Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury

Minor delay in progression of glycolytic type 2x fiber switching in TA of Myf5/<i>Fktn</i> KO mice normalizes by 8 weeks.

<p>Glycolytic intermediate-twitch 2x fibers in Myf5/<i>Fktn</i>-deficient (KO) and control (LC) (A) iliopsoas and (B) TA muscles. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with antibody detecting all myosin isoforms except type 2x (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Unstained (negative) fibers were counted to measure type 2x. Scale bar = 100μm. n = 4 for all 2 and 4 wko measurements (except Ilio 4wko LC, n = 3); n = 5 per 8 wko group.</p

Frequencies of type 2a oxidative and type 2b glycolytic fast-twitch fibers are unchanged in iliopsoas and TA between Myf5/<i>Fktn</i> KO and LC mice.

<p>(A) Oxidative type 2a fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. (B) Glycolytic type 2b fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn-</i>deficient (KO) and control (LC) mice. *, p<0.05; two-way ANOVA with Bonferroni’s post-test. n = 4 for all 2 and 4 wko experimental groups (except Ilio 4 wko LC, n = 3); n = 5 per 8 wko group.</p

Both presynaptic and postsynaptic components are present at neuromuscular junctions in 14 d regenerated muscle of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs.

<p>(A) Proportion of NMJs positive for synaptophysin and BGTX in saline- or CTX-injected TA of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs or grouped Myf5/Tam LCs. (B) Representative images showing localization of NMJ pre-synaptic (synaptophysin, red) and post-synaptic markers (BGTX, green) in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> KO and LC TAs injected with saline or CTX. Nuclei stained with DAPI (blue). Scale bar = 100μm. n = 7, LC; n = 4, Myf5 KO; n = 6 Tam KO.</p

Slow oxidative fibers are decreased in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> LC mice following CTX injection.

<p>(A) Quantification of slow type 1 oxidative fibers in TA muscle of saline- or CTX-injected Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> LC or KO mice. *, p<0.05; **, p<0.01; ***, p < .001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (B) Whole tissue maps of CTX-injected TA muscle stained with anti-myosin heavy chain type 1 antibody (red), with sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Frequency of type 1 oxidative fibers decreases with age in iliopsoas and TA.

<p>Oxidative type 1 fibers in (A) iliopsoas and (B) TA of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with anti-myosin heavy chain type 1 antibody (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 4 for all 2 and 4 wko measurements (except Ilio LC, n = 3); n = 5 for all 8 wko measurements.</p

Glycolytic type 2x, but not type 2b, fibers decrease following muscle regeneration.

<p>Glycolytic type 2b (A) or type 2x (B) fiber proportions in muscle-specific (Myf5) or whole-body (Tam) <i>Fktn</i> KO and LC muscle injected with saline or CTX. *, p<0.05; ***, p<0.001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (C) Whole tissue maps of TA muscle from Myf5/<i>Fktn</i> or Tam/<i>Fktn</i> KO and LC muscle injected with saline or CTX and stained with an antibody detecting all myosin heavy chain isoforms except type 2x. Unstained (negative) fibers were counted to measure type 2x. Scale bar = 100 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Fast oxidative fibers are increased in Tam/<i>Fktn</i> KO mice following CTX injection.

<p>(A) Quantification of fast type 2a oxidative fibers in TA muscle of saline- or CTX-injected Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> LC or KO mice. *, p<0.05; **, p<0.01; ***, p < .001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (B) Whole tissue maps of CTX-injected TA muscle stained with anti-myosin heavy chain type 2a antibody (red), with sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 200 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism

Background: Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. Methods: In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant (Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. Results: Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Conclusions: Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery. Electronic supplementary material The online version of this article (doi:10.1186/s13229-017-0142-z) contains supplementary material, which is available to authorized users

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism

Background: Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. Methods: In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant (Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. Results: Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Conclusions: Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery. Electronic supplementary material The online version of this article (doi:10.1186/s13229-017-0142-z) contains supplementary material, which is available to authorized users