Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

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

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

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

A Novel Form of Memory for Auditory Fear Conditioning at a Low-Intensity Unconditioned Stimulus

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short-tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus

Herbivore benefits from vectoring plant virus through reduction of period of vulnerability to predation

Herbivores can profit from vectoring plant pathogens because the induced defence of plants against pathogens sometimes interferes with the induced defence of plants against herbivores. Plants can also defend themselves indirectly by the action of the natural enemies of the herbivores. It is unknown whether the defence against pathogens induced in the plant also interferes with the indirect defence against herbivores mediated via the third trophic level. We previously showed that infection of plants with Tomato spotted wilt virus (TSWV) increased the developmental rate of and juvenile survival of its vector, the thrips Frankliniella occidentalis. Here, we present the results of a study on the effects of TSWV infections of plants on the effectiveness of three species of natural enemies of F. occidentalis: the predatory mites Neoseiulus cucumeris and Iphiseius degenerans, and the predatory bug Orius laevigatus. The growth rate of thrips larvae was positively affected by the presence of virus in the host plant. Because large larvae are invulnerable to predation by the two species of predatory mites, this resulted in a shorter period of vulnerability to predation for thrips that developed on plants with virus than thrips developing on uninfected plants (4.4 vs. 7.9 days, respectively). Because large thrips larvae are not invulnerable to predation by the predatory bug Orius laevigatus, infection of the plant did not affect the predation risk of thrips larvae from this predator. This is the first demonstration of a negative effect of a plant pathogen on the predation risk of its vector

Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior

Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C−terminal domains (CTDs). To identify shared ancestral functions and diversified subunit−specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock−in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit−specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long−term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 gene

Ablation of NG2 Proteoglycan Leads to Deficits in Brown Fat Function and to Adult Onset Obesity

Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O2 consumption and CO2 production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes

The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity