Characterization of Dysferlin Deficient SJL/J Mice to Assess Preclinical Drug Efficacy: Fasudil Exacerbates Muscle Disease Phenotype

The dysferlin deficient SJL/J mouse strain is commonly used to study dysferlin deficient myopathies. Therefore, we systematically evaluated behavior in relatively young (9–25 weeks) SJL/J mice and compared them to C57BL6 mice to determine which functional end points may be the most effective to use for preclinical studies in the SJL/J strain. SJL/J mice had reduced body weight, lower open field scores, higher creatine kinase levels, and less muscle force than did C57BL6 mice. Power calculations for expected effect sizes indicated that grip strength normalized to body weight and open field activity were the most sensitive indicators of functional status in SJL/J mice. Weight and open field scores of SJL/J mice deteriorated over the course of the study, indicating that progressive myopathy was ongoing even in relatively young (<6 months old) SJL/J mice. To further characterize SJL/J mice within the context of treatment, we assessed the effect of fasudil, a rho-kinase inhibitor, on disease phenotype. Fasudil was evaluated based on previous observations that Rho signaling may be overly activated as part of the inflammatory cascade in SJL/J mice. Fasudil treated SJL/J mice showed increased body weight, but decreased grip strength, horizontal activity, and soleus muscle force, compared to untreated SJL/J controls. Fasudil either improved or had no effect on these outcomes in C57BL6 mice. Fasudil also reduced the number of infiltrating macrophages/monocytes in SJL/J muscle tissue, but had no effect on muscle fiber degeneration/regeneration. These studies provide a basis for standardization of preclinical drug testing trials in the dysferlin deficient SJL/J mice, and identify measures of functional status that are potentially translatable to clinical trial outcomes. In addition, the data provide pharmacological evidence suggesting that activation of rho-kinase, at least in part, may represent a beneficial compensatory response in dysferlin deficient myopathies

Impact of Polymorphisms in PTK2 on Intrinsic Muscle Strength

Abstract Title: Impact of Polymorphisms in PTK2 on Intrinsic Muscle Strength Primary Presenter Full Name: Zachary Zeller Co-presenter Full Name(s): Click here to enter text. Co-author Full Name(s): Mohamed Al-Amoodi, Whitney Jones, Danny Lee, Steven Mckenzie, Helen Miller, Seth Stubblefied, Susan Knoblach, Heather Gordish-Dressman, Dustin Hittel, Laura L. Tosi Abstract Text (should not exceed 400 words): Recent studies have begun to search for correlations between genetic variations and muscle strength. One such study by Stebbings et al.1 examined two single nucleotide polymorphisms (SNPs)—rs7843014 and rs7460—on the PTK2 gene. The study found that genetic variation in the PTK2 gene impacts muscle-specific force, which is the force generated per unit of cross-sectional area of muscle. Muscle-specific force ultimately represents the intrinsic strength of a muscle and is a key determinant of functional capacity and mobility. This study sought to expand on prior research by looking for associations between genetic variants of PTK2 and measures of grip strength, as well as general anthropomorphic measures, in a cohort of healthy young adults. Our study assessed phenotypes for height, weight, VO2 max, max grip strength, and body mass index (BMI) using the Assessing Inherited Markers of Metabolic Syndrome in the Young (AIMMY) University of Calgary subset of 190 healthy, primarily Caucasian, individuals between the ages of 18 and 35. DNA samples were genotyped using ThermoFisher Taqman SNP genotype assays, and underwent the Applied Biosystems 7900HT real-time polymerase chain reaction (PCR) process. Analysis of covariance (ANCOVA) models were used to perform statistical analysis to look for genotype-phenotype associations. Unlike the findings by Stebbings et al.1 an association between the PTK2 genotypes and grip strength was not found. This could be due to the lower statistical power in the grip strength test, thus potentially indicating that grip strength and muscle-specific force do not measure similar parameters of muscle strength. Genetic variation in PTK2 has also been previously associated with VO2 max, but no association was found in the current study. Positive associations were found between genetic variants rs7843014 and rs7460 in PTK2 and BMI, and between genetic variant rs7843014 and height. High levels of functioning PTK2 have been found to have increased strength due to increased costamere density, resulting in more muscle myofibrils, and therein larger, presumably heavier muscles. However, this finding was only observed in males, and could be attributed to differential acquisition and maintenance of muscle mass based on sex. We identified a potentially novel association between genetic variants in PTK2 and anthropomorphic phenotypes. However, we were unable to confirm the effects of genetic variants on measures of intrinsic muscle strength, namely max grip strength or VO2 max in terms of functional capacity. Further research is needed to confirm this newly identified role for PTK2

