Untranslated element in neurofilament mRNA has neuropathic effect on motor neurons of transgenic mice

Abstract

Studies of experimental motor neuron degeneration attributable to expression of neurofilament light chain (NF-L) transgenes have raised the possibility that the neuropathic effects result from overexpression of NF-L mRNA, independent of NF-L protein effects (Cañete-Soler et al., 1999). The present study was undertaken to test for an RNA-mediated pathogenesis. Transgenic mice were derived using either an enhanced green fluorescent protein reporter construct or modified chimeric constructs that differ only in their 3' untranslated regions (UTRs). Motor function and spinal cord histology were normal in mice expressing the unmodified reporter transgene. In mice expressing a chimeric transgene in which sequence of NF-L 3' UTR was inserted into the 3' UTR of the reporter transgene, we observed growth retardation and reduced kinetic activity during postnatal development. Older mice developed impairment of motor function and atrophy of nerve fibers in the ventral roots. A similar but more severe phenotype was observed when the chimeric transgene contained a 36 bp c-myc insert in an mRNA destabilizing element of the NF-L sequence. Our results suggest that neuropathic effects of overexpressing NF-L can occur at the level of transgene RNA and are mediated by sequences in the NF-L 3' UTR.

Fig. 1.

Construction of transgenes with NF-L sequence in 3′ UTR. A, Schematic diagram of the 5′ UTR coding region and 3′ UTR of the EGFP/wt, EFGP/NF-L, and EGFP/NF-L/c-myc transgenes. Each transgene contains a CMV promoter (+8/+613) in the 5′ UTRs, EGFP cDNA (+613/+1408) in the coding region, and SV40 polyadenylation sequence (+1408/+1642) in the 3′ UTR. A 680 nt segment of mouse NF-L cDNA (NF-L-A680) was inserted into the proximal 3′ UTR of EGFP/NF-L, and the same sequence with a 36 nt c-myc tag (NF-L-A680/c-myc) was inserted into the proximal 3′ UTR of EGFP/NF-L/c-myc. Nucleotide numbering is from the parent pEGFP-C1 vector (Clontech). Locations of primers for RT-PCR are also shown. B, C, RT-PCR with primers A and D (B) or primers C and E (C) amplify RNA products of expected size in COS cells transfected with EFGP/wt, EGFP/NF-L, and EGFP/NF-L/c-myc transgenes. D, Anti-EGFP immunoblot of lysates from transfected COS cells showing expression of EGFP protein (29 kDa) from EFGP/wt, EGFP/NF-L, and EGFP/NF-L/c-myc constructs. E,F, Detection of anti-EGFP immunoreactivity in blood smears from an EGFP/NF-L transgenic pup (E) but not from a nontransgenic littermate (F). Scale bars, 100 μm. MWM, Molecular weight markers.

Fig. 2.

Retarded growth and impaired kinetic activity in EGFP/NF-L transgenic pups. A, Reduced size of a white EGFP/NF-L transgenic Fo pup compared with three nontransgenic agouti littermates at 18 d. B, Small EGFP/NF-L transgenic Fo pup showing awkward positioning of hindlimbs and forelimbs in front of a nontransgenic littermate at 16 d. C, Small white EGFP/NF-L transgenic F1 pup with nontransgenic agouti littermates at 18 d. D, EGFP/NF-L transgenic F1 pup with awkward extension of hindlimb and distal phalanges.

Fig. 3.

Age-dependent impairment of motor function of EGFP/NF-L transgenic mice. A, EGFP/NF-L F1 transgenic mouse at 12 months showing arched posture with hindlimb extension and widening of base. B, EGFP/NF-L Fo mouse at 18 months showing inability to elevate during locomotion. C, Abnormal limb flexion of EGFP/NF-L F1 mouse (right) compared with age-matched EGFP/wt mouse (left) during tail-suspension test. D, EGFP/NF-L F1 mouse with flexed and pronated phalanges unable to grasp rotarod surface.E, Results of rotarod performance tests of 12- to 16-month-old transgenic mice showing reduced retention times of EGFP/NF-L transgenic mice. Tests 1 and 2 were conducted at 7 d intervals.

Fig. 4.

EGFP/NF-L phenotype is not associated with altered expression of NF proteins. A–J, Serial sections of motor neurons in lumbar spinal cords of 14-month-old EGFP/NF-L mouse with age-dependent motor impairment (A,C, E, G,I) and of age-matched EGFP/wt mouse (B, D, F,H, J) immunostained with antibodies to EGFP (A, B), phosphorylated NF-H (C, D), nonphosphorylated NF-H (E, F), NF-M (G,H), and NF-L (I,J). K, Western blots of brain, spinal cord, dorsal root ganglia (DRG), and spinal motor nerve roots of EGFP/NF-L and EGFP/wt transgenic mice immunoreacted with the same anti-NF antibodies. Anti-actin immunoreactivity was used as control. Tissues were pooled from age-matched mice showing anti-EGFP immunoreactivities in motor neurons. Scale bars, 100 μm.

Fig. 5.

Atrophy of L4–L5 ventral spinal nerve roots in EGFP/NF-L transgenic mice with age-dependent motor impairment.A–D, Representative cross-section of L4–L5 ventral (A, B) and dorsal (C,D) spinal nerve roots from EGFP/NF-L (A,C) and age-matched EGFP/wt (B, D) transgenic mice showing loss of large myelinated fibers in motor roots, but not in sensory roots, of EGFP/NF-L transgenic mouse. Scale bars, 100 μm. E, Quantitation of myelinated nerve fiber distribution in L4–L5 ventral nerve roots of EGFP/NF-L transgenic mice with age-dependent motor impairment and age-matched EGFP/wt transgenic mice. Analyses were conducted on six sets of nerve roots from 12- to 16-month-old mice.

Fig. 6.

Cytoplasmic vacuolization in neurons of EGFP/NF-L/c-myc and EGFP/NF-L transgenic mice. A, Severe vacuolar changes in two lateral groups of motor neurons (arrows) and less severe changes in neurons of the medial anterior horn (arrowhead) in the lumbar spinal cord of C3 EGFP/NF-L/c-myc Fo mouse. H & E-stained section. Scale bar, 100 μm. B, Cytoplasmic vacuolar changes in motor neurons (arrows) of the cervical spinal cord of C3 EGFP/NF-L c-myc mouse, with inset showing enlargement of vacuolated neuron. H & E-stained section. Scale bar, 100 μm.C, Vacuolar changes in enteric neurons (arrow) of the myenteric plexus in the small intestine of C3 EGFP/NF-L/c-myc mouse. H & E-stained section. Scale bar, 100 μm. D, Similar cytoplasmic vacuolar changes in motor neurons (arrows) in lumbar spinal cord of 16-month-old EGFP/NF-L mouse with age-dependent impairment of motor activity and atrophy of L4–L5 motor nerve root fibers. Higher magnifications of vacuolated motor neuron is shown in the inset. H & E-stained sections. Scale bar, 100 μm.