players in the formation of this specialized peripheral synapse. Yet, the individual and collaborative functions of Wnts as well as their downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain of function studies in culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. In contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR clustering and mRNA downstream activation of the beta-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the Planar Cell Polarity (PCP) pathway, which accumulates at embryonic NMJ. Moreover, mice bearing a Vangl2 loss of function mutation (looptail) exhibit a decreased number of AChR clusters and overgrowth of motor axons bypassing AChR clusters. Taken together, our results provide genetic and biochemical evidences that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.

22/11/2016 | Cereb Cortex   IF 8.3
Activity-Dependent Neuroplasticity Induced by an Enriched Environment Reverses Cognitive Deficits in Scribble Deficient Mouse.
Hilal ML, Moreau MM, Racca C, Pinheiro VL, Piguel NH, Santoni MJ, Dos Santos Carvalho S, Blanc JM, Abada YK, Peyroutou R, Medina C, Doat H, Papouin T, Vuillard L, Borg JP, Rachel R, Panatier A, Montcouquiol M, Oliet SHR, Sans N

Planar cell polarity (PCP) signaling is well known to play a critical role during prenatal brain development; whether it plays specific roles at postnatal stages remains rather unknown. Here, we investigated the role of a key PCP-associated gene scrib in CA1 hippocampal structure and function at postnatal stages. We found that Scrib is required for learning and memory consolidation in the Morris water maze as well as synaptic maturation and NMDAR-dependent bidirectional plasticity. Furthermore, we unveiled a direct molecular interaction between Scrib and PP1/PP2A phosphatases whose levels were decreased in postsynaptic density of conditional knock-out mice. Remarkably, exposure to enriched environment (EE) preserved memory formation in CaMK-Scrib-/- mice by recovering synaptic plasticity and maturation. Thus, Scrib is required for synaptic function involved in memory formation and EE has beneficiary therapeutic effects. Our results demonstrate a distinct new role for a PCP-associated protein, beyond embryonic development, in cognitive functions during adulthood.

Extensive evidence suggests that long term dietary n-3 polyunsaturated fatty acids (PUFAs) deficiency results in altered emotional behaviour. We have recently demonstrated that n-3 PUFAs deficiency induces emotional alterations through abnormal corticosterone secretion which leads to altered dendritic arborisation in the prefrontal cortex (PFC). Here we show that hypothalamic-pituitary-adrenal (HPA) axis feedback inhibition was not compromised in n-3 deficient mice. Rather, glucocorticoid receptor (GR) signaling pathway was inactivated in the PFC but not in the hippocampus of n-3 deficient mice. Consequently, only dendritic arborisation in PFC was affected by dietary n-3 PUFAs deficiency. In addition, occlusion experiment with GR blockade altered GR signaling in the PFC of control mice, with no further alterations in n-3 deficient mice. In conclusion, n-3 PUFAs deficiency compromised PFC, leading to dendritic atrophy, but did not change hippocampal GR function and dendritic arborisation. We argue that this GR sensitivity contributes to n-3 PUFAs deficiency-related emotional behaviour deficits.

18/02/2015 | J Neurosci   IF 6.3
Microglial activation enhances associative taste memory through purinergic modulation of glutamatergic neurotransmission.
Delpech JC, Saucisse N, Parkes SL, Lacabanne C, Aubert A, Casenave F, Coutureau E, Sans N, Laye S, Ferreira G, Nadjar A

The cerebral innate immune system is able to modulate brain functioning and cognitive processes. During activation of the cerebral innate immune system, inflammatory factors produced by microglia, such as cytokines and adenosine triphosphate (ATP), have been directly linked to modulation of glutamatergic system on one hand and learning and memory functions on the other hand. However, the cellular mechanisms by which microglial activation modulates cognitive processes are still unclear. Here, we used taste memory tasks, highly dependent on glutamatergic transmission in the insular cortex, to investigate the behavioral and cellular impacts of an inflammation restricted to this cortical area in rats. We first show that intrainsular infusion of the endotoxin lipopolysaccharide induces a local inflammation and increases glutamatergic AMPA, but not NMDA, receptor expression at the synaptic level. This cortical inflammation also enhances associative, but not incidental, taste memory through increase of glutamatergic AMPA receptor trafficking. Moreover, we demonstrate that ATP, but not proinflammatory cytokines, is responsible for inflammation-induced enhancement of both associative taste memory and AMPA receptor expression in insular cortex. In conclusion, we propose that inflammation restricted to the insular cortex enhances associative taste memory through a purinergic-dependent increase of glutamatergic AMPA receptor expression at the synapse.

01/02/2015 | Development   IF 6.5
Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.
May-Simera HL, Petralia RS, Montcouquiol M, Wang YX, Szarama KB, Liu Y, Lin W, Deans MR, Pazour GJ, Kelley MW

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP.

10/11/2014 | Nat Neurosci   IF 15
Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1 mice.
Zhang Y*, Bonnan A*, Bony G*, Ferezou I, Pietropaolo S, Ginger M, Sans N, Rossier J, Oostra B, Lemasson G, Frick A

Hypersensitivity in response to sensory stimuli and neocortical hyperexcitability are prominent features of Fragile X Syndrome (FXS) and autism spectrum disorders, but little is known about the dendritic mechanisms underlying these phenomena. We found that the primary somatosensory neocortex (S1) was hyperexcited in response to tactile sensory stimulation in Fmr1-/y mice. This correlated with neuronal and dendritic hyperexcitability of S1 pyramidal neurons, which affect all major aspects of neuronal computation, from the integration of synaptic input to the generation of action potential output. Using dendritic electrophysiological recordings, calcium imaging, pharmacology, biochemistry and a computer model, we found that this defect was, at least in part, attributable to the reduction and dysfunction of dendritic h- and BKCa channels. We pharmacologically rescued several core hyperexcitability phenomena by targeting BKCa channels. Our results provide strong evidence pointing to the utility of BKCa channel openers for the treatment of the sensory hypersensitivity aspects of FXS.

