Induction of axon growth arrest without growth cone collapse through the <i>N</i>‐terminal region of four‐transmembrane glycoprotein M6a

Sato, Yasufumi; Mita, Sakura; Fukushima, Nanae; Fujisawa, Hajime; Saga, Yumiko; Hirata, Tatsumi

doi:10.1002/dneu.20941

Cited by 20 publications

(33 citation statements)

References 62 publications

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“…Furthermore, in the previous study, we found notable axon behavior involving M6a; when anti-M6a antibody binds to M6a protein in culture, the axons stalls the elongation (Sato et al, 2011a). Interestingly, during this reaction, the axons maintain the actin-rich growth cones exhibiting highly motile movement.…”

Section: Introductionmentioning

confidence: 67%

“…For analysis of expression of M6 proteins, ICR wild-type mice were purchased from Japan SLC, Inc. M6a knockout mice in the C57BL/6 background were produced previously (Sato et al, 2011a), and maintained in the animal facility at the National Institute of Genetics. Drs.…”

Section: Animalsmentioning

confidence: 99%

“…The cells were plated at a density of 6×10 3 cells/cm 2 on the laminin-coated 8-well chamber slides (Nalge Nunc) and cultured in Neurobasal medium (Life Technologies) supplemented with B27 and N2 supplements (Life Technologies) for 72 h at 37°C in a 5% CO2 atmosphere. Primary olfactory bulb neurons were cultured as described previously (Sato et al, 2011a).…”

Section: Dissociated Culture Of Cortical Neuronsmentioning

confidence: 99%

“…We already had anti-M6a antibody (Sato et al, 2011a), but I need to prepare the antibody for M6b. The anti-M6b antibody was designed against the intracellular C-terminus (CMQ AYQ…”

Section: Overlapping Expression Of M6a and M6b In The Central Nervousmentioning

confidence: 99%

“…For the formation of filopodia/spine, the second extracellular domain (C174 and C192) is essential (Fuchsova et al, 2009). When the anti-M6a antibody stalls axon alongation, a short N-terminus sequence (amino acid 1-25) is important (Sato et al, 2011a).…”

Section: Functional Domains Of M6a For the Axonal Growthmentioning

confidence: 99%

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M6 proteins regulate axon outgrowth in mouse callosal neurons

Mita

Saga

Werner³

et al. 2010

Neuroscience Research

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Corpus callosum (CC) is the largest fiber tract in the human brain that connects the left and right cerebral hemispheres. The absence of the CC results in impairment of higher-order cognitive functions because interhemispheric transfer of information is disrupted. The formation of the CC is intricately regulated by a large number of molecules, which have not been fully characterized, while actively searched. In this study, I showed that additional molecules, M6 proteins, are also involved in the CC development.M6 proteins consist of M6a and M6b, highly homologous four-transmembrane proteins. M6a was originally found by the antibody screening for candidate molecules that are involved in guidance of axons. Previously, we found that when anti-M6a antibody binds to M6a protein in cultured neurons, the axons stall the elongation. Therefore, it seemed that M6a is involved in axon elongation of developing neurons. However, no study has so far examined the function of M6 proteins in vivo. To address this issue, I analyzed brain morphology and axonal projections in knockout mice for the M6a and M6b genes.First, to assess whether the two M6 proteins have redundant functions, I investigated expression patterns of M6a and M6b proteins in the wildtype mouse brains during developmental stages, using a newly prepared anti-M6b specific antibody. M6a was expressed mainly in growing axons as previously reported. The new anti-M6b antibody revealed that M6b was well co-localized with M6a in these axons. The expression level of M6b on the axons appeared lower at E14.5 compared with M6a, and subsequently intensified at E16.5 and P0, displaying comparable overlapping patterns with M6a. I also examined the subcellular distribution of M6 proteins in neurons in dissociated culture. As described previously, M6a protein was enriched in the edge of growth cones and the shaft filopodia of axons in cultured neurons. M6b protein was also expressed in the growth cones and the shaft filopodia, and co- However, the size of the CC bundle was abnormally small in the M6a-/-M6b-/-knockout mice.This hypoplasia in the CC was not clear at early developmental stages (E14.5, E16.5 and P0), but became obvious by P7 and throughout life. I quantified the size of the CC in wild-type, single and double mutant mice at P7, and found that the defect was most pronounced in the double knockout mice, indicating the redundant function of the two M6 proteins.I considered the following three possibilities to explain the smaller CC bundles in the double knockout mice. 1) the number of the callosal neurons is reduced.2) The axons of the callosal neurons are misdirected from callosal pathway.3) The axon outgrowth is impaired in the callosal neurons.First, to evaluate the possibility 1), I measured the density of cortical neurons in the mutant cortex in the motor, somatosensory, and visual areas. The neural density of the double mutant cortex was comparable to that of the wild-type. This result suggests that the number of callosal neurons was not reduced in the M6a...

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Section: Introductionmentioning

confidence: 67%

Section: Animalsmentioning

confidence: 99%

Section: Dissociated Culture Of Cortical Neuronsmentioning

confidence: 99%

“…We already had anti-M6a antibody (Sato et al, 2011a), but I need to prepare the antibody for M6b. The anti-M6b antibody was designed against the intracellular C-terminus (CMQ AYQ…”

Section: Overlapping Expression Of M6a and M6b In The Central Nervousmentioning

confidence: 99%

Section: Functional Domains Of M6a For the Axonal Growthmentioning

confidence: 99%

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M6 proteins regulate axon outgrowth in mouse callosal neurons

Mita

Saga

Werner³

et al. 2010

Neuroscience Research

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Tyrosine 251 at the C‐terminus of neuronal glycoprotein M6a is critical for neurite outgrowth

Formoso

Billi

Frasch

et al. 2014

J of Neuroscience Research

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Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension.

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Different modes of growth cone collapse in NG 108-15 cells

et al. 2013

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In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-013-0907-z) contains supplementary material, which is available to authorized users.

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Induction of axon growth arrest without growth cone collapse through the N‐terminal region of four‐transmembrane glycoprotein M6a

Cited by 20 publications

References 62 publications

M6 proteins regulate axon outgrowth in mouse callosal neurons

M6 proteins regulate axon outgrowth in mouse callosal neurons

Tyrosine 251 at the C‐terminus of neuronal glycoprotein M6a is critical for neurite outgrowth

Different modes of growth cone collapse in NG 108-15 cells

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