The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7ß1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Benfotiamine improves dystrophic pathology and exercise capacity in mdx mice by reducing inflammation and fibrosis.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease. Cycles of myofibre degeneration and regeneration are hallmarks of the disease where immune cells infiltrate to repair damaged skeletal muscle. Benfotiamine is a lipid soluble precursor to thiamine, shown clinically to reduce inflammation in diabetic related complications. We assessed whether benfotiamine administration could reduce inflammation related dystrophic pathology. Benfotiamine (10 mg/kg/day) was fed to male mdx mice (n = 7) for 15 weeks from 4 weeks of age. Treated mice had an increased growth weight (5-7 weeks) and myofibre size at treatment completion. Markers of dystrophic pathology (area of damaged necrotic tissue, central nuclei) were reduced in benfotiamine mdx quadriceps. Grip strength was increased and improved exercise capacity was found in mdx treated with benfotiamine for 12 weeks, before being placed into individual cages and allowed access to an exercise wheel for 3 weeks. Global gene expression profiling (RNAseq) in the gastrocnemius revealed benfotiamine regulated signalling pathways relevant to dystrophic pathology (Inflammatory Response, Myogenesis) and fibrotic gene markers (Col1a1, Col1a2, Col4a5, Col5a2, Col6a2, Col6a2, Col6a3, Lum) towards wildtype levels. In addition, we observed a reduction in gene expression of inflammatory gene markers in the quadriceps (Emr1, Cd163, Cd4, Cd8, Ifng). Overall, these data suggest that benfotiamine reduces dystrophic pathology by acting on inflammatory and fibrotic gene markers and signalling pathways. Given benfotiamine's excellent safety profile and current clinical use, it could be used in combination with glucocorticoids to treat DMD patients.

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways.

BACKGROUND: The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdxTG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression. METHODS: The tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdxTG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators. RESULTS: Through our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdxTG muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with ß-spectrin in mdxTG samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt. CONCLUSIONS: Our findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

Implementation of an electronic hand hygiene monitoring system: Learnings on how to maximize the investment.

BACKGROUND: Electronic monitoring systems (EMS) for measuring hand hygiene performance have many advantages. Previous studies have shared results of EMS in individual units or single institutions, without many details of implementation. The implementation steps for house wide use of an EMS in 12 hospitals are described. METHODS: Hospital resources used in this 3-year implementation included those for installation activities, initial education about the components and function of the EMS, evaluation of healthcare professionals' processes related to hand hygiene, routine data feedback in a variety of methods, continuous coaching and training on the EMS, incentive programs and strong leadership support. RESULTS: Continual process improvement activities resulted in a 23% increase in hand hygiene performance, from 53% at baseline, to 76%. DISCUSSION/CONCLUSION: Implementation of an EMS required many resources beyond those for the technology, but resulted in measurable improvement in hand hygiene performance.

Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin.

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg-1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

Loss of sarcospan exacerbates pathology in mdx mice, but does not affect utrophin amelioration of disease.

The dystrophin-glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the sarcolemma by linking the myocyte's internal cytoskeleton and external extracellular matrix. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive muscle damage. Utrophin (UTRN), an autosomal homolog of dystrophin, is upregulated in dystrophic muscle and partially compensates for the loss of dystrophin in muscle from patients with DMD. Here, we examine the interaction between Utr and sarcospan (SSPN), a small transmembrane protein that is a core component of both UTRN-glycoprotein complex (UGC) and DGC. We show that additional loss of SSPN causes an earlier onset of disease in dystrophin-deficient mdx mice by reducing the expression of the UGC at the sarcolemma. In order to further evaluate the role of SSPN in maintaining therapeutic levels of Utr at the sarcolemma, we tested the effect of Utr transgenic overexpression in mdx mice lacking SSPN (mdx:SSPN -/-:Utr-Tg). We found that overexpression of Utr restored SSPN to the sarcolemma in mdx muscle but that the ablation of SSPN in mdx muscle reduced Utr at the membrane. Nevertheless, Utr overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that high levels of Utr transgenic overexpression ameliorate the mdx phenotype independently of SSPN expression but that loss of SSPN may impair Utr-based mechanisms that rely on lower levels of Utr protein.