11/2014 | Med Sci (Paris)
[The multiple links between cilia and planar cell polarity].
Ezan J, Montcouquiol M

Since our seminal study in 2003, much has been written about core planar cell polarity (core PCP) signaling and the inner ear. In just a few years, and using the inner ear as a model system, our understanding of the molecular basis of this signaling pathway and how it can influence the development of tissues in mammals has increased considerably. Recently, a number of studies using various animal models of development have uncovered original relationships between the cilia and PCP, and the study of the hair cells of the inner ear has helped elucidating one of these links. In this review, we highlight the differences of PCP signaling between mammals and invertebrates. In the light of recent results, we sum up our current knowledge about PCP signaling in the mammalian cochlear epithelium and we discuss the impact of recent data in the field. We focus our attention on the interrelationship between asymmetric polarity complexes and the position of the cilium, which is essential for the establishment of the overall tissue polarity.

23/10/2014 | Cell Rep   IF 7.2
Scribble1/AP2 complex coordinates NMDA receptor endocytic recycling.
Piguel NH, Fievre S, Blanc JM, Carta M, Moreau MM, Moutin E, Pinheiro VL, Medina C, Ezan J, Lasvaux L, Loll F, Durand CM, Chang K, Petralia RS, Wenthold RJ, Stephenson FA, Vuillard L, Darbon H, Perroy J, Mulle C, Montcouquiol M, Racca C, Sans N

The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.

29/07/2014 | Proc Natl Acad Sci U S A
A dual role for planar cell polarity genes in ciliated cells.
Boutin C, Labedan P, Dimidschstein J, Richard F, Cremer H, Andre P, Yang Y, Montcouquiol M, Goffinet AM, Tissir F

In the nervous system, cilia dysfunction perturbs the circulation of the cerebrospinal fluid, thus affecting neurogenesis and brain homeostasis. A role for planar cell polarity (PCP) signaling in the orientation of cilia (rotational polarity) and ciliogenesis is established. However, whether and how PCP regulates cilia positioning in the apical domain (translational polarity) in radial progenitors and ependymal cells remain unclear. By analysis of a large panel of mutant mice, we show that two PCP signals are operating in ciliated cells. The first signal, controlled by cadherin, EGF-like, laminin G-like, seven-pass, G-type receptor (Celsr) 2, Celsr3, Frizzled3 (Fzd3) and Van Gogh like2 (Vangl2) organizes multicilia in individual cells (single-cell polarity), whereas the second signal, governed by Celsr1, Fzd3, and Vangl2, coordinates polarity between cells in both radial progenitors and ependymal cells (tissue polarity). Loss of either of these signals is associated with specific defects in the cytoskeleton. Our data reveal unreported functions of PCP and provide an integrated view of planar polarization of the brain ciliated cells.

2014 | Transl Psychiatry   IF 4.4
Nutritional omega-3 modulates neuronal morphology in the prefrontal cortex along with depression-related behaviour through corticosterone secretion.
Larrieu T, Hilal ML, Fourrier C, De Smedt-Peyrusse V, Sans N, Capuron L, Laye S

Understanding how malnutrition contributes to depression is building momentum. In the present study we unravel molecular and cellular mechanisms by which nutritional disturbances lead to impaired emotional behaviour in mice. Here we report that nutritional n-3 polyunsaturated fatty acids (PUFA) deficiency induces a chronic stress state reflected by disrupted glucocorticoid receptor (GR)-mediated signalling pathway along with hypothalamic-pituitary-adrenal (HPA) axis hyperactivity. This hyperactivity in turn resulted in neuronal atrophy in the dorsolateral (dl)- and dorsomedial (dm)- prefrontal cortex (PFC) and subsequent mood-related behaviour alterations, similarly to chronic social defeat stress. Supplementation of n-3 PUFA prevented detrimental chronic social defeat stress-induced emotional and neuronal impairments by impeding HPA axis hyperactivity. These results indicate a role for dietary n-3 PUFA in the prevention of HPA axis dysfunction associated with the development of some neuropsychiatric disorders including depression.

2014 | Front Cell Neurosci   IF 4.2
ER to synapse trafficking of NMDA receptors.
Horak M, Petralia RS, Kaniakova M, Sans N

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. There are three distinct subtypes of ionotropic glutamate receptors (GluRs) that have been identified including 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptors (AMPARs), N-methyl-D-aspartate receptors (NMDARs) and kainate receptors. The most common GluRs in mature synapses are AMPARs that mediate the fast excitatory neurotransmission and NMDARs that mediate the slow excitatory neurotransmission. There have been large numbers of recent reports studying how a single neuron regulates synaptic numbers and types of AMPARs and NMDARs. Our current research is centered primarily on NMDARs and, therefore, we will focus in this review on recent knowledge of molecular mechanisms occurring (1) early in the biosynthetic pathway of NMDARs, (2) in the transport of NMDARs after their release from the endoplasmic reticulum (ER); and (3) at the plasma membrane including excitatory synapses. Because a growing body of evidence also indicates that abnormalities in NMDAR functioning are associated with a number of human psychiatric and neurological diseases, this review together with other chapters in this issue may help to enhance research and to gain further knowledge of normal synaptic physiology as well as of the etiology of many human brain diseases.

09/2013 | Mol Cell Proteomics   IF 7.3
The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions.
Belotti E, Polanowska J , Daulat AM , Audebert S , Thome V , Lissitzky JC , Lembo F , Blibek K , Omi S , Lenfant N , Gangar A , Montcouquiol M , Santoni MJ , Sebbagh M , Aurrand-Lions M , Angers S , Kodjabachian L , Reboul J , Borg JP

Protein-protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRbeta, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.

09/2013 | Nat Cell Biol   IF 20.8
Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton.
Ezan J , Lasvaux L , Gezer A , Novakovic A , May-Simera H , Belotti E , Lhoumeau AC , Birnbaumer L , Beer-Hammer S , Borg JP , Le Bivic A , Nurnberg B , Sans N , Montcouquiol M

In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Galphai3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Galphai3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.

