Increased oxygen stimulation promotes chemoresistance and phenotype shifting through PLCB1 in gliomas.

Gliomas, the most common CNS (central nerve system) tumors, face poor survival due to severe chemoresistance exacerbated by hypoxia. However, studies on whether altered hypoxic conditions benefit for chemo-sensitivity and how gliomas react to increased oxygen stimulation are limited. In this study, we demonstrated that increased oxygen stimulation promotes glioma growth and chemoresistance. Mechanically, increased oxygen stimulation upregulates miR-1290 levels. miR-1290, in turn, downregulates PLCB1, while PLCB1 facilitates the proteasomal degradation of ß-catenin and active-ß-catenin by increasing the proportion of ubiquitinated ß-catenin in a destruction complex-independent mechanism. This process inhibits PLCB1 expression, leads to the accumulation of active-ß-catenin, boosting Wnt signaling through an independent mechanism and ultimately promoting chemoresistance in glioma cells. Pharmacological inhibition of Wnt by WNT974 could partially inhibit glioma volume growth and prolong the shortened survival caused by increased oxygen stimulation in a glioma-bearing mouse model. Moreover, PLCB1, a key molecule regulated by increased oxygen stimulation, shows promising predictive power in survival analysis and has great potential to be a biomarker for grading and prognosis in glioma patients. These results provide preliminary insights into clinical scenarios associated with altered hypoxic conditions in gliomas, and introduce a novel perspective on the role of the hypoxic microenvironment in glioma progression. Furthermore, the outcomes reveal the potential risks of utilizing hyperbaric oxygen treatment (HBOT) in glioma patients, particularly when considering HBOT as a standalone option to ameliorate neuro-dysfunctions or when combining HBOT with a single chemotherapy agent without radiotherapy.

The involvement of CaMKKI in activating AMPKα in yesso scallop Patinopecten yessoensis under high temperature stress.

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5'-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca2+). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.

Involvement of metabotropic glutamate receptors in regulation of immune response in the Pacific oyster Crassostrea gigas.

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks.

Genome-wide identification of the HSP70 genes in Pacific oyster Magallana gigas and their response to heat stress.

Heat shock protein 70 (HSP70), the most prominent and well-characterized stress protein in animals, plays an important role in assisting animals in responding to various adverse conditions. In the present study, a total of 113 HSP70 gene family members were identified in the updated genome of Magallana gigas (designated MgHSP70) (previously known as Crassostrea gigas). There were 75, 12, 11, and 8 HSP70s located in the cytoplasm, nucleus, mitochondria, and endoplasmic reticulum, respectively, and 7 HSP70s were located in both the nucleus and cytoplasm. Among 113 MgHSP70 genes, 107 were unevenly distributed in 8 chromosomes of M. gigas with the greatest number in chromosome 07 (61 genes, 57.01%). The MgHSP70 gene family members were mainly assigned into five clusters, among which the HSPa12 subfamily underwent lineage-specific expansion, consisting of 89 members. A total of 68 MgHSP70 genes (60.18%) were tandemly duplicated and formed 30 gene pairs, among which 14 gene pairs were under strong positive selection. In general, the expression of MgHSP70s was tissue-specific, with the highest expression in labial palp and gill and the lowest expression in adductor muscle and hemocytes. There were 35, 31, and 47 significantly upregulated genes at 6, 12, and 24 h after heat shock treatment (28 °C), respectively. The expression patterns of different tandemly duplicated genes exhibited distinct characteristics after shock treatment, indicating that these genes may have different functions. Nevertheless, genes within the same tandemly duplicated group exhibit similar expression patterns. Most of the tandemly duplicated HSP70 gene pairs showed the highest expression levels at 24 h. This study provides a comprehensive description of the MgHSP70 gene family in M. gigas and offers valuable insights into the functions of HSP70 in the mollusc adaptation of oysters to environmental stress.

Hypothermia protects the integrity of corticospinal tracts and alleviates mitochondria injury after intracerebral hemorrhage in mice.

Disruption of corticospinal tracts (CST) is a leading factor for motor impairments following intracerebral hemorrhage (ICH) in the striatum. Previous studies have shown that therapeutic hypothermia (HT) improves outcomes of ICH patients. However, whether HT has a direct protection effect on the CST integrity and the underlying mechanisms remain largely unknown. In this study, we employed a chemogenetics approach to selectively activate bilateral warm-sensitive neurons in the preoptic areas to induce a hypothermia-like state. We then assessed effects of HT treatment on the integrity of CST and motor functional recovery after ICH. Our results showed that HT treatment significantly alleviated axonal degeneration around the hematoma and the CST axons at remote midbrain region, ultimately promoted skilled motor function recovery. Anterograde and retrograde tracing revealed that HT treatment protected the integrity of the CST over an extended period. Mechanistically, HT treatment prevented mitochondrial swelling in degenerated axons around the hematoma, alleviated mitochondrial impairment by reducing mitochondrial ROS accumulation and improving mitochondrial membrane potential in primarily cultured cortical neurons with oxyhemoglobin treatment. Serving as a proof of principle, our study provided novel insights into the application of HT to improve functional recovery after ICH.

