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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 various durations  and under various experimental conditions. Each column represents the  mean accumulation ratio for the number of cells indicated and the error bars  represent the standard deviation about  the mean. An accumulation ratio >1  indicates an accumulation of neurofilament protein in the constricted axon  relative to the control sister axon and  an accumulation ratio <1 indicates a  depletion. (A) Time course of accumulation. For the sham experiment, no  constriction was performed so as to allow comparison of the normal variability between sister axons. For the other  experiments, the axons were constricted for either 5 s, 30 min, or 2 h.  The mean proximal accumulation ratio  after 2 h was significantly greater than  the corresponding distal accumulation ratio (P = 0.01, t test), and significantly greater than the proximal accumulation ratios after 5 s  (P = 0.01, t test) and 30 min (P = 0.04, t test). All other accumulation ratios were not significantly different from each other. (B) Constriction in the presence of an inhibitor of glycolysis. Cells were preincubated in medium containing 2 mM sodium iodoacetate for 1 h  and then constricted for 2 h in the same medium. The mean proximal accumulation ratio was significantly less than for axons constricted  for 2 h in the absence of inhibitor (P = 0.02, t test). (C) Permeabilization of constricted axons with detergent to extract soluble neurofilament protein. Cells were constricted for 2 h and then extracted with 0.02% saponin before fixation as described in Materials and Methods. The mean proximal accumulation ratio was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test)  and significantly greater than the proximal accumulation ratio in unextracted axons after constriction for 5 s (P = 0.01, t test).
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Figure 5: Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for various durations and under various experimental conditions. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean. An accumulation ratio >1 indicates an accumulation of neurofilament protein in the constricted axon relative to the control sister axon and an accumulation ratio <1 indicates a depletion. (A) Time course of accumulation. For the sham experiment, no constriction was performed so as to allow comparison of the normal variability between sister axons. For the other experiments, the axons were constricted for either 5 s, 30 min, or 2 h. The mean proximal accumulation ratio after 2 h was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test), and significantly greater than the proximal accumulation ratios after 5 s (P = 0.01, t test) and 30 min (P = 0.04, t test). All other accumulation ratios were not significantly different from each other. (B) Constriction in the presence of an inhibitor of glycolysis. Cells were preincubated in medium containing 2 mM sodium iodoacetate for 1 h and then constricted for 2 h in the same medium. The mean proximal accumulation ratio was significantly less than for axons constricted for 2 h in the absence of inhibitor (P = 0.02, t test). (C) Permeabilization of constricted axons with detergent to extract soluble neurofilament protein. Cells were constricted for 2 h and then extracted with 0.02% saponin before fixation as described in Materials and Methods. The mean proximal accumulation ratio was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test) and significantly greater than the proximal accumulation ratio in unextracted axons after constriction for 5 s (P = 0.01, t test).

Mentions: Fig. 5 A shows the mean proximal and distal accumulation ratios after constriction for different durations. To establish the extent of variation in neurofilament protein content between sister axons, we performed sham experiments in which neither sister axon was constricted. This yielded mean accumulation ratios of 1.0 (range = 0.7–2.1, n = 7) for the proximal measurement window and 0.9 (range = 0.4–1.4, n = 7) for the distal measurement window, indicating that sister axons were not significantly different from each other in their neurofilament content (P = 0.6, t test). These data confirm the validity of using sister axon comparisons in our quantitative analyses. After constriction of axons for 5 s, there was no detectable change in the amount of neurofilament protein either proximal to the constriction (mean accumulation ratio = 1.0, range = 0.5–1.4, n = 5) or distal to the constriction (mean accumulation ratio = 0.9, range = 0.6–1.5, n = 5). This confirms that there was no redistribution of neurofilament protein as an immediate consequence of compression of the axon with the glass fiber. After constriction for 30 min, the average amount of neurofilament protein was 110% higher proximally (mean accumulation ratio = 2.1, range = 0.6– 4.3, n = 4) and 60% higher distally (mean accumulation ratio = 1.6, range = 1.0–2.6, n = 4) but these values were not significantly different from axons constricted for 5 s (P = 0.3 and 0.1 respectively, t test). After constriction for 2 h, the average amount of neurofilament protein was 630% higher proximally (mean accumulation ratio = 7.3, range = 2.0–12.4, n = 6), which represented a statistically significant increase compared with axons constricted for 5 s (P = 0.01, t test) and 30 min (P = 0.04, t test). In contrast, the average amount of neurofilament protein distal to the constriction increased by only 30% (mean accumulation ratio = 1.3, range = 0.4–2.3, n = 6), and was not significantly different from axons constricted for 5 s (P = 0.3, t test) and 30 min (P = 0.6, t test). These data indicate that there was a marked time-dependent accumulation of neurofilament protein proximal to the constrictions, which suggests that neurofilament protein is transported anterogradely in these axons.


