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1. IGF-1 receptor regulates dynamic changes in neuronal polarity during cerebral cortical migration

Author

Alvaro F. Nieto Guil, Mariana Oksdath, Linnea A. Weiss, Diego J. Grassi, Lucas J. Sosa, Marta Nieto, and Santiago Quiroga

Subjects
Medicine, Science
Abstract

Abstract During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development. Neurons electroporated with a shRNA targeting IGF-1 receptor failed to migrate to the upper cortical layers and accumulated at the ventricular/subventricular zones. Co-electroporation with a constitutively active form of PI3K rescued migration. The change of the morphology from multipolar to bipolar cells was also attenuated. Cells lacking the IGF-1 receptor remain arrested as multipolar forming a highly disorganized tissue. The typical orientation of the migrating neurons with the Golgi complex oriented toward the cortical upper layers was also affected by electroporation with shRNA targeting IGF-1 receptor. Finally, cells electroporated with the shRNA targeting IGF-1 receptor were unable to form an axon and, therefore, neuron polarity was absent.

Published
2017
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2. Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy

Author

Ruby E. Dawson, Alvaro F. Nieto Guil, Louise J. Robertson, Sandra G. Piltz, James N. Hughes, and Paul Q. Thomas

Subjects
Neurodevelopment, Epilepsy, Focal cortical dysplasia, Molecular genetics, Functional testing, Developmental genetics, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract

Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

Published
2020
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3. Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

Author

Maria Eugenia Bernis, Mariana eOksdath, Sebastian eDupraz, Alvaro F. Nieto Guil, Marisa M. Fernandez, Emilio L. Malchiodi, Silvana B. Rosso, and Santiago eQuiroga

Subjects
neuronal polarity, axonal outgrowth, Wnt3a, IGF-1 receptor pathway, Frizzled-7, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7.

Published
2013
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4. Disrupted Excitatory Synaptic Contacts and Altered Neuronal Network Activity Underpins the Neurological Phenotype in PCDH19-Clustering Epilepsy (PCDH19-CE)

Author

Jozef Gecz, Stefka Mincheva-Tasheva, Paul Q. Thomas, Claire C. Homan, Alvaro F. Nieto Guil, Mincheva-Tasheva, Stefka, Nieto Guil, Alvaro F, Homan, Claire C, Gecz, Jozel, and Thomas, Paul Q

Subjects
0301 basic medicine, PCDH19-Clustering Epilepsy (PCDH19-CE), PCDH19, Neurite, Neuroscience (miscellaneous), Synaptogenesis, protocadherin 19, Biology, medicine.disease_cause, 03 medical and health sciences, Cellular and Molecular Neuroscience, 0302 clinical medicine, medicine, Biological neural network, Premovement neuronal activity, Mutation, Phenotype, 030104 developmental biology, medicine.anatomical_structure, Neurology, Excitatory postsynaptic potential, epilepsy, synapses, Neuron, Neuroscience, 030217 neurology & neurosurgery
Abstract

PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE. Refereed/Peer-reviewed

Published
2021
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5. Disrupted Excitatory Synaptic Contacts and Altered Neuronal Network Activity Underpins the Neurological Phenotype in PCDH19-Clustering Epilepsy (PCDH19-CE)

Author

Stefka, Mincheva-Tasheva, Alvaro F, Nieto Guil, Claire C, Homan, Jozef, Gecz, and Paul Q, Thomas

Subjects
Mice, Knockout, Heterozygote, Mice, Epilepsy, Mosaicism, Mutation, Synapses, Animals, Nerve Net, Cadherins, Axons, Protocadherins
Abstract

PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE.

Published
2020

6. The motor KIF5C links the requirements of stable microtubules and IGF-1 receptor membrane insertion for neuronal polarization

Author

Santiago Quiroga, Lucas J. Sosa, Diego Grassi, Alvaro F. Nieto Guil, Mariana Oksdath, Oksdath, Mariana, Guil, Álvaro F Nieto, Grassi, Diego, Sosa, Lucas J, and Quiroga, Santiago

Subjects
0301 basic medicine, IGF-1 receptor, Cytochalasin D, Paclitaxel, Neurite, Otras Ciencias Biológicas, Neuroscience (miscellaneous), Kinesins, Biology, neuronal polarization, Hippocampus, Microtubules, Syntaxin 6, Receptor, IGF Type 1, R-SNARE Proteins, Ciencias Biológicas, 03 medical and health sciences, Cellular and Molecular Neuroscience, chemistry.chemical_compound, Microtubule, Cell polarity, Neurites, medicine, Animals, Syntaxin, Axon, KIF5C, neuronal differentiation, Cells, Cultured, Nucleic Acid Synthesis Inhibitors, Neurons, Qa-SNARE Proteins, Nocodazole, Cell Membrane, Neurosciences, Cell Polarity, Tubulin Modulators, Rats, Cell biology, Vesicular transport protein, 030104 developmental biology, medicine.anatomical_structure, Neurology, chemistry, STABLE MICROTUBULES, Kinesin, stable microtubles, CIENCIAS NATURALES Y EXACTAS
Abstract