An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG.

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

PyLKB1 regulates glucose transport via activating PyAMPKα in Yesso Scallop Patinopecten yessoensis under high temperature stress.

Liver kinase B1 (LKB1) is a classical serine/threonine protein kinase and plays an important role in maintaining energy homeostasis through phosphorylate AMP-activated protein kinase α subunit (AMPKα). In this study, a homologous molecule of LKB1 with a typical serine/threonine kinase domain and two nuclear localization sequences (NLSs) was identified in Yesso Scallop Patinopecten yessoensis (PyLKB1). The mRNA transcripts of PyLKB1 were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in hepatopancreas. PyLKB1 was mainly located in cytoplasm and nucleus of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyLKB1, PyAMPKα, and PyGLUT1 in hepatopancreas, the phosphorylation level of PyAMPKα at Thr170 in hepatopancreas, the positive fluorescence signals of PyLKB1 in haemocytes, glucose analogue 2-NBDG content in haemocytes, and glucose content in hepatopancreas, haemocytes and serum all increased significantly (p < 0.05) compared to blank group (15 °C). However, there was no significant difference at the protein level of PyLKB1 and PyAMPKα. After PyLKB1 was knockdown by siRNA, the mRNA expression level of PyGLUT1, and the glucose content in hepatopancreas and serum were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. However, there were no significant difference in PyGLUT1 expression, glucose content and glucose analogue 2-NBDG content in haemocytes. These results collectively suggested that PyLKB1-PyAMPKα pathway was activated to promote glucose transport by regulating PyGLUT1 in response to high temperature stress. These results would be helpful for understanding the function of PyLKB1-PyAMPKα pathway in regulating glucose metabolism and maintaining energy homeostasis under high temperature stress in scallops.

Chemogenetic stimulation of intact corticospinal tract during rehabilitative training promotes circuit rewiring and functional recovery after stroke.

BACKGROUND: Neuromodulatory techniques have been proven to enhance functional recovery after stroke in patients and animals, such as repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS). However, the success and feasibility of these approaches were often variable, largely due to a lack of target specificity. OBJECTIVE: We explored the effects of specific chemogenetic stimulation of intact corticospinal tract during rehabilitative training on functional recovery after stroke in mice. METHODS: We developed a viral-based intersectional targeting approach that allows specific chemogentic activation of contralateral hindlimb corticospinal neurons (CSNs) in a photothrombotic stroke model. RESULTS: We demonstrated that specific chemogenetic activation of CSNs, when combined with daily rehabilitation training, leads to significant skilled motor functional recovery via promoting corticospinal tract (CST) axons midline crossing sprouting from intact to the denervated spinal hemicord, and rewiring new functional circuits by new synapse formation. Mechanistically, we revealed that combined chemogenetic stimulation of CSNs and daily rehabilitation training significantly enhanced the mTOR activity of CSNs. CONCLUSIONS: Our findings highlight the great potential of specific neural activation protocols in combination with motor training for the recovery of skilled motor functions after stroke.

Applications of Induced Pluripotent Stem Cell-Derived Glia in Brain Disease Research and Treatment.

Glia are integral components of neural networks and are crucial in both physiological functions and pathological processes of the brain. Many brain diseases involve glial abnormalities, including inflammatory changes, mitochondrial damage, calcium signaling disturbance, hemichannel opening, and loss of glutamate transporters. Induced pluripotent stem cell (iPSC)-derived glia provide opportunities to study the contributions of glia in human brain diseases. These cells have been used for human disease modeling as well as generating new therapies. This chapter introduces glial involvement in brain diseases, then summarizes different methods of generating iPSC-derived glia disease models of these cells. Finally, strategies for treating disease using iPSC-derived glia are discussed. The goal of this chapter is to provide an overview and shed light on the applications of iPSC-derived glia in brain disease research and treatment.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

The intestinal bacterial community over seasons and its relationship with physiological status of Yesso scallop Patinopecten yessoensis.

