scholarly journals Neurabin-I Is Phosphorylated by Cdk5: Implications for Neuronal Morphogenesis and Cortical Migration

2007 ◽  
Vol 18 (11) ◽  
pp. 4327-4342 ◽  
Author(s):  
Frédéric Causeret ◽  
Tom Jacobs ◽  
Mami Terao ◽  
Owen Heath ◽  
Mikio Hoshino ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Pyramidal Neurons ◽  
Normal Development ◽  
Phosphorylation Site ◽  
Neuronal Morphology ◽  
Actin Binding ◽  
Neuronal Morphogenesis ◽  
And Migration

The correct morphology and migration of neurons, which is essential for the normal development of the nervous system, is enabled by the regulation of their cytoskeletal elements. We reveal that Neurabin-I, a neuronal-specific F-actin–binding protein, has an essential function in the developing forebrain. We show that gain and loss of Neurabin-I expression affect neuronal morphology, neurite outgrowth, and radial migration of differentiating cortical and hippocampal neurons, suggesting that tight regulation of Neurabin-I function is required for normal forebrain development. Importantly, loss of Neurabin-I prevents pyramidal neurons from migrating into the cerebral cortex, indicating its essential role during early stages of corticogenesis. We demonstrate that in neurons Rac1 activation is affected by the expression levels of Neurabin-I. Furthermore, the Cdk5 kinase, a key regulator of neuronal migration and morphology, directly phosphorylates Neurabin-I and controls its association with F-actin. Mutation of the Cdk5 phosphorylation site reduces the phenotypic consequences of Neurabin-I overexpression both in vitro and in vivo, suggesting that Neurabin-I function depends, at least in part, on its phosphorylation status. Together our findings provide new insight into the signaling pathways responsible for controlled changes of the F-actin cytoskeleton that are required for normal development of the forebrain.

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

scholarly journals Acceleration of conduction velocity linked to clustering of nodal components precedes myelination

2015 ◽  
Vol 112 (3) ◽  
pp. E321-E328 ◽  
Author(s):  
Sean A. Freeman ◽  
Anne Desmazières ◽  
Jean Simonnet ◽  
Marie Gatta ◽  
Friederike Pfeiffer ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Pyramidal Neurons ◽  
Axonal Conduction ◽  
Electrophysiological Recordings ◽  
Single Axon ◽  
Physiological Relevance ◽  
Saltatory Conduction ◽  
Insight Into

High-density accumulation of voltage-gated sodium (Nav) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Nav channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Nav isoforms Nav1.1, Nav1.2, and Nav1.6 are detected at prenodes, with Nav1.6 progressively replacing Nav1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Nav1.1 and Nav1.2 but not of Nav1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.

scholarly journals Repeated Neonatal Sevoflurane Induced Neurocognitive Impairment Through NF-κB-Mediated Pyroptosis

2021 ◽  
Author(s):  
Jing Dai ◽  
Xue Li ◽  
Cai Wang ◽  
Shuxin Gu ◽  
Lei Dai ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Biochemical Analysis ◽  
Neurological Diseases ◽  
Neurocognitive Impairment ◽  
Postnatal Period ◽  
Neuronal Morphology ◽  
Clinical Settings

Abstract Background: Exposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis, a novel type of programmed cell death that along with inflammation, plays an important role in the mechanism of diverse neurological diseases. Nevertheless, its role in GA-induced neuroinflammation and neurocognitive impairment in developing brain has not been investigated. Methods: Rats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro, received 3% sevoflurane for 2 hours daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)‑κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analysis were performed to assess the pyroptosis and neuronal and synaptic damages both in vivo and in vitro. In addition, behavioral tests were performed to evaluate the neurocognitive ability in rats.Results: Repeated sevoflurane exposures activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus of developing rats, caused damages in neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082, the inhibitor of NF-κB, suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damages, and ameliorated the neurocognitive impairment induced by repeated sevoflurane in the developing rats.Conclusions: These results demonstrated that repeated sevoflurane GA might induce neuroinflammation and cognitive impairment in developing rats via activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role for pyroptosis as a potential therapeutic target in neuroinflammation to repeated neonatal GA.

scholarly journals Transient oxytocin signaling primes the development and function of excitatory hippocampal neurons

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Silvia Ripamonti ◽  
Mateusz C Ambrozkiewicz ◽  
Francesca Guzzi ◽  
Marta Gravati ◽  
Gerardo Biella ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Pyramidal Neurons ◽  
Hippocampal Pyramidal Neurons ◽  
Excitatory Transmission ◽  
Oxytocin Receptors ◽  
Synapse Density ◽  
Protein Components ◽  
And Function

