White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARγ in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.

L-3-18 F-a-methyl tyrosine ( 18 F-FAMT) has been developed as a PET radiotracer for tumor imaging. Clinical studies have demonstrated the usefulness of 18 F-FAMT PET for the prediction of prognosis and the differentiation of malignant tumors and benign lesions. 18 F-FAMT exhibits higher cancer specificity in peripheral organs than other amino acid PET tracers and 18 F-FDG. The accumulation of 18 F-FAMT is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L highly upregulated in cancers. In this study, we examined the interaction of 3-fluoro-L-a-methyltyrosine (FAMT) with amino acid transporters to assess the mechanisms of 18 F-FAMT uptake in PET. Methods: We applied in vitro assays using established mammalian cell lines stably expressing LAT1 or a non-cancer-type system L isoform LAT2. The inhibitory effect on L-14 C-leucine uptake and the induction effect on efflux of preloaded L-14 C-leucine were examined for FAMT and other amino acid tracers. FAMT transport was compared among cell lines with varied LAT1 expression level. Results: FAMT prominently inhibited LAT1-mediated L-14 C-leucine uptake in a competitive manner but had less of an effect on LAT2. In the efflux experiments, FAMT induced the efflux of preloaded L-14 C-leucine through LAT1, indicating that FAMT is transported by LAT1 and not by LAT2. Among amino acid-related compounds examined in this study, including those used for PET tracers, the compounds with an a-methyl group such as FAMT, 2-fluoro-L-a-methyl-tyrosine, 3-iodo-L-a-methyl-tyrosine, and L-a-methyl-tyrosine were well transported by LAT1 but not by LAT2. However, L-methionine, L-tyrosine, 3-fluoro-L-tyrosine, 2-fluoro-L-tyrosine, and O-(2-fluoroethyl)-L-tyrosine were transported by both LAT1 and LAT2, suggesting that the a-methyl moiety is responsible for the LAT1 selectivity of FAMT. FAMT transport rate and LAT1 protein level were well correlated, supporting the importance of LAT1 for the cellular uptake of FAMT. Conclusion: Distinct from other amino acid PET tracers, because of its a-methyl moiety, FAMT is selective to LAT1 and not transported by LAT2. This property of FAMT is proposed to contribute to highly tumorspecific accumulation of 18 F-FAMT in PET.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretchinduced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

Background: The formation of long-term memory (LTM) and the late phase of long-term potentiation (L-LTP) depend on macromolecule synthesis, translation, and transcription in neurons. vesl-1S (VASP/Ena-related gene upregulated during seizure and LTP, also known as homer-1a) is an LTP-induced immediate early gene. The short form of Vesl (Vesl-1S) is an alternatively spliced isoform of the vesl-1 gene, which also encodes the long form of the Vesl protein (Vesl-1L). Vesl-1L is a postsynaptic scaffolding protein that binds to and modulates the metabotropic glutamate receptor 1/5 (mGluR1/5), the IP 3 receptor, and the ryanodine receptor. Vesl-1 null mutant mice show abnormal behavior, which includes anxiety-and depression-related behaviors, and an increase in cocaine-induced locomotion; however, the function of the short form of Vesl in behavior is poorly understood because of the lack of short-form-specific knockout mice.