␥-Secretase inhibitors (GSIs) reduce amyloid-␤ (A␤) peptides but inevitably increase the ␤-C-terminal fragment (␤-CTFSubchronic dosing with either GSI rather impaired normal cognition in 3-month-old Tg2576 mice, with no inhibition on the processing of other ␥-secretase substrates, such as Notch, N-cadherin, or EphA4, in the brain. LY450139 also impaired normal cognition in wild-type mice; however, the potency was 10-fold lower than that in Tg2576 mice, indicating an APP-dependent mechanism likely with ␤-CTF accumulation. Immunofluorescence studies revealed that the ␤-CTF accumulation was localized in the presynaptic terminals of the hippocampal stratum lucidum and dentate hilus, implying an effect on presynaptic function in the mossy fibers. In contrast, both acute and subchronic dosing with GSM-2 significantly ameliorated memory deficits in Tg2576 mice and did not affect normal cognition in wild-type mice. We demonstrated a clear difference between GSI and GSM in effects on functional consequences, providing new insights into strategies for developing these drugs against Alzheimer's disease.

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.P lasma membrane fusion is a fundamental cellular process required for the conception, development, and physiology of multicellular organisms. Membrane fusion occurs during viral infection of a host cell, between intracellular membranes, and between two plasma membranes to form syncytial tissues (1). Cell-cell fusion is critical for a wide array of cellular processes, including sperm-egg fertilization, macrophage function, bone and placental development, and skeletal muscle formation. This form of fusion must be precisely controlled to prevent inappropriate cellular mixing.The fusion of myoblasts requires cell recognition, migration, adhesion, signaling, and finally, membrane coalescence (2). Much of our knowledge about myoblast fusion has originated from studies performed in Drosophila. In this system, intracellular signaling results in cytoskeletal alterations and actin polymerization, which drive the formation of cellular projections that invade neighboring cells to cause fusion (3). Recent evidence also indicates a critical function for branched actin polymerization during Drosophila indirect flight muscle fusion (4). The essential role of the cytoskeleton in fusion is conserved in mammals, in which the actin regulators Rac1, cdc42, and N-WASp are required for muscle development (5-8). In addition to actin dynamics, numerous proteins that regulate diverse cellular processes have been associated with myoblast fusion (9-13).We recently discovered a muscle-specific membrane protein named myomaker [annotated as Transmembrane protein 8c (TMEM8c)] that is absolutely required for skeletal myocyte fusion in the mouse (14,15). To begin to decipher the mechanisms whereby myomaker controls cell-cell fusion, we designed a heterologous cell-cell fusion assay to monitor the ability of a series of myomaker mutants to direct the fusion of lab...

Central adenosine A 2A receptor is a promising target for drugs to treat Parkinson's disease (PD), and the central blockade of adenosine A 1 receptor improves cognitive function. In the present study, we investigated the effect of a novel adenosine A 1 and A 2A dual antagonist, 5-[5-amino-3-(4-fluorophenyl) pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in animal models of PD and cognition. The binding affinities of ASP5854 for human A 1 and A 2A receptors were 9.03 and 1.76 nM, respectively, with higher specificity and no species differences. ASP5854 also showed antagonistic action on A 1 and A 2A agonist-induced increases of intracellular Ca 2ϩ concentration. ASP5854 ameliorated A 2A agonist 2-[p-(2-carboxyethyl) phenethylamino]-5Ј-N-ethylcarboxamidoadenosine (CGS21680)-and haloperidol-induced catalepsy in mice, with the minimum effective doses of 0.32 and 0.1 mg/kg, respectively, and it also improved haloperidol-induced catalepsy in rats at doses higher than 0.1 mg/kg. In unilateral 6-hydroxydopamine-lesioned rats, ASP5854 significantly potentiated l-dihydroxyphenylalanine (L-DOPA)-induced rotational behavior at doses higher than 0.032 mg/kg. ASP5854 also significantly restored the striatal dopamine content reduced by 1-metyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in mice at doses higher than 0.1 mg/ kg. Furthermore, in the rat passive avoidance test, ASP5854 significantly reversed the scopolamine-induced memory deficits, whereas the specific adenosine A 2A antagonist 8-((E)-2-(3,4-dimethoxyphenyl)ethenyl)-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002; istradefylline) did not. Scopolamine-or 5H-dibenzo [a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801)-induced impairment of spontaneous alternation in the mouse Y-maze test was ameliorated by ASP5854, whereas KW-6002 did not exert improvement at therapeutically relevant dosages. These results demonstrate that the novel, selective, and orally active dual adenosine A 1 and A 2A receptors antagonist ASP5854 improves motor impairments, is neuroprotective via A 2A antagonism, and also enhances cognitive function through A 1 antagonism.Adenosine is a ubiquitous neuromodulator in the central nervous system. Its major role in the central nervous system is to modulate neurotransmitter release, the postsynaptic components, and also the nonsynaptic components such as glial cell signaling. Adenosine exerts these diverse physiological actions through activation of specific G protein-coupled receptors termed A 1 , A 2A , A 2B , and A 3 (Fredholm et al., 1994).Adenosine A 2A receptors are specifically localized in the striatum (Svenningsson et al., 1999), where they are coexpressed with dopamine D 2 receptors in the GABAergic striaArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.121962. ABBREVIATIONS:CGS21680, 2-[p-(2-carboxyethyl)phenethylamino]-5Ј-N-ethylcarboxamidoadenosine; NECA, N-ethylcarboxamidoadenosine; KW-6002, 7-methyl-3,7-dihydro-1H-purine-2,6-dion...

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MScan be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.