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. 2016 Sep;204(1):191-203.
doi: 10.1534/genetics.116.186932. Epub 2016 Jul 8.

Axonopathy in the Central Nervous System Is the Hallmark of Mice With a Novel Intragenic Null Mutation of Dystonin

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Free PMC article

Axonopathy in the Central Nervous System Is the Hallmark of Mice With a Novel Intragenic Null Mutation of Dystonin

Frauke Seehusen et al. Genetics. .
Free PMC article

Abstract

Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary sensory autonomic neuropathy. Mutated mice show severe movement disorders and die at the age of 3-4 weeks. This study describes the discovery and molecular, clinical, as well as pathological characterization of a new spontaneously occurring mutation in the dystonin gene in C57BL/6N mice. The mutation represents a 40-kb intragenic deletion allele of the dystonin gene on chromosome 1 with exactly defined deletion borders. It was demonstrated by Western blot, mass spectrometry, and immunohistology that mice with a homozygous mutation were entirely devoid of the dystonin protein. Pathomorphological lesions were restricted to the brain stem and spinal cord and consisted of swollen, argyrophilic axons and dilated myelin sheaths in the white matter and, less frequently, total chromatolysis of neurons in the gray matter. Axonal damage was detected by amyloid precursor protein and nonphosphorylated neurofilament immunohistology. Axonopathy in the central nervous system (CNS) represents the hallmark of this disease. Mice with the dystonin mutation also showed suppurative inflammation in the respiratory tract, presumably due to brain stem lesion-associated food aspiration, whereas skeletal muscles showed no pathomorphological changes. This study describes a novel mutation in the dystonin gene in mice leading to axonopathy in the CNS. In further studies, this model may provide new insights into the pathogenesis of neurodegenerative diseases and may elucidate the complex interactions of dystonin with various other cellular proteins especially in the CNS.

Keywords: axonopathy; dystonia musculorum; dystonin deficiency; genomic deletion; spontaneous mutation.

