scholarly journals Morphological Neurite Changes Induced by Porcupine Inhibition Are Rescued by Wnt Ligands

2020 ◽  
Author(s):  
Juan A. Godoy ◽  
Jasson Espinoza-Caicedo ◽  
Nibaldo C Inestrosa
Keyword(s):  
Hippocampal Neurons ◽  
Neurological Diseases ◽  
Dendritic Tree ◽  
Immunoblot Analysis ◽  
Neuronal Morphology ◽  
Sprague Dawley ◽  
Dendrite Morphology ◽  
Wnt Ligands ◽  
Dendritic Arbors

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 young hippocampal neurons (DIV 4). Under these conditions, the morphology and length of the neurites and the complexity of the dendritic tree and axonal polarity were evaluated. Methods: Cultured primary young 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 and neurons were fixed on DIV 4. 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 young hippocampal neurons, with decreases -catenin and Wnt3a and an increase in GSK-3 (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 Wnt concentrations. Conclusions: Our results indicated that all 3 Wnt ligands restored dendritic arbor complexity with recovery of secondary and tertiary projections in young hippocampal neurons. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat neurological diseases.

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

2021 ◽  
Vol 19 (1) ◽  
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 ◽  
Gsk 3Β

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 evaluated Methods 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 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. 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, β-catenin and GSK-3β (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 β-catenin and Wnt3a and an apparent increase in GSK-3β (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 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 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 Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro

2020 ◽  
Vol 21 (3) ◽  
pp. 856
Author(s):  
David Wegrzyn ◽  
Christine Wegrzyn ◽  
Kerry Tedford ◽  
Klaus-Dieter Fischer ◽  
Andreas Faissner
Keyword(s):  
Hippocampal Neurons ◽  
Synapse Formation ◽  
Synergistic Effects ◽  
Neuronal Morphology ◽  
Network Activity ◽  
Nucleotide Exchange ◽  
Double Knockout ◽  
Nucleotide Exchange Factor ◽  
Key Events

Vav proteins activate GTPases of the RhoA subfamily that regulate the cytoskeleton and are involved in adhesion, migration, differentiation, polarity and the cell cycle. While the importance of RhoA GTPases for neuronal morphology is undisputed, their regulation is less well understood. In this perspective, we studied the consequences of the deletion of Vav2, Vav3 and Vav2 and 3 (Vav2−/−, Vav3−/−, Vav2−/−/3−/−) for the development of embryonic hippocampal neurons in vitro. Using an indirect co-culture system of hippocampal neurons with primary wild-type (WT) cortical astrocytes, we analysed axonal and dendritic parameters, structural synapse numbers and the spontaneous network activity via immunocytochemistry and multielectrode array analysis (MEA). Here, we observed a higher process complexity in Vav3−/−, but not in Vav2−/− neurons after three and five days in vitro (DIV). Furthermore, an enhanced synapse formation was observed in Vav3−/− after 14 days in culture. Remarkably, Vav2−/−/3−/− double knockout neurons did not display synergistic effects. Interestingly, these differences were transient and compensated after a cultivation period of 21 days. Network analysis revealed a diminished number of spontaneously occurring action potentials in Vav3−/− neurons after 21 DIV. Based on these results, it appears that Vav3 participates in key events of neuronal differentiation.

scholarly journals The Protein Dendrite Arborization and Synapse Maturation 1 (Dasm-1) Is Dispensable for Dendrite Arborization

2008 ◽  
Vol 28 (8) ◽  
pp. 2782-2791 ◽  
Author(s):  
Archana Mishra ◽  
Boris Knerr ◽  
Sónia Paixão ◽  
Edgar R. Kramer ◽  
Rüdiger Klein
Keyword(s):  
Hippocampal Neurons ◽  
Critical Role ◽  
Dendritic Tree ◽  
Dendrite Growth ◽  
Immunoglobulin Superfamily ◽  
Genetic Ablation ◽  
Neuronal Connections ◽  
Synapse Maturation

ABSTRACT The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization.

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 Fluorometric Determination of Glucose Utilization in Neurons in Vitro and in Vivo

2004 ◽  
Vol 24 (9) ◽  
pp. 993-1003 ◽  
Author(s):  
Yoshiaki Itoh ◽  
Takato Abe ◽  
Rie Takaoka ◽  
Norio Tanahashi
Keyword(s):  
Energy Source ◽  
Hippocampal Neurons ◽  
Glucose Utilization ◽  
Adult Brain ◽  
Sprague Dawley ◽  
Extracellular Lactate ◽  
Major Energy Source ◽  
Cerebellar Purkinje Cells

Glucose is the major energy source the adult brain utilizes under physiologic conditions. Recent findings, however, have suggested that neurons obtain most of their energy from the oxidation of extracellular lactate derived from astroglial metabolism of glucose transported into the brain from the blood. In the present studies we have used 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a fluorescent analogue of 2-deoxyglucose, which is often used to trace glucose utilization in neural tissues, to examine glucose metabolism in neurons in vitro and in vivo. Cultured neurons and astroglia were incubated with 2-NBDG for up to 15 minutes, and nonmetabolized 2-NBDG was washed out. We found that fluorescence intensity increased linearly with incubation time in both neurons and astroglia, indicating that both types of brain cells could utilize glucose as their energy source in vitro. To determine if the same were true in vivo, Sprague-Dawley rats were injected intravenously with a pulse bolus of 2-NBDG and decapitated 45 minutes later. Examination of brain sections demonstrated that phosphorylated 2-NBDG accumulated in hippocampal neurons and cerebellar Purkinje cells, indicating that neurons can utilize glucose in vivo as energy source.

scholarly journals Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Anna Rocchi ◽  
Silvio Sacchetti ◽  
Antonio De Fusco ◽  
Silvia Giovedi ◽  
Barbara Parisi ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Neurological Diseases ◽  
Synaptic Function ◽  
Inhibitory Synapses ◽  
Synapsin I ◽  
Loss Of Function ◽  
Reduced Density ◽  
Cytoplasmic Side

AbstractSynapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient’s autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by FcγII/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies.

scholarly journals Amot regulates neuronal dendritic tree through Yap1

2018 ◽  
Author(s):  
Katarzyna O. Rojek ◽  
Joanna Krzemień ◽  
Hubert Doleżyczek ◽  
Paweł M. Boguszewski ◽  
Leszek Kaczmarek ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Hippo Pathway ◽  
Dendritic Tree ◽  
Cell Contact ◽  
Cellular Polarity ◽  
Dendritic Trees ◽  
Conditional Deletion ◽  
The Central Nervous System ◽  
Cerebellar Size

ABSTRACTThe Amot-Yap1 complex plays a major role in the regulation of cell contact inhibition, cellular polarity and growth. However, the function of Angiomotin (Amot) and Hippo pathway transcription co-activator Yap1 in the central nervous system remains unclear. In this study, we demonstrate that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing hippocampal neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. Conditional deletion of Amot or Yap1 in neurons leads to impaired morphogenesis of Purkinje cell dendritic trees, decreased cerebellar size, and causes defects in locomotor coordination of mutant animals. Thus, our studies identified Amot and Yap1 as novel regulators of dendritic tree morphogenesis.

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