Sensory-motor circuit is a therapeutic target for dystonia musculorum mice, a model of hereditary sensory and autonomic neuropathy 6

Abstract

Mutations in Dystonin (DST), which encodes cytoskeletal linker proteins, cause hereditary sensory and autonomic neuropathy 6 (HSAN-VI) in humans and the dystonia musculorum (dt) phenotype in mice; however, the neuronal circuit underlying the HSAN-VI and dt phenotype is unresolved. dt mice exhibit dystonic movements accompanied by the simultaneous contraction of agonist and antagonist muscles and postnatal lethality. Here, we identified the sensory-motor circuit as a major causative neural circuit using a gene trap system that enables neural circuit-selective inactivation and restoration of Dst by Cre-mediated recombination. Sensory neuron-selective Dst deletion led to motor impairment, degeneration of proprioceptive sensory neurons, and disruption of the sensory-motor circuit. Restoration of Dst expression in sensory neurons using Cre driver mice or a single postnatal injection of Cre-expressing adeno-associated virus ameliorated sensory degeneration and improved abnormal movements. These findings demonstrate that the sensory-motor circuit is involved in the movement disorders in dt mice and that the sensory circuit is a therapeutic target for HSAN-VI.

Fig. 1.. Wnt1-lineage selective deletion and restoration of Dst expression.

(A) The multipurpose gene trap cassette locates in the intron of the Dst locus. The gene trap cassette contains splice acceptor (SA) sequence, the reporter gene βgeo, and poly-A (pA) termination signal. In this FLEX system, the gene trap cassette is flanked by pairs of inversely oriented target sites of FLP recombinase (Frt and F3: half circles) and Cre recombinase (loxP and lox5171: triangles). FLP- or Cre-mediated recombination irreversibly switches mutant Dst^Gt allele to functional Dst^Gt-inv allele and functional Dst^Gt-inv allele to mutant Dst^Gt-DO allele. (B) Mating scheme to generate Wnt1-Cre;Dst cGT and Wnt1-Cre;Dst cRescue mice. (C) X-gal staining visualized gene trap cassette inversions in 1-month-old Wnt1-Cre;Dst^Gt-inv/wt mice. βGeo (blue) was expressed in dorsal root ganglion (DRG) and sympathetic ganglion (SG), while the spinal cord (SC) lacked βgeo expression. In the brain, βgeo was expressed in the cerebellum (CB), pontine nucleus (Pn), and midbrain, while scarcely detected in the cerebral cortex (Ctx), diencephalon (Die), and medulla oblongata (MO). The dotted line indicates the edge of the SC. Boxed area is enlarged to show the DRG. DCN, deep cerebellar nucleus; SN, substantia nigra. (D) In situ hybridization in each group at 3 to 4 weeks of age. Dst mRNA was lost in DRG neurons of Wnt1-Cre;Dst cGT mice but restored in DRG neurons of Wnt1-Cre;Dst cRescue mice. (E) The number of Dst-positive neurons in the DRG [Ctrl (n = 8 mice); Wnt1-Cre;Dst cGT (n = 3); Wnt1-Cre;Dst cRescue (n = 6)]. (F) Western blotting for Dst and β-actin (Actb) in the DRG and SC of 2-week-old mice. (G) Quantification of Dst levels normalized to Actb (n = 3 mice). Scale bars, 200 μm [(C) and (D)]. Data are presented as mean ± SE. P > 0.05 [not significant (ns)] and ***P < 0.005, using analysis of variance (ANOVA) with Tukey’s test in (E) and (G). WT, wild type.

Fig. 2.. Phenotypic characterization of Wnt1-Cre;Dst cGT mice and Wnt1-Cre;Dst cRescue mice.