Enhanced Methods for Needle Biopsy and Cryopreservation of Skeletal Muscle in Older Adults.

Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. The VACORA vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in embedding medium for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples. The VACORA Biopsy System provided as many as four skeletal muscle core samples from a single biopsy site. Biopsy samples from 12 older adults weighed an average of 147.5 ± 11 mg each and had a consistent size and shape. There were no complications, and the residual scar is less than 10 mm. The freezing method using standard tissue cassettes with direct freezing in liquid nitrogen yielded high quality cryopreserved muscle tissue suitable for histological analysis without the need for isopentane and with little to no freeze-thaw damage. These enhancements have streamlined and improved the consistency of our muscle biopsy protocol and provide sufficient high-quality sample for multi-dimensional downstream studies of human muscle in aging and disease.

Large in-frame 5' deletions in DMD associated with mild Duchenne muscular dystrophy: Two case reports and a review of the literature.

Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype. In this study, we describe two individuals with large in-frame 5' deletions (exon 3-23 and exon 3-28) that remove the majority of the N-terminal region, including part of the actin binding and central rod domains. Both patients had progressive muscle weakness during childhood but are observed to have a relatively mild disease course compared to typical Duchenne. We show that in muscle biopsies from both patients, truncated dystrophin is expressed at the sarcolemma. We have additionally developed a patient-specific fibroblast-derived cell model, which can be inducibly reprogrammed to form myotubes that largely recapitulate biopsy findings for the patient with the exon 3-23 deletion, providing a culture model for future investigation of this unusual case. We discuss these mutations in the context of previously reported 5' in-frame DMD deletions and relevant animal models, and review the spectrum of phenotypes associated with these deletions.

Effective and Novel Application of Hydrodynamic Voltammetry to the Study of Superoxide Radical Scavenging by Natural Phenolic Antioxidants.

The reactions of antioxidants with superoxide radical were studied by cyclic voltammetry (CV)-and hydrodynamic voltammetry at a rotating ring-disk electrode (RRDE). In both methods, the superoxide is generated in solution from dissolved oxygen and then measured after being allowed to react with the antioxidant being studied. Both methods detected and measured the radical scavenging but the RRDE was able to give detailed insight into the antioxidant behavior. Three flavonoids, chrysin, quercetin and eriodictyol, were studied, their scavenging activity of superoxide was assessed and the molecular structure of each flavonoid was related to its scavenging capability. From our improved and novel RRDE method, we determine the ability of these 3 antioxidants to react with superoxide radical in a more quantitative manner than the classical CV. Density Functional Theory (DFT) and single crystal X-ray diffraction data provide structural information that assists in clarifying the scavenging molecular mechanism. Hydroxyls associated with the A ring, as found in chrysin, scavenge superoxide in a different manner than those found in the B ring of flavonoids, as those in quercetin and eriodictyol.

Thinking Critically: How to Teach Translational Medicine.

Translational Medicine (TM) is a comparatively new field of study that focusses on the continuum of activities from the conception of an idea, to advanced clinical testing and the development of a new medical technology or drug. In recent years, graduate education programs have been established internationally to train a new generation of professionals with specific skills necessary to navigate the translational landscape. Literature in the area highlights the importance of integrating specific competencies relevant to translational medicine as part of curriculum development. In addition to developing a working understanding of core knowledge (e.g., ethics, funding, regulation, policy, etc.), skills including effective communication, reflection, interdisciplinary, and interprofessional collaboration are critical components of a skilled TM professional. Curriculum development must focus on content, while carefully selecting the teaching strategies that are most effective to achieve the desired outcomes, which is for learners to comprehend the complex material. The following publication presents a series of vignettes that describe the experiences of an associate professor of molecular biology, who is looking to explore her role in translational medicine and develop skills for an innovative approach to problem-solving. The vignettes are focused on a variety of teaching and learning strategies that can be used to teach translational medicine. Each vignette includes a description of the experience from the perspective of the learner and the faculty as it pertains to the teaching strategy, method of delivery, and learning outcomes. TM is as complex to teach as it is to learn. The specialized skills and knowledges that are part of the TM toolbox cannot all be taught in a lecture format. Educators must consider multiple strategies and select those which are most effective for achieving the learning outcomes.

Multidepartmental Response to a Duodenoscope Used on a CRE Patient: A Case Study.