Beyond its role in parturition and lactation, oxytocin influences higher brain processes that control social behavior of mammals, and perturbed oxytocin signaling has been linked to the pathogenesis of several psychiatric disorders. However, it is still largely unknown how oxytocin exactly regulates neuronal function. We show that early, transient oxytocin exposure in vitro inhibits the development of hippocampal glutamatergic neurons, leading to reduced dendrite complexity, synapse density, and excitatory transmission, while sparing GABAergic neurons. Conversely, genetic elimination of oxytocin receptors increases the expression of protein components of excitatory synapses and excitatory synaptic transmission in vitro. In vivo, oxytocin-receptor-deficient hippocampal pyramidal neurons develop more complex dendrites, which leads to increased spine number and reduced γ-oscillations. These results indicate that oxytocin controls the development of hippocampal excitatory neurons and contributes to the maintenance of a physiological excitation/inhibition balance, whose disruption can cause neurobehavioral disturbances.

scholarly journals Enhanced Neuronal Damage After Ischemic Insults in Mice Lacking Kir6.2-Containing ATP-Sensitive K+ Channels

2006 ◽  
Vol 95 (4) ◽  
pp. 2590-2601 ◽  
Author(s):  
Hong-Shuo Sun ◽  
Zhong-Ping Feng ◽  
Takashi Miki ◽  
Susumu Seino ◽  
Robert J. French
Keyword(s):  
Hippocampal Neurons ◽  
Pyramidal Neurons ◽  
Neuronal Damage ◽  
Focal Cerebral Ischemia ◽  
Hippocampal Slices ◽  
Immunocytochemical Staining ◽  
Sulfonylurea Receptor ◽  
Ca1 Pyramidal Neurons

Adenosine triphosphate (ATP)–sensitive potassium (KATP) channels, incorporating Kir6.x and sulfonylurea receptor subunits, are weak inward rectifiers that are thought to play a role in neuronal protection from ischemic insults. However, the involvement of Kir6.2-containing KATP channel in hippocampus and neocortex has not been tested directly. To delineate the physiological roles of Kir6.2 channels in the CNS, we used knockout (KO) mice that do not express Kir6.2. Immunocytochemical staining demonstrated that Kir6.2 protein was expressed robustly in hippocampal neurons of the wild-type (WT) mice and absent in the KO. To examine neuronal sensitivity to metabolic stress in vitro, and to ischemia in vivo, we 1) exposed hippocampal slices to transient oxygen and glucose deprivation (OGD) and 2) produced focal cerebral ischemia by middle cerebral artery occlusion (MCAO). Both slice and whole animal studies showed that neurons from the KO mice were severely damaged after anoxia or ischemia, whereas few injured neurons were observed in the WT, suggesting that Kir6.2 channels are necessary to protect neurons from ischemic insults. Membrane potential recordings from the WT CA1 pyramidal neurons showed a biphasic response to OGD; a brief hyperpolarization was followed by a small depolarization during OGD, with complete recovery within 30 min after returning to normoxic conditions. By contrast, CA1 pyramidal neurons from the KO mice were irreversibly depolarized by OGD exposure, without any preceding hyperpolarization. These data suggest that expression of Kir6.2 channels prevents prolonged depolarization of neurons resulting from acute hypoxic or ischemic insults, and thus protects these central neurons from the injury.

scholarly journals Morphological neurite changes induced by porcupine inhibition are rescued by Wnt ligands

2021 ◽  
Author(s):  
Juan A. Godoy ◽  
Jasson Espinoza-Caicedo ◽  
Nibaldo C Inestrosa
Keyword(s):  
Hippocampal Neurons ◽  
Dendritic Tree ◽  
Immunoblot Analysis ◽  
Neuronal Morphology ◽  
Sprague Dawley ◽  
Dendrite Morphology ◽  
Wnt Ligands ◽  
Dendritic Arbors ◽  
And Migration

Abstract Background: Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/β-catenin-dependent or β-catenin-independent or "non-canonical", both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluatedMethods: Cultured primary embryonic hippocampal neurons obtained from Sprague-Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24h, Wnt ligands were added to the cultures on DIV 3 for 24h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, b-catenin and GSK-3b (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin.Results: We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases b-catenin and Wnt3a and an apparent increase in GSK-3b (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. Conclusions: Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases.

