Axonal transport declines with age in two distinct phases separated by a period of relative stability

Abstract

Axonal transport is critical for supplying newly synthesized proteins, organelles, mRNAs, and other cargoes from neuronal cell bodies into axons. Its impairment in many neurodegenerative conditions appears likely to contribute to pathogenesis. Axonal transport also declines during normal aging, but little is known about the timing of these changes, or about the effect of aging on specific cargoes in individual axons. This is important for understanding mechanisms of age-related axon loss and age-related axonal disorders. Here we use fluorescence live imaging of peripheral nerve and central nervous system tissue explants to investigate vesicular and mitochondrial axonal transport. Interestingly, we identify 2 distinct periods of change, 1 period during young adulthood and the other in old age, separated by a relatively stable plateau during most of adult life. We also find that after tibial nerve regeneration, even in old animals, neurons are able to support higher transport rates of each cargo for a prolonged period. Thus, the age-related decline in axonal transport is not an inevitable consequence of either aging neurons or an aging systemic milieu.

Keywords: Aging; Axon regeneration; Axonal transport; Fluorescence live imaging; Mitochondrial transport; Nicotinamide mononucleotide Adenylyltransferase 2.

Fig. 1

Age-associated changes in NMNAT2-Venus axonal transport in sciatic nerve axons. (A) Representative straightened axon, kymograph, and kymograph of tracked particles of NMNAT2-Venus transport in sciatic nerves of 1.5- and 24-month-old NMNAT2-Venus (line 8) mice. The straightened axon represents the first frame of the time lapse recording (total 240 frames; frame rate 2 fps) that was used to generate the original kymograph. Moving particles were tracked using the ImageJ Difference Tracker set of plugins (see Table 2 for analysis parameters) and another kymograph generated to show successfully tracked particles. (B–E) Quantification of axonal transport parameters in sciatic nerve explants from NMNAT2-Venus line 8 animals of indicated ages. For all graphs, each data point represents the mean value obtained for 1 animal (5 fields of view and, on average, 14 axons per animal). Horizontal bar indicates mean, error bars are SEM. *Statistically significant difference between indicated ages or groups of ages. (*p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001; 1-way analysis of variance with Tukey multiple comparisons post-test or Student t test). The following parameters are shown: (B) anterograde particle velocity, (C) retrograde particle velocity, (D) anterograde particle count, and (E) retrograde particle count. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

Fig. 2

Age-associated changes in mitochondrial transport in sciatic nerve axons. (A) Representative straightened axon, kymograph, and kymograph of tracked particles of mitochondrial transport in sciatic nerves of 3- and 12-month-old MitoS mice. The straightened axon represents the first frame of the time lapse recording (total 360 frames; frame rate 2 fps) that was used to generate the original kymograph. Moving particles were tracked using the ImageJ Difference Tracker set of plugins (see Table 2 for analysis parameters) and another kymograph generated to show successfully tracked particles. (B–E) Quantification of axonal transport parameters in sciatic nerve explants from MitoS animals of indicated ages. For all graphs, each data point represents the mean value obtained for 1 animal (5 fields of view and, on average, 19 axons per animal). Horizontal bar indicates mean and error bars standard error of the mean. Statistically significant differences between ages or groups of ages are indicated as follows: ∗∗ p < 0.01; ∗∗∗p < 0.001 (Student t test). The following parameters are shown: (B) anterograde particle count, (C) retrograde particle count, (D) anterograde particle velocity, and (E) retrograde particle velocity. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

Fig. 3

Age-associated changes in NMNAT2-Venus axonal transport in optic nerve. (A) Representative straightened axon, kymograph, and kymograph of tracked particles of NMNAT2-Venus transport in optic nerve of 1.5- and 24-month-old NMNAT2-Venus (line 8) mice. The straightened axon represents the first frame of the time lapse recording (total 120 frames; frame rate 2 fps) that was used to generate the original kymograph. Moving particles were tracked using the ImageJ Difference Tracker set of plugins (see Table 2 for analysis parameters) and another kymograph generated to show successfully tracked particles. (B, C) Quantification of axonal transport parameters in optic nerve explants from NMNAT2-Venus line 8 animals of indicated ages. Each data point represents the mean value obtained for 1 animal (7 fields of view and, on average, 12 axons per animal). Horizontal bar indicates mean and error bars standard error of the mean. *Statistically significant difference between indicated ages or groups of ages. (*p < 0.05, *** p < 0.001; 1-way analysis of variance with Tukey multiple comparisons post-test). The following parameters are shown: (B) total particle count, (C) particle velocity. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

Fig. 4

Age-associated changes in NMNAT2-Venus axonal transport in fimbria. (A-H) Dissection of fimbria for live imaging. (A) Mouse brain in oxygenated Neurobasal A medium with cortex and striatum removed unilaterally. (B) After removal of cortex and striatum on both sides, hippocampi are visible (arrows). (C) Removal of hippocampi exposes the right and left side fimbria (arrows) and the body of fornix (arrowhead). (D) Illustration of dissection to remove the fimbria (right-side fimbria, indicated by arrow) from remainder of the brain. (E) Fimbria and body of fornix after removal from the brain; several pieces of gray matter are still attached and need to be removed before imaging (asterisks). (F) Final step of the clean-up procedure—removal of gray matter (triangular septal nuclei) from the dorsal side of the fornix. (G) Ventral view of the completed explant; this side will be used for live imaging. (H) Dorsal view of completed explant. (I) Representative straightened axon, kymograph, and kymograph of tracked particles of NMNAT2-Venus transport in fimbria of 1.5- and 24-month-old NMNAT2-Venus (line 8) mice. The straightened axon represents the first frame of the time lapse recording (total 120 frames; frame rate 2 fps) that was used to generate the original kymograph. Moving particles were tracked using the ImageJ Difference Tracker set of plugins (see Table 2 for parameters) and another kymograph generated to show tracked particles. (J, K) Quantification of axonal transport parameters in fimbria explants from NMNAT2-Venus line 8 animals of indicated ages. Each data point represents the mean value obtained for 1 animal (5 fields of view and, on average, 17 axon tracts per animal). Horizontal bar indicates mean and error bars standard error of the mean. *Indicate statistically significant difference between indicated ages or groups of ages (**p < 0.01, ***p < 0.001; 1-way analysis of variance with Tukey multiple comparisons post-test). The following parameters are shown: (J) total particle count, (K) particle velocity. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

Fig. 5

Partial reversal of age-associated drop in axonal transport of NMNAT2-Venus and mitochondria after nerve crush. (A) Semi-thin sections (500 nm) of control and crushed/regenerated tibial nerves 8 weeks after surgery. White arrows indicate myelin invaginations in all samples. (B, D) Anterograde and (C, E) retrograde particle counts in tibial nerve explants from MitoS (B, C) and NMNAT2-Venus line 8 animals (D, E) at 25 months of age. Eight weeks before imaging, 1 tibial nerve in each animal was crushed (at the level of the distal sciatic nerve) and allowed to regenerate. The contralateral uninjured nerve was used as a control. For all graphs, each data point represents the mean value obtained for 1 animal (6 fields of view and, on average, 15 axons per nerve). Horizontal bar indicates group mean and error bars indicate standard error of the mean. Control and crushed/regenerated nerves from the same animal are connected to indicate matching values. *Statistically significant difference between control and crushed/regenerated nerves (*p < 0.05; paired t test). (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)