The Daam2-VHL-Nedd4 axis governs developmental and regenerative oligodendrocyte differentiation.

Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.

Daam2 phosphorylation by CK2α negatively regulates Wnt activity during white matter development and injury.

Wnt signaling plays an essential role in developmental and regenerative myelination in the central nervous system. The Wnt signaling pathway is composed of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte (OL) development remains unclear. Here, we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt activity during OL development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of OL development, accelerating early differentiation followed by decelerating maturation and myelination. Application toward white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a unique regulatory node in the Wnt pathway that regulates OL development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration.

Daam2 Regulates Myelin Structure and the Oligodendrocyte Actin Cytoskeleton through Rac1 and Gelsolin.

Myelin is essential to neuronal health and CNS function, and oligodendrocytes (OLs) undergo a complex process of cytoskeletal remodeling to form compact myelin sheaths. We previously discovered that a formin protein, Dishevelled associated activator of morphogenesis 2 (Daam2), suppresses OL differentiation through Wnt signaling; however, its role in cytoskeletal control remains unknown. To investigate this, we used OL-specific Daam2 conditional knockout (Daam2 cKO) mice of either sex and found myelin decompaction during an active period of myelination in postnatal development and motor coordination deficits in adulthood. Using primary OL cultures, we found Daam2-depleted OLs showed morphologic dysregulation during differentiation, suggesting that Daam2 regulates the OL cytoskeleton. In vivo screening identified the actin regulators Rac1 and Gelsolin as possible effectors in Daam2-deficient OL cytoskeletal regulation. Using gain-of-function and loss-of-function (LOF) experiments in primary OLs, we found that Rac1 and Gelsolin operate downstream of Daam2 in OL differentiation, with Gelsolin and Daam2 promoting and inhibiting membrane spreading during late differentiation, respectively. In vivo experiments using Daam2 cKO mice revealed increased protein levels of Gelsolin in the developing white matter with no change in RNA levels, suggesting that Daam2 acts in a posttranslational manner to suppress Gelsolin levels. In vitro biochemical studies show Daam2 induces Gelsolin ubiquitination and degradation in OLs. Together, our studies show Daam2 is essential for formation of functional myelin through modulation of Gelsolin levels to regulate the OL cytoskeleton. These findings further demonstrate the critical role of cytoskeletal dynamics in myelination and reveal novel avenues for treatment of a variety of white matter diseases.SIGNIFICANCE STATEMENT Proper myelin formation is essential to CNS function, and oligodendrocytes (OLs) require extensive changes in the actin cytoskeleton to form myelin sheaths. Here, we show that the formin protein Dishevelled associated activator of morphogenesis 2 (Daam2) is necessary for myelin compaction during development and motor learning in adulthood. Further, we demonstrate that Daam2 regulates OL differentiation and morphology through actin regulators Rac1 and Gelsolin. Lastly, we find that Daam2 may control myelin compaction by modulating the ubiquitination and degradation of Gelsolin through recruitment of the E3 ubiquitin ligase Nedd4. These findings reveal novel pathways for regulating myelin structure and function during white matter development.

Daam2 couples translocation and clustering of Wnt receptor signalosomes through Rac1.

Wnt signaling plays a critical role in development across species and is dysregulated in a host of human diseases. A key step in signal transduction is the formation of Wnt receptor signalosomes, during which a large number of components translocate to the membrane, cluster together and amplify downstream signaling. However, the molecular processes that coordinate these events remain poorly defined. Here, we show that Daam2 regulates canonical Wnt signaling via the PIP2-PIP5K axis through its association with Rac1. Clustering of Daam2-mediated Wnt receptor complexes requires both Rac1 and PIP5K, and PIP5K promotes membrane localization of these complexes in a Rac1-dependent manner. Importantly, the localization of Daam2 complexes and Daam2-mediated canonical Wnt signaling is dependent upon actin polymerization. These studies - in chick spinal cord and human and monkey cell lines - highlight novel roles for Rac1 and the actin cytoskeleton in the regulation of canonical Wnt signaling and define Daam2 as a key scaffolding hub that coordinates membrane translocation and signalosome clustering.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function.

Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.

Magnetically Modulated Fluorescence of Nitrogen-Vacancy Centers in Nanodiamonds for Ultrasensitive Biomedical Analysis.

