Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene

Abstract

Mice heterozygous for the radiation-induced Sprawling (Swl) mutation display an early-onset sensory neuropathy with muscle spindle deficiency. The lack of an H reflex despite normal motor nerve function in the hindlimbs of these mutants strongly suggests defective proprioception. Immunohistochemical analyses reveal that proprioceptive sensory neurons are severely compromised in the lumbar dorsal root ganglia of newborn Swl/+ mice, whereas motor neuron numbers remain unaltered even in aged animals. We have used positional cloning to identify a nine base-pair deletion in the cytoplasmic dynein heavy chain 1 gene (Dync1h1) in this mutant. Furthermore, we demonstrate that Loa/+ mice, which have previously been shown to carry a missense point mutation in Dync1h1 that results in late-onset motor neuron loss, also present with a severe, early-onset proprioceptive sensory neuropathy. Interestingly, in contrast to the Loa mutation, the Swl mutation does not delay disease progression in a motor neuron disease mouse model overexpressing a human mutant superoxide dismutase (SOD1(G93A)) transgene. Together, we provide in vivo evidence that distinct mutations in cytoplasmic dynein can either result in a pure sensory neuropathy or in a sensory neuropathy with motor neuron involvement.

Figure 1.

Swl/+ mice display abnormal hindlimb function. Abnormal postures of Swl/+ mice while being lifted by the tail (A), during locomotion (B), and being placed on a wired structure (C). D, Motor function examined by a grip strength test shows no difference in forelimb between male Swl/+ mice and WT mice, whereas hindlimb grip strength is reduced by 50% in Swl/+ mice (*p < 0.01, Student's t test). E, Nociceptive function measured by a tail flick test is comparable between Swl/+ mice and WT mice. F, H reflex, which represents the integrity of peripheral proprioception and spinal monosynaptic pathway, is absent in Swl/+ mice, whereas it was presented in WT mice.

Figure 2.

Proprioceptive sensory neurons and muscle spindles are marked reduced in adult Swl/+ mice. A, Mature muscle spindles, stained by S46 antibody (green) and collagen IV antibody (ColgIV, red), are primarily absent in the Swl/+ hindlimbs. B, Quantitative analysis of muscle spindles demonstrates that Swl/+ mice have 88% fewer muscle spindles than those of WT mice. *p < 0.01 (Student's t test). C, Representative immunostained sections show that proprioceptive neurons (PV⁺) are more severely compromised than nociceptive neurons (CGRP⁺) in the lumbar Swl/+ DRGs. Scale bars (in A, C), 100 μm. D, Quantitative analysis indicates that the total number of DRG neurons is significantly reduced in the lumbar (L4) DRGs but is not significantly reduced in the cervical (C4) DRGs of Swl/+ mice. *p < 0.01 (Student's t test). E, Quantitative analysis of subpopulation in the L4 DRGs demonstrates that the number of proprioceptive neurons (PV⁺) is reduced by 69% in Swl/+ mice (*p < 0.01, Student's t test), whereas only a 31% reduction of the number of nociceptive neurons (CGRP⁺) is detected in Swl/+ mice (p > 0.05, Student's t test).

Figure 3.

Swl/+ mice exhibit lumbar proprioceptive DRG neuron and muscle spindle degeneration during development. A, Immunofluorescence stainings of developing (Egr3⁺) and mature (S46⁺) muscle spindles show that the gross morphology of muscle spindle during different stages is comparable between Swl/+ mice and WT littermate mice. Scale bar, 50 μm. B, Quantitative analysis of muscle spindle indicates that the spindle count in the Swl/+ hindlimbs is comparable with those in WT hindlimbs at E15.5 (p > 0.05, Student's t test). However, at P0.5 and P91, the number of muscle spindles is reduced by 87 and 88%, respectively, in Swl/+ mice compared with those in WT mice. *p < 0.01 (Student's t test). C, Quantitative analysis of L4 PV⁺ DRG neurons reveals no significant change in the number of the lumbar proprioceptive DRG neurons in Swl/+ mice at E15.5 (p > 0.05, Student's t test) but a 69% reduction in the number of Swl/+ lumbar proprioceptive DRG neurons at both P0.5 and P91 in Swl/+ mice compared with those of WT mice. *p < 0.05 (Student's t test).