05/2013 | Semin Cell Dev Biol   IF 6.2
Revisiting planar cell polarity in the inner ear.
Ezan J, Montcouquiol M

Since the first implication of the core planar cell polarity (PCP) pathway in stereocilia orientation of sensory hair cells in the mammalian cochlea, much has been written about this subject, in terms of understanding how this pathway can shape the mammalian hair cells and using the inner ear as a model system to understand mammalian PCP signaling. However, many conflicting results have arisen, leading to puzzling questions regarding the actual mechanism and roles of core PCP signaling in mammals and invertebrates. In this review, we summarize our current knowledge on the establishment of PCP during inner ear development and revisit the contrast between wing epithelial cells in Drosophila melanogaster and sensory epithelia in the mammalian cochlea. Notably, we focus on similarities and differences in the asymmetric distribution of core PCP proteins in the context of cell autonomous versus non-autonomous role of PCP signaling in the two systems. Additionally, we address the relationship between the kinocilium position and PCP in cochlear hair cells and increasing results suggest an alternate cell autonomous pathway in regulating PCP in sensory hair cells.

10/2012 | Development   IF 6.2
Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear.
Giese AP*, Ezan J*, Wang L, Lasvaux L, Lembo F, Mazzocco C, Richard E, Reboul J, Borg JP, Kelley MW, Sans N, Brigande J, Montcouquiol M

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.

03/09/2012 | J Cell Biol   IF 10.8
Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity.
Mahuzier A , Gaude HM , Grampa V , Anselme I , Silbermann F , Leroux-Berger M , Delacour D , Ezan J , Montcouquiol M , Saunier S , Schneider-Maunoury S , Vesque C

Cilia are at the core of planar polarity cellular events in many systems. However, the molecular mechanisms by which they influence the polarization process are unclear. Here, we identify the function of the ciliopathy protein Rpgrip1l in planar polarity. In the mouse cochlea and in the zebrafish floor plate, Rpgrip1l was required for positioning the basal body along the planar polarity axis. Rpgrip1l was also essential for stabilizing dishevelled at the cilium base in the zebrafish floor plate and in mammalian renal cells. In rescue experiments, we showed that in the zebrafish floor plate the function of Rpgrip1l in planar polarity was mediated by dishevelled stabilization. In cultured cells, Rpgrip1l participated in a complex with inversin and nephrocystin-4, two ciliopathy proteins known to target dishevelled to the proteasome, and, in this complex, Rpgrip1l prevented dishevelled degradation. We thus uncover a ciliopathy protein complex that finely tunes dishevelled levels, thereby modulating planar cell polarity processes.

01/2012 | Mol Psychiatry   IF 14.9
SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism.
Durand CM, Perroy J, Loll F, Perrais D, Fagni L, Bourgeron T, Montcouquiol M, Sans N

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies.

2012 | PLoS ONE   IF 3.7
Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
Belotti E, Puvirajesinghe TM , Audebert S , Baudelet E , Camoin L , Pierres M , Lasvaux L , Ferracci G , Montcouquiol M , Borg JP

BACKGROUND: Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results. METHODOLOGY AND MAIN FINDINGS: A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2(Lp) protein in mutant mice compared to the wild type mice. CONCLUSION: Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.

The avian inner ear possesses a remarkable ability to regenerate sensory hair cells after ototoxic injury. Regenerated hair cells possess phenotypes and innervation that are similar to those found in the undamaged ear, but little is known about the signaling pathways that guide hair cell differentiation during the regenerative process. The aim of the present study was to examine the factors that specify the orientation of hair cell stereocilia bundles during regeneration. Using organ cultures of the chick utricle, we show that hair cells are properly oriented after having regenerated entirely in vitro and that orientation is not affected by surgical removal of the striolar reversal zone. These results suggest that the orientation of regenerating stereocilia is not guided by the release of a diffusible morphogen from the striolar reversal zone but is specified locally within the regenerating sensory organ. In order to determine the nature of the reorientation cues, we examined the expression patterns of the core planar cell polarity molecule Vangl2 in the normal and regenerating utricle. We found that Vangl2 is asymmetrically expressed on cells within the sensory epithelium and that this expression pattern is maintained after ototoxic injury and throughout regeneration. Notably, treatment with a small molecule inhibitor of c-Jun-N-terminal kinase disrupted the orientation of regenerated hair cells. Both of these results are consistent with the hypothesis that noncanonical Wnt signaling guides hair cell orientation during regeneration.

21/07/2010 | J Neurosci   IF 7.5
The planar polarity protein Scribble1 is essential for neuronal plasticity and brain function.
Moreau MM, Piguel N, Papouin T, Koehl M, Durand CM, Rubio ME, Loll F, Richard EM, Mazzocco C, Racca C, Oliet SH, Abrous DN, Montcouquiol M, Sans N

Scribble (Scrib) is a key regulator of apicobasal polarity, presynaptic architecture, and short-term synaptic plasticity in Drosophila. In mammals, its homolog Scrib1 has been implicated in cancer, neural tube closure, and planar cell polarity (PCP), but its specific role in the developing and adult nervous system is unclear. Here, we used the circletail mutant, a mouse model for PCP defects, to show that Scrib1 is located in spines where it influences actin cytoskeleton and spine morphing. In the hippocampus of these mutants, we observed an increased synapse pruning associated with an increased number of enlarged spines and postsynaptic density, and a decreased number of perforated synapses. This phenotype was associated with a mislocalization of the signaling pathway downstream of Scrib1, leading to an overall activation of Rac1 and defects in actin dynamic reorganization. Finally, Scrib1-deficient mice exhibit enhanced learning and memory abilities and impaired social behavior, two features relevant to autistic spectrum disorders. Our data identify Scrib1 as a crucial regulator of brain development and spine morphology, and suggest that Scrib1(crc/+) mice might be a model for studying synaptic dysfunction and human psychiatric disorders.