Figures

Figure 1
Figure 1
Structure of different murine dystonin isoforms. The structure of the different dystonin isoforms is shown (adapted from Goryunov et al. 2007). ABD, actin-binding domain; CC, coiled-coil domain; EF hands, EF hand-calcium binding domains; IFBD1/2, intermediate filament binding domain 1/2; MTBD, microtubule binding domain; SR, spectrin rod domain.
Figure 2
Figure 2
Clinical findings in dt-MP mice. Affected mice show a severe neuromuscular disorder beginning with a wrist flexion of the forelimbs (arrows) (A and B). The hindlimbs are splayed (B). The animals have severe difficulties in turning after being placed on their backs (C). Note that the left forelimb (arrow) is extended along the body axis instead of being used for turning over the left shoulder.
Figure 3
Figure 3
(A) Molecular identification of the deletion area. PCR on genomic DNA of control and dt-MP mice. Lanes 1 and 2 show exon 43 is amplified in control, but not in dt-MP mice. Lanes 3 and 4 show primers spanning over the deletion (from exon 39 to intron 61) do not amplify genomic DNA in control, but in dt-MP mice. Lanes 5 and 6 show dystonin nt 272159–272908 as internal control. (B) Structure of dystonin alleles. Structure of the genomic region of mouse dystonin as obtained with WebScipio (Hatje et al. 2013). Dark gray and light gray boxes represent exons and introns, respectively. Introns have been scaled down by a factor of 12.9 for clarity. ATG and TAG represent start and stop codons, respectively, of the indicated isoforms. Functional domains are illustrated in the wild-type allele on top for better orientation (ABD, actin-binding domain; IFBD, intermediate filament binding domain; MTBD, microtubule-binding domain; SR, spectrin repeats). The scheme on the bottom represents the genomic region of the dt-MP allele that misses 39,553 bp in the middle of the gene including the dystonin-e/n-specific stop codon. For nucleotide and amino acid sequences in the wild-type mouse dystonin and dt-MP alleles, see Figure S2. (C) Genotyping of the dystonin locus. A 700-bp band is amplified from the wild-type allele only. A 1200-bp band characterizes the mutant allele.
Figure 4
Figure 4
Detection of the dystonin isoforms in cDNA of different tissues. (A) In the brain stem of control (+/+) animals dystonin-a could be strongly detected, whereas dystonin-b was present very weakly. From control heart cDNA, both dystonin-a and -b were amplified with the b isoform giving the stronger band. In skin tissue, dystonin-e/n was clearly and dystonin-a weakly present. (B) In brain stem, heart, and skin of dt-MP (dt/dt) animals, neither dystonin isoform was detected. Controls in A and B: β-actin.
Figure 5
Figure 5
Western blot with the polyclonal antibody 18024 on brain stem tissue. Two high molecular weight bands of >200 kDa are detectable in tissue of a control (dt/+) animal, but missing in tissue of a dt/dt littermate. Since the antibody 18024 is directed against the plakin domain, other plakin family proteins than dystonin might be detected.
Figure 6
Figure 6
Dystonin immunohistology. Immunohistological investigation with the polyclonal anti-dystonin antibody LS-C123425/70642 on brain tissue, peripheral nerve, haired skin, and skeletal muscle of dystonin control mice (+/+) and dt-MP (dt/dt) animals. (A) Bulbus olfactorius of a control (+/+) animal with positively stained cytoplasm of neurons. Bar, 50 μm. (B) Bulbus olfactorius of a dt-MP mouse with only a weak cytoplasmic signal. Bar, 50 μm. (C) Sciatic nerve of a control (+/+) animal with positively stained nerve fibers. Bar, 20 μm. (D) Sciatic nerve of a dt-MP mouse without immunoreactivity. Bar, 20 μm. (E) Haired skin of a control (+/+) animal with positively stained keratinocytes and follicular epithelial cells. Bar, 50 μm. (F) Haired skin of a dt-MP mouse with only a weak cytoplasmic immunoreactivity of superficial keratinocytes. Bar, 50 μm. (G) Skeletal muscle of a control (+/+) animal with positively stained muscle fibers. Bar, 50 μm. (H) Skeletal muscle of a dt-MP mouse with only a multifocal weak cytoplasmic signal of myocytes. Bar, 50 μm.
Figure 7
Figure 7
Affected regions in the brain stem and spinal cord of dt-MP (dt/dt) mice. (A and B) H&E stain of murine brain and (C) thoracic spinal cord. (A) One (1), anterior pretectal nucleus; 2, retroethmoid thalamic nucleus. Bar, 1000 µm. (B) Three (3), trigeminal nuclei; 4, spinal trigeminal nucleus; 5, pontine nuclei; 6, gigantocellular reticular nucleus; 7, raphe nuclei. Bar, 1000 µm. (C) Eight (8), ventral horns; 9, dorsal root ganglia. Bar, 500 µm.
Figure 8
Figure 8
Histological and immunohistological findings showing neurodegeneration in dt-MP (dt/dt) animals. (A and B) H&E stain of the reticular formation of a dt-MP (dt/dt) mouse. Bar, 100 μm. (A) Degenerated neurons with chromatolysis (black arrows) and margination of the nucleus (white arrow). (B) Spheroids (arrows) representing swollen axons in dilated myelin sheaths. Asterisk: dilated myelin sheath with possible axonal dropout. (C) Normal staining of axons in the white matter of the reticular formation of a +/+ mouse; Bielschowsky’s silver stain. Bar, 50 μm. (D) Severe accumulation of argyrophilic material in spheroids in the reticular formation of a dt-MP (dt/dt) mouse; Bielschowsky’s silver stain (arrows). Bar, 50 μm. (E) Immunohistological staining of nonphosphorylated neurofilament (n-NF) of the cervical spinal cord in a +/+ mouse. Bar, 50 μm. (F) Marked n-NF accumulation in spheroids of the cervical spinal cord in a dt-MP (dt/dt) mouse (arrows). Bar, 50 µm. (G) Lack of axonal β-APP staining in a +/+ animal. Bar, 50 µm. (H) Immunohistological staining of β-amyloid precursor protein (β-APP) in a spheroid in a dt-MP (dt/dt) mouse (arrow). Bar, 50 μm.
Figure 9
Figure 9
Electron microscopy of the central nervous system. (A) Control animal (+/+) with numerous myelinated axons. Magnification, 8000-fold. Bar, 1000 nm. (B) Hyper- and dysmyelination of axons in the spinal cord of a dt-MP (dt/dt) mouse characterized by thickening and folding of the myelin sheath. Magnification, 8000-fold. Bar, 1000 nm. (C) Spinal cord of dt-MP (dt/dt) mouse showing reduction in axon density and spheroid formation with enlarged diameter of damaged axon and accumulation of numerous cell organelles (mitochondria, dense bodies, and tubulovesicular profiles) as well as a thin myelin sheath. Magnification, 20,000-fold. Bar, 435 nm.
Figure 10
Figure 10
Electron microscopy of the peripheral nervous system. (A) Dystonin wild type (+/+) animal with numerous myelinated axons. Magnification, 4000-fold. Bar, 2000 nm. (B) Variation of axonal diameter and reduction in axon density in the sciatic nerve of a dt-MP (dt/dt) mouse. Magnification, 4000-fold. Bar, 2000 nm. (C) Sciatic nerve of dt-MP (dt/dt) mouse showing axon with enlarged diameter (spheroid). Magnification, 16,000-fold. Bar, 500 nm.

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