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Slow axonal transport of neurofilament protein in cultured neurons.

Koehnle TJ, Brown A - J. Cell Biol. (1999)

Bottom Line: The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form.These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation.This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.

ABSTRACT
We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

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Quantitative analysis of neurofilament protein  accumulation proximal and  distal to the constriction site  after constriction for 2 h in  the presence or absence of  metabolic substrates. Each  column represents the mean  accumulation ratio for the  number of cells indicated and  the error bars represent the  standard deviation about the  mean as described in the legend to Fig. 5. For these experiments, the L-15-based  culture medium was replaced  with a simpler Dulbecco's  PBS-based medium with or  without 0.6% glucose and  0.055% sodium pyruvate  (fed and starved, respectively). The mean proximal  accumulation ratio in the presence of glucose and pyruvate was  lower than for axons constricted for the same period of time in  L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced  significantly when these two substrates were omitted (P = 0.01,  t test).
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Figure 6: Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for 2 h in the presence or absence of metabolic substrates. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean as described in the legend to Fig. 5. For these experiments, the L-15-based culture medium was replaced with a simpler Dulbecco's PBS-based medium with or without 0.6% glucose and 0.055% sodium pyruvate (fed and starved, respectively). The mean proximal accumulation ratio in the presence of glucose and pyruvate was lower than for axons constricted for the same period of time in L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced significantly when these two substrates were omitted (P = 0.01, t test).

Mentions: To confirm the dependence of neurofilament protein accumulation on glycolysis, we also constricted axons in a medium that lacked metabolic substrates. For these experiments, we replaced the L-15-based culture medium with a simpler Dulbecco's PBS-based medium, either without any metabolic substrates or with glucose and pyruvate as the sole metabolic substrates. In the presence of glucose and pyruvate (fed), axons exhibited apparently normal swellings proximal and distal to the constriction and neurofilament protein was observed to accumulate proximally (Fig. 6). However, the magnitude of the accumulation was lower than for axons constricted in the L-15-based medium and this suggests that the physiology of these cells was compromised somewhat in the simpler PBS-based medium, which lacked serum as well as numerous other components that are present in the L-15 formulation (see Materials and Methods). In the absence of glucose and pyruvate (starved) the axons did not swell either proximal or distal to the constriction and the accumulation of neurofilament protein was reduced significantly (P = 0.01, t test). Collectively, these data indicate that the anterograde axonal transport of neurofilament protein is an active process that is dependent, directly or indirectly, on nucleoside triphosphates.


Slow axonal transport of neurofilament protein in cultured neurons.

Koehnle TJ, Brown A - J. Cell Biol. (1999)

Quantitative analysis of neurofilament protein  accumulation proximal and  distal to the constriction site  after constriction for 2 h in  the presence or absence of  metabolic substrates. Each  column represents the mean  accumulation ratio for the  number of cells indicated and  the error bars represent the  standard deviation about the  mean as described in the legend to Fig. 5. For these experiments, the L-15-based  culture medium was replaced  with a simpler Dulbecco's  PBS-based medium with or  without 0.6% glucose and  0.055% sodium pyruvate  (fed and starved, respectively). The mean proximal  accumulation ratio in the presence of glucose and pyruvate was  lower than for axons constricted for the same period of time in  L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced  significantly when these two substrates were omitted (P = 0.01,  t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132919&req=5

Figure 6: Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for 2 h in the presence or absence of metabolic substrates. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean as described in the legend to Fig. 5. For these experiments, the L-15-based culture medium was replaced with a simpler Dulbecco's PBS-based medium with or without 0.6% glucose and 0.055% sodium pyruvate (fed and starved, respectively). The mean proximal accumulation ratio in the presence of glucose and pyruvate was lower than for axons constricted for the same period of time in L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced significantly when these two substrates were omitted (P = 0.01, t test).
Mentions: To confirm the dependence of neurofilament protein accumulation on glycolysis, we also constricted axons in a medium that lacked metabolic substrates. For these experiments, we replaced the L-15-based culture medium with a simpler Dulbecco's PBS-based medium, either without any metabolic substrates or with glucose and pyruvate as the sole metabolic substrates. In the presence of glucose and pyruvate (fed), axons exhibited apparently normal swellings proximal and distal to the constriction and neurofilament protein was observed to accumulate proximally (Fig. 6). However, the magnitude of the accumulation was lower than for axons constricted in the L-15-based medium and this suggests that the physiology of these cells was compromised somewhat in the simpler PBS-based medium, which lacked serum as well as numerous other components that are present in the L-15 formulation (see Materials and Methods). In the absence of glucose and pyruvate (starved) the axons did not swell either proximal or distal to the constriction and the accumulation of neurofilament protein was reduced significantly (P = 0.01, t test). Collectively, these data indicate that the anterograde axonal transport of neurofilament protein is an active process that is dependent, directly or indirectly, on nucleoside triphosphates.

Bottom Line: The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form.These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation.This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.

ABSTRACT
We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 micrometer/h (3.1 mm/d), and approximately 90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.

Show MeSH
Related in: MedlinePlus