Emerging evidence indicates that the intestinal bacterial communities associated with eukaryotes play critical roles in the physiological activities and health of their hosts. Yesso scallop Patinopecten yessoensis, one of the cold-water aquaculture species in the North Yellow Sea of China, has suffered from massive mortality in recent years. In the present study, P. yessoensis were collected from Zhangzi Island, Dalian from March 2021 to January 2022 to investigate the intestinal bacterial community and physiological indices. 16S rRNA gene sequencing data revealed that the diversity of intestinal bacteria changed significantly over seasons, with the highest Chao1 (237.42) and Shannon (6.13) indices detected in January and the lowest Chao1 (115.44) and Shannon (2.73) indices detected in July. Tenericutes, Proteobacteria and Firmicutes were dominant phyla in the intestinal bacteria of P. yessoensis, among which Firmicutes and Proteobacteria significantly enriched in August and January, respectively. Mycoplasma was the most abundant genus during the sampling period, which exhibited the highest abundance in October (75.26%) and lowest abundance in August (13.15%). The functional profiles of intestinal bacteria also exhibited seasonal variation, with the pathways related to pentose phosphate and deoxyribonucleotides biosynthesis enriched in August while the glycogen biosynthesis pathway enriched in October. Redundancy analysis showed that seawater pH, dissolved inorganic nitrogen and silicate were major environmental factors driving the temporal succession of scallop intestinal bacteria. Correlation clustering analysis suggested that the relative abundances of Endozoicomonas and Vibrio in the intestine were positively correlated with superoxide dismutase activity in hepatopancreas while negatively correlated with malondialdehyde content in hepatopancreas and glycogen content in adductor muscle. All the results revealed that the intestine harbored a lower bacterial diversity and a higher abundance of Vibrio in August, compared to January, which were closely related to the oxidative stress status of scallop in summer. These findings will advance our understanding of the relationship between seasonal alteration in the intestinal bacteria and the physiological status of scallops.

A neural cell adhesion molecule from oyster Crassostrea gigas: Molecular identification and immune functional characterization.

Neural cell adhesion molecules (NCAMs) are large cell-surface glycoproteins playing important roles in cell-cell and cell-extracellular matrix interactions in nervous system. Recent study identified a homologue of NCAM (CgNCAM) from the Pacific oyster Crassostrea gigas. Its ORF was of 2634 bp which encodes a protein (877 amino acids) consisting of five immunoglobulin domains and two fibronectin type III domains. CgNCAM transcripts were broadly distributed in oyster tissues especially in mantle, labial palp and haemolymph. CgNCAM showed up-regulated expression in haemocytes of oysters after Vibrio splendidus and Staphylococcus aureus stimulation. The recombinant CgNCAM protein (rCgNCAM) was able to bind manose, lipopolysaccharide and glucan, as well as different microbes including Gram-negative bacteria and fungi. rCgNCAM displayed bacterial agglutination and hemagglutination activity. CgNCAM improved the phagocytosis of haemocytes towards V. splendidus by regulating the expression of CgIntegrin, CgRho J and CgMAPKK. Moreover, CgNCAM was involved in the extracellular trap establishment of haemocytes after V. splendidus stimulation. The results collectively indicated that CgNCAM acted as a recognition receptor executing multiple immune functions to recognize and eliminate invading microorganisms in innate immunity of oysters.

Serum-based Raman spectroscopic diagnosis of blast-induced brain injury in a rat model.

The diagnosis of blast-induced traumatic brain injury (bTBI) is of paramount importance for early care and clinical therapy. Therefore, the rapid diagnosis of bTBI is vital to the treatment and prognosis in clinic. In this paper, we reported a new strategy for label-free bTBI diagnosis through serum-based Raman spectroscopy. The Raman spectral characteristics of serum in rat were investigated at 3 h, 24 h, 48 h and 72 h after mild and moderate bTBIs. It has been demonstrated that both the position and intensity of Raman characteristic peaks exhibited apparent differences in the range of 800-3000cm-1 compared with control group. It could be inferred that the content, structure and interaction of biomolecules in the serum were changed after blast exposure, which might help to understand the neurological syndromes caused by bTBI. Furthermore, the control group, mild and moderate bTBIs at different times (a total of 9 groups) were automatically classified by combining principal component analysis and four machine learning algorithms (quadratic discriminant analysis, support vector machine, k-nearest neighbor, neural network). The highest classification accuracy, sensitivity and precision were up to 95.4%, 95.9% and 95.7%. It is suggested that this method has great potential for high-sensitive, rapid, and label-free diagnosis of bTBI.

Effect of high temperature stress on glycogen metabolism in gills of Yesso scallop Patinopecten yessoensis.

