Phytocannabinoids, such as the principal bioactive component of marijuana, ⌬ 9 -tetrahydrocannabinol, have been used for thousands of years for medical and recreational purposes. ⌬ 9 -Tetrahydrocannabinol and endogenous cannabinoids (e.g., anandamide) initiate their agonist properties by stimulating the cannabinoid family of G protein-coupled receptors (CB 1 and CB2). The biosynthesis and physiology of anandamide is well understood, but its mechanism of uptake (resulting in signal termination by fatty acid amide hydrolase) has been elusive. Mounting evidence points to the existence of a specific anandamide transport protein; however, no direct evidence for this protein has been provided. Here, we use a potent, competitive small molecule inhibitor of anandamide uptake (LY2318912, IC 50 7.27 ؎ 0.510 nM) to identify a high-affinity, saturable anandamide transporter binding site (LY2318912; K d ‫؍‬ 7.62 ؎ 1.18 nM, Bmax ‫؍‬ 31.6 ؎ 1.80 fmol͞mg protein) that is distinct from fatty acid amide hydrolase. Systemic administration of the inhibitor into rodents elevates anandamide levels 5-fold in the brain and demonstrates efficacy in the formalin paw-licking model of persistent pain with no obvious adverse effects on motor function. Identification of the anandamide transporter binding site resolves a missing mechanistic link in endocannabinoid signaling, and in vivo results suggest that endocannabinoid transporter antagonists may provide a strategy for positive modulation of cannabinoid receptors.anandamide ͉ fatty acid amide hydrolase ͉ cannabinoid ͉ marijuana ͉ transporter E ndocannabinoids are recognized as significant intracellular lipid signaling molecules in the central nervous system with extensive control of physiological and behavioral mood and affect. Increases in endocannabinoid neurotransmission have broad therapeutic potential, including reduction of nausea and emesis (1), appetite stimulation (2), analgesia (3), anxiolytic activity (4), antispasmodic activity (5), and lowering of intraocular pressure in glaucoma (6). Identification of a specific binding site for the phytocannabinoid, ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC) (7), cloning of the cannabinoid receptors (CB 1 and CB 2 ) (8, 9), and the identification of an endogenous ligand, anandamide (N-arachidonoylethanolamide) (10), provided evidence of an endogenous cannabinoid system. Anandamide represents a class of lipid neurotransmitters that stimulate not only presynaptic and postsynaptic CB 1 receptors but also TRPV1 ion channels (11, 12), 5-hydroxytryptamine receptors (13-16), and possibly other receptors, as well as CB 2 receptors in the periphery (10,(17)(18)(19). More recently, the enzymes that are responsible for anandamide synthesis (phospholipase D) and catabolism (fatty acid amide hydrolase, FAAH) have been identified and characterized (20,21). Unlike typical neurotransmitter molecules, anandamide is synthesized in the membrane bilayer, resulting in the phospholipid precursor of anandamide, Narachidonoylphosphatidylethanolamine (22-25). Calciumacti...

Mutations in the human LMNA gene, which encodes the nuclear envelope proteins lamins A and C, cause autosomal dominant Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, limb-girdle muscular dystrophy, and other diseases collectively known as laminopathies. The molecular mechanisms responsible for these diseases remain incompletely understood, but the muscle-specific defects suggest that mutations may render nuclei more susceptible to mechanical stress. Using three mouse models of muscle laminopathies, we found that Lmna mutations caused extensive nuclear envelope damage, consisting of chromatin protrusions and transient rupture of the nuclear envelope, in skeletal muscle cells in vitro and in vivo. The nuclear envelope damage was associated with progressive DNA damage, activation of DNA damage response pathways, and reduced viability. Intriguingly, nuclear envelope damage resulted from nuclear movement in maturing skeletal muscle cells, rather than actomyosin contractility, and was reversed by either depletion of kinesin-1 or stabilization of microtubules. Depletion of kinesin-1 also rescued DNA damage, indicating that DNA damage is the result of nuclear envelope damage. The extent of nuclear envelope damage and DNA damage in the different Lmna mouse models strongly correlated with the disease onset and severity in vivo, and inducing DNA damage in wild-type muscle cells was sufficient to phenocopy the reduced cell viability of lamin A/C-deficient muscle cells, suggesting a causative role of DNA damage in disease pathogenesis. Corroborating the mouse model data, muscle biopsies from patients with LMNA associated muscular dystrophy similarly revealed significant DNA damage compared to age-matched controls, particularly in severe cases of the disease. Taken together, these findings point to a new and important role of DNA damage as a pathogenic contributor for these skeletal muscle diseases.

BackgroundThere is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.ResultsA total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.ConclusionsThe CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

Autosomal recessive limb girdle muscular dystrophies 2C-2F represent a family of diseases caused by primary mutations in the sarcoglycan genes. We show that sarcospan, a novel tetraspan-like protein, is also lost in patients with either a complete or partial loss of the sarcoglycans. In particular, sarcospan was absent in a gamma-sarcoglycanopathy patient with normal levels of alpha-, beta- and delta-sarcoglycan. Thus, it is likely that assembly of the complete, tetrameric sarcoglycan complex is a prerequisite for membrane targeting and localization of sarcospan. Based on our findings that sarcospan is integrally associated with the sarcoglycans, we screened >50 autosomal recessive muscular dystrophy cases for mutations in sarcospan. Although we identified three intragenic polymorphisms, we did not find any cases of muscular dystrophy associated with primary mutations in the sarcospan gene. Finally, we have identified an important case of limb girdle muscular dystrophy and cardiomyopathy with normal expression of sarcospan. This patient has a primary mutation in the gamma-sarcoglycan gene, which causes premature truncation of gamma-sarcoglycan without affecting assembly of the mutant gamma-sarcoglycan into a complex with alpha-, beta- and delta-sarcoglycan and sarcospan. This is the first demonstration that membrane expression of a mutant sarcoglycan-sarcospan complex is insufficient in preventing muscular dystrophy and cardiomyopathy and that the C-terminus of gamma-sarcoglycan is critical for the functioning of the entire sarcoglycan-sarcospan complex. These findings are important as they contribute to a greater understanding of the structural determinants required for proper sarcoglycan-sarcospan expression and function.

Cannabinoids, in the form of marijuana plant extracts, have been used for thousands of years for a wide variety of medical conditions, ranging from general malaise and mood disorders to more specific ailments, such as pain, nausea, and muscle spasms. The discovery of tetrahydrocannabinol, the active principal in marijuana, and the identification and cloning of two cannabinoid receptors (i.e., CB1 and CB2) has subsequently led to biomedical appreciation for a family of endocannabinoid lipid transmitters. The biosynthesis and catabolism of the endocannabinoids and growing knowledge of their broad physiological roles are providing insight into potentially novel therapeutic targets. Compounds directed at one or more of these targets may allow for cannabinoid-based therapeutics with limited side effects and abuse liability.