The association of polymorphism rs3736228 within the LRP5 gene with Bone Mineral Density in a Cohort of Caucasian Young Adults

INTRODUCTION: Osteoporosis is a significant burden for our aging population. Developing a better understanding of the genetic underpinnings of poor bone quality may assist in the future development of prevention strategies. Correa-Rodriguez et al. have identified a group of single nucleotide polymorphisms (SNPs) that were associated with bone mineral density (BMD) in a population of Spanish Caucasians. In particular, they found that SNP rs3736228 in the low-density lipoprotein receptor related protein 5 (LRP5) gene had an influence on BMD. While the role of LRP5 in the Wnt canonical pathway has been fairly well characterized, its association with phenotypic BMD and osteoporosis has only been explored in a limited fashion. The aim of this study is to expand on this, and to replicate the findings of previous studies in a cohort of healthy young adults. METHODS: Cohort: The University of Calgary cohort from the Assessing Inherited Metabolic Syndrome Markers in the Young (UC AIMMY) study. Participants included consist of 168 healthy, predominantly Caucasian young adults. Phenotypes: height, weight, BMI, and total BMD. Genotyping: Allelic discrimination was determined. Statistical Analysis: After being tested for Hardy-Weinberg equilibrium (HWE), the data was run through analysis of covariance (ANCOVA). RESULTS: Using a dominant model, we found that females with one or more copies of the risk T allele of SNP rs3736228 had a significant negative association with total BMD (p = 0.0347). However, a similar association was not seen in males in this cohort. We did not find a significant association for this polymorphism and height, weight, or BMI. DISCUSSION: Polymorphisms in rs3736228 alter the codon in position 1330, downregulating the LRP5 cell surface receptor function. The LRP5 gene has now been shown in multiple studies to be associated with bone quality measures like calcaneal Qualitative Ultrasound (QUS) and BMD. Our study suggests that SNP rs3736228 also influences BMD in healthy young females. This supports the work of Correa-Rodriguez et al that found that when stratifying by sex, females only showed a trend towards significance (p = 0.092) in QUS measures. SIGNIFICANCE: This study expands our understanding of the importance of LRP5 rs3736228 polymorphisms in BMD by extending its relationship to a cohort of predominantly Caucasian college students. While the development of BMD is polygenic, this work broadened the role of SNP rs3736228 across the age span, and the sexual dimorphism seen in musculoskeletal traits

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated. Conclusions Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

Background: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. Methods: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. Results: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. Conclusions: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Seizures enhance thyrotropin-releasing hormone (TRH) expression in brain regions associated with epilepsy and depression

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Characterization of inflammatory gene expression and galectin-3 function after spinal cord injury in mice

Inflammation has long been implicated in secondary tissue damage after spinal cord injury (SCI). Our previous studies of inflammatory gene expression in rats after SCI revealed two temporally correlated clusters: the first was expressed early after injury and the second was up-regulated later, with peak expression at 1–2 weeks and persistent up-regulation through 6 months. To further address the role of inflammation after SCI, we examined inflammatory genes in a second species, mice, through 28 days after SCI. Using anchor gene clustering analysis, we found similar expression patterns for both the acute and chronic gene clusters previously identified after rat SCI. The acute group returned to normal expression levels by 7 days post-injury. The chronic group, which included C1qB, p22(phox) and galectin-3, showed peak expression at 7 days and remained up-regulated through 28 days. Immunohistochemistry and western blot analysis showed that the protein expression of these genes was consistent with the mRNA expression. Further exploration of the role of one of these genes, galectin-3, suggests that galectin-3 may contribute to secondary injury. In summary, our findings extend our prior gene profiling data by demonstrating the chronic expression of a cluster of microglial associated inflammatory genes after SCI in mice. Moreover, by demonstrating that inhibition of one such factor improves recovery, the findings suggest that such chronic up-regulation of inflammatory processes may contribute to secondary tissue damage after SCI, and that there may be a broader therapeutic window for neuroprotection than generally accepted