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. 2014 May 15;23(10):2694-710.
doi: 10.1093/hmg/ddt663. Epub 2013 Dec 30.

Transgenic Expression of Neuronal Dystonin Isoform 2 Partially Rescues the Disease Phenotype of the Dystonia Musculorum Mouse Model of Hereditary Sensory Autonomic Neuropathy VI

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Transgenic Expression of Neuronal Dystonin Isoform 2 Partially Rescues the Disease Phenotype of the Dystonia Musculorum Mouse Model of Hereditary Sensory Autonomic Neuropathy VI

Andrew Ferrier et al. Hum Mol Genet. .
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Abstract

A newly identified lethal form of hereditary sensory and autonomic neuropathy (HSAN), designated HSAN-VI, is caused by a homozygous mutation in the bullous pemphigoid antigen 1 (BPAG1)/dystonin gene (DST). The HSAN-VI mutation impacts all major neuronal BPAG1/dystonin protein isoforms: dystonin-a1, -a2 and -a3. Homozygous mutations in the murine Dst gene cause a severe sensory neuropathy termed dystonia musculorum (dt). Phenotypically, dt mice are similar to HSAN-VI patients, manifesting progressive limb contractures, dystonia, dysautonomia and early postnatal death. To obtain a better molecular understanding of disease pathogenesis in HSAN-VI patients and the dt disorder, we generated transgenic mice expressing a myc-tagged dystonin-a2 protein under the regulation of the neuronal prion protein promoter on the dt(Tg4/Tg4) background, which is devoid of endogenous dystonin-a1 and -a2, but does express dystonin-a3. Restoring dystonin-a2 expression in the nervous system, particularly within sensory neurons, prevented the disorganization of organelle membranes and microtubule networks, attenuated the degeneration of sensory neuron subtypes and ameliorated the phenotype and increased life span in these mice. Despite these improvements, complete rescue was not observed likely because of inadequate expression of the transgene. Taken together, this study provides needed insight into the molecular basis of the dt disorder and other peripheral neuropathies including HSAN-VI.