scholarly journals SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts

2018 ◽  
Vol 29 (11) ◽  
pp. 2641-2657
Author(s):  
Chengguo Wei ◽  
Khadija Banu ◽  
Felipe Garzon ◽  
John M. Basgen ◽  
Nimrod Philippe ◽  
...  
Keyword(s):  
Rho Kinase ◽  
Protective Role ◽  
Binding Domain ◽  
Actin Binding ◽  
Src Homology 3 ◽  
Src Homology ◽  
Glomerular Development ◽  
And Migration

BackgroundWe previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin–binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development.MethodsWe used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli.ResultsExpression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3–binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration.ConclusionsWe demonstrate a novel mechanism that may explain SHROOM3’s dichotomous associations in glomerular versus nonglomerular compartments in CKD

scholarly journals Repeated neonatal sevoflurane induced neurocognitive impairment through NF-κB-mediated pyroptosis

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Jing Dai ◽  
Xue Li ◽  
Cai Wang ◽  
Shuxin Gu ◽  
Lei Dai ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Neurological Diseases ◽  
Neurocognitive Impairment ◽  
Postnatal Period ◽  
Neuronal Morphology ◽  
Clinical Settings ◽  
Biochemical Analyses

Abstract Background Exposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis is a novel type of programmed cell death that, along with inflammation, has been found to play an important role in the mechanism of diverse neurological diseases. However, its roles in GA-induced neuroinflammation and neurocognitive impairment in the developing brain have not been investigated. Methods Rats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro received 3% sevoflurane for 2 h daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)-κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analyses were performed to assess the pyroptosis as well as neuronal and synaptic damage both in vivo and in vitro. In addition, behavioral tests were performed to evaluate neurocognitive ability in rats. Results Repeated sevoflurane exposure activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus in developing rats, damaged the neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082 treatment suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damage, and ameliorated the neurocognitive impairment induced by repeated sevoflurane administration to developing rats. Conclusions Repeated sevoflurane GA may induce neuroinflammation and neurocognitive impairment in developing rats via the activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role of pyroptosis as a potential therapeutic target in neuroinflammation after repeated neonatal GA.

scholarly journals 14-3-3 shuttles Activity-dependent neuroprotective protein to the cytoplasm to promote appropriate neuronal morphogenesis, cortical connectivity and calcium signaling

2020 ◽  
Author(s):  
Sarah A. Bennison ◽  
Sara M. Blazejewski ◽  
Xiaonan Liu ◽  
Kazuhito Toyo-oka
Keyword(s):  
Calcium Signaling ◽  
Pyramidal Neurons ◽  
Autism Spectrum ◽  
Neuronal Morphology ◽  
Cortical Connectivity ◽  
Neuronal Morphogenesis ◽  
Layer 2 ◽  
Neurite Formation ◽  
Activity Dependent

AbstractNeurite formation is the earliest stage of neuronal morphogenesis, where primitive dendrites and the primitive axon emerge from a spherical neuron and begin to elongate. Defective neuritogenesis is a contributing pathogenic mechanism behind a variety of neurodevelopmental disorders. Activity-dependent neuroprotective protein (Adnp) is essential to embryonic and postnatal brain development, and mutations in ADNP are among the most frequent underlying autism spectrum disorder (ASD). We found that knockdown of Adnp in vitro and in vivo in mouse layer 2/3 pyramidal neurons leads to increased neurite initiation and defective neurite elongation, suggesting that Adnp has distinct roles in each. In vivo analysis revealed that deficits begin at P0 and are sustained throughout development, the most notable of which include increased neurite stabilization, disrupted angle of the apical dendrite, increased basal dendrite number, and increased axon length. Because small changes in neuronal morphology can have large-scale effects on neuronal function and connectivity, we performed ex vivo calcium imaging to assess spontaneous function of layer 2/3 pyramidal neurons deficient in Adnp. This revealed that Adnp deficient neurons had a greater spontaneous calcium influx and a higher proportion of cells firing action potentials. Next, we utilized GRAPHIC, a novel synaptic tracing technology, to assess interhemispheric cortical connectivity. We found increased interhemispheric excitatory connectivity between Adnp deficient layer 2/3 pyramidal neurons. Because Adnp is a multifunctional protein with both transcription factor and cytoskeletal activity, we performed localization analysis of Adnp as neurons underwent neurite formation to probe the mechanism of our morphological defects. We found that Adnp is shuttled from the nucleus to the cytoplasm upon differentiation and this shuttling can be blocked via application of a global 14-3-3 inhibitor, difopein. Furthermore, we found that Adnp binds nuclear-cytoplasmic shuttle 14-3-3ε. We conclude that Adnp is shuttled from the nucleus to the cytoplasm by 14-3-3ε, where it regulates neuronal morphology, maturation, cortical connectivity, and calcium signaling.

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