The negatively charged nitrogen-vacancy center in fluorescent nanodiamonds (FNDs) is a point defect with unique magneto-optical properties. It emits far-red fluorescence at ∼700 nm, and its intensity can be magnetically modulated with a depth of more than 10% at a field strength of 30 mT. We have closely examined this property and illustrated its practical use in biomedicine by applying a periodic, time-varying magnetic field to FNDs deposited on a surface or dispersed in a solution with a lock-in detection method. We achieved selective and sensitive detection of 100 nm FNDs on a nitrocellulose membrane at a particle density of 0.04 ng/mm2 (or ∼2 × 104 particles/mm2) and in an aqueous solution with a particle concentration of 1 ng/mL (or ∼1 fM) in 10 s as the detection limits. The utility and versatility of the technique were demonstrated with an application to background-free detection of FNDs as reporters for FND-based lateral flow immunoassays as well as selective quantification of FNDs in tissue digests for in vivo studies.

The selective lipoprotein-associated phospholipase A2 inhibitor darapladib triggers irreversible actions on glioma cell apoptosis and mitochondrial dysfunction.

Although the development of a therapeutic strategy for glioblastoma multiforme (GBM), the most aggressive type of brain tumor in adults, is in progress, the prognosis is still limited. In this study, we evaluated the anti-glioma effects of darapladib, a selective reversible inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2) that is encoded by the PLA2G7 gene and serves as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases. The three glioma cell lines (rat C6 glioma cell line, human U87MG, and human U251MG) and an ex vivo brain tissue slice-glioma cell co-culture system were used to validate the inhibitory effect of darapladib on the expansion of glioma cells. Exposure to darapladib at doses higher than 5 µM induced profound cytotoxicity in C6, U87MG, and U251MG. Moreover, the colony formation ability of the glioma cell lines was significantly repressed after the addition of darapladib. Although darapladib did not reduce the generation of the Lp-PLA2 downstream molecule, arachidonic acid (AA), in the glioma cells, this small compound triggered mitochondrial membrane depolarization and cell apoptosis in these glioma cells. In addition, transient exposure to darapladib induced the upregulation of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) levels, but reduced phosphorylation of AKT/PKB (protein kinase B). The results from an ex vivo brain slice culture system further confirmed the effective inhibition of darapladib on the expansion of glioma cells. In conclusion, darapladib acts as a potential anti-glioma compound via the induction of mitochondrial membrane depolarization and cell apoptosis, and the inhibition of AKT signaling in glioma cells.

BRCA1/BRCA2-containing complex subunit 3 controls oligodendrocyte differentiation by dynamically regulating lysine 63-linked ubiquitination.

Oligodendrocytes (OLs) provide the myelin sheath surrounding axons that propagates action potentials in the central nervous system (CNS). The metabolism of myelinated membranes and proteins is strictly regulated in the OLs and is closely associated with OL differentiation and maturation. The ubiquitination-associated proteasome and endosomal system have not yet been well studied during OL differentiation and maturation. Here, we determined the functions of the Lys63-linked ubiquitination (K63Ub) and K63-specific deubiquitination (DUB) systems regulated by BRCA1/BRCA2-containing complex subunit 3 (BRCC3) during OL differentiation. The competitive inhibition of K63Ub by overexpression of mutant ubiquitin (K63R) in oligodendrocyte precursor cells (OPCs) indicated that the two major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), were upregulated in OLs derived from K63R OPCs. In contrast, the knockdown of BRCC3 (BRCC3-KD) through the application of lentivirus-mediated shRNA delivery system into OPCs suppressed OL differentiation by decreasing MBP expression and PLP production. Further immunoprecipitation assays revealed higher levels of sphingolipid GalC, MBP, and PLP, which were associated with K63Ub-immunoprecipitants and detected in endosome/lysosomal compartments, in BRCC3-KD OLs than those in OLs transfected with the scrambled shRNA (scramble OLs). The differentiation of OLs from BRCC3-KD OPCs was impaired in the demyelinating corpus callosum of rats receiving a cuprizone-containing diet. In the demyelinating tissues from human patients suffering from multiple sclerosis, we detected a decreased number of BRCC3-expressing OLs at the lesion site, accompanied by a greater number of OLs expressing EEA1 and K63Ub at high levels. Altogether, the counterbalance of the K63Ub machinery and BRCC3-triggered DUB machinery are important for the cellular trafficking of myelin proteins and OL differentiation.

Down-regulation of interleukin-33 expression in oligodendrocyte precursor cells impairs oligodendrocyte lineage progression.