(A) Survival curve indicates a shorter life span of Wnt1-Cre;Dst cGT mice (yellow dashed line, n = 18 mice) than Ctrl mice (black solid line, n = 36). Wnt1-Cre;Dst cRescue mice (blue solid line, n = 19) showed a longer life span than Dst GT mice (red dotted line, n = 16). (B) Body weight (grams) of male mice at 3 weeks of age [Ctrl (n = 23 mice); Wnt1-Cre;Dst cGT (n = 7); Dst GT (n = 16); Wnt1-Cre;Dst cRescue (n = 9)]. Wnt1-Cre;Dst cGT mice showed significant decrease in body weight compared with Ctrl mice. Body weight of Wnt1-Cre;Dst cRescue mice was also light compared with Ctrl mice and not significantly different from Dst GT mice. (C) In the rotarod test, latency to fall (seconds) was measured to assess motor performance in each group at 3 to 4 weeks of age [Ctrl (n = 28 mice); Wnt1-Cre;Dst cGT (n = 8); Dst GT (n = 4); Wnt1-Cre;Dst cRescue (n = 16)]. The motor performance of Wnt1-Cre;Dst cGT mice was more impaired than Ctrl mice. The motor performance of Wnt1-Cre;Dst cRescue mice was improved than Dst GT mice. (D) Tail suspension test in each mouse. Ctrl mice maintained a normal posture during tail suspension. Wnt1-Cre;Dst cGT mice displayed abnormal postures such as hyperextended and clasped hindlimbs and truncal twists similar to Dst GT mice. On the other hand, Wnt1-Cre;Dst cRescue mice maintained a normal posture similarly to Ctrl mice. Data are presented as mean ± SE. P > 0.05 (ns), *P < 0.05, and ***P < 0.005, using ANOVA with Tukey’s test in (B) and (C).

Fig. 3.. EMG analysis of simultaneous contractions in Wnt1-Cre;Dst cGT mice and Wnt1-Cre;Dst cRescue mice.

(A) Rectified EMG of the triceps (blue) and biceps (magenta) brachii muscles in Ctrl, Wnt1-Cre;Dst cGT, Dst GT, and Wnt1-Cre;Dst cRescue mice at 6 to 12 weeks of age. Co-contraction between the triceps and biceps brachii muscles (represented by black vertical lines based on Co_Tri) was frequently observed in Dst GT mice (frequency of co-contraction = 61.0%), less frequently in Wnt1-Cre;Dst cGT (39.0%) and Wnt1-Cre;Dst cRescue mice (33.5%), and least frequently in Ctrl mice (26.5%). (B) Cross-correlograms between triceps and biceps brachii muscle activity were calculated from rectified EMG of Ctrl, Wnt1-Cre;Dst cGT, Dst GT, and Wnt1-Cre;Dst cRescue mice. Synchronization between triceps and biceps brachii muscle activity was observed as a hump at around 0 ms in Dst GT mice but not in other mice.

Fig. 4.. Histological analysis of sensory-motor circuit in Wnt1-Cre;Dst cGT and Wnt1-Cre;Dst cRescue mice.

(A) In Dst GT and Wnt1-Cre;Dst cGT mice, ATF3 was expressed in some DRG neurons together with neurofilament (NF) accumulation (arrowheads) at 3 to 4 weeks of age. Increases in ATF3 and NF were rarely observed in Wnt1-Cre;Dst cRescue mice. The dotted line indicates the edge of the DRG. (B) VGluT1-positive axon terminals were observed in the lumbar SC. In the anterior horn (AH), ChAT-positive motor neurons were surrounded by VGluT1-positive axon terminals. There were fewer VGluT1-positive axon terminals in the AH of Dst GT and Wnt1-Cre;Dst cGT mice than in Ctrl mice, but they were normally distributed in Wnt1-Cre;Dst cRescue mice. The dotted line indicates the edge of the SC. (C) Quantitative data showed a significant increase in numbers of ATF3-positive cells and NF-accumulating cells in Dst GT and Wnt1-Cre;Dst cGT mice [ATF3, Ctrl (n = 5 mice); Wnt1-Cre;Dst cGT (n = 3); Dst GT (n = 5); Wnt1-Cre;Dst cRescue (n = 6); NF, Ctrl (n = 6); Wnt1-Cre;Dst cGT (n = 3); Dst GT (n = 4); Wnt1-Cre;Dst cRescue (n = 3)]. Quantitative data showed a significant decrease in number of VGluT1-positive axon terminals in the AH of Wnt1-Cre;Dst cGT mice. VGluT1-positive axon terminals were significantly increased in Wnt1-Cre;Dst cRescue mice than Dst GT mice [Ctrl (n = 12); Wnt1-Cre;Dst cGT (n = 3); Dst GT (n = 6); Wnt1-Cre;Dst cRescue (n = 5)]. The number of ChAT-positive cells was statistically same between groups [Ctrl (n = 8); Wnt1-Cre;Dst cGT (n = 3); Dst GT (n = 5); Wnt1-Cre;Dst cRescue (n = 5)]. Scale bars, 50 μm (A) and 200 μm (B). Data are presented as mean ± SE. P > 0.05 (ns), *P < 0.05, **P < 0.01, and ***P < 0.005, using ANOVA with Tukey’s test in (C).

Fig. 5.. Functional analysis of the sensory-motor circuit in Wnt1-Cre;Dst cGT and Wnt1-Cre;Dst cRescue mice.

