Functional rescue in a mouse model of congenital muscular dystrophy with megaconial myopathy.

Congenital muscular dystrophy with megaconial myopathy (MDCMC) is an autosomal recessive disorder characterized by progressive muscle weakness and wasting. The observation of megamitochondria in skeletal muscle biopsies is exclusive to this type of MD. The disease is caused by loss of function mutations in the choline kinase beta (CHKB) gene which results in dysfunction of the Kennedy pathway for the synthesis of phosphatidylcholine. We have previously reported a rostrocaudal MD (rmd) mouse with a deletion in the Chkb gene resulting in an MDCMC-like phenotype, and we used this mouse to test gene therapy strategies for the rescue and alleviation of the dystrophic phenotype. Introduction of a muscle-specific Chkb transgene completely rescues motor and behavioral function in the rmd mouse model, confirming the cell-autonomous nature of the disease. Intramuscular gene therapy post-disease onset using an adeno-associated viral 6 (AAV6) vector carrying a functional copy of Chkb is also capable of rescuing the dystrophy phenotype. In addition, we examined the ability of choline kinase alpha (Chka), a gene paralog of Chkb, to improve dystrophic phenotypes when upregulated in skeletal muscles of rmd mutant mice using a similar AAV6 vector. The sum of our results in a preclinical model of disease suggest that replacement of the Chkb gene or upregulation of endogenous Chka could serve as potential lines of therapy for MDCMC patients.

Hanging on for the ride: adhesion to the extracellular matrix mediates cellular responses in skeletal muscle morphogenesis and disease.

Skeletal muscle specification and morphogenesis during early development are critical for normal physiology. In addition to mediating locomotion, skeletal muscle is a secretory organ that contributes to metabolic homeostasis. Muscle is a highly adaptable tissue, as evidenced by the ability to increase muscle cell size and/or number in response to weight bearing exercise. Conversely, muscle wasting can occur during aging (sarcopenia), cancer (cancer cachexia), extended hospital stays (disuse atrophy), and in many genetic diseases collectively known as the muscular dystrophies and myopathies. It is therefore of great interest to understand the cellular and molecular mechanisms that mediate skeletal muscle development and adaptation. Muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction. This process requires carefully orchestrated interactions between cells and their extracellular matrix microenvironment. These interactions are dynamic, allowing muscle cells to sense biophysical, structural, organizational, and/or signaling changes within their microenvironment and respond appropriately. In many musculoskeletal diseases, these cell adhesion interactions are disrupted to such a degree that normal cellular adaptive responses are not sufficient to compensate for accumulating damage. Thus, one major focus of current research is to identify the cell adhesion mechanisms that drive muscle morphogenesis, with the hope that understanding how muscle cell adhesion promotes the intrinsic adaptability of muscle tissue during development may provide insight into potential therapeutic approaches for muscle diseases. Our objectives in this review are to highlight recent studies suggesting conserved roles for cell-extracellular matrix adhesion in vertebrate muscle morphogenesis and cellular adaptive responses in animal models of muscle diseases.

Choline kinase beta is required for normal endochondral bone formation.

BACKGROUND: Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb(-/-) mice display neonatal forelimb bone deformations. METHODS: To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb(-/-) mice. RESULTS: The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb(-/-) mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb(-/-) mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb(-/-) mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb(-/-) mice contained fewer osteoclasts along the cartilage/bone interface. CONCLUSIONS: Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice. GENERAL SIGNIFICANCE: Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis.

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.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy.

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.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilized human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence dependent on S100A9 and stress factors. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Identification of quantitative trait loci for survival in the mutant dynactin p150Glued mouse model of motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5-10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness.

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse.

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase beta is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase alpha is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb(-/-) mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A(2) in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb(-/-) mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb(-/-) mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb(-/-) mice is due to abundant choline kinase alpha and the stable homeostasis of phosphatidylcholine.

Effect of genetic background on phenotype variability in transgenic mouse models of amyotrophic lateral sclerosis: a window of opportunity in the search for genetic modifiers.

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.

Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice.

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb -/- mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP: phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A2 are increased. The mitochondria in Chkb -/- mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb -/- mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb -/- mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb -/- mice. We conclude that the hindlimb muscular dystrophy in Chkb -/- mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.

Author Correction: NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

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.

The interaction of genetics and environmental toxicants in amyotrophic lateral sclerosis: results from animal models.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in the progressive death of motor neurons, leading to paralysis and eventual death. There is presently no cure for ALS, and only two drugs are available, neither of which provide significant extension of life. The wide variation in onset and progression of the disease, both in sporadic and even in strongly penetrant monogenic familial forms of ALS, indicate that in addition to background genetic variation impacting the disease process, environmental exposures are likely contributors. Epidemiological evidence worldwide implicates exposures to bacterial toxins, heavy metals, pesticides, and trauma as probable environmental factors. Here, we review current advances in gene-environment interactions in ALS animal models. We report our recent discoveries in a zebrafish model of ALS in relation to exposure to the cyanobacterial toxin BMAA, and discuss several results from mouse models that show interactions with exposure to mercury and statin drugs, both leading to acceleration of the disease process. The increasing research into this combinatorial gene-environment process is just starting, but shows early promise to uncover the underlying biochemical pathways that instigate the initial motor neuron defects and lead to their rapidly progressive dysfunction.

