Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase

Abstract

To investigate the role of neurofilaments in motor neuron disease caused by superoxide dismutase (SOD1) mutations, transgenic mice expressing a amyotrophic lateral sclerosis-linked SOD1 mutant (SOD1(G37R)) were mated with transgenic mice expressing human neurofilament heavy (NF-H) subunits. Unexpectedly, expression of human NF-H transgenes increased by up to 65%, the mean lifespan of SOD1(G37R) mice. Microscopic examination corroborated the protective effect of NF-H protein against SOD1 toxicity. Although massive neurodegeneration occurred in 1-yr-old mice expressing SOD1(G37R) alone, spinal root axons and motor neurons were remarkably spared in doubly SOD1(G37R);NF-H-transgenic littermates.

Figure 1

Levels of neurofilament proteins in spinal cord and ventral roots. Coomassie stained SDS/PAGE gel of cytoskeletal-enriched preparations from the spinal cord (A) and lumbar ventral roots (B) of 4-mo-old mice. (A) The level of NF-H protein is increased by ≈twofold in the spinal cord of transgenic mice heterozygous for the NF-H⁴⁴ (lane 4) or NF-H⁴³ (lane 5) transgenes as compared with normal mice (lane 2). The NF-H overexpression caused a reduction in the levels of NF-M. Expression of SOD1^G37R did not affect the levels of neurofilament proteins in the doubly SOD1^G37R;NF-H⁴⁴-transgenic mice (lane 3). (B) The levels of neurofilament proteins were reduced markedly in ventral root axons of NF-H⁴³ mice (lane 5) in contrast to NF-H⁴⁴ mice (lane 4) or normal mice (lane 2).

Figure 2

Light micrographs of ventral roots, dorsal roots and spinal cord from NF-H-transgenic mice. Transverse sections of L5 ventral roots, dorsal roots, and spinal cord from normal mice (A, B, and C), NF-H⁴⁴ mice (D, E, and F), and NF-H⁴³ mice (G, H, and I) (12-mo-old). Mice overexpressing human NF-H⁴³ showed more prominent neurofilamentous accumulations in spinal motor neurons (I) than mice overexpressing NF-H⁴⁴ (F). The axonal atrophy in ventral and dorsal roots is also more severe in mice expressing NF-H⁴³ (G and H) than in those expressing NF-H⁴⁴ (D and E). The arrows point to neurofilament accumulations. (I, Bar = 10 μm.)

Figure 3

Expression of human NF-H and SOD1^G37R proteins in transgenic mice. (A) Immunodetection of human NF-H protein in spinal cord extracts from normal mice (lane 1), transgenic mice bearing the SOD1^G37R (lane 2), doubly SOD1^G37R;NF-H⁴⁴-transgenic mice (lane 3), and transgenic mice expressing the human NF-H⁴⁴ protein (lane 4). (B) Immunodetection of human mutant and mouse SOD1 proteins in spinal cord extracts from normal mice (lane 1), transgenic mice for SOD1^G37R(lane 2), SOD1^G37R;NF-H⁴⁴-transgenic mice (lane 3), and transgenic mice expressing the human NF-H⁴⁴ protein (lane 4).

Figure 4

Increased lifespan of SOD1^G37R-transgenic mice by NF-H overexpression. Survival curves of transgenic mice expressing SOD1^G37R alone or together with NF-H transgenes coding for the NF-H⁴³ or NF-H⁴⁴ proteins. The survival probability of transgenic mice is plotted as a function of their age in months. It is remarkable that expression of the human NF-H⁴⁴ transgene increased the mean longevity of SOD1^G37R by ≈6 mo.

Figure 5

Lessen neurodegeneration in SOD1^G37R-transgenic mice coexpressing human NF-H proteins. Light micrographs show the lumbar (L5) ventral root axons, dorsal root axons, and motor neurons from a normal mouse (A, B, and C), a singly SOD1^G37R-transgenic mouse (D, E, and F), a doubly SOD1^G37R;NF-H⁴⁴-transgenic mouse (G, H, and I), and a doubly SOD1^G37R;NF-H⁴³-transgenic mouse (J, K, and L). Neurofilamentous swellings are indicated by arrows in L. Tissues were embedded in Epon and 1 μm sections were stained with toluidine blue. (Bar = 50 μm.)