RESUMO
BACKGROUND: Sleep/wake disturbances (e.g., insomnia and sleep fragmentation) are common in neurodegenerative disorders, especially Alzheimer's disease (AD) and frontotemporal dementia (FTD). These symptoms are somewhat reminiscent of narcolepsy with cataplexy, caused by the loss of orexin-producing neurons. A bidirectional relationship between sleep disturbance and disease pathology suggests a detrimental cycle that accelerates disease progression and cognitive decline. The accumulation of brain tau fibrils is a core pathology of AD and FTD-tau and clinical evidence supports that tau may impair the orexin system in AD/FTD. This hypothesis was investigated using tau mutant mice. OBJECTIVE: To characterize orexin receptor mRNA expression in sleep/wake regulatory brain centers and quantify noradrenergic locus coeruleus (LC) and orexinergic lateral hypothalamus (LH) neurons, in tau transgenic rTg4510 and tau-/- mice. METHODS: We used i n situ hybridization and immunohistochemistry (IHC) in rTg4510 and tau-/- mice. RESULTS: rTg4510 and tau-/- mice exhibited a similar decrease in orexin receptor 1 (OX1R) mRNA expression in the LC compared with wildtype controls. IHC data indicated this was not due to decreased numbers of LC tyrosine hydroxylase-positive (TH) or orexin neurons and demonstrated that tau invades TH LC and orexinergic LH neurons in rTg4510 mice. In contrast, orexin receptor 2 (OX2R) mRNA levels were unaffected in either model. CONCLUSION: The LC is strongly implicated in the regulation of sleep/wakefulness and expresses high levels of OX1R. These findings raise interesting questions regarding the effects of altered tau on the orexin system, specifically LC OX1Rs, and emphasize a potential mechanism which may help explain sleep/wake disturbances in AD and FTD.
Assuntos
Nível de Alerta , Locus Cerúleo/metabolismo , Receptores de Orexina/metabolismo , Proteínas tau/metabolismo , Animais , Feminino , Região Hipotalâmica Lateral/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/metabolismoRESUMO
Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
Assuntos
Encéfalo/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Mutação , Animais , Cristalografia por Raios X , Proteínas de Fluorescência Verde/genética , Ligação de Hidrogênio , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Conformação Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Transdução GenéticaRESUMO
Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-ß (GSK3ß) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3ß kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3ß-dependent phosphorylation in SH-SY5Y cells.