(A) A diagram of the H-reflex mediated by proprioceptive sensory neurons and motor neurons. The M response was evoked by electronic stimulation of motor fibers in the tibial nerve of mice. The H-reflex is transmitted via Ia afferent fibers derived from proprioceptive neurons. (B) Representative EMG images of M response and H-reflex. In Ctrl mice, electrical stimulation (Sti) evoked M response and H-reflex sequentially (black solid line). In Wnt1-Cre;Dst cGT mice, H-reflex was attenuated and delayed (yellow dashed line). In Wnt1-Cre;Dst cRescue mice, stimulation normally induced H-reflex (blue solid line), whereas it was attenuated and delayed in Dst GT mice (red dashed line). (C) Quantitative data of H/M amplitude ratio, H-reflex latency (milliseconds), and M response latency (milliseconds) in each mouse group at 3 to 4 weeks of age. H/M amplitude ratio was significantly decreased in Dst GT mice and Wnt1-Cre;Dst cGT mice compared to Ctrl mice, whereas Wnt1-Cre;Dst cRescue mice had a normal level of H/M amplitude ratio as in Ctrl mice [H/M amplitude ratio and H-reflex latency, Ctrl (n = 29); Wnt1-Cre;Dst cGT (n = 6); Dst GT (n = 7); Wnt1-Cre;Dst cRescue (n = 6); sample sizes are numbers of successful evocation of H-reflex]. The latencies of H-reflex and M response in Dst Gt mice and Wnt1-Cre;Dst cGT mice were significantly longer than in Ctrl mice, while latencies of both responses in Wnt1-Cre;Dst cRescue were statistically same as in Ctrl mice [M response latency, Ctrl (n = 32); Wnt1-Cre;Dst cGT (n = 11); Dst GT (n = 12); Wnt1-Cre;Dst cRescue (n = 6); sample sizes are numbers of measurement of M response]. Data are presented as mean ± SE. P > 0.05 (ns) and ***P < 0.005, using ANOVA with Tukey’s test in (C).

Fig. 6.. Manipulation of Dst expression separately in sensory neurons and cerebellar circuits.

(A) During tail suspension, Avil-Cre;Dst cGT mice showed hindlimb clasping at 3 to 4 weeks of age, while Avil-Cre;Dst cRescue mice maintained normal postures. En1-Cre;Dst cGT and En1-Cre;Dst cRescue mice neither developed nor rescued abnormal postures, respectively. (B) In a rotarod test, Avil-Cre;Dst cGT mice displayed shorter latency to fall (seconds) than Ctrl. Avil-Cre;Dst cRescue mice performed better than Dst GT mice [Ctrl (n = 14 mice); Avil-Cre;Dst cGT (n = 4); Dst GT (n = 4); Avil-Cre;Dst cRescue (n = 8)]. En1-Cre;Dst cGT and En1-Cre;Dst cRescue mice showed no differences compared to Ctrl and Dst GT mice, respectively [Ctrl (n = 6); En1-Cre;Dst cGT (n = 7); Dst GT (n = 4); En1-Cre;Dst cRescue (n = 3)]. (C) In the lumbar SC of each group at 3 months old, VGluT1-labeled terminals were decreased in Avil-Cre;Dst cGT mice than Ctrl but normal in Avil-Cre;Dst cRescue mice. (D) Quantitative data on numbers of VGluT1-positive terminals [Ctrl (n = 6 mice); Avil-Cre;Dst cGT (n = 3); Avil-Cre;Dst cRescue (n = 3)]. (E) Quantification of electrophysiological data in mice at 3 months of age. H/M amplitude ratio was reduced in Avil-Cre;Dst cGT mice than Ctrl but normal in Avil-Cre;Dst cRescue mice [H/M amplitude ratio and H-reflex latency, Ctrl (n = 12); Avil-Cre;Dst cGT (n = 5); Avil-Cre;Dst cRescue (n = 8); sample sizes are numbers of successful evocation of H-reflex]. Avil-Cre;Dst cRescue mice show delayed M response and H-reflex [M response latency, Ctrl (n = 13); Avil-Cre;Dst cGT (n = 12); Avil-Cre;Dst cRescue (n = 8); sample sizes are numbers of measurement of M response]. Scale bars, 100 μm (C). Data are presented as mean ± SE. P > 0.05 (ns), *P < 0.05, **P < 0.01, and ***P < 0.005, using ANOVA with Tukey’s test in (B), (D), and (E).

Fig. 7.. Presymptomatic restoration of Dst expression using viral vectors rescued disease phenotypes.