06/2010 | Nat Neurosci   IF 15.7
Lack of cadherins Celsr2 and Celsr3 impairs ependymal ciliogenesis, leading to
Tissir F , Qu Y , Montcouquiol M , Zhou L , Komatsu K , Shi D , Fujimori T , Labeau J , Tyteca D , Courtoy P , Poumay Y , Uemura T , Goffinet AM

Ependymal cells form the epithelial lining of cerebral ventricles. Their apical

04/2010 | Development   IF 7.3
Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b
Wansleeben C, Feitsma H, Montcouquiol M, Kroon C, Cuppen E, Meijlink F

04/2010 | Nat Cell Biol   IF 17.6
Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia
Guirao B, Meunier A, Mortaud S, Aguilar A, Corsi JM, Strehl L, Hirota Y, Desoeuvre A, Boutin C, Han YG, Mirzadeh Z, Cremer H, Montcouquiol M*, Sawamoto K*, Spassky N*

15/11/2008 | Hum Mol Genet
Scrib regulates PAK activity during the cell migration process
Nola S, Sebbagh M, Marchetto S, Osmani N, Nourry C, Audebert S, Navarro C, Rachel R, Montcouquiol M, Sans N, Etienne-Manneville S, Borg J P, Santoni M J

Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.

25/06/2008 | J Neurosci
Jxc1/Sobp, encoding a nuclear zinc finger protein, is critical for cochlear growth, cell fate, and patterning of the organ of corti
Chen Z, Montcouquiol M, Calderon R, Jenkins N A, Copeland N G, Kelley M W, Noben-Trauth K

The mouse cochlea emerges from the ventral pole of the otocyst to form a one and three-quarter coil. Little is known about the factors that control the growth of the cochlea. Jackson circler (jc) is a recessive mutation causing deafness resulting from a growth arrest of the cochlea duct at day 13.5 of embryonic development. Here, we identify the vertebrate homolog of the Drosophila Sobp (sine oculis-binding protein) gene (named Jxc1) in the jc locus. Jxc1 encodes a nuclear protein that has two FCS-type zinc finger domains (PS51024) and bears nuclear localization signals and highly conserved sequence motifs. Transiently expressed wild-type protein is targeted to the nucleus, but mutant isoforms were mislocalized in the cytoplasm. In jc mutants, the cellular patterning of the organ of Corti is severely disrupted, exhibiting supernumerary hair cells at the apex, showing mirror-image duplications of tunnel of Corti and inner hair cells, and expressing ectopic vestibular-like hair cells within Kolliker's organ. Jxc1 mRNA was detected in inner ear sensory hair cells, supporting cells, and the acoustic ganglia. Expression was also found in the developing retina, olfactory epithelium, trigeminal ganglion, and hair follicles. Collectively, our data support a role for Jxc1 in controlling a critical step in cochlear growth, cell fate, and patterning of the organ of Corti.

2008 | Methods Mol Biol
Detection of planar polarity proteins in mammalian cochlea.
Montcouquiol M, Jones JM, Sans N

The 'core genes' were identified as a group of genes believed to function as a conserved signaling cassette for the specification of planar polarity in Drosophila Melanogaster, and includes frizzled (fz), van gogh (vang) or strabismus (stbm), prickle (Pk), dishevelled (dsh), flamingo (fmi), and diego. The mutation of each of these genes not only causes the disruption of planar polarity within the wing or the eye of the animal, but also affects the localization of all the other protein members of the core group. These properties emphasize the importance of the interrelations between the proteins of this group. All of these core genes have homologs in vertebrates. Studies in Danio Rerio (zebrafish) and Xenopus laevis (frog) have uncovered other roles for some of these molecules in gastrulation and neurulation, during which the shape of a given tissue will undergo major transformation through cell movements. A disruption in these processes can lead to severe neural tube defects in diverse organisms, including humans. In fact, a large body of evidence suggests that planar polarity proteins are not involved in one specific cascade but in many different ones and many different mechanisms such as, but not limited to, hair or cilia orientation, asymmetric division, cellular movements, or neuronal migration. In mice cochleae, mutations in planar polarity genes lead to defects in the orientation of the stereociliary bundles at the apex of each hair cell. This phenotype established the cochlea as one of the clearest examples of planar polarity in mammals. Although significant progress has been made toward understanding the molecular basis required for the development of planar polarity in invertebrates, similar advances in vertebrates are more recent and rely mainly on the identification of a group of mammalian mutants that affect hair cell stereociliary bundle orientation. These include mutation of vangl2, scrb1, celsr1, PTK-7, dvl1-2, and more recently fz3 and fz6. In this chapter, we describe how to use the mammalian cochlea, which represents one of the best systems to study planar polarity in mammals, to identify planar polarity mutants, study protein distribution, do in vitro analysis, and perform Western blots to analyze putative planar polarity proteins.

24/10/2007 | J Neurosci
The role of the PDZ protein GIPC in regulating NMDA receptor trafficking
Yi Z, Petralia R S, Fu Z, Swanwick C C, Wang Y X, Prybylowski K, Sans N, Vicini S, Wenthold R J

The NMDA receptor is an important component of excitatory synapses in the CNS. In addition to its synaptic localization, the NMDA receptor is also present at extrasynaptic sites where it may have functions distinct from those at the synapse. Little is known about how the number, composition, and localization of extrasynaptic receptors are regulated. We identified a novel NMDA receptor-interacting protein, GIPC (GAIP-interacting protein, C terminus), that associates with surface as well as internalized NMDA receptors when expressed in heterologous cells. In neurons, GIPC colocalizes with a population of NMDA receptors on the cell surface, and changes in GIPC expression alter the number of surface receptors. GIPC is mainly excluded from the synapse, and changes in GIPC expression do not change the total number of synaptic receptors. Our results suggest that GIPC may be preferentially associated with extrasynaptic NMDA receptors and may play a role in the organization and trafficking of this population of receptors.

