The +TIP Navigator-1 is an actin–microtubule crosslinker that regulates axonal growth cone motility

Microtubule (MT) plus-end tracking proteins (+TIPs) are central players in the coordination between the MT and actin cytoskeletons in growth cones (GCs) during axon guidance. The +TIP Navigator-1 (NAV1) is expressed in the developing nervous system, yet its neuronal functions remain poorly elucidated. Here, we report that NAV1 controls the dynamics and motility of the axonal GCs of cortical neurons in an EB1-dependent manner and is required for axon turning toward a gradient of netrin-1. NAV1 accumulates in F-actin–rich domains of GCs and binds actin filaments in vitro. NAV1 can also bind MTs independently of EB1 in vitro and crosslinks nonpolymerizing MT plus ends to actin filaments in axonal GCs, preventing MT depolymerization in F-actin–rich areas. Together, our findings pinpoint NAV1 as a key player in the actin–MT crosstalk that promotes MT persistence at the GC periphery and regulates GC steering. Additionally, we present data assigning to NAV1 an important role in the radial migration of cortical projection neurons in vivo.

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A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

The Journal of Cell Biology ◽

10.1083/jcb.201311003 ◽

2014 ◽

Vol 205 (2) ◽

pp. 217-232 ◽

Cited By ~ 52

Author(s):

Cortney C. Winkle ◽

Leslie M. McClain ◽

Juli G. Valtschanoff ◽

Charles S. Park ◽

Christopher Maglione ◽

...

Keyword(s):

Plasma Membrane ◽

Cortical Neurons ◽

Direct Interaction ◽

Vesicle Fusion ◽

Dependent Manner ◽

Axon Branching ◽

Spatial Regulation ◽

Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

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Histidine–Tryptophan-–Ketoglutarate Solution as a Neuroprotective Against Ischemia/Reperfusion Injury

10.20944/preprints201810.0105.v1 ◽

2018 ◽

Author(s):

Jiun Hsu ◽

Chih-Hsien Wang ◽

Shu-Chien Huang ◽

Yung-Wei Chen ◽

Shengpin Yu ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

In Vitro Study ◽

Dependent Manner ◽

Sterile Saline ◽

Nadph Oxidase 4 ◽

H2o2 Level

Ischemic neuron loss contributes to brain dysfunction in patients with cardiac arrest (CA). Histidine–tryptophan–ketoglutarate (HTK) solution is a preservative used during organ transplantation. Can HTK also protect neurons from severe hypoxia (SH) following CA? We isolated rat primary cortical neurons and induced SH with or without HTK. Changes in caspase-3, hypoxia-inducible factor 1-alpha (HIF-1α), and NADPH oxidase-4 (NOX4) expression were evaluated at different time points till 72 h. Using a rat asphyxia model, we induced CA-mediated brain damage and then completed resuscitation. HTK or sterile saline was administered into the left carotid artery. Neurological deficit scoring and mortality were evaluated for 3 days. Then the rats were sacrificed for evaluating NOX4 and H2O2 level in blood and brain. In the in vitro study, HTK attenuated SH- and H2O2-mediated cytotoxicity in a volume- and time-dependent manner, associated with persisted HIF-1α expression, reductions in procaspase-3 activation and NOX4 expression. The inhibition of HIF-1α abrogated HTK’s effect on NOX4. In the in vivo study, neurological scores were significantly improved by HTK. H2O2 level, NOX4 activity and NOX4 gene expression were all decreased in the brain specimen of HTK-treated rats. Our results suggest that HTK acts as an effective neuroprotective solution.

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Fezf2 transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

10.1101/2020.08.13.242230 ◽

2020 ◽

Author(s):

Cameron Sadegh ◽

Wataru Ebina ◽

Anthony C. Arvanites ◽

Lance S. Davidow ◽

Lee L. Rubin ◽

...

Keyword(s):

Stem Cell ◽

Cortical Neurons ◽

Embryonic Stem ◽

Induced Pluripotent Stem Cell ◽

Sirtuin 1 ◽

Projection Neurons ◽

Specific Expression ◽

Specific Differentiation

AbstractDuring late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

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The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments

Journal of Cell Science ◽

10.1242/jcs.112.22.3943 ◽

1999 ◽

Vol 112 (22) ◽

pp. 3943-3954 ◽

Cited By ~ 1

Author(s):

V. Assmann ◽

D. Jenkinson ◽

J.F. Marshall ◽

I.R. Hart

Keyword(s):

Actin Filaments ◽

Human Cancer ◽

Cell Surface Receptor ◽

Dependent Manner ◽

Interaction Domain ◽

Microtubule Binding ◽

Projection Domain ◽

Hyaluronan Receptor

We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca(2+)-dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574–602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.

