RESUMEN
Patients with dementia are increasing with the aging of the population, and dementia has become a disease with high unmet medical needs. Glucagon-like peptide-1 (GLP-1), a neuropeptide, has been reported to improve learning and memory following intracerebroventricular administration. We focused on intranasal administration, which can deliver drugs noninvasively and efficiently to the brain. Although much of the human nasal mucosa is occupied by respiratory epithelium, many capillaries are present in the paracellular route of respiratory epithelium. Therefore, to incorporate GLP-1 into cells, we created a GLP-1 derivative by adding cell-penetrating peptides (CPP) and penetration accelerating sequences (PAS) to GLP-1. We investigated in vitro and in vivo function of PAS-CPP-GLP-1 to enable the translocation of GLP-1 directly from nose to brain. PAS-CPP-GLP-1 enhanced cellular uptake by macropinocytosis with CPP, efficiently escaped from the endosomes due to PAS, and exited the cells. PAS-CPP-GLP-1 also transited trigeminal nerve cells through axon transport and migrated to the adjacent trigeminal nerve cell. Moreover, PAS-CPP-GLP-1 showed significant improvement in learning memory in mice within 20 min of intranasal administration. These results suggested CPP and PAS may be important for the efficient transfer of GLP-1 to the site of action in the brain following intranasal administration.
Asunto(s)
Demencia , Péptido 1 Similar al Glucagón , Ratones , Humanos , Animales , Encéfalo , Administración Intranasal , Demencia/tratamiento farmacológico , NeuronasRESUMEN
The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.
Asunto(s)
Fases del Sueño , Factor de Transcripción AP-2/genética , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Femenino , Intrones , Masculino , Ratones , Mutación Puntual , Factor de Transcripción AP-2/metabolismoRESUMEN
A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.