Transmission of carbapenem-resistant Enterobacteriaceae (CRE) via duodenoscopes, specialized endoscopes used during endoscopic retrograde cholangiopancreatography (ERCP) procedures, has attracted media attention since early 2015. This attention has placed increasing focus on the reprocessing of duodenoscopes. Current reprocessing recommendations for these endoscopes require either high-level disinfection or ethylene oxide sterilization. While reprocessing duodenoscopes, staff at endoscopy locations within the Mercy health system perform a single high-level disinfection cycle that is preceded by two cycles of manual cleaning. The Mercy system has 37 locations for gastrointestinal endoscopic procedures and nine that can accommodate patients requiring ERCP. In early 2016, the Mercy Oklahoma City location performed an ERCP on a patient known prior to the case to be a carrier of CRE. After the case, multiple departments located in both the Oklahoma City and St. Louis facilities partnered to culture and sterilize the duodenoscope used in that case to ensure its safety for use on subsequent patients. This case study presents the situation and discusses culturing of endoscopes. In light of the evidence presented, the importance of enhanced communication and cooperation to achieve patient safety should be paramount to all other factors.

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal's free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Primary cilia in neurodevelopmental disorders.

Primary cilia are generally solitary organelles that emanate from the surface of almost all vertebrate cell types. Until recently, details regarding the function of these structures were lacking; however, extensive evidence now suggests that primary cilia have critical roles in sensing the extracellular environment, and in coordinating developmental and homeostatic signalling pathways. Furthermore, disruption of these functions seems to underlie a diverse spectrum of disorders, known as primary ciliopathies. These disorders are characterized by wide-ranging clinical and genetic heterogeneity, but with substantial overlap among distinct conditions. Indeed, ciliopathies are associated with a large variety of manifestations that often include distinctive neurological findings. Herein, we review neurological features associated with primary ciliopathies, highlight genotype-phenotype correlations, and discuss potential mechanisms underlying these findings.

The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish.

DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

Analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations.

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 µm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies.

A new and exciting phase of muscle disease research has recently been entered. The application of next generation sequencing technology has spurred an unprecedented era of gene discovery for both myopathies and muscular dystrophies. Gene-based therapies for Duchenne muscular dystrophy have entered clinical trial, and several pathway-based therapies are doing so as well for a handful of muscle diseases. While many factors have aided the extraordinary developments in gene discovery and therapy development, the zebrafish model system has emerged as a vital tool in these advancements. In this review, we will highlight how the zebrafish has greatly aided in the identification of new muscle disease genes and in the recognition of novel therapeutic strategies. We will start with a general introduction to the zebrafish as a model, discuss the ways in which muscle disease can be modeled and analyzed in the fish, and conclude with observations from recent studies that highlight the power of the fish as a research tool for muscle disease.

Hemimegalencephaly, a paradigm for somatic postzygotic neurodevelopmental disorders.

PURPOSE OF REVIEW: Combining human genomics and molecular biology, recent studies have made pivotal progress toward understanding the cause of hemimegalencephaly (HME) and other cerebral megalencephaly syndromes. The present article highlights recent advances of the genetic cause of these conditions, and considers the role of somatic postzygotic genetic lesions in brain maldevelopment. RECENT FINDINGS: Studies over the past 12 months have identified de-novo somatic mutations as one possible cause in HME. The gene mutations involve components of the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B)-mammalian target of rapamycin (mTOR) pathway and include PIK3CA, PIK3R2, AKT3, and MTOR. These mutations were identified by comparing genomic data obtained from surgically resected brain tissue with nondiseased tissue, and by single-neuron sequencing in combination with molecular biology techniques. The association between the somatic mutations and downstream activation of the PI3K-mTOR pathway suggests that HME is a neurodevelopmental disease caused by gain-of-function activation of the PI3K-AKT-mTOR pathway. SUMMARY: The studies reviewed suggest that somatic mutations of the PI3K-AKT-mTOR pathway limited to the brain may represent one cause of HME. Dysregulation of this pathway has possible therapeutic potential in the identification of HME. Somatic mutations may be an important yet underappreciated disease mechanism in developmental neurological diseases.

Two dynamin-2 genes are required for normal zebrafish development.

Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.