Editor's Highlight: Embryonic Exposure to the Environmental Neurotoxin BMAA Negatively Impacts Early Neuronal Development and Progression of Neurodegeneration in the Sod1-G93R Zebrafish Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to progressive paralysis and death within 2-5 years after diagnosis. Sporadic cases (SALS) comprise approximately 90% of cases with the remaining 10% familial (FALS) caused by mutations in approximately 27 genes. The vast heterogeneity seen in age and location of disease onset, rate of progression, and duration of disease has been linked with genetic and environmental influences in both SALS and FALS cases. Increased ALS incidence clusters in Guam, southern France, and Maryland have been linked with exposure to Beta-methylamino-L-alanine (BMAA), a nonproteinogenic amino acid produced by cyanobacteria, dinoflaggelates, and diatoms. We embryonically exposed zebrafish, Danio rerio, (transgenically overexpressing a FALS-causing SOD1-G93R mutation) to BMAA to investigate early motor neuron outgrowth in larvae and endurance and fatigability in 5-month adults. SOD1-G93R zebrafish showed decreased embryonic nerve length with increased BMAA dose, a phenotypic change mirrored in 5-month performance measures of weaker swimming and increased fatigability. In contrast, transgenic fish overexpressing wild-type SOD1 were resistant to phenotypic changes, indicating a potential neuroprotective function of healthy SOD1. We show that the etiology of genetic ALS animal models can be influenced by environmental exposures, and that embryonic toxin exposures can result in changes to both early and adult measures. We demonstrate that zebrafish can be a robust model for investigating causes of ALS heterogeneity. Establishing these links between developmental and adult ALS-like symptoms in the zebrafish increases the power of this model for toxicological and drug screens.

Genetic background effects on disease onset and lifespan of the mutant dynactin p150Glued mouse model of motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease primarily affecting motor neurons in the central nervous system. Although most cases of ALS are sporadic, about 5-10% of cases are familial (FALS) with approximately 20% of FALS caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. We have reported that hSOD1-G93A transgenic mice modeling this disease show a more severe phenotype when the transgene is bred on a pure SJL background and a milder phenotype when bred on a pure B6 background and that these phenotype differences link to a region on mouse Chromosome 17.To examine whether other models of motor neuron degeneration are affected by genetic background, we bred the mutant human dynactin p150Glued (G59S-hDCTN1) transgene onto inbred SJL and B6 congenic lines. This model is based on an autosomal dominant lower motor neuron disease in humans linked to a mutation in the p150Glued subunit of the dynactin complex. As seen in hSOD1-G93A mice, we observed a more severe phenotype with earlier disease onset (p<0.001) and decreased survival (p<0.00001) when the G59S-hDCTN1 transgene was bred onto the SJL background and delayed onset (p<0.0001) with increased survival (p<0.00001) when bred onto the B6 background. Furthermore, B6 mice with an SJL derived chromosome 17 interval previously shown to delay disease onset in hSOD1-G93A mice also showed delays onset in G59S-hDCTN1 mice suggesting that at least some genetic modifiers are shared. We have shown that genetic background influences phenotype in G59S-hDCTN1 mice, in part through a region of chromosome 17 similar to the G93-hSOD1 ALS mouse model. These results support the presence of genetic modifiers in both these models some of which may be shared. Identification of these modifiers will highlight intracellular pathways involved in motor neuron disease and provide new therapeutic targets that may be applicable to motor neuron degeneration.

A major QTL on mouse chromosome 17 resulting in lifespan variability in SOD1-G93A transgenic mouse models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a late-onset degenerative disease affecting motor neurons in the spinal cord, brainstem, and motor cortex. There is great variation in the expression of ALS symptoms even between siblings who both carry the same Cu/Zn superoxide dismutase (SOD1) mutations. One important use of transgenic mouse models of SOD1-ALS is the study of genetic influences on ALS severity. We utilized multiple inbred mouse strains containing the SOD1-G93A transgene to demonstrate a major quantitative trait locus (QTL) on mouse chromosome 17 resulting in a significant shift in lifespan. Reciprocal crosses between long- and short-lived strains identified critical regions, and we have narrowed the area for potential genetic modifier(s) to < 2Mb of the genome. Results showed that resequencing of this region resulted in 28 candidate genes with potentially functional differences between strains. In conclusion, these studies provide the first major modifier locus affecting lifespan in this model of FALS and, once identified, these candidate modifier genes may provide insight into modifiers of human disease and, most importantly, define new targets for the development of therapies.

Rhabdomyosarcomas in aging A/J mice.

Rhabdomyosarcomas (RSCs) are skeletal muscle neoplasms found in humans and domestic mammals. The A/J inbred strain developed a high frequency (between 70-80%) of adult pleomorphic type (APT) RSC at >20 months of age while BALB/cByJ also develop RSC but less frequently. These neoplasms invaded skeletal muscle surrounding either the axial or proximal appendicular skeleton and were characterized by pleomorphic cells with abundant eosinophilic cytoplasm, multiple nuclei, and cross striations. The diagnosis was confirmed by detection of alpha-sarcomeric actin and myogenin in the neoplastic cells using immunocytochemistry. The A/J strain, but not the related BALB/c substrains, is also characterised by a progressive muscular dystrophy homologous to limb-girdle muscular dystrophy type 2B. The association between the development of RSC in similar muscle groups to those most severely affected by the progressive muscular dystrophy suggested that these neoplasms developed from abnormal regeneration of the skeletal muscle exacerbated by the dysferlin mutation. Transcriptome analyses of RSCs revealed marked downregulation of genes in muscular development and function signaling networks. Non-synonymous coding SNPs were found in Myl1, Abra, Sgca, Ttn, and Kcnj12 suggesting these may be important in the pathogenesis of RSC. These studies suggest that A strains of mice can be useful models for dissecting the molecular genetic basis for development, progression, and ultimately for testing novel anticancer therapeutic agents dealing with rhabdomyosarcoma.

A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis.

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 (alpha and beta) 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.

Gait analysis detects early changes in transgenic SOD1(G93A) mice.

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.