Interleukin-33 (IL-33), a member of the IL1 family, has been found to be expressed in oligodendrocytes (OLGs) and released as an alarmin from injured OLGs to work on other glial cell-types in the central nervous system. However, its functional role in OLGs remains unclear. Herein, we present that IL-33 was mainly expressed in the nucleus of CC1+ -oligodendrocytes (OLGs) in mouse and rat corpus callosum, as well as NG2+ -oligodendrocyte precursor cells (OPCs). The in vitro study indicated that the amount of IL-33 expressing in OPCs was higher when compared to that detected in OLGs. Results from the experiments using lentivirus-mediated shRNA delivery against IL-33 expression (IL33-KD) in OPCs showed that IL33-KD reduced the differentiation of OLGs into mature OLGs along with the down-regulation of OLG differentiation-related genes and mature OLG marker proteins, myelin basic protein (MBP) and proteolipid protein (PLP). Alternatively, we observed reduced differentiation of OLGs that were prepared from the brains of IL-33 gene knockout (IL33-KO) mice with anxiolytic-like behavior. Observations were correlated with the results showing lower levels of MBP and PLP in IL33-KO cultures than those detected in the control cultures prepared from wildtype (WT) mice. Transmission Electron Microscopy (TEM) analysis revealed that the myelin structures in the corpus callosum of the IL33-KO mice were impaired compared to those observed in the WT mice. Overall, this study provides important evidence that declined expression of IL-33 in OPCs suppresses the maturation of OLGs. Moreover, gene deficiency of IL-33 can disrupt OLG maturation and interfere with myelin compaction. Cover Image for this issue: doi: 10.1111/jnc.14522.

In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified ( n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

MicroRNA-212 inhibits oligodendrocytes during maturation by down-regulation of differentiation-associated gene expression.

MicroRNA-212 (mir-212) has been reported to regulate neuronal development and functioning. However, its expression and function in glia are not yet known. Here, we demonstrate that the level of microRNA-212 (mir-212) was reduced in spinal cord lesion site at 1 week and 1 month after a contusive spinal cord injury. In addition to its expression in neurons, mir-212 expression was detected in oligodendrocytes (OLGs) and glial progenitor cells (GPCs) in adult CNS. The addition of antagomir-212 to reduce mir-212 expression enabled to improve the cell process outgrowth of OLGs along with the up-regulation of the genes associated with OLG differentiation and maturation, including OLIG1, SOX10, myelin basic protein (MBP), and proteolipid protein 1 (PLP1). In contrast, these genes were significantly down-regulated by an increased expression of mir-212 in GPCs or in OLG progenitor cells (OPCs) through lentivirus-mediated gene delivery approach. Moreover, we found that PLP1 was the direct target molecule of mir-212. Furthermore, mir-212 over-expression diminished the protein production of OLGs markers including 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), MBP, and PLP. Additionally, mir-212 over-expression decreased the number of mature OLGs expressing MBP, and the expression of galactocerebroside (GC). Complementary studies in a hippocampal neuron-OLG co-culture model and an ex vivo cerebellar slice system indicated that OLGs derived from GPCs with mir-212 over-expression exhibited decreased ability to interact with neuronal axons. Collectively, our findings demonstrate that mir-212 repressed the expression of OLG maturation-associated proteins and inhibited OLG cell process extension, indicating that mir-212 has negative regulatory effect on OLG lineage progression.

Reduction of CD200 expression in glioma cells enhances microglia activation and tumor growth.

CD200, a type I transmembrane glycoprotein, can interact with its receptor CD200R, which plays an inhibitory role in the activation of microglia-the resident macrophages of the central nervous system. In this study, the rat C6 glioma cell line (C6-1) that was previously characterized with high in vivo tumorigenicity was found to generate CD200 mRNA abundantly. However, CD200 expression was barely detected in another C6 glioma cell clone (C6-2) that was previously found to display low tumorigenic behavior. The results from CD200 immunohistochemistry on human glioma tissue array also showed that tumor cells in Grade I-II astrocytoma expressed a lower level of CD200 immunoreactivity than those detected in Grade III-IV glioblastoma multiforme. C6-1 transfectants with stable downregulation of CD200 gene expression using lentivirus knockdown approach were generated (C6-KD). Microglia and iNOS+ cells were increased when microglia were co-cultured with C6-KD cells. The colony formation of C6-KD was also augmented when those cells were co-cultured with microglia. Yet, increased colony formation of C6-KD transfectants in the co-culture with microglia was effectively suppressed by interleukin (IL)-4 and IL-10. The in vivo results indicated that the tumor formation of C6-1 cells in rat brain was promoted after CD200 gene knockdown. Moreover, CD11b+ activated microglia and iNOS+ microglia were highly accumulated in the tumor site formed by C6-KD. In conclusion, our findings demonstrate that the downregulation of CD200 expression in CD200-rich glioma cells could foster the formation of an activated microglia-associated tumor microenvironment, leading to glioma progression. © 2016 Wiley Periodicals, Inc.