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity. Fil: Oksdath Mansilla, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Grassi, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Sosa, Lucas Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Published
2017

7. Protein interacting with NIMA (never in mitosis A)-1 regulates axonal growth cone adhesion and spreading through myristoylated alanine-rich C kinase substrate isomerization

Author

Mariana Oksdath, James S. Malter, Alvaro F. Nieto Guil, Lucas J. Sosa, Karl H. Pfenninger, Jie Hu, Santiago Quiroga, Florentyna Bustos Plonka, Sosa, Lucas J, Malter, James S, Hu, Jie, Plonka, Florentyna Bustos, Oksdath, Mariana, Nieto Guil, Alvaro F, Quiroga, Santiago, and Pfenninger, Karl H

Subjects
0301 basic medicine, MARCKS, Biochemistry & Molecular Biology, Otras Ciencias Biológicas, Growth Cones, brain development, Biology, Biochemistry, Axonal growth cone, isomerization, Rats, Sprague-Dawley, Ciencias Biológicas, corpus callosum, Mice, 03 medical and health sciences, Cellular and Molecular Neuroscience, Organ Culture Techniques, Pin1, Isomerism, Pregnancy, Cell Adhesion, medicine, Animals, NIMA-Interacting Peptidylprolyl Isomerase, Myristoylated Alanine-Rich C Kinase Substrate, Cell adhesion, Growth cone, Cells, Cultured, Cerebral Cortex, Mice, Knockout, growth cone adhesion, Intracellular Signaling Peptides and Proteins, Neurosciences, Membrane Proteins, Axons, Rats, Cell biology, 030104 developmental biology, medicine.anatomical_structure, Cerebral cortex, PIN1, Phosphorylation, Female, CIENCIAS NATURALES Y EXACTAS
Abstract

Axonal growth cone motility requires precise regulation of adhesion to navigate the complex environment of the nervous system and reach its target. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is enriched in the developing brain and plays an important, phosphorylation-dependent role in the modulation of axonal growth cone adhesion. The ratio of phospho-MARCKS (MARCKS-P) to total MARCKS controls adhesion modulation and spreading of the axonal growth cone. Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that recognizes and binds to phosphorylated serine/threonine residues preceded by a proline (pSer/Thr-Pro) is also expressed in the developing brain. Here, we show that Pin1 is present in the growth cone, interacts with MARCKS-P, and regulates its dephosphorylation. We also described morphological alterations in the corpus callosum and cerebral cortex fibers of the Pin1 knockout mouse brain that may be caused by the misregulation of MARCKS-P and alterations of neuronal adhesion. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment. Fil: Sosa, Lucas Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Malter, James S.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Hu, Jie. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Bustos Plonka, Florentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Oksdath Mansilla, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Pfenninger, Karl H.. University of Colorado. School of Medicine. Research Center. Department of Pediatrics and Colorado Intellectual and Developmental Disabilities; Estados Unidos

Published
2016

8. Selected SNARE proteins are essential for the polarized membrane insertion of igf-1 receptor and the regulation of initial axonal outgrowth in neurons

Author

Santiago Quiroga, Alvaro F. Nieto Guil, Florentyna Bustos Plonka, Mariana Oksdath, Diego Grassi, Lucas J. Sosa, Grassi, Diego, Plonka, Florentyna Bustos, Oksdath, Mariana, Guil, Álvaro Nieto, Sosa, Lucas J, and Quiroga, Santiago

Subjects
IGF-1 receptor, NEURONAL DIFFERENTIATION, Sintaxyn6, Otras Ciencias Biológicas, neuronal polarization, Biology, Hippocampal formation, Biochemistry, Article, Exocytosis, purl.org/becyt/ford/1 [https], Ciencias Biológicas, membrane expansion, SNAP23, Genetics, medicine, Gene silencing, MEMBRANE EXPANSION, Axon, purl.org/becyt/ford/1.6 [https], Receptor, Molecular Biology, VAMP2, Vesicle, IGF-1 RECEPTOR, Cell Biology, NEURONAL POLARIZATION, Cell biology, medicine.anatomical_structure, Neuronal differentiation, nervous system, VAMP4, SINTAXYN6, CIENCIAS NATURALES Y EXACTAS
Abstract

