The effective treatment or cure of motoneuron disease will require understanding the disease processes that precede irreversible cell loss. To study these early stages, and to evaluate potential treatments in relevant animal models, requires a sensitive functional assay. To this end, we sought to determine whether the gait pattern of SOD1 transgenic mice changed prior to overt symptoms. Using a simplified video-based approach we compared the treadmill gait of C57BL/6J and B6.SOD1 transgenic mice at 8 and 10 weeks of age. B6.SOD1 mice had significantly longer stride and stance times than controls by 8 weeks. Consistent with disease progression, hindpaw measures of B6.SOD1 mice showed larger changes than front paws. Differences between control and B6.SOD1 mice increased at 10 weeks, but only because repeat testing caused habituation in control mice to a greater extent than in B6.SOD1 mice. Together the results demonstrate that simplified gait analysis is sensitive to early processes of motor system disease in mice. Keywordsamyotrophic lateral sclerosis; gait dynamics; mice; SOD1; transgenic Abbreviations ALS, amyotrophic lateral sclerosis; B6, C57BL/6J; B6.SOD1, B6.Cg-Tg(SOD1-G93A); B6SJL, B6SJL-TgN(SOD1-G93A)1GUR; SOD1, superoxide dismutase 1In amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, functional deficits are preceded for an uncertain, but variable, interval by pathological changes. It is critical to understand these early disease processes so that optimal therapeutic targets, which not only arrest the disease but also prevent the development of irreversible functional deficits, can be identified.Measurement and analysis of gait has been successfully applied to every common laboratory species and many others as well. 5,24 Moreover, it has provided a detailed basic understanding of human and quadruped locomotion and is a now a common cross-species clinical tool that is sensitive to relatively minor changes associated with disease, injury, or rehabilitation. 2,8,22,23 In its most sophisticated form, analysis of gait can include synchronized collection of ground reaction forces and kinematic and electromyographic data. 13 The difficulty of scaling and applying these techniques to mice has limited their use despite a steady increase in studies of the murine motor system. 15,16Correspondence to: K. Seburn; e-mail:kevins@jax.org. NIH Public Access NIH-PA Author ManuscriptA simple but related method that has been successfully used in mice is footprint analysis. This requires application of ink to the animal's paws and then measurement of static gait parameters (e.g., stride length) from the resulting footprints. 7,9,20 Such an approach is simple and reasonably sensitive but has significant practical limitations. We have extended it by using digital video capture of paw placements of mice during treadmill locomotion and then generating standard gait measures from the video images.Using this method we describe the earliest functional deficits (8 weeks) that have been reported for S...

Muscular dystrophies include a diverse group of genetically heterogeneous disorders that together affect 1 in 2000 births worldwide. The diseases are characterized by progressive muscle weakness and wasting that lead to severe disability and often premature death. Rostrocaudal muscular dystrophy (rmd) is a new recessive mouse mutation that causes a rapidly progressive muscular dystrophy and a neonatal forelimb bone deformity. The rmd mutation is a 1.6-kb intragenic deletion within the choline kinase beta (Chkb) gene, resulting in a complete loss of CHKB protein and enzymatic activity. CHKB is one of two mammalian choline kinase (CHK) enzymes (␣ and ␤) that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid phosphatidylcholine. While mutant rmd mice show a dramatic decrease of CHK activity in all tissues, the dystrophy is only evident in skeletal muscle tissues in an unusual rostral-to-caudal gradient. Minor membrane disruption similar to dysferlinopathies suggest that membrane fusion defects may underlie this dystrophy, because severe membrane disruptions are not evident as determined by creatine kinase levels, Evans Blue infiltration, and unaltered levels of proteins in the dystrophin-glycoprotein complex. The rmd mutant mouse offers the first demonstration of a defect in a phospholipid biosynthetic enzyme causing muscular dystrophy, representing a unique model for understanding mechanisms of muscle degeneration.Muscular dystrophies are a variable class of more than 20 human disorders characterized by progressive muscle wasting and weakness resulting from myofiber degeneration and regeneration. Histologically, variation in myofiber size with centrally localized nuclei, fibrosis, and fatty infiltration are common features (1, 2). Despite their common pathologies, the genetic causes, severity, age of onset, and inheritance patterns vary widely among the dystrophies. The Muscular Dystrophy Association currently lists over 40 neuromuscular diseases as targets for its research programs and categorizes them by phenotypic characteristics such as age of onset, affected muscle groups, and inheritance pattern (Muscular Dystrophy Association, www.mdausa.org). In the last 10 years, the genetic mapping and identification of novel skeletal muscle genes, including cytoskeletal, cytosolic, nuclear membrane, sarcolemmal and extracellular matrix proteins, has dramatically changed this phenotype-based classification and provided clues as to the molecular basis of these disorders (3). What was once considered a single disease entity such as limb-girdle muscular dystrophy (LGMD) 4 has now been subdivided into seven different molecularly defined autosomal dominant (LGMD1A-1G) and ten autosomal recessive (LGMD2A-2J) diseases. Not surprisingly, many of these genes have converged to define pathways critical for the normal functioning and maintenance of skeletal muscles. The fact that many muscular dystrophy cases exist in which mutations to known dystrophy-causing genes have...

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

Transgenic (Tg) mouse models of FALS containing mutant human SOD1 genes (G37R, G85R, D90A, or G93A missense mutations or truncated SOD1) exhibit progressive neurodegeneration of the motor system that bears a striking resemblance to ALS, both clinically and pathologically. The most utilized and best characterized Tg mice are the G93A mutant hSOD1 (Tg(hSOD1-G93A)1GUR mice), abbreviated G93A. In this review we highlight what is known about background-dependent differences in disease phenotype in transgenic mice that carry mutated human or mouse SOD1. Expression of G93A-hSOD1Tg in congenic lines with ALR, NOD.Rag1KO, SJL or C3H backgrounds show a more severe phenotype than in the mixed (B6xSJL) hSOD1Tg mice, whereas a milder phenotype is observed in B6, B10, BALB/c and DBA inbred lines. We hypothesize that the background differences are due to disease-modifying genes. Identification of modifier genes can highlight intracellular pathways already suspected to be involved in motor neuron degeneration; it may also point to new pathways and processes that have not yet been considered. Most importantly, identified modifier genes provide new targets for the development of therapies.

Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.

The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, "donut" shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology.

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway—Ribosome-associated Quality Control (RQC)—by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF’s role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.