Efficacious and safe orotracheal intubation for laboratory mice using slim torqueable guidewire-based technique: comparisons between a modified and a conventional method.

BACKGROUND: Tracheal intubation of laboratory mice remains essential yet challenging for most researchers. The aim of this study was to investigate whether this procedure can be more efficiently and safely accomplished by a novel method using slim and torqueable guidewires to guide access to the trachea. METHODS: This study was carried out in an animal laboratory affiliated to a tertiary medical center. Mice weighing 22 to 28 g were subjected to various open-chest experiments after being anesthetized with intraperitoneal ketamine (100 mg/kg) and lidocaine hydrochloride (10 mg/kg). The oropharyngeal cavity was opened with angled tissue forceps, and the trachea was transilluminated using an external light. The vocal cords were then crossed using either the Conventional method with a 38-mm-long, end-blunted stiff needle as a guide for insertion of a 22-gauge, 25-mm-long intravenous catheter into the trachea, or the Modified method utilizing using a 0.014-inch-thin torqueable wire as the guide to introduce an identical tube over it into the trachea. The epithelial integrity of the trachea was later examined histologically when the animals were sacrificed either immediately after the surgery or at 28 days post-surgery, depending on the corresponding research protocols. RESULTS: Orotracheal intubation was successfully completed in all mice using either the Conventional (N = 42) or the Modified method (N = 50). With the Modified method, intubation took less time (1.73 vs. 2.17 min, Modified vs. Conventional, p < 0.001) and fewer attempts (1.0 vs. 1.33, p < 0.001), and there were fewer procedural difficulties (0% vs. 16.7%, p = 0.009) and complications (0% vs. 11.9%, p = 0.041) compared with the Conventional method. Histological analysis revealed a significantly lower incidence of immediate (0% vs. 39%, p < 0.001) and late (0% vs. 58%, p < 0.001) injuries to the tracheal epithelial lining with the Modified method compared to the Conventional method. CONCLUSIONS: Tracheal intubation for laboratory mice can be completed efficiently, safely and atraumatically using the proposed Modified method employing readily available inexpensive instruments.

MicroRNA-145 as one negative regulator of astrogliosis.

Astrogliosis occurs at the lesion site within days to weeks after spinal cord injury (SCI) and involves the proliferation and hypertrophy of astrocytes, leading to glia scar formation. Changes in gene expression by deregulated microRNAs (miRNAs) are involved in the process of central nervous system neurodegeneration. Here, we report that mir-145, a miRNA enriched in rat spinal neurons and astrocytes, was downregulated at 1 week and 1 month after SCI. Our in vitro studies using astrocytes prepared from neonatal spinal cord tissues indicated that potent inflammagen lipopolysaccharide downregulated mir-145 expression in astrocytes, suggesting that SCI-triggered inflammatory signaling pathways could play the inhibitory role in astrocytic mir-145 expression. To induce overexpression of mir-145 in astrocytes at the spinal cord lesion site, we developed a lentivirus-mediated pre-miRNA delivery system using the promoter of glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament. The results indicated that astrocyte-specific overexpression of mir-145 reduced astrocytic cell density at the lesion border of the injured spinal cord. In parallel, overexpression of mir-145 reduced the size of astrocytes and the number of related cell processes, as well as cell proliferation and migration. Through a luciferase reporter system, we found that GFAP and c-myc were the two potential targets of mir-145 in astrocytes. Together, the findings demonstrate the novel role of mir-145 in the regulation of astrocytic dynamics, and reveal that the downregulation of mir-145 in astrocytes is a critical factor inducing astrogliosis after SCI. GLIA 2015;63:194-205.

Interleukin-6 Inhibits Expression of miR-204-5p, a Regulator of Oligodendrocyte Differentiation: Involvement of miR-204-5p in the Prevention of Chemical-Induced Oligodendrocyte Impairment.