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 various durations  and under various experimental conditions. Each column represents the  mean accumulation ratio for the number of cells indicated and the error bars  represent the standard deviation about  the mean. An accumulation ratio >1  indicates an accumulation of neurofilament protein in the constricted axon  relative to the control sister axon and  an accumulation ratio <1 indicates a  depletion. (A) Time course of accumulation. For the sham experiment, no  constriction was performed so as to allow comparison of the normal variability between sister axons. For the other  experiments, the axons were constricted for either 5 s, 30 min, or 2 h.  The mean proximal accumulation ratio  after 2 h was significantly greater than  the corresponding distal accumulation ratio (P = 0.01, t test), and significantly greater than the proximal accumulation ratios after 5 s  (P = 0.01, t test) and 30 min (P = 0.04, t test). All other accumulation ratios were not significantly different from each other. (B) Constriction in the presence of an inhibitor of glycolysis. Cells were preincubated in medium containing 2 mM sodium iodoacetate for 1 h  and then constricted for 2 h in the same medium. The mean proximal accumulation ratio was significantly less than for axons constricted  for 2 h in the absence of inhibitor (P = 0.02, t test). (C) Permeabilization of constricted axons with detergent to extract soluble neurofilament protein. Cells were constricted for 2 h and then extracted with 0.02% saponin before fixation as described in Materials and Methods. The mean proximal accumulation ratio was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test)  and significantly greater than the proximal accumulation ratio in unextracted axons after constriction for 5 s (P = 0.01, t test).
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Figure 5: Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for various durations and under various experimental conditions. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean. An accumulation ratio >1 indicates an accumulation of neurofilament protein in the constricted axon relative to the control sister axon and an accumulation ratio <1 indicates a depletion. (A) Time course of accumulation. For the sham experiment, no constriction was performed so as to allow comparison of the normal variability between sister axons. For the other experiments, the axons were constricted for either 5 s, 30 min, or 2 h. The mean proximal accumulation ratio after 2 h was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test), and significantly greater than the proximal accumulation ratios after 5 s (P = 0.01, t test) and 30 min (P = 0.04, t test). All other accumulation ratios were not significantly different from each other. (B) Constriction in the presence of an inhibitor of glycolysis. Cells were preincubated in medium containing 2 mM sodium iodoacetate for 1 h and then constricted for 2 h in the same medium. The mean proximal accumulation ratio was significantly less than for axons constricted for 2 h in the absence of inhibitor (P = 0.02, t test). (C) Permeabilization of constricted axons with detergent to extract soluble neurofilament protein. Cells were constricted for 2 h and then extracted with 0.02% saponin before fixation as described in Materials and Methods. The mean proximal accumulation ratio was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test) and significantly greater than the proximal accumulation ratio in unextracted axons after constriction for 5 s (P = 0.01, t test).
Mentions: Fig. 5 A shows the mean proximal and distal accumulation ratios after constriction for different durations. To establish the extent of variation in neurofilament protein content between sister axons, we performed sham experiments in which neither sister axon was constricted. This yielded mean accumulation ratios of 1.0 (range = 0.7–2.1, n = 7) for the proximal measurement window and 0.9 (range = 0.4–1.4, n = 7) for the distal measurement window, indicating that sister axons were not significantly different from each other in their neurofilament content (P = 0.6, t test). These data confirm the validity of using sister axon comparisons in our quantitative analyses. After constriction of axons for 5 s, there was no detectable change in the amount of neurofilament protein either proximal to the constriction (mean accumulation ratio = 1.0, range = 0.5–1.4, n = 5) or distal to the constriction (mean accumulation ratio = 0.9, range = 0.6–1.5, n = 5). This confirms that there was no redistribution of neurofilament protein as an immediate consequence of compression of the axon with the glass fiber. After constriction for 30 min, the average amount of neurofilament protein was 110% higher proximally (mean accumulation ratio = 2.1, range = 0.6– 4.3, n = 4) and 60% higher distally (mean accumulation ratio = 1.6, range = 1.0–2.6, n = 4) but these values were not significantly different from axons constricted for 5 s (P = 0.3 and 0.1 respectively, t test). After constriction for 2 h, the average amount of neurofilament protein was 630% higher proximally (mean accumulation ratio = 7.3, range = 2.0–12.4, n = 6), which represented a statistically significant increase compared with axons constricted for 5 s (P = 0.01, t test) and 30 min (P = 0.04, t test). In contrast, the average amount of neurofilament protein distal to the constriction increased by only 30% (mean accumulation ratio = 1.3, range = 0.4–2.3, n = 6), and was not significantly different from axons constricted for 5 s (P = 0.3, t test) and 30 min (P = 0.6, t test). These data indicate that there was a marked time-dependent accumulation of neurofilament protein proximal to the constrictions, which suggests that neurofilament protein is transported anterogradely in these axons.

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