Nitric oxide (NO) produced by inducible NO synthase contributes to ischemic brain damage. However, the role of inducible NO synthase-derived NO on neonatal hypoxic-ischemic encephalopathy has not been clarified. We demonstrate here that aminoguanidine, a relatively selective inhibitor of inducible NO synthase, ameliorated neonatal hypoxic-ischemic brain damage and that temporal profiles of NO correlated with the neuroprotective effect of aminoguanidine. Seven-day-old Wister rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure (8% oxygen). Infarct volumes (cortical and striatal) were assessed 72 h after the onset of hypoxia-ischemia by planimetric analysis of coronal brain slices stained with hematoxylin-eosin. Aminoguanidine (300 mg/kg i.p.), administered once before the onset of hypoxia-ischemia and then three times daily, significantly ameliorated infarct volume (89% reduction in the cerebral cortex and 90% in the striatum; p<0.001). NO metabolites were measured by means of chemiluminescence using an NO analyzer. In controls, there was a significant biphasic increase in NO metabolites in the ligated side at 1 h (during hypoxia) and at 72 h after the onset of hypoxia (p<0.05). Aminoguanidine did not suppress the first peak but significantly reduced the second one (p<0.05), and markedly reduced infarct size in a neonatal ischemic rat model. Suppression of NO production after reperfusion is a likely mechanism of this neuroprotection.
Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ-tailed AChE on muscle sections of Colq(-/-) mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C-terminal domain (CTD) abrogate anchoring ColQ-tailed AChE to the NMJ. In vitro plate-binding assay similarly demonstrated that the three mutants inhibit binding of ColQ-tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq(-/-) mice with adeno-associated virus serotype 8 carrying mutant COLQ-p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ-tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq(-/-) mice similarly failed to anchor ColQ-tailed AChE at the NMJ. We proved that the missense mutations in ColQ-CTD cause endplate AChE deficiency by compromising ColQ-MuSK interaction at the NMJ.
Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.
Although embryonic stem (ES) cells are capable of unlimited proliferation and pluripotent differentiation, effective preparation of neural stem cells from ES cells are not achieved. Here, we have directly generated under the coculture with dissociated primary neurosphere cells in serum-free medium and the same effect was observed when ES cells were cultured with conditioned medium of primary neurosphere culture (CMPNC). ES-neural stem cells (NSCs) could proliferate for more than seven times and differentiate into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. The responsible molecule in CMPNC was confirmed by matrix-assisted laser desorption͞ionization time-of-flight mass spectrometry, which turned out to be cystatin C. Purified cystatin C in place of the CMPNC could generate ES-NSCs efficiently with self-renewal and multidifferentiation potentials. These results reveal the validity of cystatin C for generating NSCs from ES cells.coculture system ͉ conditioned medium of primary neurosphere culture N eurogenesis is considered to be the most complex event of organogenesis during embryonic development and involves a precise signaling, along with cellular interaction cascade, to generate the functional cellular networks. The embryonic organizer allows cells in its vicinity to execute their default neural program by emitting bone morphogenetic protein antagonists (1, 2). However, other works suggest a more complex mechanism (3-5).Neural stem cells (NSCs) are the self-renewal, multipotent cells that generate neurons, astrocytes, and oligodendrocytes (6, 7). They have great potential as a therapeutic tool for the repair of a number of central nervous system (CNS) disorders. Several in vitro systems allowing derivation of neuronal progeny from embryonic stem (ES) cells, which differentiate into all of the cell fates in a developing embryo, have been described. However, attempts to exclusively generate NSCs or neural progenitor cells from ES cells are restricted. It has been shown that neural fates emerge from ES cells in the serum-free conditions (8, 9). Although these procedures are noteworthy, both the quality and the quantity of ES-derived neural cells are not sufficient for further examination or clinical applications. Higher levels of neural differentiation are achieved by treatment of embryoid bodies with retinoic acid (RA) in the presence of FCS (10, 11) or by coculture with particular stromal cell lines (12, 13). The action of RA is pleiotropic (14, 15), whereas the effect of several stromal cell lines is attributed to an undefined neural inducing activity. These factors severely restrict the ability to use cells cultured with FCS or with particular stromal feeder cells for therapeutic treatments.Here, we developed an efficient system for the generation of ES cell-derived NSCs (ES-NSCs) during coculture with dissociated neurosphere cells without a need for FCS or feeder cells, which creates a bottleneck for therapeutic methods. We demonstrated that neurosphere-derived factor (NDF), which induces...
Objective Duchenne muscular dystrophy (DMD) is a progressive muscular disease characterized by chronic cycles of inflammatory and necrotic processes. Prostaglandin D2 (PGD2) is produced by hematopoietic PGD synthase (HPGDS), which is pathologically implicated in muscle necrosis. This randomized, double‐blind, placebo‐controlled early phase 2 study (NCT02752048) aimed to assess the efficacy and safety of the novel selective HPGDS inhibitor, TAS‐205, with exploratory measures in male DMD patients aged ≥5 years. Methods Patients were randomized 1:1:1 to receive low‐dose TAS‐205 (6.67–13.33 mg/kg/dose), high‐dose TAS‐205 (13.33–26.67 mg/kg/dose), or placebo. The primary endpoint was the change from baseline in a 6‐minute walk distance (6MWD) at Week 24. Results Thirty‐six patients were enrolled, of whom 35 patients were analysed for safety. The mean (standard error) changes from baseline to Week 24 in 6MWD were −17.0 (17.6) m in the placebo group (n = 10), −3.5 (20.3) m in the TAS‐205 low‐dose group (n = 11), and −7.5 (11.2) m in the TAS‐205 high‐dose group (n = 11). The mean (95% confidence interval) difference from the placebo group was 13.5 (−43.3 to 70.2) m in the TAS‐205 low‐dose group and 9.5 (−33.3 to 52.4) m in the TAS‐205 high‐dose group. No obvious differences were observed in the incidences of adverse events between treatment groups. No adverse drug reactions specific to TAS‐205 treatment were observed. Interpretation The HPGDS inhibitor TAS‐205 showed a favorable safety profile in DMD patients. Further research is required to examine the effectiveness of TAS‐205 in a larger trial.
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