With Alzheimer's disease (AD) exhibiting reduced ability of neural stem cell renewal, we hypothesized that de novo mutations controlling embryonic development, in the form of brain somatic mutations instigate the disease. A leading gene presenting heterozygous dominant de novo autism-intellectual disabilities (ID) causing mutations is activity-dependent neuroprotective protein (ADNP), with intact ADNP protecting against AD-tauopathy. We discovered a genomic autism ADNP mutation (c.2188C>T) in postmortem AD olfactory bulbs and hippocampi. RNA-Seq of olfactory bulbs also identified a novel ADNP hotspot mutation, c.2187_2188insA. Altogether, 665 mutations in 596 genes with 441 mutations in AD patients (389 genes, 38% AD-exclusive mutations) and 104 genes presenting disease-causing mutations (OMIM) were discovered. OMIM AD mutated genes converged on cytoskeletal mechanisms, autism and ID causing mutations (about 40% each). The number and average frequencies of AD-related mutations per subject were higher in AD subjects compared to controls. RNA-seq datamining (hippocampus, dorsolateral prefrontal cortex, fusiform gyrus and superior frontal gyrus-583 subjects) yielded similar results. Overlapping all tested brain areas identified unique and shared mutations, with ADNP singled out as a gene associated with autism/ID/AD and presenting several unique aging/AD mutations. The large fusiform gyrus library (117 subjects) with high sequencing coverage correlated the c.2187_2188insA ADNP mutation frequency to Braak stage (tauopathy) and showed more ADNP mutations in AD specimens. In cell cultures, the ADNP-derived snippet NAP inhibited mutated-ADNP-microtubule (MT) toxicity and enhanced Tau-MT association. We propose a paradigm-shifting concept in the perception of AD whereby accumulating mosaic somatic mutations promote brain pathology.

• MeSH
• Alzheimerova nemoc * genetika   MeSH
• autistická porucha * genetika   MeSH
• homeodoménové proteiny genetika   MeSH
• lidé MeSH
• mentální retardace * MeSH
• mozek metabolismus   MeSH
• mutace MeSH
• proteiny nervové tkáně genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADNP protein, human MeSH Prohlížeč
• homeodoménové proteiny MeSH
• proteiny nervové tkáně MeSH

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane β-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of β-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that β-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that β-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and β-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

• Klíčová slova
• ERK1/2, TRPV1, biased signaling, receptor lateral mobility, β-arrestin 2, μ-opioid receptor,
• MeSH
• arrestiny metabolismus   MeSH
• beta arrestin 2 metabolismus   fyziologie   MeSH
• beta arrestiny metabolismus   MeSH
• buněčná membrána metabolismus   fyziologie   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV metabolismus   fyziologie   MeSH
• lidé MeSH
• MAP kinasový signální systém fyziologie   MeSH
• morfin metabolismus   MeSH
• opioidní analgetika metabolismus   MeSH
• receptory opiátové mu metabolismus   fyziologie   MeSH
• receptory opiátové metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arrestiny MeSH
• beta arrestin 2 MeSH
• beta arrestiny MeSH
• kationtové kanály TRPV MeSH
• morfin MeSH
• opioidní analgetika MeSH
• receptory opiátové mu MeSH
• receptory opiátové MeSH
• TRPV1 protein, human MeSH Prohlížeč

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin β signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin β and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin β in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

• Klíčová slova
• RanGTP, importazole, importin β, meiosis I, oocyte,
• MeSH
• aparát dělícího vřeténka fyziologie   MeSH
• beta karyoferiny genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• meióza fyziologie   MeSH
• mikrotubuly metabolismus   MeSH
• mutace MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• Ran protein vázající GTP genetika   metabolismus   MeSH
• segregace chromozomů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• beta karyoferiny MeSH
• jaderné proteiny MeSH
• proteiny buněčného cyklu MeSH
• Ran protein vázající GTP MeSH
• Rcc1 protein, mouse MeSH Prohlížeč
• výměnné faktory guaninnukleotidů MeSH