08/2007 | Development
Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea.
Jacques BE, Montcouquiol ME, Layman EM, Lewandoski M, Kelley MW

The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate (PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them potential targets for regenerative therapies.

Millions of lives are affected by hearing and balance deficits that arise as a consequence of sensory hair cell loss. Those deficits affect mammals permanently, but hearing and balance recover in nonmammals after epithelial supporting cells divide and produce replacement hair cells. Hair cells are not effectively replaced in mammals, but balance epithelia cultured from the ears of rodents and adult humans can respond to hair cell loss with low levels of supporting cell proliferation. We have sought to stimulate vestibular proliferation; and we report here that treatment with glial growth factor 2 (rhGGF2) yields a 20-fold increase in cell proliferation within sheets of pure utricular hair cell epithelium explanted from adult rats into long-term culture. In epithelia from neonates, substantially greater proliferation responses are evoked by rhGGF2 alone, insulin alone and to a lesser degree by serum even during short-term cultures, but all these responses progressively decline during the first 2 weeks of postnatal maturation. Thus, sheets of utricular epithelium from newborn rats average > 40% labelling when cultured for 72 h with bromo-deoxyuridine (BrdU) and either rhGGF2 or insulin. Those from 5- and 6-day-olds average 8-15%, 12-day-olds average < 1% and after 72 h there is little or no labelling in epithelia from 27- and 35-day-olds. These cells are the mammalian counterparts of the progenitors that produce replacement hair cells in nonmammals, so the postnatal quiescence described here is likely to be responsible for at least part of the mammalian ear's unique vulnerability to permanent sensory deficits.

02/2007 | Eur J Neurosci
Lhx3, a LIM domain transcription factor, is regulated by Pou4f3 in the auditory but not in the vestibular system
Hertzano R, Dror A A, Montcouquiol M, Ahmed Z M, Ellsworth B, Camper S, Friedman T B, Kelley M W, Avraham K B

A dominant mutation of the gene encoding the POU4F3 transcription factor underlies human non-syndromic progressive hearing loss DFNA15. Using oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3(ddl/ddl) mutant and wild-type mice, we have identified and validated Lhx3, a LIM domain transcription factor, as an in vivo target gene regulated by Pou4f3. Lhx3 is a hair cell-specific gene expressed in all hair cells of the auditory and vestibular system as early as embryonic day 16. The level of Lhx3 mRNA is greatly reduced in the inner ears of embryonic Pou4f3 mutant mice. Our data also show that the expression of Lhx3 is regulated differently in auditory and vestibular hair cells. This is the first example of a hair cell-specific gene expressed both in auditory and in vestibular hair cells, with differential regulation of expression in these two closely related systems.

Planar cell polarity (PCP) genes were originally identified in invertebrates

10/05/2006 | J Neurosci
Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals.
Montcouquiol M , Sans N , Huss D , Kach J , Dickman JD , Forge A , Rachel RA , Copeland NG , Jenkins NA , Bogani D , Murdoch J , Warchol ME , Wenthold RJ , Kelley MW

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

The basic helix-loop-helix (bHLH) transcription factor Math1 (also called Atoh1) is both necessary and sufficient for hair cell development in the mammalian cochlea (Bermingham et al., 1999; Zheng and Gao, 2000). Previous studies have demonstrated that a dynamic pattern of Math1 expression plays a key role in regulating the number and position of mechanosensory hair cells. However, the factors that regulate the temporal and spatial expression of Math1 within the cochlea are unknown. The bHLH-related inhibitors of differentiation and DNA binding (Id) proteins are known to negatively regulate many bHLH transcription factors, including Math1, in a number of different systems. Therefore, Id proteins are good candidates for regulating Math1 in the cochlea. Results from PCR and in situ hybridization indicate that Id1, Id2, and Id3 are expressed within the cochlear duct in a pattern that is consistent with a role in regulation of hair cell development. In particular, expression of Ids and Math1 overlapped in cochlear progenitor cells before cellular differentiation, but a specific downregulation of Id expression was observed in individual cells that differentiated as hair cells. In addition, progenitor cells in which the expression of Ids was maintained during the time period for hair cell differentiation were inhibited from developing as hair cells. These results indicate a key role for Ids in the regulation of expression of Math1 and hair cell differentiation in the developing cochlea.

2006 | Annu Rev Neurosci
Noncanonical Wnt signaling and neural polarity.
Montcouquiol M, Crenshaw EB 3rd, Kelley MW

The Wnt signaling pathway regulates multiple events in development and disease in both vertebrates and invertebrates. Recently, the noncanonical Wnt signaling cascades, those that do not signal through beta-catenin, have gained prominence for their role in the regulation of cellular polarity. It is not surprising that cellular polarization influences a number of different developmental events within the nervous system, including neurulation and neural tube closure, cellular migration, and uniform orientation of cells within an epithelial plane (planar cell polarity). In this review, we describe the differences between the canonical and noncanonical pathways, summarize recent data illustrating the roles of the noncanonical Wnt pathway in different polarizing events during neural development, and discuss the potential molecular mechanisms that underlie the generation of cellular asymmetry and polarity.

12/2005 | Nat Cell Biol
mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression
Sans N, Wang P Y, Du Q, Petralia R S, Wang Y X, Nakka S, Blumer J B, Macara I G, Wenthold R J

15/09/2005 | Neuron
The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2
Prybylowski K, Chang K, Sans N, Kan L, Vicini S, Wenthold R J

Neurone glial-related cell adhesion molecule (NrCAM) is a member of the L1 family of transmembrane cell adhesion receptors which are involved in the development and function of the mammalian nervous system. How these receptors interact with intracellular signalling pathways is not understood. To date the only identified binding partner to the cytoplasmic terminus of NrCAM is ankyrin G. We screened a developing rat brain cDNA yeast two-hybrid library with the cytoplasmic domain of NrCAM to identify further intracellular binding partners. We identified synapse associated protein 102 (SAP102) as a new binding partner for NrCAM. The interaction was confirmed biochemically using glutathione S-transferase (GST)-pull-down and tandem affinity purification, and also immunocytochemically as NrCAM and SAP102 co-localized in COS-7 and cerebellar granule cells. Binding was specific to NrCAM as neither neurofascin nor L1 bound SAP102, and this interaction was reliant on the last three amino acids of NrCAM. Additionally, NrCAM constructs whose last three amino acids had been deleted appeared to have a dominant negative effect on neurite extension of cerebellar granule cells. This is the first interaction reported for NrCAM, and its association with SAP102 suggests that it is part of a larger complex which can interact with many different signalling pathways.

