Argonaute 2 is lost from neuromuscular junctions affected with amyotrophic lateral sclerosis in SOD1G93A mice.

miRNAs are necessary for neuromuscular junction (NMJ) health; however, little is known about the proteins required for their activity in this regard. We examined expression of Argonaute 2 (Ago2) and miRNA biogenesis genes in skeletal muscles during development, following nerve injury and in the SOD1G93A ALS mouse model. We found that these genes are enriched in neonate muscles and in adult muscles following nerve injury. Despite widespread NMJ deterioration, these genes were not increased in muscles of SOD1G93A mice. We also found that Ago2 distribution is linked to maturation, innervation, and health of NMJs. Ago2 increasingly concentrates in synaptic regions during NMJ maturation, disperses following experimental denervation and reconcentrates at the NMJ upon reinnervation. Similar to experimentally denervated muscles, a homogenous distribution of Ago2 was observed in SOD1G93A muscle fibers. To determine if Ago2 is necessary for the health of adult muscles, we excised Ago2 from Ago2fl/fl mice using adeno-associated virus mediated Cre recombinase expression. We observed modest changes in muscle histology after 3 months of Ago2 knockdown. Together, these data provide critical insights into the role of Ago2 and miRNA biogenesis genes in healthy and ALS-afflicted skeletal muscles and NMJs.

Hydrophobic and Hydrogel-Based Methods for Passive Tissue Clearing.

Optical tissue clearing enables the precise imaging of cellular and subcellular structures in whole organs and tissues without the need for physical tissue sectioning. By combining tissue clearing with confocal or lightsheet microscopy, 3D images can be generated of entire specimens for visualization and large-scale data analysis. Here we demonstrate two different passive tissue clearing techniques that are compatible with immunofluorescent staining and lightsheet microscopy: PACT, an aqueous hydrogel-based clearing method, and iDISCO+, an organic solvent-based clearing method.

KLF15 overexpression in myocytes fails to ameliorate ALS-related pathology or extend the lifespan of SOD1G93A mice.

Amyotrophic Lateral Sclerosis (ALS) is a currently incurable disease that causes progressive motor neuron loss, paralysis and death. Skeletal muscle pathology occurs early during the course of ALS. It is characterized by impaired mitochondrial biogenesis, metabolic dysfunction and deterioration of the neuromuscular junction (NMJ), the synapse through which motor neurons communicate with muscles. Therefore, a better understanding of the molecules that underlie this pathology may lead to therapies that slow motor neuron loss and delay ALS progression. Kruppel Like Factor 15 (KLF15) has been identified as a transcription factor that activates alternative metabolic pathways and NMJ maintenance factors, including Fibroblast Growth Factor Binding Protein 1 (FGFBP1), in skeletal myocytes. In this capacity, KLF15 has been shown to play a protective role in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), however its role in ALS has not been evaluated. Here, we examined whether muscle-specific KLF15 overexpression promotes the health of skeletal muscles and NMJs in the SOD1G93A ALS mouse model. We show that muscle-specific KLF15 overexpression did not elicit a significant beneficial effect on skeletal muscle atrophy, NMJ health, motor function, or survival in SOD1G93A ALS mice. Our findings suggest that, unlike in mouse models of DMD and SMA, KLF15 overexpression has a minimal impact on ALS disease progression in SOD1G93A mice.

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss.

The muscular dystrophy X-linked mouse (mdx) is the most commonly used preclinical model for Duchenne muscular dystrophy. Although disease progression in the mouse does not perfectly model the human disease, it shares many pathological features. Early characterizations of the model reported severe pathology through early adulthood followed by disease stabilization. As a result, research in the mdx mouse has largely focused on early adulthood. The overarching goal of this study is to improve the understanding of the mdx mouse model by tracking pathological features of the disease throughout life. We performed a thorough characterization of myofiber pathology in mdx mice from 2 weeks to 2 years of age. We report that individual mdx muscle fibers undergo progressive hypertrophy that continues through the lifespan. Despite massive hypertrophy on the myofiber level, we report no hypertrophy on the muscle level. These seemingly contradictory findings are explained by previously underappreciated myofiber loss in mdx mice. We conclude that due to myofiber loss, in combination with the progressive nature of other pathological features, aged mdx muscle tissue provides reliable benchmarks for disease progression that may be valuable in testing the efficacy of therapeutics for Duchenne muscular dystrophy.

Exclusive vital labeling of myonuclei for studying myonuclear arrangement in mouse skeletal muscle tissue.

BACKGROUND: The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health, but there is a dearth of in vivo exploration of potential effects of myonuclear organization on the function and regeneration of skeletal muscle because traditional nuclear stains are performed on postmortem tissue. Therefore, we sought a transgenic method to produce a selective and persistent myonuclear label in whole muscles of living mice. METHODS: We bred together a mouse line with skeletal muscle fiber-selective expression of Cre recombinase and a second mouse line with a Cre-inducible fluorescently tagged histone protein to generate a mouse line that produces a myonuclear label suitable for vital imaging and histology of fixed tissue. We tested the effectiveness of this vital label in three conditions known to generate abnormal myonuclear positioning. First, we injured myofibers of young mice with cardiotoxin. Second, this nuclear label was bred into a murine model of Duchenne muscular dystrophy. Finally, we examined old mice from this line that have undergone the natural aging process. Welch's t test was used to compare wild type and transgenic mice. RESULTS: The resulting mouse line transgenically produces a vital red fluorescent label of myonuclei, which facilitates their in vivo imaging in skeletal muscle tissue. Transgenic fluorescent labeling of myonuclei has no significant effect on skeletal muscle function, as determined by twitch and tetanic force recordings. In each muscle examined, including those under damaged, dystrophic, and aged conditions, the labeled myonuclei exhibit morphology consistent with established literature, and reveal a specialized arrangement of subsynaptic myonuclei at the neuromuscular junction. CONCLUSIONS: Taken together, our results demonstrate that this mouse line provides a versatile tool to selectively visualize myonuclei within both living and fixed preparations of healthy, injured, diseased, and aged muscles.