TRESK Background K+ Channel Is Inhibited by PAR-1/MARK Microtubule Affinity-Regulating Kinases in Xenopus Oocytes

TRESK (TWIK-related spinal cord K+ channel, KCNK18) is a major background K+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279) in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel

SOIL SCIENTIFIC EVALUATION OF HAJDÚNÁNÁS-ZAGOLY ETA-01 KURGAN

Publisher Copyright: © 2022 Szent István University, Institute of Nature Conservation and Landscape Management. All rights reserved.Burial mounds, known as kurgans, are characteristic landscape monuments of the Central and Eastern European plains, including the Great Hungarian Plain. These formations are not only highly important from the archaeological and cultural heritage protection perspective. Their nature conservation value is also outstanding, as in many cases they are home to valuable flora and fauna elements. They are time capsules that hide invaluable information about the burial practice and the human remains placed in the grave. They are also unique in respect of environmental history. In the case of the kurgan, excavated at Zagolya-dulo near Hajdúnánás, the phenomenon of cultural recycling could be identified. The original Yamnaya burial mound was re-used many times by later cultures. Imprints of the Baden inhabitation, and footprints of much later cultures of the Migration Period and the Árpád Ages were also detected. In this paper we summarize the results of the systematic stratigraphic and the related archaeological features' soil analyses. In addition to the observations of the on-site soil examination (soil morphology), we also present the soil physical and chemical data obtained by high-resolution sampling and laboratory analyses. Data of the field survey and laboratory tests are supplemented by macroarchaeobotanical and phytolith analyses as well. By this interdisciplinary approach, we not only provide a detailed soil and sediment description of the kurgan, but also highlight the advantages of conjoint methodologies.Peer reviewe

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles

Expression of iNOS, CD163 and ARG-1 taken as M1 and M2 markers of microglial polarization in human glioblastoma and the surrounding normal parenchyma

Microglia and macrophages appear to be the most common cells in the GBM microenvironment. In the present study we investigated the status of macrophages/microglia activation in surgical specimens from 41 patients diagnosed with grade IV GBM. For each patient we analyzed both the center of tumor and the parenchyma surrounding the tumor. The specimens were stained for: i) IBA1, a 17-kDa EF hand protein specifically expressed in microglia/macrophages ii) CD163, a cell surface antigen associated with M2 phenotype; iii) iNOS, taken as a functional marker of M1 phenotype, and iv) ARG-I, taken as a functional marker of M2 phenotype. Staining was scored in a double-blinded score on a scale from 0 to 5. Our results suggest that CD163 expression is higher within the tumor than in surrounding periphery in both male and female patients; while iNOS is higher within the tumor in males, no significant difference was found for ARG-1. In addition, analyzing the data in TGCA database, we found that CD163 expression was significantly and inversely correlated with mean survival times, with average survival times ranging from 448 days in patients having low expression, to 319 in mid, and 353 in patients with high CD163 expressing tumors. In contrast, no significant association was found between survival time and ARG-1 or iNOS expression

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders