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N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons.

Islam MA, Sharif SR, Lee H, Moon IS - Mol. Cells (2015)

Bottom Line: We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature).In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth.The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, College of Medicine Dongguk University, Gyeongju 780-714, Korea.

ABSTRACT
N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of 'DYNLRB1 (74-96)', a small peptide derived from DYNLRB1's C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

No MeSH data available.


Expression of NAGK in developmental stage 4 (dendritic growth) and 5 (mature) neurons. (A) Stage 4 neurons. Epifluorescence microscopic images showing the expression profile of NAGK in sub-stages of Stage 4. In early Stage 4 (DIV7; a), the axonal growth cone is large (arrow) and NAGK levels in the axonal shaft (arrowheads) and growth cone remain high. However, as neurons matured (DIV10, b), the sizes of axonal growth cones (arrow) reduced and shafts (arrowheads) tightened. Note that the expression level of NAGK is significantly lower in (A-b) than in (A-a). In late stage 4 neurons (DIV14, c), the axonal expression of NAGK was reduced further. (B) A mature stage 5 neuron (DIV21). The axon was identified by staining with an antibody against ankyrin G (Ank G). Note that the expression level of NAGK is very low in the axon initial segment (arrow) and in its shaft (arrowheads). Scale bar, 30 μm.
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f2-molce-38-10-876: Expression of NAGK in developmental stage 4 (dendritic growth) and 5 (mature) neurons. (A) Stage 4 neurons. Epifluorescence microscopic images showing the expression profile of NAGK in sub-stages of Stage 4. In early Stage 4 (DIV7; a), the axonal growth cone is large (arrow) and NAGK levels in the axonal shaft (arrowheads) and growth cone remain high. However, as neurons matured (DIV10, b), the sizes of axonal growth cones (arrow) reduced and shafts (arrowheads) tightened. Note that the expression level of NAGK is significantly lower in (A-b) than in (A-a). In late stage 4 neurons (DIV14, c), the axonal expression of NAGK was reduced further. (B) A mature stage 5 neuron (DIV21). The axon was identified by staining with an antibody against ankyrin G (Ank G). Note that the expression level of NAGK is very low in the axon initial segment (arrow) and in its shaft (arrowheads). Scale bar, 30 μm.

Mentions: In stage 4 (dendritic outgrowth) neurons, dendrites start to grow in length and axons mature to produce their characteristic shape. At this stage, we fixed hippocampal neurons at three different culture time points (i.e., DIV7, DIV10, and DIV14), and double-labeled with anti-NAGK and anti-tubulin antibodies. At DIV7, we observed NAGK signals in axonal shafts (Fig. 2A–a, arrowheads) and growth cones (box with an arrow). At DIV 10 (Fig. 2A–b), axons had extended further and growth cones were smaller (arrow). During maturation, NAGK expression in axons decreased significantly (Fig. 2A–b, arrowheads), but it did not do so in the somatodendritic domain (Fig. 2A–b, short arrows). Later in stage 4 (DIV14), NAGK-IR was barely seen in axons (Fig. 2A–c, arrowheads), which by then had developed typical axon-like features. This observed gradual decline in NAGK expression in axons suggests that NAGK’s function in this compartment becomes less important as neuronal development progresses.


N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons.

Islam MA, Sharif SR, Lee H, Moon IS - Mol. Cells (2015)

Expression of NAGK in developmental stage 4 (dendritic growth) and 5 (mature) neurons. (A) Stage 4 neurons. Epifluorescence microscopic images showing the expression profile of NAGK in sub-stages of Stage 4. In early Stage 4 (DIV7; a), the axonal growth cone is large (arrow) and NAGK levels in the axonal shaft (arrowheads) and growth cone remain high. However, as neurons matured (DIV10, b), the sizes of axonal growth cones (arrow) reduced and shafts (arrowheads) tightened. Note that the expression level of NAGK is significantly lower in (A-b) than in (A-a). In late stage 4 neurons (DIV14, c), the axonal expression of NAGK was reduced further. (B) A mature stage 5 neuron (DIV21). The axon was identified by staining with an antibody against ankyrin G (Ank G). Note that the expression level of NAGK is very low in the axon initial segment (arrow) and in its shaft (arrowheads). Scale bar, 30 μm.
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Related In: Results  -  Collection

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f2-molce-38-10-876: Expression of NAGK in developmental stage 4 (dendritic growth) and 5 (mature) neurons. (A) Stage 4 neurons. Epifluorescence microscopic images showing the expression profile of NAGK in sub-stages of Stage 4. In early Stage 4 (DIV7; a), the axonal growth cone is large (arrow) and NAGK levels in the axonal shaft (arrowheads) and growth cone remain high. However, as neurons matured (DIV10, b), the sizes of axonal growth cones (arrow) reduced and shafts (arrowheads) tightened. Note that the expression level of NAGK is significantly lower in (A-b) than in (A-a). In late stage 4 neurons (DIV14, c), the axonal expression of NAGK was reduced further. (B) A mature stage 5 neuron (DIV21). The axon was identified by staining with an antibody against ankyrin G (Ank G). Note that the expression level of NAGK is very low in the axon initial segment (arrow) and in its shaft (arrowheads). Scale bar, 30 μm.
Mentions: In stage 4 (dendritic outgrowth) neurons, dendrites start to grow in length and axons mature to produce their characteristic shape. At this stage, we fixed hippocampal neurons at three different culture time points (i.e., DIV7, DIV10, and DIV14), and double-labeled with anti-NAGK and anti-tubulin antibodies. At DIV7, we observed NAGK signals in axonal shafts (Fig. 2A–a, arrowheads) and growth cones (box with an arrow). At DIV 10 (Fig. 2A–b), axons had extended further and growth cones were smaller (arrow). During maturation, NAGK expression in axons decreased significantly (Fig. 2A–b, arrowheads), but it did not do so in the somatodendritic domain (Fig. 2A–b, short arrows). Later in stage 4 (DIV14), NAGK-IR was barely seen in axons (Fig. 2A–c, arrowheads), which by then had developed typical axon-like features. This observed gradual decline in NAGK expression in axons suggests that NAGK’s function in this compartment becomes less important as neuronal development progresses.

Bottom Line: We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature).In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth.The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, College of Medicine Dongguk University, Gyeongju 780-714, Korea.

ABSTRACT
N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of 'DYNLRB1 (74-96)', a small peptide derived from DYNLRB1's C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

No MeSH data available.