Figure 4.

Positional cloning of the Swl mutation identifies a 9 bp deletion in Dync1h1. A, Genetic linkage mapping defined D12Mit123 as the proximal flanking polymorphic marker by a recombinant frequency of 0.86% (7 of 816) and D12Mit20 as the distal flanking polymorphic marker by a recombinant frequency of 1.84% (15 of 816) based on the analysis of 816 backcross progeny. Filled and open boxes indicate the presence and absence, respectively, of the C57BL/6J allele. B, Region of mouse chromosome 12 surrounding the critical interval. Distance between markers is drawn to scale of physical distance. Among 82 known genes and expressed sequence tags within the 4.6 Mb critical interval, Dync1h1 was selected as a candidate gene for the Swl mutation. C, Genomic structure of Dync1h1. Position of 9 bp deletion in Swl mutants is indicated. D, Chromatogram shows the 9 bp deletion in exon 12 of Dync1h1 in homozygous and heterozygous Swl mice.

Figure 5.

Expression of Dync1h1 is not altered in Swl mice. A, Graphical illustration of the domain structure of the DYNC1H1 protein, adapted from the publication of Hafezparast et al. (2006), shows the relative position of the Swl, Loa, and Cra1 mutations. The cargo-binding domain and motor domain of DYNC1H1 are shown by the black box and the gray box, respectively. HC, Heavy chain binding site; IC, intermediate chain binding site; LIC, light intermediate chain binding site. B, RT-PCR analysis indicates that Dync1h1 mRNA expression is not altered in Swl/Swl mutants (E7.5) or Swl/+ mutants (E7.5 and P91) compared with WT (+/+) animals. Cyclophilin is amplified separately as a positive control. C, No differences in the level or localization of DYNC1H1 is detected by using immunofluorescence staining of dynein heavy chain antibody (R-325) in E7.5 Swl/Swl embryos compared with E7.5 WT (+/+) embryos. Scale bar, 100 μm.

Figure 6.

Proprioceptive sensory deficit is a common feature in Loa and Swl Dync1h1 mutations. A, Hindlimb grip strength is reduced by 52% in 13-week-old male Loa/+ mice, whereas no difference is detected in their forelimbs. *p < 0.01 (Student's t test). B, Quantitative analysis of muscle spindles demonstrates that Loa/+ mice have 86% fewer muscle spindles than WT mice. *p < 0.01 (Student's t test). C, Quantitative analysis of DRG neurons indicates that the number of DRG neurons is significantly reduced in L4 segment but not in C4 segment of Loa/+ mice. *p < 0.01 (Student's t test). D, In contrast to the ventral root (VR), the dorsal root (DR) and the sciatic nerve (SN) are markedly thinner in Swl/+ and Loa/+ mice compared with the WT littermate mice. Scale bar, 100 μm. E, The H reflex is absent in Loa/+ mice (n = 4) but present in three of four WT littermates.

Figure 7.

The Swl Dync1h1 mutation does not delay disease progression in SOD1^G93A mice. A, C, E, Swl/+ × SOD1^G93A hemizygote; B, D, F, Loa/+ × SOD1^G93A hemizygote. The Kaplan–Meier survival analysis demonstrates that Swl/SOD1^G93A mice have a comparable average survival time with that of SOD1^G93A/+ mice (A), whereas the average survival time of Loa/SOD1^G93A mice is significantly delayed compared with that of SOD1^G93A/+ mice (B; p < 0.01, log-rank test for equality of survival curves). In contrast to Swl/SOD1^G93A mice (C), the body weight loss of Loa/SOD1^G93A mice is significantly delayed compared with that of SOD1^G93A/+ mice during the observation period (D; p < 0.01, Student's t test). Similarly, the motor function determined by forelimb grip strength test shows no significant difference between Swl/SOD1^G93A mice and SOD1^G93A/+ mice (E), whereas Loa/SOD1^G93A mice have a significant delay in motor dysfunction compared with that of SOD1^G93A/+ mice during the observation period (F; p < 0.01, Student's t test).