Glycogen is the main energy storage material in mollusc, and the regulation of its metabolism is essential for the response against high temperature stress. In the present study, the alternation of lactic acid (LD) content, glycogen reserves, mRNA expression level of genes encoding glycogen metabolism enzymes and activities of glycogen metabolism enzymes in gills of Yesso scallop Patinopecten yessoensis after an acute high temperature treatment at 25 °C were examined to understand the effect of high temperature on glycogen metabolism. The activity of T-ATPase in gills of scallops presented a gradual increase trend especially at 6 h after the acute high temperature treatment (p < 0.05). The glycogen reserves did not change significantly even there was a downward trend at 24 h after the acute high temperature treatment (p > 0.05). The mRNA transcripts of glycogen synthase (PyGCS) in gills of scallops decreased significantly at 1, 3, 6 and 12 h (p < 0.05), and recovered to normal level at 24 h (p > 0.05) after the acute high temperature treatment, while that of glycogen phosphorylase a (PyGPa) and phosphoenol pyruvate carboxy kinase (PyPEPCK) were both significantly down-regulated from 1 h to 24 h (p < 0.05) after the acute high temperature treatment. The activity of PyGPa at 1, 12 and 24 h and the content of LD at 3 and 24 h in gills of scallops after the acute high temperature treatment both increased significantly (p < 0.05). Furthermore, the mRNA transcripts of hexokinase (PyHK) and pyruvate kinase (PyPK) in gills of scallops increased significantly (p < 0.05) after the acute high temperature treatment, and the response of PyHK was stronger. However, there was no significant difference on the activity of PyPK in gills of scallops between the experimental samples and the blank samples (p > 0.05). In addition, the mRNA transcripts of citrate synthase (PyCS) in gills of scallops were significantly down-regulated at 6 h and 12 h (p < 0.05), and finally returned to normal level at 24 h (p > 0.05) after the acute high temperature treatment. These results collectively indicated acute high temperature stress leaded the alternation of glycogen metabolism in the gills of Yesso scallop, glycogenesis, gluconeogenesis and TCA cycle were inhibited, and the glycolysis pathway of glycogen was enhanced to produce more energy for coping with environmental pressure.

Characterization of the role of TMEM175 in an in vitro lysosomal H+ fluxes model.

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.

Functional characterization and in vitro pharmacological rescue of KCNQ2 pore mutations associated with epileptic encephalopathy.

Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.

Acetylated α-tubulin alleviates injury to the dendritic spines after ischemic stroke in mice.

BACKGROUND AND AIM: Functional recovery is associated with the preservation of dendritic spines in the penumbra area after stroke. Previous studies found that polymerized microtubules (MTs) serve a crucial role in regulating dendritic spine formation and plasticity. However, the mechanisms that are involved are poorly understood. This study is designed to understand whether the upregulation of acetylated α-tubulin (α-Ac-Tub, a marker for stable, and polymerized MTs) could alleviate injury to the dendritic spines in the penumbra area and motor dysfunction after ischemic stroke. METHODS: Ischemic stroke was mimicked both in an in vivo and in vitro setup using middle cerebral artery occlusion and oxygen-glucose deprivation models. Thy1-YFP mice were utilized to observe the morphology of the dendritic spines in the penumbra area. MEC17 is the specific acetyltransferase of α-tubulin. Thy1 CreERT2-eYFP and MEC17fl/fl mice were mated to produce mice with decreased expression of α-Ac-Tub in dendritic spines of pyramidal neurons in the cerebral cortex. Moreover, AAV-PHP.B-DIO-MEC17 virus and tubastatin A (TBA) were injected into Thy1 CreERT2-eYFP and Thy1-YFP mice to increase α-Ac-Tub expression. Single-pellet retrieval, irregular ladder walking, rotarod, and cylinder tests were performed to test the motor function after the ischemic stroke. RESULTS: α-Ac-Tub was colocalized with postsynaptic density 95. Although knockout of MEC17 in the pyramidal neurons did not affect the density of the dendritic spines, it significantly aggravated the injury to them in the penumbra area and motor dysfunction after stroke. However, MEC17 upregulation in the pyramidal neurons and TBA treatment could maintain mature dendritic spine density and alleviate motor dysfunction after stroke. CONCLUSION: Our study demonstrated that α-Ac-Tub plays a crucial role in the maintenance of the structure and functions of mature dendritic spines. Moreover, α-Ac-Tub protected the dendritic spines in the penumbra area and alleviated motor dysfunction after stroke.

Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice.