There are some indications that biased μ-opioid ligands may diversely affect μ-opioid receptor (MOR) properties. Here, we used confocal fluorescence recovery after photobleaching (FRAP) to study the regulation by different MOR agonists of receptor movement within the plasma membrane of HEK293 cells stably expressing a functional yellow fluorescent protein (YFP)-tagged μ-opioid receptor (MOR-YFP). We found that the lateral mobility of MOR-YFP was increased by (D-Ala2,N-MePhe4,Gly5-ol)-enkephalin (DAMGO) and to a lesser extent also by morphine but decreased by endomorphin-2. Interestingly, cholesterol depletion strongly enhanced the ability of morphine to elevate receptor mobility but significantly reduced or even eliminated the effect of DAMGO and endomorphin-2, respectively. Moreover, the ability of DAMGO and endomorphin-2 to influence MOR-YFP movement was diminished by pertussis toxin treatment. The results obtained by agonist-stimulated [35S]GTPγS binding assays indicated that DAMGO exhibited higher efficacy than morphine and endomorphin-2 did and that the efficacy of DAMGO, contrary to the latter agonists, was enhanced by cholesterol depletion. Overall, our study provides clear evidence that biased MOR agonists diversely affect receptor mobility in plasma membranes as well as MOR/G protein coupling and that the regulatory effect of different ligands depends on the membrane cholesterol content. These findings help to delineate the fundamental properties of MOR regarding their interaction with biased MOR ligands and cognate G proteins.

• Klíčová slova
• Biased agonists *, Cholesterol *, FRAP *, G protein coupling *, μ-Opioid receptor *,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• cholesterol nedostatek   MeSH
• enkefalin, Ala(2)-MePhe(4)-Gly(5)- metabolismus   farmakologie   MeSH
• FRAP MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• HEK293 buňky MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• ligandy MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• morfin metabolismus   farmakologie   MeSH
• narkotika - antagonisté farmakologie   MeSH
• oligopeptidy metabolismus   farmakologie   MeSH
• pertusový toxin farmakologie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go metabolismus   MeSH
• receptory opiátové mu agonisté   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• transfekce MeSH
• transport proteinů MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cholesterol MeSH
• endomorphin 2 MeSH Prohlížeč
• enkefalin, Ala(2)-MePhe(4)-Gly(5)- MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• ligandy MeSH
• luminescentní proteiny MeSH
• morfin MeSH
• narkotika - antagonisté MeSH
• oligopeptidy MeSH
• pertusový toxin MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
• receptory opiátové mu MeSH
• rekombinantní fúzní proteiny MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• analýza rozptylu MeSH
• bromodeoxyuridin MeSH
• chromatinová imunoprecipitace MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• fluorescence MeSH
• galaktosidy MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny metabolismus   MeSH
• hybridizace in situ MeSH
• imunohistochemie MeSH
• indoly MeSH
• koncové značení zlomů DNA in situ MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• metoda rotující tyčky MeSH
• mikročipová analýza MeSH
• morfogeneze genetika   fyziologie   MeSH
• mozeček embryologie   MeSH
• myši transgenní MeSH
• myši MeSH
• nervové kmenové buňky metabolismus   fyziologie   MeSH
• počítačové zpracování obrazu MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• Purkyňovy buňky metabolismus   MeSH
• restrukturace chromatinu fyziologie   MeSH
• toloniumchlorid MeSH
• transmisní elektronová mikroskopie MeSH
• vývojová regulace genové exprese genetika   fyziologie   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• 5-bromo-4-chloro-3-indolyl beta-galactoside MeSH Prohlížeč
• adenosintrifosfatasy MeSH
• bromodeoxyuridin MeSH
• chromozomální proteiny, nehistonové MeSH
• En1 protein, mouse MeSH Prohlížeč
• galaktosidy MeSH
• histony MeSH
• homeodoménové proteiny MeSH
• indoly MeSH
• Smarca5 protein, mouse MeSH Prohlížeč
• toloniumchlorid MeSH