Figure 1.
Schematic representation of domains within the dystonin protein and the relative location of mutations for some dt alleles.
The dttg4 mutant line has a 45-kb transgene insertion at the 5′ region of the gene, resulting in deletion of coding regions upstream the actin binding domain (ABD). The dtAlb allele is a spontaneous mutant line resulting in a deletion within the central region of the gene (plakin domain [PD] and intermediate filament binding domain [IFBD]). The dttm1efu (Bpag1 knock out [KO]) was generated through homologous recombination. All three dt alleles affect all known neuronal and muscle dystonin transcripts and are effectively null mutations. The Dst gene includes coding sequences for an ABD, PD, IFBD, Spectrin Repeats (S.R.), and Microtubule Binding Domain (MTBD).
Table 1.
Primary Antibodies.
Table 2.
Secondary Antibodies.
Figure 2.
The number of alpha and gamma motor neurons is reduced in dt27J L1 spinal cord.
A–B. A graphical representation showing the number of neurons per defined area of the ventral horn of the L1 spinal cord from wild type (n = 5) and severely affected dt27J (n = 5) mice at P15. There is a significant difference in the number of alpha motor neurons and gamma motor neurons between wild type and dt27J mice (**p<0.01, student t-test). C. No significant difference was observed between wild type and dt27J mice in the number of interneurons per defined area of the ventral horn of the L1 spinal cord (p>0.05, student t-test). D. The total number of motor axons is significantly reduced in dt27J mice (*p<0.05, student t-test). E. The percentage of large caliber motor axons is reduced in dt27J ventral roots by comparison to wild type ventral roots (*p<0.05, student t-test).
Figure 3.
Increase in the number of eccentric alpha motor neuron nuclei in dt27J L1 spinal cord compared to wild type L1 spinal cord.
A. Normal alpha motor neurons were observed by light microscopy after Pyronine Y staining of the ventral horn of the L1 spinal cord from a wild type mouse. B. Eccentric motor nucleus (black arrow) observed in L1 spinal cord from a dt27J mouse. Scale bar, 10 µm (A, B). C. Dot plot graph showing the percentage of eccentric alpha motor neuron nuclei in the L1 spinal cord region as observed in individual wild type (n = 5) and dt27J (n = 5) mice (**p<0.01, student t-test).
Figure 4.
Analysis of neurofilament and alpha-internexin immunostaining in the dorsal root ganglion and ventral horn of the L1 spinal cord of wild type and dt27J mice.
A. Alpha-internexin staining (green) in cell bodies within the DRG of wild type mice. RT-97 staining of phosphorylated neurofilaments (red) is limited to a few cells. B. The DRG from dt27J mice is smaller and has fewer ganglion cells per DRG compared to wild type DRGs. RT-97 staining of phosphorylated neurofilaments (red) can be readily seen within the perikarya of many ganglion cells while alpha-internexin staining (green) is also present in a few cells. Scale bar, 10 µm (A, B). C. Ventral horn region of the L1 spinal cord from a wild type mouse showing no accumulation of phosphorylated neurofilaments within the perikarya of motor neurons (white arrows). Alpha-internexin staining (green) is observed throughout the dendrites and axons of the motor neurons. D. Ventral horn region of the L1 spinal cord from a dt27J mouse showing abnormal accumulation of phosphorylated neurofilaments within the perikarya (white arrows) of motor neurons. In comparison, alpha-internexin staining (green) is observed in dendrites and axons. Scale bar, 20 µm (C, D). E–G. TUNEL assay showed no labeling in cells in the L1 spinal cords of WT and dt27J mice (F–G). DNase treated L1 spinal cords were used as a positive control (E). Scale bar 10 µm.
Figure 5.
Defects in dt27J ventral motor and dorsal sensory spinal roots.
Toluidine blue staining of transverse sections of dorsal (dr) (A–B) and ventral (vr) roots (C–D). A. Dorsal sensory root from wild type mice showing many myelinated axons. B. Dorsal sensory root from dt27J mice showing several abnormalities including fewer axons, axons undergoing degeneration, and axonal swellings (sa). The dt dorsal sensory root is also smaller than the wild type counterpart. C. Ventral motor root from wild type mice showing many myelinated axons of different calibers. D. Ventral motor root from dt27J mice showing a mixture of myelinated and amyelinated axons of different calibers. Several large and intermediate caliber amyelinated axons are detected. The dt ventral motor root is smaller than the wild type counterpart and the axons are more compacted. Scale bar, 5 µm (all panels).