(A) Intraperitoneal (ip) injection of AAV9-Cre or AAV9-GFP into neonatal Dst^Gt/Gt mice. (B) A reconstructed transparent SC image from AAV9-GFP–injected wild-type mice at 2 months of age. GFP labeled the DRG and sensory tract in the dorsal funiculus (DF, arrow). The dotted line indicates the edge of DRG. (C) GFP-labeled axons are distributed in the dorsal horn (DH). In the AH, axons of proprioceptive neurons were also labeled with GFP. (D) The survival curve indicates a life extension of AAV9-Cre–treated Dst^Gt/Gt mice (blue solid line, n = 8 mice) compared with Dst^Gt/Gt mice (red dashed line, n = 12). (E) AAV9-GFP–treated Dst^Gt/Gt mice exhibited twist movements with hyperextension of forelimbs and hindlimbs (arrowheads). AAV9-Cre–treated Dst^Gt/Gt mice showed normal posture 3 weeks after injection. (F) Motor performance was assessed by counting number of footfalls on the horizontal ladder floor. Impaired motor performance of Dst^Gt/Gt mice was represented with many footfalls compared with Ctrl mice. AAV9-Cre treatment significantly improved motor performance of Dst^Gt/Gt mice at 3 weeks of age while not reaching the Ctrl level [Ctrl (n = 3 mice); Dst^Gt/Gt (n = 3); Dst^Gt/Gt + AAV9-Cre (n = 4)]. (G) In situ hybridization image in the DRG from each group at 3 weeks of age. Dst mRNA was detected in DRG neurons of Dst^Gt/Gt mice injected with AAV9-Cre, but Dst mRNA was diminished in DRG neurons of Dst^Gt/Gt mice injected with AAV9-GFP. (H) Quantitative data on numbers of Dst-positive neurons in the DRG [Ctrl (n = 3 mice); Dst^Gt/Gt (n = 4); Dst^Gt/Gt + AAV9-Cre (n = 5)]. Scale bars, 500 μm (B), 50 μm (C), and 100 μm (G). Data are presented as mean ± SE. P > 0.05 (ns), *P < 0.05, and ***P < 0.005, using ANOVA with Tukey’s test in (F) and (H).

Fig. 8.. Presymptomatic treatment with AAV rescued the sensory-motor circuit of Dst^Gt mice.

(A) Histological analysis of neurodegeneration in each group at 3 weeks of age. In DRG of Dst^Gt/Gt mice injected with AAV9-GFP, ATF3 and NF accumulation was abundantly observed, and there were few VGluT1-positive axon terminals around motor neurons in the AH of lumbar SC. In DRG of Dst^Gt/Gt mice injected with AAV9-Cre, ATF3 and NF accumulation was scarcely observed, and VGluT1-positive axon terminals were abundant. PV-positive proprioceptive neurons were few in the DRG of Dst^Gt/Gt mice injected with AAV9-GFP while normally observed in Dst^Gt/Gt mice injected with AAV9-Cre. (B) Quantification of numbers of ATF3-positive cells [Ctrl (n = 4 mice); Dst^Gt/Gt (n = 4); Dst^Gt/Gt + AAV9-Cre (n = 5)], NF-accumulating cells [Ctrl (n = 3); Dst^Gt/Gt (n = 3); Dst^Gt/Gt + AAV9-Cre (n = 5)], PV-positive cells [Ctrl (n = 4); Dst^Gt/Gt (n = 5); Dst^Gt/Gt + AAV9-Cre (n = 5)], and VGluT1-positive axon terminals [Ctrl (n = 3); Dst^Gt/Gt (n = 3); Dst^Gt/Gt + AAV9-Cre (n = 5)]. (C) Quantification of electrophysiological data in mice at 2 to 3 weeks of age. H/M amplitude ratio and H-reflex latency in Dst^Gt/Gt mice were rescued by injection with AAV9-Cre. [H/M amplitude ratio and H-reflex latency, Ctrl (n = 8); Dst^Gt/Gt (n = 9); Dst^Gt/Gt + AAV9-Cre (n = 4); sample sizes are numbers of successful evocation of H-reflex; M response latency, n = 10 Ctrl; n = 16 Dst^Gt/Gt; n = 6 Dst^Gt/Gt + AAV9-Cre; sample sizes are numbers of measurement of M response]. Scale bars, 50 μm (A). Data are presented as mean ± SE. P > 0.05 (ns), *P < 0.05, and ***P < 0.005, using ANOVA with Tukey’s test in (B) and (C).