Figures

Figure 1.
Figure 1.
Generation of the PrP-dystonin-a2 transgenic mice. (A) Schematic representation of the N-terminal regions of the major dystonin neuronal isoforms (dystonin-a1, -a2 and -a3). The actin binding domain (ABD) of both dystonin-a1 and -a2 contains a pair of calponin homology sites, while dystonin-a3 contains a single calponin homology site. The plakin domain is common to all three isoforms. The differentiating feature of all dystonin-a isoforms lies in the initial N-terminal segment. Dystonin-a2 harbors a highly conserved transmembrane (TM) domain, which aids its localization to the membrane of organelles. Dystonin-a3 contains a myristoylation motif that localizes it to the plasma membrane. The C-termini of these proteins contain a MTBD, consisting of EF hands, a Gas2-related domain and a glycine–serine–arginine rich domain (data not shown). (B) Schematic representation of the PrP-dystonin-a2 cDNA construct used to generate transgenic mice. The construct harbors full-length dystonin-a2 cDNA in frame with a myc/his tag under the regulation of a strong neuronal promoter [prion protein promoter (PrP)]. (C) Validation of PrP-dystonin-a2 transgene expression in F11 sensory neurons. Antigenic labeling of the PrP-dystonin-a2 myc/his tag (using anti-c-myc) 48 h post-transfection in F11 sensory neurons produced a perinuclear/cytoplasmic-staining pattern expected for the dystonin-a2 isoform. Cells were counterstained with DAPI to label the nuclei (scale bar = 20 μm). (D) Schematic representation of the location of oligonucleotide primers used to amplify the endogenous Dst gene and the PrP-dystonin-a2 transgene. Arrows indicate the position of primers used to amplify between exons 7 and 8 of the Dst gene. Amplification of the endogenous locus yields a 318 bp fragment due to the presence of an intron, whereas amplification of the PrP-dystonin-a2 transgene gives rise to a 220 bp product. (E) PCR screening of the F0 generation indicates five offspring positive for the PrP-dystonin-a2 transgene (220 bp). Founder lines 542 and 559 were bred and established for further analysis.
Figure 2.
Figure 2.
The PrP-dystonin-a2 transgene is expressed in neuronal tissues of transgenic lines 559 and 542. (A) Schematic representation of the location of oligonucleotide primers for the amplification of PrP-dystonin-a2 derived transcripts. Primers amplify a 62 bp fragment that includes the myc/his tag coding sequence. (B and C) RT–PCR analysis of RNA derived from P7 neuronal tissues (DRG, spinal cord and brain) of heterozygous transgenic mice from lines 559 and 542 indicates PrP-dystonin-a2 transgene expression (PrP, +RT lanes). Transgene expression was not evident in tibialis anterior (TA) muscle of either transgenic line. Controls: P7 wild-type (WT) cDNA of neuronal tissues, PrP (no RT, minus reverse transcriptase), (+) and (−) cDNA of F11 sensory neurons transfected or non-transfected with PrP-dystonin-a2 construct, respectively. Actin mRNA amplification served as a positive control for RNA.
Figure 3.
Figure 3.
Immunohistochemical staining demonstrating robust PrP-dystonin-a2 transgene expression in neuronal tissues. (A) Representative tissue sections from brain (P10) and DRGs (P10 and P58) of homozygous PrP-dystonin-a2/PrP-dystonin-a2 (PrP/PrP) transgenic mice. Immunohistochemical staining with anti-c-myc antibody produced a peri-nuclear/cytoplasmic staining pattern in P10 cortical brain tissue, while a strong peri-nuclear staining pattern was observed in P10 and P58 DRGs. As expected, DRGs from wild-type (WT) non-transgenic mice stained with anti-c-myc revealed no specific staining (scale bars = 20 μm). (B) Analysis of transgene expression in cultured DRG neurons. Again, as expected, there was no anti-c-myc staining in P10 dtTg4/Tg4 sensory neurons (left panel), whereas staining was present in large-, medium- and small-sized sensory neurons of P10 PrP-dystonin-a2 mice (right panel). Scale bar = 50 μm. Sections or cells were counterstained with DAPI to label the nuclei.
Figure 4.
Figure 4.
PrP-dystonin-a2 transgene product is expressed in DRG neurons and is organized into a perinuclear pattern. (A and B) Representative micrograph of anti-c-myc immunohistochemical labeling of P10 wild-type (WT) non-transgenic (A) and P10 PrP/PrP;dtTg4/Tg4 (B) DRG tissue sections. (C) Immunohistochemical staining with anti-c-myc antibody of P10 PrP/PrP;dtTg4/Tg4 sensory neurons in primary culture. PrP-dystonin-a2 expression is observed in the cell body in a perinuclear pattern. Images were captured by confocal microscopy. Scale bar = 20 μm.
Figure 5.
Figure 5.