07/2005 | Mol Cell Neurosci
Ontogeny of postsynaptic density proteins at glutamatergic synapses
Petralia R S, Sans N, Wang Y X, Wenthold R J

In glutamatergic synapses, glutamate receptors (GluRs) associate with many other proteins involved in scaffolding and signal transduction. The ontogeny of these postsynaptic density (PSD) proteins involves changes in their composition during development, paralleling changes in GluR type and function. In the CA1 region of the hippocampus, at postnatal day 2 (P2), many synapses already have a distinct PSD. We used immunoblot analysis, subcellular fractionation, and quantitative immunogold electron microscopy to examine the distribution of PSD proteins during development of the hippocampus. Synapses at P2 contained substantial levels of NR1 and NR2B and most GluR-associated proteins, including SAP102, SynGAP, the chain of proteins from GluRs/SAP102 through GKAP/Shank/Homer and metabotropic glutamate receptors, and the adhesion factors, cadherin, catenin, neuroligin, and Nr-CAM. Development was marked by substantial decreases in NR2B and SAP102 and increases in NR2A, PSD-95, AMPA receptors, and CaMKII. Other components showed more moderate changes.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) type of ionotropic glutamate receptor is the major mediator of fast neurotransmission in the brain and spinal cord. Most AMPA receptors are impermeable to calcium because they contain the GluR2 subunit. However, some AMPA receptors lack GluR2 and pass calcium which can mediate synaptic plasticity and, in excess, neurotoxicity. Previously, we showed a decrease in the density of synaptic AMPA receptors in the hippocampus of mice lacking GluR2. In this study, using these GluR2-lacking mice, we examined other areas of the brain that differ in the amount of GluR2 normally present. Like hippocampal spines, cerebellar Purkinje spines normally express AMPA receptors with high GluR2 and showed a decrease in synaptic AMPA receptors in mutant mice. In contrast, neurons that normally express AMPA receptors with little or no GluR2, such as in the anteroventral cochlear nucleus, showed no decrease in AMPA receptors and even showed an increase in one AMPA receptor subunit. These two different patterns may relate to preadaptations to prevent calcium neurotoxicity; such mechanisms might be absent in Purkinje and hippocampal spines so that these neurons must decrease their total expression of synaptic AMPA receptors (calcium permeable in mutant mice) to prevent calcium neurotoxicity. In addition, we found that another glutamate receptor, GluRdelta2, which is abundant only in parallel fibre synapses on Purkinje cells and in the dorsal cochlear nucleus, is up-regulated at these synapses in mutant mice; this probably reflects some change in GluRdelta2 targeting to these synapses.

2004 | Nat Neurosci
Math1 regulates development of the sensory epithelium in the mammalian cochlea
Woods C*, Montcouquiol M*, Kelley MW

The transcription factor Math1 (encoded by the gene Atoh1, also called Math1) is required for the formation of mechanosensory hair cells in the inner ear; however, its specific molecular role is unknown. Here we show that absence of Math1 in mice results in a complete disruption of formation of the sensory epithelium of the cochlea, including the development of both hair cells and associated supporting cells. In addition, ectopic expression of Math1 in nonsensory regions of the cochlea is sufficient to induce the formation of sensory clusters that contain both hair cells and supporting cells. The formation of these clusters is dependent on inhibitory interactions mediated, most probably, through the Notch pathway, and on inductive interactions that recruit cells to develop as supporting cells through a pathway independent of Math1. These results show that Math1 functions in the developing cochlea to initiate both inductive and inhibitory signals that regulate the overall formation of the sensory epithelia.

2004 | Hum Mol Genet
Transcription profiling of inner ears from Pou4f3(ddl/ddl) identifies Gfi1 as a target of the Pou4f3 deafness gene.
Hertzano R, Montcouquiol M, Rashi-Elkeles S, Elkon R, Yücel R, Frankel WN, Rechavi G, Möröy T, Friedman TB, Kelley MW, Avraham KB

08/2003 | biochem soc trans
Early events in the trafficking of N-methyl-D-aspartate (NMDA) receptors.
Wenthold RJ, Sans N, Standley S, Prybylowski K, Petralia RS

The N-methyl-D-aspartate (NMDA) receptor plays a central role at excitatory synapses where it has been implicated in multiple functions associated with synaptic plasticity. While this receptor has been intensely studied with respect to its physiology and pharmacology, its cell-biological properties, such as subunit assembly, post-translational processing and trafficking in neurons, are only beginning to be addressed. Critical to many of the functions of the NMDA receptor are the multiple proteins with which it interacts. While these interactions have been most thoroughly studied with respect to the receptor at the synapse, the same proteins may also interact with the receptor much earlier in its biosynthetic pathway and play important roles in receptor trafficking from the endoplasmic reticulum to the synapse.

06/2003 | Development
Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea.
Dabdoub A , Donohue MJ , Brennan A , Wolf V , Montcouquiol M , Sassoon DA , Hseih JC , Rubin JS , Salinas PC , Kelley MW

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.

2003 | J Neurosci
Aberrant formation of glutamate receptor complexes in hippocampal neurons of mice lacking the GluR2 AMPA receptor subunit.
Sans N, Vissel B, Petralia RS, Wang YX, Chang K, Royle GA, Wang CY, O'Gorman S, Heinemann SF, Wenthold RW

The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knock-out mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.