The establishment of polarity necessitates initial axonal outgrowth and, therefore, the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles (PPVs) primarily at the neuronal growth cone. Little is known about the SNAREs family proteins involved in the regulation of PPV fusion with the neuronal plasmalemma at early stages of differentiation. We show here that five SNARE proteins (VAMP2, VAMP4, VAMP7, Syntaxin6 and SNAP23) were expressed by hippocampal pyramidal neurons before polarization. Expression silencing of three of these proteins (VAMP4, Syntaxin6 and SNAP23) repressed axonal outgrowth and the establishment of neuronal polarity, by inhibiting IGF-1 receptor exocytotic polarized insertion, necessary for neuronal polarization. In addition, stimulation with IGF-1 triggered the association of VAMP4, Syntaxin6 and SNAP23 to vesicular structures carrying the IGF-1 receptor and overexpression of a negative dominant form of Syntaxin6 significantly inhibited exocytosis of IGF-1 receptor containing vesicles at the neuronal growth cone. Taken together, our results indicated that VAMP4, Syntaxin6 and SNAP23 functions are essential for regulation of PPV exocytosis and the polarized insertion of IGF-1 receptor and, therefore, required for initial axonal elongation and the establishment of neuronal polarity. Fil: Grassi, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Plonka, Florentyna Bustos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Oksdath Mansilla, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Sosa, Lucas Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Published
2015
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9. Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

Author

Santiago Quiroga, Emilio L. Malchiodi, Mariana Oksdath, Alvaro F. Nieto Guil, Marisa M. Fernández, Sebastian Dupraz, Silvana B. Rosso, Maria Eugenia Bernis, Bernis, Maria E, Oksdath, Mariana, Dupraz, Sebastian, Guil, Álvaro Nieto, Fernandez, Marisa M, Malchiodi, Emilio L, Rosso, Silvana B, and Quiroga, Santiago

Subjects
neuronal polarity, animal structures, IGF-1 receptor pathway, medicine.medical_treatment, Stimulation, Factores de Crecimiento, Biology, lcsh:RC321-571, Frizzled-7, Polaridad Neuronal, Ciencias Biológicas, purl.org/becyt/ford/1 [https], Insulin-like growth factor, chemistry.chemical_compound, Cellular and Molecular Neuroscience, Wnt, medicine, ddc:610, Phosphatidylinositol, Original Research Article, Growth cone, Receptor, purl.org/becyt/ford/1.6 [https], Protein kinase B, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, PI3K/AKT/mTOR pathway, Wnt signaling pathway, Neurosciences, Wnt3a, Bioquímica y Biología Molecular, Cell biology, body regions, chemistry, axonal outgrowth, embryonic structures, IGF-1, receptor pathway, Neuroscience, CIENCIAS NATURALES Y EXACTAS
Abstract

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7. Fil: Bernis, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Oksdath Mansilla, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Dupraz, Sebastian, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Fernández, Marisa Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Rosso, Silvana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Published
2013
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10. The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons

Author

Sebastian Dupraz, Diana Karnas, Alvaro F. Nieto Guil, Santiago Quiroga, Diego Grassi, and David Hicks

Subjects
Central Nervous System, Retinal Ganglion Cells, Anatomy and Physiology, Polyphosphoinositide Signaling Cascade, medicine.medical_treatment, lcsh:Medicine, Retinal ganglion cells, Receptor, IGF Type 1, purl.org/becyt/ford/1 [https], Phosphatidylinositol 3-Kinases, Ciencias Naturales y Exactas, Molecular Cell Biology, Neurobiology of Disease and Regeneration, Morphogenesis, Axon, Insulin-Like Growth Factor I, lcsh:Science, Phosphoinositide-3 Kinase Inhibitors, Neurons, Multidisciplinary, Neuronal Morphology, Bioquímica y Biología Molecular (ídem 3.1.10), Signaling Cascades, Cell biology, medicine.anatomical_structure, Axonal regeneration, Signal Transduction, Research Article, Transcriptional Activation, IGF-1 receptor, medicine.medical_specialty, Morpholines, Central nervous system, Biology, Retinal ganglion, Signaling Pathways, Neurological System, Ciencias Biológicas, Developmental Neuroscience, Internal medicine, medicine, Animals, Humans, Regeneration, Rats, Wistar, Growth cone, purl.org/becyt/ford/1.6 [https], PI3K/AKT/mTOR pathway, Retina, Regeneration (biology), Growth factor, lcsh:R, Adult CNS, Molecular Development, Axons, Signaling, Rats, Endocrinology, Gene Expression Regulation, nervous system, Chromones, Cellular Neuroscience, lcsh:Q, Molecular Neuroscience, Developmental Biology, Neuroscience
Abstract

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma. Fil: Dupraz, Sebastian, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina; Fil: Grassi, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina; Fil: Karnas, Diana. Rhythms, Life and Death in the Retina. Centre National de la Recherche Scientifique (CNRS). Université de Strasbourg. Institut des Neurosciences Cellulaires et Intégratives; France; Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina; Fil: Hicks, David. Rhythms, Life and Death in the Retina. Centre National de la Recherche Scientifique (CNRS). Université de Strasbourg. Institut des Neurosciences Cellulaires et Intégratives; France; Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina

Published
2013

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