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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 using 200 μM 9d does not alter the development of neuronal polarity. (A) Western blot (WB) analysis of O-GlcNAc modifications in cultured forebrain neurons either untreated or 9d treated for 8 and 48 h of in vitro development. Treatment with 9d greatly increased the levels of O-GlcNAc-modified proteins as early as 8 h. Ponceau protein stain was used to determine relative loading across samples. (B) 9d did not affect the development of neuronal polarity (χ2 test, p > 0.3). The experiment was performed in quadruplicate with ≥100 neurons scored per experiment. (C) 9d did not alter the total length of axons (primary axon + axon branches; 2-tailed Welch t-test). n = 90–94 axons per group. (D) 9d did not change the branching frequency of axons (2-tailed Welch t-test). n = 155–173 axons per group. All data obtained from 48 h old cultures.
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fig03: Inhibition of O-GlcNAcase using 200 μM 9d does not alter the development of neuronal polarity. (A) Western blot (WB) analysis of O-GlcNAc modifications in cultured forebrain neurons either untreated or 9d treated for 8 and 48 h of in vitro development. Treatment with 9d greatly increased the levels of O-GlcNAc-modified proteins as early as 8 h. Ponceau protein stain was used to determine relative loading across samples. (B) 9d did not affect the development of neuronal polarity (χ2 test, p > 0.3). The experiment was performed in quadruplicate with ≥100 neurons scored per experiment. (C) 9d did not alter the total length of axons (primary axon + axon branches; 2-tailed Welch t-test). n = 90–94 axons per group. (D) 9d did not change the branching frequency of axons (2-tailed Welch t-test). n = 155–173 axons per group. All data obtained from 48 h old cultures.

Mentions: 9d is a selective inhibitor of O-GlcNAcase, and has been used to specifically increase levels of O-GlcNAc modifications on proteins in cell culture (Macauley et al., 2005). We first verified that 9d increased the levels of O-GlcNAc modifications on proteins in our culturing system using Western blot analysis. Forebrain cultures were treated for 8 or 48 h with 9d starting at the time of plating, and equal levels of protein from cell lysates were analyzed by Western blotting using an O-GlcNAc specific antibody. We observed multiple O-GlcNAc positive bands in control neurons at both 8 and 48 h [Fig. 3(A)]. Although the overall levels of O-GlcNAc reactivity at 8 and 48 h of control cells did not change appreciably, the intensity of O-GlcNAc reactivity in several specific bands was altered between 8 and 48 h, suggesting potential dynamic O-GlcNAc modification of some proteins between 8 and 48 h in vitro development [Fig. 3(A)]. Treatment with 9d elevated O-GlcNAc levels in the majority of protein bands at 8 h, and this elevation persisted through 48 h [see Fig. 3(A)]. The importance in demonstrating maximal O-GlcNAc elevation at 8 h is that by this time point in culture, neurons have attached to the substratum but not yet elaborated processes. Thus, 9d increased O-GlcNAc levels in neurons during the time period when the neurons extend processes.


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 using 200 μM 9d does not alter the development of neuronal polarity. (A) Western blot (WB) analysis of O-GlcNAc modifications in cultured forebrain neurons either untreated or 9d treated for 8 and 48 h of in vitro development. Treatment with 9d greatly increased the levels of O-GlcNAc-modified proteins as early as 8 h. Ponceau protein stain was used to determine relative loading across samples. (B) 9d did not affect the development of neuronal polarity (χ2 test, p > 0.3). The experiment was performed in quadruplicate with ≥100 neurons scored per experiment. (C) 9d did not alter the total length of axons (primary axon + axon branches; 2-tailed Welch t-test). n = 90–94 axons per group. (D) 9d did not change the branching frequency of axons (2-tailed Welch t-test). n = 155–173 axons per group. All data obtained from 48 h old cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Inhibition of O-GlcNAcase using 200 μM 9d does not alter the development of neuronal polarity. (A) Western blot (WB) analysis of O-GlcNAc modifications in cultured forebrain neurons either untreated or 9d treated for 8 and 48 h of in vitro development. Treatment with 9d greatly increased the levels of O-GlcNAc-modified proteins as early as 8 h. Ponceau protein stain was used to determine relative loading across samples. (B) 9d did not affect the development of neuronal polarity (χ2 test, p > 0.3). The experiment was performed in quadruplicate with ≥100 neurons scored per experiment. (C) 9d did not alter the total length of axons (primary axon + axon branches; 2-tailed Welch t-test). n = 90–94 axons per group. (D) 9d did not change the branching frequency of axons (2-tailed Welch t-test). n = 155–173 axons per group. All data obtained from 48 h old cultures.
Mentions: 9d is a selective inhibitor of O-GlcNAcase, and has been used to specifically increase levels of O-GlcNAc modifications on proteins in cell culture (Macauley et al., 2005). We first verified that 9d increased the levels of O-GlcNAc modifications on proteins in our culturing system using Western blot analysis. Forebrain cultures were treated for 8 or 48 h with 9d starting at the time of plating, and equal levels of protein from cell lysates were analyzed by Western blotting using an O-GlcNAc specific antibody. We observed multiple O-GlcNAc positive bands in control neurons at both 8 and 48 h [Fig. 3(A)]. Although the overall levels of O-GlcNAc reactivity at 8 and 48 h of control cells did not change appreciably, the intensity of O-GlcNAc reactivity in several specific bands was altered between 8 and 48 h, suggesting potential dynamic O-GlcNAc modification of some proteins between 8 and 48 h in vitro development [Fig. 3(A)]. Treatment with 9d elevated O-GlcNAc levels in the majority of protein bands at 8 h, and this elevation persisted through 48 h [see Fig. 3(A)]. The importance in demonstrating maximal O-GlcNAc elevation at 8 h is that by this time point in culture, neurons have attached to the substratum but not yet elaborated processes. Thus, 9d increased O-GlcNAc levels in neurons during the time period when the neurons extend processes.

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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