PrP-dystonin-a2 transgene transcript is expressed at lower levels than the endogenous dystonin-a2 transcript in DRGs from P15 mice. (A) Amplification curves from real-time RT–qPCR for dystonin-a2 (red—wild-type; green—PrP/PrP;dtTg4/Tg4; and dtTg4/Tg4—no detectable amplification). Curves shown represent n = 3 in technical triplicates. (B) Agarose gel electrophoresis of RT–qPCR products. Lane 1 = 100 bp ladder; lanes 2–4, products from dystonin-a2 mRNA amplification reactions (736 bp product) with lane 2 = wild-type, lane 3 = dtTg4/Tg4 and lane 4 = PrP/PrP;dtTg4/Tg4; lane 5 = blank; lanes 6–8, products from actin mRNA amplification reactions (407 bp product) with lane 6 = wild-type, lane 7 = dtTg4/Tg4 and lane 8 = PrP/PrP;dtTg4/Tg4; lane 9 = no template control (dystonin-a2 primers); lane 10 = no template control (actin primers).
Figure 6.
Figure 6.
Postnatal characteristics of PrP-dystonin-a2/PrP-dystonin-a2;dtTg4/Tg4 transgenic rescue mice. The different groups analyzed were: control (PrP/PrP, n = 10), mutant (dtTg4/Tg4, n = 7) and transgenic rescue (PrP/PrP;dtTg4/Tg4, n = 10). (A) Kaplan–Meier survival curve analysis indicates a significant increase in lifespan of PrP/PrP;dtTg4/Tg4 mice (median lifespan, 55 days) when compared with dtTg4/Tg4 mice (median lifespan, 21 days) (****P < 0.0001). A significant decrease in lifespan was also observed between PrP/PrP;dtTg4/Tg4 mice and PrP/PrP mice (****P < 0.0001), indicating that although there was some rescue, it was not complete. (B) Postnatal growth curve analysis suggests that PrP/PrP;dtTg4/Tg4 mice have a progressive gain in weight between postnatal days P16 and P52, and plateau thereafter. PrP/PrP;dtTg4/Tg4 mice do not show comparable increases in weight to that of control PrP/PrP mice between P30 and P123 (**P < 0.01, ****P < 0.0001). (C) PrP/PrP;dtTg4/Tg4 mice show significant increases in weight compared with dtTg4/Tg4 mice at postnatal days P16 (**P < 0.01), P18 (**P < 0.01) and P20 (****P < 0.0001). This increase in weight at early postnatal days is comparable to PrP/PrP control mice. Further comparative weight analysis was not feasible for dtTg4/Tg4 mice as these mice die at ∼ P20. Two-way ANOVA, Bonferroni posttest, data are represented as mean ± SEM. (D) Performance analysis of PrP/PrP control, dtTg4/Tg4 and PrP/PrP;dtTg4/Tg4 mice in the pen test at various post-natal days between P23 and P123. PrP/PrP;dtTg4/Tg4 mice show a decreased latency to fall and as such are unable to effectively balance and/or grip to remain on the suspended pen compared with PrP/PrP control mice (****P < 0.0001). (E) PrP/PrP;dtTg4/Tg4 mice show improvements in balance and grip at early postnatal days (P18 and P20) compared with dtTg4/Tg4 mice (*P < 0.05). Despite this improvement, PrP/PrP control mice manifest superior balance and grip at P18 and P20 compared with PrP/PrP;dtTg4/Tg4 mice (***P < 0.001). Two-way ANOVA, Bonferroni posttest, data are represented as mean ± SEM. (F) Representative images of hind limb clasping during tail suspension, a hallmark dt phenotype. P15 dtTg4/Tg4 mice consistently displayed limb clasping (arrow), while P15 PrP/PrP;dtTg4/Tg4 mice manifested splayed hind limbs, reminiscent of wild-type or PrP/PrP control mice. Note that limb clasping was detectable in P21 PrP/PrP;dtTg4/Tg4 mice. (G) Representative examples of PrP/PrP (P20); dtTg4/Tg4 (P20) and PrP/PrP;dtTg4/Tg4 (P20) mouse paw prints used for gait analysis (red = front paws, red arrow; blue = back paws, blue arrows). Note the similar gait between P20 PrP/PrP and P20 PrP/PrP;dtTg4/Tg4 mice and the abnormal gait exhibited by P20 dtTg4/Tg4 mice.
Figure 7.
Figure 7.
The PrP-dystonin-a2 transgene confers protection in dtTg4/Tg4 sensory neurons by delaying cellular demise. (AE) Representative TUNEL labeling images of lumbar DRG tissue sections. (C) P15 dtTg4/Tg4 DRG tissue sections displayed significantly more TUNEL-positive cells (mean cell death, 37.6%) then P15 PrP/PrP;dtTg4/Tg4 (D) (mean cell death, 7.3%) and P21 PrP/PrP;dtTg4/Tg4 cells (E) (mean cell death, 18%). The decrease observed in TUNEL-positive cells for P15 PrP/PrP;dtTg4/Tg4 DRGs compared with P15 dtTg4/Tg4 was transient as P21 PrP/PrP;dtTg4/Tg4 DRGs showed an increase in cellular death. PrP/PrP control DRGs showed few TUNEL-positive cells, while DRG tissue sections that were DNase treated were replete with TUNEL-positive cells. (F) Quantification of the TUNEL analysis in DRG sections. ANOVA, post hoc Tukey, ***P < 0.001, *P < 0.05, n = 3/genotype. Scale bar = 20 μm.
Figure 8.
Figure 8.
Exogenous expression of dystonin-a2 partially rescues the number of myelinated axons in the dorsal sensory roots of dtTg4/Tg4 mice. Toluidine blue staining of transverse sections of dorsal sensory roots at P15. (A) Dorsal sensory roots from wild-type (WT) mice showing numerous myelinated axons (high magnification in A′). (B) Dorsal sensory roots from dtTg4/Tg4 mice are generally smaller than their wild-type counterparts, showing fewer axons, axons undergoing degeneration and axonal swellings (sa) (higher magnification in B′). (C) Dorsal sensory roots from PrP/PrP;dtTg4/Tg4 mice are generally larger than those from dtTg4/Tg4 mice, although they still remain smaller than wild-type dorsal roots. They display an increase in the number of myelinated axons when compared with dtTg4/Tg4 dorsal roots, but axonal swellings (sa) are still present (see higher magnification in C′). Scale bars = 50 μm (A–C) and 10 μm (A′–C′).