Figure 6.
Ultrastructural analysis of ventral motor roots of the L1 spinal nerve of wild type and dt27J mice.
Transverse sections of ventral motor roots from P15 wild type and dt27J mice were prepared for electron microscopy. A. Ventral motor root from wild type mice showing normal myelinated axons of different calibers. B. Ventral motor root from dt27J mice showing hypomyelinated and amyelinated axons of large and intermediate caliber. Scale bar, 1 µm.
Figure 7.
Endplates from dt27J mice are poorly developed.
Representative photomicrographs showing NMJ endplate morphology in TA muscle at P15 from wild type (A–E) and dt27J (A'–E') mice. dt27J TA muscles show more immature NMJs (A'–B') when compared to wild type (A–E). The endplate morphology included two different patterns frequently observed – an elongated form with domains (D and D') and oval-like form with domains (E and E'). Scale bar, 15 µm. F. Graphical representation showing the percentage of immature endplates in TA and EDL muscles from wild type (n = 5) and dt27J (n = 5) mice at P15 (**p<0.01, student t-test).
Figure 8.
Pre-synaptic axonal swellings and reduced axonal sprouting at TA and EDL NMJs from dt27J pre-synaptic terminals.
A–L. Representative confocal images of wild type (A–C) and dt27J (D–F) TA NMJs and wild type (G–I) and dt27J (J–L) EDL NMJs. NMJs are labeled with alpha-bungarotoxin (BTX, red in the merged image), the axons with SMI-312 (green) and the synaptic vesicles with SV2 (green). Scale bar, 50 µm. dt27J pre-synaptic terminals display fewer arbors and increased bulb-like swellings (F and L) whereas wild type pre-synaptic terminals exhibit extensive axon branching with no bulb-like swellings (C and I). dt27J endplates (D and J) appear less mature (fewer perforations) in comparison to wild type littermate endplates (A and G). M. The percentage of pre-synaptic swellings at TA/EDL NMJs in wild type (n = 3) and dt27J (n = 3) mice (TA NMJ, **p<0.01; EDL NMJ, *p<0.05, student t-test). N. Quantification of axon sprouting between wild type (n = 3) and dt27J (n = 3) at TA and EDL NMJs indicates reduced axon sprouting in dt27J NMJs (TA/EDL NMJ, **p<0.01, student t-test).
Figure 9.
TA myofiber atrophy in dt27J mice at P15.
TA myofibers in cross-section were stained with hematoxylin and eosin. A. Healthy myofibers from TA wild type muscle were observed under light microscopy. B. Myofiber atrophy in TA dt27J muscle is observed. Myofibers are smaller and many more nuclei are visible. C. A graphical representation showing the TA muscle fiber area in µm2 from wild type (n = 6) and dt27J (n = 6) mice revealed muscle atrophy in dt27J mice (** p<0.01, student t-test). Scale bars (A, B), 100 µm.
Figure 10.
Motor neuron degeneration in the brainstem of dt27J mice.
Sagittal sections from mouse brainstem (trigeminal nerve of the pons) identified in (A) of P15 wild type (B) and dt27J (C) mice were stained for degenerating neurons with Fluorojade B. D. Fold change in neurodegeneration in dt27J brainstem relative to wild type littermates is depicted (** p<0.01, student t-test). Sagittal sections from mouse brainstem of P15 wild type (E) and dt27J (F) mice were antigenically labeled for mature neurons with NeuN. Alpha motor neurons are identifiable by their larger size (greater than 9 µm, arrow) relative to other neuronal cell types (arrowhead). Nuclei were counter stained with DAPI to facilitate quantification. G. Quantification of percent alpha motor neurons yielded a decrease in neuron number in the trigeminal nerve of dt27J mice relative to wild type littermates (**p<0.01, student t-test). Scale bars, 10 µm.
Table 3.
Results from behavioral tests performed on wt and dt27J mice at P15.