Oligodendrocytes (OLs) form myelin sheaths around axons in the central nervous system (CNS) facilitate the propagation of action potentials. The studies have shown that the differentiation and maturation of OLs involve microRNA (miR) regulation. The recent findings have addressed that miR-204 regulates OL differentiation in culture. In this study, through in situ hybridization in combination with immunohistochemistry, we showed that microRNA-204-5p in the corpus callosum was mainly expressed in OLs immunoreactive with adenomatous polyposis coli (APC), an OL marker. We also found miR-204-5p expression in mature OLs was suppressed by the addition of interleukin-6 (IL-6). Moreover, IL-6-induced inhibition of miR-204-5p expression was blocked by the addition of the inhibitors specific for p38 mitogen-activated protein kinase (p38MAPK) or phosphatidylinositol 3-kinase (PI3K) pathway. We further utilized a rat model by feeding cuprizone (CPZ)-containing diet for 3 weeks to induce demyelination and gliosis in the corpus callosum, as well as the upregulation of IL-6 gene expression significantly. Despite that miR-204-5p expression in the corpus callosum was not altered after feeding by CPZ for 3 weeks, its expression was increased and IL-6 transcription was decreased in the corpus callosum of the recovery group that was fed by CPZ for the first 2 weeks and by the regular diet for one more week. Our data demonstrate that miR-204-5p expression in OLs declined under the influence of the inflamed microenvironment. The findings that an increase in miR-204-5p and declined IL-6 expression observed in the recovery group might be involved with OL repair in the corpus callosum, and also shed light on a potential role for miR-204-5p in OL homeostasis following the white matter injury.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests that ion homeostasis is a cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption, which was rescued by pharmacological or genetic inhibition of the CCL2-CCR2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-CCL2 and endothelial CCR2 axis as a mechanism controlling BBB integrity and repair, while providing insights for a therapeutic approach against BBB-related CNS disorders.

Valproic acid attenuates microgliosis in injured spinal cord and purinergic P2X4 receptor expression in activated microglia.

Peripheral injection with a high dose of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, into animals with mild or moderate spinal cord injury (SCI) for 1 week can reduce spinal cord tissue loss and promote hindlimb locomotor recovery. A purinergic adenosine triphosphate (ATP) receptor subtype, P2X4 receptor (P2X4 R), has been considered as a potential target to diminish SCI-associated inflammatory responses. In this study, using a minipump-based infusion system, we found that intraspinal infusion with VPA for 3 days into injured spinal cord significantly improved hindlimb locomotion of rats with severe SCI induced by a 10-g NYU impactor dropping from the height of 50 mm onto the spinal T9/10 segment. The neuronal fibers in the injured spinal cord tissues were significantly preserved in VPA-treated rats compared with those observed in vehicle-treated animals. Moreover, the accumulation of microglia/macrophages and astrocytes in the injured spinal cord was attenuated in the animal group receiving VPA infusion. VPA also significantly reduced P2X4 R expression post-SCI. Furthermore, in vitro study indicated that VPA, but not the other HDAC inhibitors, sodium butyrate and trichostatin A (TSA), caused downregulation of P2X4 R in microglia activated with lipopolysaccharide (LPS). Moreover, p38 mitogen-activated protein kinase (MAPK)-triggered signaling was involved in the effect of VPA on the inhibition of P2X4 R gene expression. In addition to the findings from others, our results also provide important evidence to show the inhibitory effect of VPA on P2X4 R expression in activated microglia, which may contribute to reduction of SCI-induced gliosis and subsequently preservation of spinal cord tissues. © 2013 Wiley Periodicals, Inc.

CK2α-dependent regulation of Wnt activity governs white matter development and repair.

Wnt signaling plays an essential role in developmental and regenerative myelination in the CNS. The Wnt signaling pathway is comprised of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte development remains unclear. Here we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt-activity during oligodendrocyte development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of oligodendrocyte development, accelerating early differentiation followed by decelerating maturation and myelination. Application towards white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a novel regulatory node in the Wnt pathway that regulates oligodendrocyte development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration. Significance: Wnt signaling plays a vital role in OL development and has been implicated as an adverse event for myelin repair after white matter injury. Emerging studies have shed light on multi-modal roles of Wnt effectors in the OL lineage, but the underlying molecular mechanisms and modifiable targets in OL remyelination remain unclear. Using genetic mouse development and injury model systems, we delineate a novel stage-specific function of Daam2 in Wnt signaling and OL development via a S704/T7-5 phosphorylation mechanism, and determine a new role of the kinase CK2α in contributing to OL development. In-depth understanding of CK2α-Daam2 pathway regulation will allow us to precisely modulate its activity in conjunction with Wnt signaling and harness its biology for white matter pathology.

Transcatheter Edge-to-Edge Repair for a Patient With Severe Mitral Regurgitation of Carpentier IIIa Classification.

Whether patients diagnosed with mitral regurgitation of Carpentier class IIIa (rheumatic origin) can possibly be treated with balloon mitral commissurotomy followed by transcatheter edge-to-edge repair remains unclear. Here, we report on such a case who was successfully treated with balloon mitral commissurotomy and then transcatheter edge-to-edge repair without aggravating mitral stenosis. (Level of Difficulty: Intermediate.).