Figure 9.
Figure 9.
Endplates from dtTg4/Tg4 mice are poorly developed and this defect is partially rescued in PrP-dystonin-a2/PrP-dystonin-a2;dtTg4/Tg4 mice. Representative photomicrographs showing NMJ endplate morphology in TA myofibers isolated from P15 wild-type (AN), dtTg4/Tg4 (A′–N′) and PrP/PrP;dtTg4/Tg4 (A″–N″) mice. The endplates from dtTg4/Tg4 myofibers were less developed than those from wild-type (as indicated by asterisks and double asterisks). In addition to having an immature morphology, the endplates indicated by the double asterisks display neurofilament accumulation on the presynaptic side. Interestingly, both of these abnormal features were reduced in the PrP/PrP;dtTg4/Tg4 mice. Scale bar = 10 μm.
Figure 10.
Figure 10.
PrP-dystonin-a2 transgene rescues muscle spindle degeneration in dtTg4/Tg4 mice. (AC and A′–C′) Paraffin sections from the tibialis anterior muscle of P15 mice were stained with hematoxylin and eosin. (A and A′) Analysis of wild-type samples demonstrate a normal muscle spindle with its intrafusal muscle and its Ia afferent sensory nerve. (B and B′) Evidence of muscle spindle degeneration in dtTg4/Tg4 muscle at P15 (black arrow) and lack of detectable axons in the spindle (white arrow). (C and C′) Muscle spindle structure appears normal in PrP/PrP;dtTg4/Tg4 mice, with a normal encapsulated muscle spindle with its innervating sensory nerve shown. Scale bars = 10 μm. (D) Quantification of muscle spindle degeneration in tibialis anterior muscle at P15. There is a statistically significant difference between wild-type and dtTg4/Tg4 (***P < 0.001), wild-type and PrP/PrP;dtTg4/Tg4 (Prp/dt) (**P < 0.01), and between dtTg4/Tg4 and PrP/PrP;dtTg4/Tg4 (***P < 0.001) in the % of muscle spindles degenerating. (D′) Quantification of the number of muscle spindles by section of muscle. There is a statistically significant difference between wild-type and dtTg4/Tg4 (**P < 0.01). No significant difference was observed between wild-type and PrP/PrP;dtTg4/Tg4 in the average number of muscle spindles.
Figure 11.
Figure 11.
PrP-dystonin-a2 transgene imparts neuro-protection in small and medium caliber sensory neurons but not in large caliber sensory neurons. (AC) Primary DRG sensory neuron cultures were established from P5 mice of various genetic backgrounds [PrP/PrP (A), PrP/PrP;dtTg4/Tg4 (B), dtTg4/Tg4 (C)], and cultured for 5 days in vitro. Cells were challenged with serum-free media for 24 h and antigenic labeling of β-III tubulin (neuronal marker) and the apoptotic marker caspase-3 was conducted thereafter. Note the accumulation of β-III tubulin in axons of dtTg4/Tg4 sensory neurons (arrowheads in C). (D) The average number of small (soma area, 100–400 μm2) and medium caliber sensory neurons (400–700 μm2) was not significantly different between genotypes. (E) A significant increase in caspase-3 staining was observed in small- and medium-sized dtTg4/Tg4 sensory neurons compared with PrP/PrP (***P < 0.001) and PrP/PrP;dtTg4/Tg4 (**P < 0.01), indicating the transgene confers protection in these cell types. (F) The average number of large caliber sensory neurons (700–1300 μm2) is significantly different between genotypes PrP/PrP and PrP/PrP;dtTg4/Tg4 (*P < 0.05); PrP/PrP and dtTg4/Tg4 (** P < 0.01). However, there was no difference between PrP/PrP;dtTg4/Tg4 and dtTg4/Tg4. (G) A significant increase in caspase-3 staining was observed in dtTg4/Tg4 large caliber sensory neurons compared with PrP/PrP large caliber sensory neurons (*P < 0.05). No significant difference in capase-3 staining was observed between PrP/PrP and PrP/PrP;dtTg4/Tg4 large caliber sensory neurons. Statistics: ANOVA, post hoc Tukey, n = 3/genotype. Scale bar = 20 μm.
Figure 12.
Figure 12.
PrP-dystonin-a2 transgene prevents the alteration of ER and Golgi membranes. Representative electron micrographs show dilated ER and Golgi membranes (B and B′, respectively) in P5 dtTg4/Tg4 sensory neurons. Arrows depict organelles, whereas asterisks depict areas of dilation. In contrast to dtTg4/Tg4 sensory neurons, ER and Golgi membranes within PrP/PrP control (A and A′, respectively) and PrP/PrP;dtTg4/Tg4 (C and C′ respectively) sensory neurons do not display dilated membranes. (D and E) Quantitative analyses of ER and Golgi ultrastructure. ANOVA, post hoc Tukey, **P < 0.01, (5 cells examined per animal, n = 3/genotype).

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