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A Critical Temporal Requirement for the Retinoblastoma Protein Family During Neuronal Determination

The Journal of Cell Biology ◽

10.1083/jcb.140.6.1497 ◽

1998 ◽

Vol 140 (6) ◽

pp. 1497-1509 ◽

Cited By ~ 66

Author(s):

Ruth S. Slack ◽

Hiba El-Bizri ◽

Josée Wong ◽

Daniel J. Belliveau ◽

Freda D. Miller

Keyword(s):

Cell Cycle ◽

Nervous System ◽

Cortical Neurons ◽

Adenovirus E1a ◽

Neuronal Gene ◽

Developing Neocortex ◽

Postmitotic Neurons ◽

Developing Nervous System

In this report, we have examined the requirement for the retinoblastoma (Rb) gene family in neuronal determination with a focus on the developing neocortex. To determine whether pRb is required for neuronal determination in vivo, we crossed the Rb−/− mice with transgenic mice expressing β-galactosidase from the early, panneuronal Tα1 α-tubulin promoter (Tα1:nlacZ). In E12.5 Rb−/− embryos, the Tα1:nlacZ transgene was robustly expressed throughout the developing nervous system. However, by E14.5, there were perturbations in Tα1:nlacZ expression throughout the nervous system, including deficits in the forebrain and retina. To more precisely define the temporal requirement for pRb in neuronal determination, we functionally ablated the pRb family in wild-type cortical progenitor cells that undergo the transition to postmitotic neurons in vitro by expression of a mutant adenovirus E1A protein. These studies revealed that induction of Tα1:nlacZ did not require proteins of the pRb family. However, in their absence, determined, Tα1:nlacZ-positive cortical neurons underwent apoptosis, presumably as a consequence of “mixed signals” deriving from their inability to undergo terminal mitosis. In contrast, when the pRb family was ablated in postmitotic cortical neurons, there was no effect on neuronal survival, nor did it cause the postmitotic neurons to reenter the cell cycle. Together, these studies define a critical temporal window of requirement for the pRb family; these proteins are not required for induction of neuronal gene expression or for the maintenance of postmitotic neurons, but are essential for determined neurons to exit the cell cycle and survive.

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Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells

PLoS ONE ◽

10.1371/journal.pone.0254113 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0254113

Author(s):

Cameron Sadegh ◽

Wataru Ebina ◽

Anthony C. Arvanites ◽

Lance S. Davidow ◽

Lee L. Rubin ◽

...

Keyword(s):

Stem Cell ◽

Small Molecule ◽

Cortical Neurons ◽

Embryonic Stem ◽

Sirtuin 1 ◽

Projection Neurons ◽

Specific Expression ◽

Specific Differentiation

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

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LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e14-01-0675 ◽

2014 ◽

Vol 25 (11) ◽

pp. 1755-1768 ◽

Cited By ~ 19

Author(s):

Tetsuya Takano ◽

Tomoki Urushibara ◽

Nozomu Yoshioka ◽

Taro Saito ◽

Mitsunori Fukuda ◽

...

Keyword(s):

Cortical Neurons ◽

Phosphorylation Site ◽

Dendrite Growth ◽

Endosomal Trafficking ◽

Dependent Manner ◽

Dendrite Development ◽

Membrane Components ◽

And Function

Neurons extend two types of neurites—axons and dendrites—that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth.

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Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy syndrome

eLife ◽

10.7554/elife.51037 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Georgia Panagiotakos ◽

Christos Haveles ◽

Arpana Arjun ◽

Ralitsa Petrova ◽

Anshul Rana ◽

...

Keyword(s):

Calcium Channel ◽

Autism Spectrum ◽

Projection Neurons ◽

Dependent Manner ◽

Loss Of Function ◽

Timothy Syndrome ◽

Cortical Projection ◽

Embryonic Mouse ◽

Syndromic Asd

The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.

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Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions

Journal of Cell Science ◽

10.1242/jcs.111.12.1695 ◽

1998 ◽

Vol 111 (12) ◽

pp. 1695-1706 ◽

Cited By ~ 2

Author(s):

B. Constantin ◽

K. Meerschaert ◽

J. Vandekerckhove ◽

J. Gettemans

Keyword(s):

Actin Cytoskeleton ◽

Cell Morphology ◽

Actin Filaments ◽

Mammalian Cells ◽

Actin Binding ◽

General Mechanism ◽

Resting Cells ◽

Dependent Manner

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

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COMPASS Family Histone Methyltransferase ASH2L Mediates Corticogenesis via Transcriptional Regulation of Wnt Signalling

10.1101/413674 ◽

2018 ◽

Author(s):

Liang Li ◽

Xiangbin Ruan ◽

Chang Wen ◽

Pan Chen ◽

Wei Liu ◽

...

Keyword(s):

Cell Fate ◽

Neural Progenitor Cells ◽

Histone Methyltransferase ◽

Projection Neurons ◽

Cell Fate Specification ◽

Cortical Projection ◽

Proliferation Ability ◽

Late Stages

AbstractCell fate specification in neural progenitor cells (NPCs) is orchestrated via extrinsic and intrinsic molecular programs, and histone methylation in these decisions has been ascribed to a crucial function regulating gene expression. Here, we show that the COMPASS family histone methyltransferase co-factor ASH2L is required in NPCs proliferation and upper layer cortical projection neurons production and position. Deletion of Ash2l impairs trimethylation of H3K4 and transcriptional machinery specifically for subsets of Wnt-β-catenin signalling, disrupting their transcription and consequently inhibiting the proliferation ability of NPCs in late stages of neurogenesis. Consistently, Ash2l conditional mutants exhibit thinning neocortex with reduced upper layer neurons and altered neuronal position. Moreover, overexpressing β-catenin after Ash2l elimination or knockdown can rescue the proliferation deficiency of NPCs both in vivo and in vitro. These results demonstrate an essential and highly specific role for Ash2l in controlling NPCs proliferation and late-born neurons lamination in corticogenesis via transcriptionally regulating Wnt-β-catenin signalling, and provide clues to how the COMPASS family epigenetic factors coordinate cell fate determination during cortex development.

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