Neurofilaments are nonessential to the pathogenesis of toxicant-induced axonal degeneration

Abstract

Axonal neurofilament (NF) accumulations occur before development of symptoms and before other pathological changes among idiopathic neurodegenerative diseases and toxic neuropathies, suggesting a cause-effect relationship. The dependence of symptoms and axonal degeneration on neurofilament accumulation has been tested here in a transgenic mouse model (Eyer and Peterson, 1994) lacking axonal NFs and using two prototypic toxicant models. Chronic acrylamide (ACR) or 2,5-hexanedione exposure resulted in progressive and cumulative increases in sensorimotor deficits. Neurobehavioral tests demonstrated similar expression of neurotoxicity in transgenic (T) mice and their nontransgenic (NT) littermates (containing normal numbers of axonal NFs). Axonal lesions were frequently observed after exposure to either toxicant. Quantitation of ACR-induced lesions demonstrated the distal location of pathology and equal susceptibility of T and NT axons. We conclude that axonal NFs have no effect on neurotoxicity and the pattern of pathology in these mammalian toxic neuropathies. These results also suggest that the role of neurofilament accumulation in the pathogenesis of neurodegenerative diseases requires careful evaluation.

Fig. 1.

A, B, Animal weights (shown as % of starting weight) for saline-treated (n = 8 T and 8 NT), ACR-treated (n = 11 T and 11 NT), and propionamide-treated (n = 8 T and 8 NT) groups of T (A) and NT (B) mice over the 18 d treatment period are shown. ACR-injected T and NT mice failed to gain weight after experimental day 12; however, no significant weight differences were observed between any of the groups.C, D, T (C) and NT (D) mice exposed to 2,5-HD (8 mmol · kg⁻¹ · d⁻¹;n = 8 T and 8 NT) over a 19 d treatment period exhibited a statistically significant reduction in body weight (p < 0.05) compared with either saline-exposed (n = 6 T and 6 NT) or 3,4-HD-exposed (n = 6 T and 6 NT) mice. No difference was found between saline- and 3,4-HD-treated mice. Error bars are not included for appearance and because most were within the font size of the datapoints. Statistical differences were determined using a two-way ANOVA for repeated measures. Differences in weight gain of the control animals of the ACR study versus those of the HD study were attributed to differences in the starting age of the mice (ACR, 2 months; HD, 3 months). PROP, Propionamide.

Fig. 2.

Effects of ACR and 2,5-HD on rotarod performance. Animals were tested daily for their ability to remain on a rotating, 1-inch-diameter cylinder rotating at a speed of 10 rpm for 30 sec. An animal was designated as failing if it fell off the rod on two trials. Data are illustrated as the percentage of animals tested that failed the test on each treatment day. Daily injections of 50 mg/kg ACR (A), equimolar propionamide (A), 8 mmol/kg 2,5-HD (B), or equimolar 3,4-HD (B) produced a similar rotarod failure in T and NT mice. T and NT ACR-exposed animals (n = 11 T and 11 NT) were significantly different from corresponding propionamide-injected (n = 8 T and 8 NT) and saline-injected (n = 8 T and 8 NT; data not shown) mice. Similarly, both T and NT 2,5-HD-exposed animals (n = 8 T and 8 NT) were significantly different from corresponding 3,4-HD-exposed (n = 6 T and 6 NT) and saline-exposed (n = 6 T and 6 NT; data not shown) animals. No statistically significant differences were found between any T and NT animals. For illustration purposes only, data from T and NT mice exposed to non-neurotoxic propionamide (A) or 3,4-HD (B) were combined. Survival analysis data do not allow the presentation of error bars. Statistical differences were determined using the Kaplan and Meier survival analysis with Breslow's statistic.

Fig. 3.

A, B, Accelerated rotarod performance of T (A) and NT (B) mice exposed to ACR (50 mg · kg⁻¹ · d⁻¹;n = 3 T and 3 NT), propionamide (Prop; 50 mg · kg⁻¹ · d⁻¹;n = 3 T and 3 NT), or saline (n= 1 T and 1 NT) is shown. ACR-treated animals were statistically different from saline- or propionamide-exposed animals (p < 0.05). C, D, T (C) and NT (D) mice receiving 2,5-HD (8 mmol · kg⁻¹ · d⁻¹;n = 3 T and 3 NT) were statistically different from 3,4-HD-treated (equimolar dose; n = 3 T and 3 NT) or saline-treated (n = 1 T and 1 NT) groups in the performance of the accelerated rotarod test at the preset significance (p < 0.05). No differences were found between T and NT mice for any experimental group. Statistical differences were determined using a two-way ANOVA for repeated measures.

Fig. 4.

A–C, Axons from ACR-injected (50 mg · kg⁻¹ · d⁻¹ for 18 d) T mice tibial nerves. Typical pathological lesions induced by ACR include accumulations of mitochondria, dense bodies, and other membrane-bound vesicles (arrows), vacuole formation (asterisks), and multilaminar bodies (arrowheads) of both myelinated and unmyelinated axons. Abnormal axoplasm is also found within glial compartments (C). Neurofilament accumulations were not observed. A–C, 21,500×. Scale bars, 1 μm.

Fig. 5.

A–C, Tibial nerve axons from NT mice injected daily with 50 mg/kg ACR for 18 d. Morphological lesions similar to those observed in other mammalian species were observed. These include the accumulation of mitochondria, dense bodies, and other membrane-bound vesicles (arrows), vacuole formation (asterisks), and multilaminar bodies (arrowhead). Numerous examples of neurofilament accumulations (B) were observed, along with abnormal organelles (B; arrow). A, C, 21,500×; B, 43,000×. Scale bars, 1 μm.

Fig. 6.

A–D, Pathological lesions in tibial nerves of T mice chronically injected with 2,5-HD (8 mmol · kg⁻¹ · d⁻¹) for 19 d. Typical lesions include accumulations of mitochondria, dense bodies, and other membrane-bound vesicles (arrows), as well as the presence of multilaminar bodies (arrowheads). These lesions are very similar to those observed in NT animals, except neurofilaments are absent from these nerve sections (see Fig. 7 to compare). These lesions are also similar to those observed with chronic ACR exposure (see Figs. 4, 5 to compare). A–D, 21,500×. Scale bars, 1 μm.

Fig. 7.

A–D, Pathological lesions in tibial nerves of NT mice chronically injected with 2,5-HD (8 mmol · kg⁻¹ · d⁻¹) for 19 d. Typical lesions include accumulations of mitochondria, dense bodies, and other membrane-bound vesicles (arrows), as well as the presence of multilaminar bodies (arrowheads) in axons and surrounding glia. Neurofilaments are present in all of these tibial nerve sections. A–D, 21,500×. Scale bars, 1 μm.

Fig. 8.

Quantitative comparison of frequency of pathological lesions within axons of sciatic and tibial nerves of T and NT mice injected with 50 mg · kg⁻¹ · d⁻¹ ACR or saline for 18 d. The number of axons counted in each group is provided above each bar. ACR significantly increased the frequency of lesions over controls. A significant difference was also observed between sciatic (proximal) and tibial (distal) nerves in both T and NT mice. No differences were found between T and NT mice under any experimental condition. Comparable data for 2,5-HD were unavailable because of an unresolvable difficulty in the perfusion of 2,5-HD-exposed mice. Statistical differences were determined using a two-way ANOVA for repeated measures followed by Tukey's highly significant differences post hoc test. *p < 0.05, significantly different from the corresponding saline control; #p < 0.05, significantly different from the corresponding sciatic nerve.