The mitochondrial permeability transition pore is a nonspecific transmembrane channel. Inhibition of mitochondrial permeability transition pore opening has been shown to alleviate mitochondrial swelling, calcium overload, and axonal degeneration. Cyclophilin D is an important component of the mitochondrial permeability transition pore. Whether cyclophilin D participates in mitochondrial impairment and axonal injury after intracerebral hemorrhage is not clear. In this study, we established mouse models of intracerebral hemorrhage in vivo by injection of autologous blood and oxyhemoglobin into the striatum in Thy1-YFP mice, in which pyramidal neurons and axons express yellow fluorescent protein. We also simulated intracerebral hemorrhage in vitro in PC12 cells using oxyhemoglobin. We found that axonal degeneration in the early stage of intracerebral hemorrhage depended on mitochondrial swelling induced by cyclophilin D activation and mitochondrial permeability transition pore opening. We further investigated the mechanism underlying the role of cyclophilin D in mouse models and PC12 cell models of intracerebral hemorrhage. We found that both cyclosporin A inhibition and short hairpin RNA interference of cyclophilin D reduced mitochondrial permeability transition pore opening and mitochondrial injury. In addition, inhibition of cyclophilin D and mitochondrial permeability transition pore opening protected corticospinal tract integrity and alleviated motor dysfunction caused by intracerebral hemorrhage. Our findings suggest that cyclophilin D is used as a key mediator of axonal degeneration after intracerebral hemorrhage; inhibition of cyclophilin D expression can protect mitochondrial structure and function and further alleviate corticospinal tract injury and motor dysfunction after intracerebral hemorrhage. Our findings provide a therapeutic target for preventing axonal degeneration of white matter injury and subsequent functional impairment in central nervous diseases.

Astrocytes exhibit diverse Ca2+ changes at subcellular domains during brain aging.

Astrocytic Ca2+ transients are essential for astrocyte integration into neural circuits. These Ca2+ transients are primarily sequestered in subcellular domains, including primary branches, branchlets and leaflets, and endfeet. In previous studies, it suggests that aging causes functional defects in astrocytes. Until now, it was unclear whether and how aging affects astrocytic Ca2+ transients at subcellular domains. In this study, we combined a genetically encoded Ca2+ sensor (GCaMP6f) and in vivo two-photon Ca2+ imaging to determine changes in Ca2+ transients within astrocytic subcellular domains during brain aging. We showed that aging increased Ca2+ transients in astrocytic primary branches, higher-order branchlets, and terminal leaflets. However, Ca2+ transients decreased within astrocytic endfeet during brain aging, which could be caused by the decreased expressions of Aquaporin-4 (AQP4). In addition, aging-induced changes of Ca2+ transient types were heterogeneous within astrocytic subcellular domains. These results demonstrate that the astrocytic Ca2+ transients within subcellular domains are affected by aging differently. This finding contributes to a better understanding of the physiological role of astrocytes in aging-induced neural circuit degeneration.

The expression profile of a multi-stress inducible transient receptor potential vanilloid 4 (TRPV4) in Pacific oyster Crassostrea gigas.

Transient receptor potential vanilloid 4 (TRPV4) is one of the major non-selective cation channel proteins, which plays a crucial role in sensing biotic and abiotic stresses, such as pathogen infection, temperature, mechanical pressure and osmotic pressure changes by regulating Ca2+ homeostasis. In the present study, a TRPV4 homologue was identified in Pacific oyster Crassostrea gigas, designated as CgTRPV4. The open reading frame (ORF) of CgTRPV4 was of 2298 bp encoding a putative polypeptide of 765 amino acid residues with three typical ankyrin domains and six conserved transmembrane domains of TRPV4 subfamily proteins, as well as multiple N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, and prokaryotic membrane lipoprotein lipid attachment site. The deduced amino acid sequence of CgTRPV4 shared 20.5%-26.2% similarity with TRPV4s from other species. During the early ontogenesis stages of oyster, the mRNA transcripts of CgTRPV4 were detectable in all the stages with the highest expression level in fertilized eggs and the lowest in D-hinged larvae. In adult oyster, the CgTRPV4 mRNA could be detected in all the examined tissues, including gill, hepatopancreas, adductor muscle, labial palp, mantle and haemocyte, with the highest expression level in gill (45.08-fold of that in hepatopancreas, p < 0.05). In immunocytochemical assay, the CgTRPV4 positive signals were distributed in both endoplasmic reticulum and cytoplasmic membrane of oyster haemocytes. The mRNA expression of CgTRPV4 in gill was significantly up-regulated after high temperature stress at 30°C (p < 0.05) and after Vibrio splendidus stimulation (p < 0.05). These results indicated that CgTRPV4 was a classical member of TRPV4 family in oyster, which was induced by either biotic or abiotic stimulations and involved in mediating the stress response of oysters.