2003 | Annu. Rev. Pharmacol. Toxicol.
Trafficking of NMDA receptors
Wenthold RW, Prybylowski K, Standley S, Sans N, Petralia RS

2003 | Nat Cell Biol
NMDA receptor trafficking through an interaction between PDZ Proteins and the exocyst complex
Sans N, Prybylowski K, Petralia RS, Chang K, Wang YX, Racca C, Vicini S, Wenthold RW

NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst-SAP102-NMDAR complex is an important component of NMDAR trafficking.

2003 | Nature
Identification of Vangl2 and Scrb1 as planar polarity genes in mammals.
Montcouquiol M*, Rachel RA*, Lanford PJ, Copeland NG, Jenkins NA, Kelley MW

We investigated whether three calcium-binding proteins, calretinin, parvalbumin, and calbindin, could identify specific aspects of the postnatal development of the rat lateral (LVN) and medial (MVN) vestibular nuclei and their vestibular and cerebellar connections. Calretinin levels in the vestibular nuclei, increased significantly between birth and postnatal day (P) 45. In situ hybridization and immunocytochemical staining showed that calretinin-immunoreactive neurons were mostly located in the parvocellular MVN at birth and that somatic and dendritic growth occurred between birth and P14. During the first week, parvalbumin-immunoreactive fibers and endings were confined to specific areas, i.e., the ventral LVN and magnocellular MVN, and identified exclusively the maturation of the vestibular afferents. Calbindin was located within the dorsal LVN and the parvocellular MVN and identified the first arrival of the corticocerebellar afferents. From the second week, in addition to labeling vestibular afferents in their specific target areas, parvalbumin was also found colocalized with calbindin in mature Purkinje cell afferents. Thus, the specific spatiotemporal distribution of parvalbumin and calbindin could correspond to two successive phases of synaptic remodeling involving integration of the vestibular sensory messages and their cerebellar control. On the basis of the sequence of distribution patterns of these proteins during the development of the vestibular nuclei, calretinin is an effective marker for neuronal development of the parvocellular MVN, parvalbumin is a specific marker identifying maturation of the vestibular afferents and endings, and calbindin is a marker of the first appearance and development of Purkinje cell afferents.

01/10/2001 | J Neurosci
Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway.
Sans N, Racca C, Petralia RS, Wang YX, McCallum J, Wenthold RJ

The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

Balance epithelia in birds closely resemble their mammalian counterparts, but their cells turnover rapidly and they quickly regenerate hair cells, leading to functional recovery from damage that would be permanent for a mammal. We isolated and cultured sheets of the chicken's utricular epithelium in bromo-deoxyuridine and specific inhibitors of different intracellular signalling pathways to identify signals that influence turnover and regeneration. Synthesis (S-phase) entry was effectively blocked by inhibition of PI3-K, TOR or MAPK, and significantly decreased by inhibitors of PKC. Comparisons indicate that activated PI3-K and TOR are required for S-phase entry in both avian and mammalian balance epithelia, but activation of the MAPK pathway appears to have a more significant role in avian utricles than in mammals. The dissimilarities in the requirements for these signalling pathways do not appear sufficient to explain the marked difference in regenerative capacity between the ears of birds and mammals.

05/2001 | Eur J Neurosci
Glutamate receptor targeting in the postsynaptic spine involves mechanisms that are independent of myosin Va.
Petralia RS, Wang YX, Sans N, Worley PF, Hammer JA 3rd, Wenthold RJ

Targeting of glutamate receptors (GluRs) to synapses involves rapid movement of intracellular receptors. This occurs in forms of synaptic upregulation of receptors, such as long-term potentiation. Thus, many GluRs are retained in a cytoplasmic pool in dendrites, and are transported to synapses for upregulation, presumably via motor proteins such as myosins travelling along cytoskeletal elements that extend up into the spine. In this ultrastructural immunogold study of the cerebellar cortex, we compared synapses between normal rats/mice and dilute lethal mutant mice. These mutant mice lack myosin Va, which has been implicated in protein trafficking at synapses. The postsynaptic spine in the cerebellum lacks the inositol trisphosphate receptor (IP3R) -laden reticular tubules that are found in normal mice and rats (Takagishi et al., Neurosci. Lett., 1996, 215, 169). Thus, we tested the hypothesis that myosin Va is necessary for transport of GluRs and associated proteins to spine synapses. We found that these spines retain a normal distribution of (i) GluRs (delta 1/2, GluR2/3 and mGluR1alpha), (ii) at least one associated MAGUK (membrane-associated guanylate kinase) protein, (iii) Homer (which interacts with mGluR1alpha and IP3Rs), (iv) the actin cytoskeleton, (v) the reticulum-associated protein BiP, and (vi) the motor-associated protein, dynein light chain. Thus, while myosin Va may maintain the IP3R-laden reticulum in the spine for proper calcium regulation, other mechanisms must be involved in the delivery of GluRs and associated proteins to synapses. Other possible mechanisms include diffusion along the extrasynaptic membrane and delivery via other motors running along the spine's actin cytoskeleton.

12/2000 | Neuron
PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants.
Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ

The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.

10/2000 | Med Sci (Paris)
Régénération postlésionnelle des cellules sensorielles vestibulaires : bilan et espoirs
Bartolami S, Montcouquiol M, Travo C, Sans A

30/09/2000 | Brain Res Dev Brain Res
Postnatal developmental changes in AMPA and NMDA receptors in the rat vestibular nuclei.
Sans NA, Montcouquiol ME, Raymond J

Changes in the expression of the AMPA receptor subunits GluR1-4 and of the NMDA receptor subunits NR1, NR2A-D were investigated in the developing rat medial and lateral vestibular nuclei. Analyses were performed using nonradioactive in situ hybridization and immunoblotting with subunit-specific antibodies. During the postnatal development, glutamatergic receptor subunits were differentially expressed in the vestibular nuclei. The level of expression of GluR1, GluR4 and NR1 subunits was higher in the developing brain as compared to the adult. We observed a gradual increase in GluR2/3, NR2A, NR2B and NR2C levels of expression in the medial and lateral vestibular nuclei during the first 3 weeks of postnatal development. In situ hybridization results were consistent with immunoblot analyses. The differential expression of AMPA and NMDA receptor subunits in immature vestibular neurons is consistent with changes in glutamate receptor properties. This may be related to the postsynaptic regulation of receptor subunits associated with the synaptic plasticity of the vestibular neuron connections during specific sequences of postnatal development.

07/2000 | j gravit physiol
Developmental study of rat vestibular neuronal circuits during a spaceflight of 17 days.
Raymond J, Dememes D, Blanc E, Sans N, Venteo S, Dechesne CJ

The aim of this study was to investigate the potential plasticity of the vestibular system, in structural and biochemical terms, at the level of the gravity receptors (the sensory hair cells), the primary neurons relaying the sensory signals (the vestibular ganglion neurons) and their projections into the vestibular nuclei. We studied the biochemical differentiation of the sensory cells and of the vestibular ganglion by investigating which calcium-binding proteins were present. We studied the development of peripheral synaptic connections of the efferent system by investigating the distribution of CGRP (calcitonin-gene related-peptide) and we also studied the cerebellar synaptic connections in the vestibular nuclei, as identified by the presence of calbindin. Putative changes were studied after a 17-day episode of microgravity (Neurolab STS-90), in developing rats between postnatal days 8 and 25. The extent to which these changes could be caused by alterations in gravity was determined by examining sensory and nervous structures not involved in gravity detection, the cochlea and the cochlear nuclei.

15/05/2000 | Neuroreport
Detection and localization of BDNF in vestibular nuclei during the postnatal development of the rat.
Montcouquiol ME, Sans NA, Travo C, Sans A, Valat J

The changes in expression and the subcellular localization of brain-derived neurotrophic factor (BDNF) protein in the rat vestibular nuclear complex (VNC), have been investigated at different postnatal stages. Immunoblotting and ELISA analyses showed a down-regulation of BDNF protein expression in VNC with age. In addition, observations by confocal microscopy revealed that BDNF is mainly located in neuronal somata at postnatal day 8 (P8) and restricted to processes by P15. These results support the idea that BDNF could have different roles in the VNC according to the stage of development The protein could act as a neurotrophic factor in embryonic and early postnatal stages whereas in later developmental stages of the VNC it could be involved in neuronal maturation and regulation of neuronal circuitry.

01/02/2000 | J Neurosci
A developmental change in NMDA receptor-associated proteins at hippocampal synapses.
Sans N, Petralia RS, Wang YX, Blahos J 2nd, Hell JW, Wenthold RJ

The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

The early development of the inner ear is largely determined by two members of the neurotrophic family: brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3). Little information is available on the role of these neurotrophins during the late stages of vestibular development in the rat which take place during the first postnatal weeks. At this period where terminal synaptogenesis and maturation occur, we have investigated the expression and the activity of BDNF, the most important neurotrophin in the vestibular system. Using different experimental approaches, we show that BDNF is released by vestibular epithelia on postnatal day 3 (P3) and continues to have a trophic effect on vestibular neurones in vitro. Immunocytochemistry coupled to confocal microscopy revealed a remarkable evolution in BDNF localization during later stages of development. Whereas BDNF is present in both supporting cells and hair cells at P3, its distribution gradually changed and is highly compartmentalized within the upper part of supporting cells at P8 and P15. In parallel, we observed the presence of a truncated form of the BDNF receptor in sensory hair cells. These results suggest an original role for supporting cells, which could be involved in the release of BDNF during the late stages of synaptogenesis in mammalian vestibular epithelia. In particular, BDNF could participate to the set up of the calyx, a specific nerve structure surrounding type I vestibular hair cells.

The effects of the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) neurotrophins on the intracellular calcium level ([Ca2+]i) were studied in vestibular ganglion neurons (VGNs) from postnatal day 3 (P3) rats cultured for 50 hr. We first assessed the expression of trkB and trkC mRNA receptors in cultured VGNs. Immunobloting and immunocytochemistry confirmed the presence of the neurotrophin receptors on neurons. Both neurotrophins induced transient [Ca2+]i elevations in VGNs: BDNF-treated neurons responded in 65% and NT-3-treated neurons in 56%. The responses could be inhibited by anti-BDNF or anti-NT-3 antibodies. The [Ca2+]i elevation was dependent on extracellular calcium since it was abolished in calcium-free medium but also implicates the release of calcium from intracellular stores as tested by prior depletion with thapsigargin. Our results suggest the implication of a short-term calcium regulation in VGNs, which could reflect specific fast effects of neurotrophins in the early postnatal rat vestibular system.

The localization of neurons expressing mRNAs for the NR1 and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NR1 and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NR1 and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process.

We examined the localization of neurons expressing mRNA for calretinin, a cytosolic EF hand calcium-binding protein, throughout the vestibular nuclei of rat and guinea pig by non-radioactive in situ hybridization, using an alkaline phosphatase labeled oligonucleotide probe. Labeled cells were particularly numerous in the medial vestibular nucleus (mVN) and their distribution was similar in rat and guinea pig, and presented a characteristic rostrocaudal and mediolateral pattern. The effects of hemilabyrinthectomy were assessed at various times post lesion from 10 h to 30 days by comparison of the pattern of labeling in the ipsi- and contra-lateral vestibular nuclei of guinea pig. After up to 48 h no modification in the calretinin mRNA distribution was detected. After 3 to 30 days of survival, there was a decrease (about 30%) of the calretinin expressing neurons in the nucleus on the side of the lesion. The unilateral sensory deprivation seemed to induce a permanent asymmetry in the expression of calretinin which was not abolished after vestibular compensation. These results suggested that the calretinin expression in these neurons depends upon the integrity and activity of sensorineuronal peripheral vestibular influences.