Madecassic Acid Reduces Fast Transient Potassium Channels and Promotes Neurite Elongation in Hippocampal CA1 Neurons

Background and Objective: Madecassic Acid (MA) is well known to induce neurite elongation. However, its correlation with the expression of fast transient potassium (AKv) channels during neuronal development has not been well studied. Therefore, the present study was designed to investigate the effects of MA on the modulation of AKv channels during neurite outgrowth. Methods: Neurite outgrowth was measured with morphometry software, and Kv4 currents were recorded by using the patch clamp technique. Results: The ability of MA to promote neurite outgrowth is dose-dependent and was blocked by using the mitogen/extracellular signal-regulated kinase (MEK) inhibitor U0126. MA reduced the peak current density and surface expression of the AKv channel Kv4.2 with or without the presence of NaN3. The surface expression of Kv4.2 channels was also reduced after MA treatment of growing neurons. Ethylene glycol tetraacetic acid (EGTA) and an N-methyl-D-aspartate (NMDA) receptor blocker, MK801 along with MA prevented the effect of MA on neurite length, indicating that calcium entry through NMDA receptors is necessary for MA-induced neurite outgrowth. Conclusion: The data demonstrated that MA increased neurite outgrowth by internalizing AKv channels in neurons. Any alterations in the precise density of ion channels can lead to deleterious consequences on health because it changes the electrical and mechanical function of a neuron or a cell. Modulating ion channel’s density is exciting research in order to develop novel drugs for the therapeutic treatment of various diseases of CNS.

Download Full-text

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

Download Full-text

Growth Arrest-Specific Gene 6 (Gas6)/Adhesion Related Kinase (Ark) Signaling Promotes Gonadotropin-Releasing Hormone Neuronal Survival via Extracellular Signal-Regulated Kinase (ERK) and Akt

Molecular Endocrinology ◽

10.1210/mend.13.2.0230 ◽

1999 ◽

Vol 13 (2) ◽

pp. 191-201 ◽

Cited By ~ 63

Author(s):

Melissa P. Allen ◽

Chan Zeng ◽

Kristina Schneider ◽

Xiaoyan Xiong ◽

Mary Kay Meintzer ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Neuronal Migration ◽

Neuronal Development ◽

Mek Inhibitor ◽

Specific Gene ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Gnrh Neurons ◽

Induced Apoptosis

Abstract We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1–7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Axl) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1–7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Axl) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.

Download Full-text

Differential Involvement of Ca2+ Channels in Survival and Neurite Outgrowth of Cultured Embryonic Cockroach Brain Neurons

Journal of Neurophysiology ◽

10.1152/jn.2002.88.3.1475 ◽

2002 ◽

Vol 88 (3) ◽

pp. 1475-1490 ◽

Cited By ~ 13

Author(s):

Pascal Benquet ◽

Janine Le Guen ◽

Yves Pichon ◽

François Tiaho

Keyword(s):

Cell Death ◽

Calcium Channels ◽

Neurite Outgrowth ◽

Calcium Current ◽

Culture Media ◽

Calf Serum ◽

Free Calcium ◽

Extracellular Potassium ◽

Patch Clamp Technique ◽

Brain Neurons

The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents. VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil. The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about −30 and −20 mV in high-potassium culture media). As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM. The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network. In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I Ba. This effect could not be prevented by addition of fetal calf serum to the culture medium. A similar effect was obtained using intra- or extracellular calcium chelating agents (10 μM BAPTA-AM or 10 mM EGTA). Quite unexpectedly, blockade of the P/Q-like (ω-Aga WA-sensitive) component of the calcium current by 500 nM of ω-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism. As expected from their lack of effect on I Ba, isradipine, nifedipine, and ω-CgTx GVIA did not induce cell death. When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion. In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM. However, these agents, as well as ω-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons. This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites. These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons. The differential contribution of the P/Q-like and R-like (ω-Aga WA-sensitive) calcium channels in these processes is discussed.

Download Full-text

Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons.

The Journal of Cell Biology ◽

10.1083/jcb.110.4.1295 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1295-1306 ◽

Cited By ~ 37

Author(s):

A M Tolkovsky ◽

A E Walker ◽

R D Murrell ◽

H S Suidan

Keyword(s):

Nerve Growth Factor ◽

Neurite Outgrowth ◽

Sympathetic Neurons ◽

Optimal Growth ◽

Normal Medium ◽

Growth And Survival ◽

Neurite Elongation ◽

Cultured Sympathetic Neurons ◽

Ca2 Channels

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth.

