Keratan sulfate phenotype in the β-1,3-N-acetylglucosaminyltransferase-7-null mouse cornea

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine–transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs

The balance between cathepsin C and cystatin F controls remyelination in the brain of Plp1-overexpressing mouse, a chronic demyelinating disease model

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp4e/-. During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process

Inhibition promotes long-term potentiation at cerebellar excitatory synapses.

The ability of the cerebellar cortex to learn from experience ensures the accuracy of movements and reflex adaptation, processes which require long-term plasticity at granule cell (GC) to Purkinje neuron (PN) excitatory synapses. PNs also receive GABAergic inhibitory inputs via GCs activation of interneurons; despite the involvement of inhibition in motor learning, its role in long-term plasticity is poorly characterized. Here we reveal a functional coupling between ionotropic GABAA receptors and low threshold CaV3 calcium channels in PNs that sustains calcium influx and promotes long-term potentiation (LTP) at GC to PN synapses. High frequency stimulation induces LTP at GC to PN synapses and CaV3-mediated calcium influx provided that inhibition is intact; LTP is mGluR1, intracellular calcium store and CaV3 dependent. LTP is impaired in CaV3.1 knockout mice but it is nevertheless recovered by strengthening inhibitory transmission onto PNs; promoting a stronger hyperpolarization via GABAA receptor activation leads to an enhanced availability of an alternative Purkinje-expressed CaV3 isoform compensating for the lack of CaV3.1 and restoring LTP. Accordingly, a stronger hyperpolarization also restores CaV3-mediated calcium influx in PNs from CaV3.1 knockout mice. We conclude that by favoring CaV3 channels availability inhibition promotes LTP at cerebellar excitatory synapses.journal article2016 Sep 192016 09 19importe

Enhancement of Both Long-Term Depression Induction and Optokinetic Response Adaptation in Mice Lacking Delphilin

In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) δ2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluRδ2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluRδ2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca2+ required for the induction of LTD appeared to be reduced in the mutant mice, while Ca2+ influx through voltage-gated Ca2+ channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning

Change in hippocampal theta oscillation associated with multiple lever presses in a bimanual two-lever choice task for robot control in rats.

Hippocampal theta oscillations have been implicated in working memory and attentional process, which might be useful for the brain-machine interface (BMI). To further elucidate the properties of the hippocampal theta oscillations that can be used in BMI, we investigated hippocampal theta oscillations during a two-lever choice task. During the task body-restrained rats were trained with a food reward to move an e-puck robot towards them by pressing the correct lever, ipsilateral to the robot several times, using the ipsilateral forelimb. The robot carried food and moved along a semicircle track set in front of the rat. We demonstrated that the power of hippocampal theta oscillations gradually increased during a 6-s preparatory period before the start of multiple lever pressing, irrespective of whether the correct lever choice or forelimb side were used. In addition, there was a significant difference in the theta power after the first choice, between correct and incorrect trials. During the correct trials the theta power was highest during the first lever-releasing period, whereas in the incorrect trials it occurred during the second correct lever-pressing period. We also analyzed the hippocampal theta oscillations at the termination of multiple lever pressing during the correct trials. Irrespective of whether the correct forelimb side was used, the power of hippocampal theta oscillations gradually decreased with the termination of multiple lever pressing. The frequency of theta oscillation also demonstrated an increase and decrease, before and after multiple lever pressing, respectively. There was a transient increase in frequency after the first lever press during the incorrect trials, while no such increase was observed during the correct trials. These results suggested that hippocampal theta oscillations reflect some aspects of preparatory and cognitive neural activities during the robot controlling task, which could be used for BMI

Ionotropic Glutamate Receptor AMPA 1 Is Associated with Ovulation Rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy

EWS/FLI Mediates Transcriptional Repression via NKX2.2 during Oncogenic Transformation in Ewing's Sarcoma

EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease