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O-GLcNAc post-translational modifications regulate the entry of neurons into an axon branching program.

Francisco H, Kollins K, Varghis N, Vocadlo D, Vosseller K, Gallo G - Dev Neurobiol (2009 Feb 1-15)

Bottom Line: Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching.Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia.These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.

ABSTRACT
Many neuronal cytosolic and nuclear proteins are post-translationally modified by the reversible addition of O-linked N-acetylglucosamine (O-GlcNAc) on serines and threonines. The cellular functions of O-GlcNAc modifications in neuronal development are not known. We report that O-GlcNAc-modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O-GlcNAcase. Western analysis revealed that O-GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology.

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Inhibition of O-GlcNAcase with 9d blocks for-skolin-induced increases in axon branching. (A) Cultures were treated with 50 μM forskolin +/− 200 μM 9d at the time of plating. Control (Cnt) cultures were treated with vehicle for the drugs. Forskolin (Forsk) increased the number of branches per unit axon length at 48 h. Cotreatment with 9d and Forsk prevented the increase in branches induced by forskolin. Comparison of Cnt with Forsk +9d did not reveal a difference (2-tailed Welch t-test). 9d treatment alone did not affect the number of branches relative to Cnt (Figure 3D). n = 70–83 axons per group. (B) Cultures were treated with 50 μM blebbistatin +/− 200 μM 9d at the time of plating. Control cultures were treated with vehicle for the drugs. Blebbistatin (Bleb) increased the number of branches at 48 h. However, 9d did not block the increase in branch number induced by Bleb. n = 84–103 axons per group.
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fig05: Inhibition of O-GlcNAcase with 9d blocks for-skolin-induced increases in axon branching. (A) Cultures were treated with 50 μM forskolin +/− 200 μM 9d at the time of plating. Control (Cnt) cultures were treated with vehicle for the drugs. Forskolin (Forsk) increased the number of branches per unit axon length at 48 h. Cotreatment with 9d and Forsk prevented the increase in branches induced by forskolin. Comparison of Cnt with Forsk +9d did not reveal a difference (2-tailed Welch t-test). 9d treatment alone did not affect the number of branches relative to Cnt (Figure 3D). n = 70–83 axons per group. (B) Cultures were treated with 50 μM blebbistatin +/− 200 μM 9d at the time of plating. Control cultures were treated with vehicle for the drugs. Blebbistatin (Bleb) increased the number of branches at 48 h. However, 9d did not block the increase in branch number induced by Bleb. n = 84–103 axons per group.

Mentions: Cyclic-AMP (cAMP) signaling is an important regulator of axon growth and branching (Weeks et al., 1991; reviewed in Cui and So, 2004). Griffith and Schmitz (1999) demonstrated a reciprocal relationship between the cAMP-protein kinase A signaling axis and O-GlcNAc levels on cytoskeletal proteins of cultured primary neurons. Given that reducing O-GlcNAc levels caused increased axon branching, we sought to determine if axon branching driven by activation of the cAMP axis would be sensitive to the levels of O-GlcNAc modifications. Culturing fore-brain neurons for 48 h in the presence of forskolin (50 μM), an activator of adenylyl cyclase resulting in increased cAMP production (Seamon and Daly, 1981) increased the number of axon branches [Fig. 5(A)], without affecting axonal filopodial numbers (data not shown). Cotreatment of cultures with forskolin and 9d blocked the increase in axon branches induced by forskolin treatment [Fig. 5(A)].


O-GLcNAc post-translational modifications regulate the entry of neurons into an axon branching program.

Francisco H, Kollins K, Varghis N, Vocadlo D, Vosseller K, Gallo G - Dev Neurobiol (2009 Feb 1-15)

Inhibition of O-GlcNAcase with 9d blocks for-skolin-induced increases in axon branching. (A) Cultures were treated with 50 μM forskolin +/− 200 μM 9d at the time of plating. Control (Cnt) cultures were treated with vehicle for the drugs. Forskolin (Forsk) increased the number of branches per unit axon length at 48 h. Cotreatment with 9d and Forsk prevented the increase in branches induced by forskolin. Comparison of Cnt with Forsk +9d did not reveal a difference (2-tailed Welch t-test). 9d treatment alone did not affect the number of branches relative to Cnt (Figure 3D). n = 70–83 axons per group. (B) Cultures were treated with 50 μM blebbistatin +/− 200 μM 9d at the time of plating. Control cultures were treated with vehicle for the drugs. Blebbistatin (Bleb) increased the number of branches at 48 h. However, 9d did not block the increase in branch number induced by Bleb. n = 84–103 axons per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2747243&req=5

fig05: Inhibition of O-GlcNAcase with 9d blocks for-skolin-induced increases in axon branching. (A) Cultures were treated with 50 μM forskolin +/− 200 μM 9d at the time of plating. Control (Cnt) cultures were treated with vehicle for the drugs. Forskolin (Forsk) increased the number of branches per unit axon length at 48 h. Cotreatment with 9d and Forsk prevented the increase in branches induced by forskolin. Comparison of Cnt with Forsk +9d did not reveal a difference (2-tailed Welch t-test). 9d treatment alone did not affect the number of branches relative to Cnt (Figure 3D). n = 70–83 axons per group. (B) Cultures were treated with 50 μM blebbistatin +/− 200 μM 9d at the time of plating. Control cultures were treated with vehicle for the drugs. Blebbistatin (Bleb) increased the number of branches at 48 h. However, 9d did not block the increase in branch number induced by Bleb. n = 84–103 axons per group.
Mentions: Cyclic-AMP (cAMP) signaling is an important regulator of axon growth and branching (Weeks et al., 1991; reviewed in Cui and So, 2004). Griffith and Schmitz (1999) demonstrated a reciprocal relationship between the cAMP-protein kinase A signaling axis and O-GlcNAc levels on cytoskeletal proteins of cultured primary neurons. Given that reducing O-GlcNAc levels caused increased axon branching, we sought to determine if axon branching driven by activation of the cAMP axis would be sensitive to the levels of O-GlcNAc modifications. Culturing fore-brain neurons for 48 h in the presence of forskolin (50 μM), an activator of adenylyl cyclase resulting in increased cAMP production (Seamon and Daly, 1981) increased the number of axon branches [Fig. 5(A)], without affecting axonal filopodial numbers (data not shown). Cotreatment of cultures with forskolin and 9d blocked the increase in axon branches induced by forskolin treatment [Fig. 5(A)].

Bottom Line: Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching.Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia.These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.

ABSTRACT
Many neuronal cytosolic and nuclear proteins are post-translationally modified by the reversible addition of O-linked N-acetylglucosamine (O-GlcNAc) on serines and threonines. The cellular functions of O-GlcNAc modifications in neuronal development are not known. We report that O-GlcNAc-modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O-GlcNAcase. Western analysis revealed that O-GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology.

Show MeSH
Related in: MedlinePlus