Download Full-text

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

PLoS ONE ◽

10.1371/journal.pone.0030684 ◽

2012 ◽

Vol 7 (1) ◽

pp. e30684 ◽

Cited By ~ 16

Author(s):

Congcong Zhang ◽

Saskia Helmsing ◽

Marta Zagrebelsky ◽

Thomas Schirrmann ◽

Andrea L. J. Marschall ◽

...

Keyword(s):

Mrna Expression ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Surface Expression ◽

Neurotrophin Receptor ◽

P75 Neurotrophin Receptor ◽

Receptor Surface Expression

Download Full-text

Ser/Thr kinase Trc controls neurite outgrowth in Drosophila by modulating microtubule-microtubule sliding

eLife ◽

10.7554/elife.52009 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Rosalind Norkett ◽

Urko del Castillo ◽

Wen Lu ◽

Vladimir I Gelfand

Keyword(s):

Neurite Outgrowth ◽

Neuronal Development ◽

Live Imaging ◽

Biochemical Methods ◽

Interphase Cells ◽

Ndr Kinase

Correct neuronal development requires tailored neurite outgrowth. Neurite outgrowth is driven in part by microtubule-sliding – the transport of microtubules along each other. We have recently demonstrated that a ‘mitotic’ kinesin-6 (Pavarotti in Drosophila) effectively inhibits microtubule-sliding and neurite outgrowth. However, mechanisms regulating Pavarotti itself in interphase cells and specifically in neurite outgrowth are unknown. Here, we use a combination of live imaging and biochemical methods to show that the inhibition of microtubule-sliding by Pavarotti is controlled by phosphorylation. We identify the Ser/Thr NDR kinase Tricornered (Trc) as a Pavarotti-dependent regulator of microtubule sliding in neurons. Further, we show that Trc-mediated phosphorylation of Pavarotti promotes its interaction with 14-3-3 proteins. Loss of 14-3-3 prevents Pavarotti from associating with microtubules. Thus, we propose a pathway by which microtubule-sliding can be up- or downregulated in neurons to control neurite outgrowth, and establish parallels between microtubule-sliding in mitosis and post-mitotic neurons.

Download Full-text

Identification of B-KSR1, a Novel Brain-Specific Isoform of KSR1 That Functions in Neuronal Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5529-5539.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5529-5539 ◽

Cited By ~ 61

Author(s):

Jürgen Müller ◽

Angela M. Cacace ◽

W. Ernest Lyons ◽

Carolyn B. McGill ◽

Deborah K. Morrison

Keyword(s):

Growth Factor ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Catalytic Domain ◽

Mek Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Loss Of Function ◽

Mapk Activity ◽

Kinase Suppressor Of Ras ◽

Mitogen Activated Protein

ABSTRACT Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we report the identification of B-KSR1, a novel splice variant of murine KSR1 that is highly expressed in brain-derived tissues. B-KSR1 protein is detectable in mouse brain throughout embryogenesis, is most abundant in adult forebrain neurons, and is complexed with activated mitogen-activated protein kinase (MAPK) and MEK in brain tissues. Expression of B-KSR1 in PC12 cells resulted in accelerated nerve growth factor (NGF)-induced neuronal differentiation and detectable epidermal growth factor (EGF)-induced neurite outgrowth. Sustained MAPK activity was observed in cells stimulated with either NGF or EGF, and all effects on neurite outgrowth could be blocked by the MEK inhibitor PD98059. In B-KSR1-expressing cells, the MAPK–B-KSR1 interaction was inducible and correlated with MAPK activation, while the MEK–B-KSR1 interaction was constitutive. Further examination of the MEK–B-KSR1 interaction revealed that all genetically identified loss-of-function mutations in the catalytic domain severely diminished MEK binding. Moreover, B-KSR1 mutants defective in MEK binding were unable to augment neurite outgrowth. Together, these findings demonstrate the functional importance of MEK binding and indicate that B-KSR1 may function to transduce Ras-dependent signals that are required for neuronal differentiation or that are involved in the normal functioning of the mature central nervous system.

Download Full-text

Golgi-resident TRIO regulates membrane trafficking during neurite outgrowth

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.007318 ◽

2019 ◽

Vol 294 (28) ◽

pp. 10954-10968 ◽

Cited By ~ 8

Author(s):

Tao Tao ◽

Jie Sun ◽

Yajing Peng ◽

Yeqiong Li ◽

Pei Wang ◽

...

Keyword(s):

Neurite Outgrowth ◽

Membrane Trafficking ◽

Rho Gtpases ◽

Neuronal Development ◽

Membrane Vesicles ◽

Cerebellar Granule Neurons ◽

Granule Neurons ◽

Nucleotide Exchange ◽

Cerebellar Granule ◽

Cytoskeletal Dynamics

Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)– and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.

Download Full-text