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The enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays a pivotal role in the regulation of nitric oxide levels by degrading the main endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Growing evidence highlight the potential implication of DDAH/ADMA axis in the etiopathogenesis of several neuropsychiatric and neurological disorders, yet the underlying molecular mechanisms remain elusive. In this study, we sought to investigate the role of DDAH1 in behavioral endophenotypes with neuropsychiatric relevance. To achieve this, a global DDAH1 knock-out (DDAH1-ko) mouse strain was employed. Behavioral testing and brain region-specific neurotransmitter profiling have been conducted to assess the effect of both genotype and sex. DDAH1-ko mice exhibited increased exploratory behavior toward novel objects, altered amphetamine response kinetics and decreased dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) level in the piriform cortex and striatum. Females of both genotypes showed the most robust amphetamine response. These results support the potential implication of the DDAH/ADMA pathway in central nervous system processes shaping the behavioral outcome. Yet, further experiments are required to complement the picture and define the specific brain-regions and mechanisms involved. [ABSTRACT FROM AUTHOR]

Purpose: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. Methods: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. Results: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. Conclusions: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period. [ABSTRACT FROM AUTHOR]

C57BL/6 mice exhibit spontaneous cerebellar malformations consisting of heterotopic neurons and glia in the molecular layer of the posterior vermis, indicative of neuronal migration defect during cerebellar development. Recognizing that many genetically engineered (GE) mouse lines are produced from C57BL/6 ES cells or backcrossed to this strain, we performed histological analyses and found that cerebellar heterotopia were a common feature present in the majority of GE lines on this background. Furthermore, we identify GE mouse lines that will be valuable in the study of cerebellar malformations including diverse driver, reporter, and optogenetic lines. Finally, we discuss the implications that these data have on the use of C57BL/6 mice and GE mice on this background in studies of cerebellar development or as models of disease.

Abstract We have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function.

Breast cancer resistance protein/ATP-binding cassette subfamily G2 (BCRP/ABCG2) is an ATP-binding cassette efflux (ABC) transporter expressed in the apical membrane of cells in tissues, such as the liver, intestine, kidney, testis, brain, and mammary gland. It is involved in xenobiotic pharmacokinetics, potentially affecting the efficacy and toxicity of many drugs. In this study, the role of ABCG2 in parasiticide monepantel (MNP) and its primary metabolite, monepantel sulfone (MNPSO 2)'s systemic distribution and excretion in milk, was tested using female and male wild‐type and Abcg2−/− mice. Liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS) was used for the analysis in a 10-min run time using positive-mode atmospheric pressure electrospray ionization (ESI+) and multiple reaction monitoring (MRM) scanning. For the primary metabolite tested, milk concentrations were 1.8-fold higher in wild-type mice than Abcg2−/− female lactating mice (P = 0.042) after intravenous administration of MNP. Finally, despite the lack of a difference between groups, we investigated potential differences in MNP and MNPSO 2 's plasma and tissue accumulation levels between wild‐type and Abcg2−/− male mice. In this study, we demonstrated that MNPSO 2 milk levels were affected by Abcg2, with potential pharmacological and toxicological consequences, contributing to the undesirable xenobiotic residues in milk. [Display omitted] • MNP and MNPSO 2 were tested as ABCG2 substrates by using wild‐type and Abcg2−/− mice. • MNPSO 2 concentration is decreased in Abcg2−/− female lactating mice milk than in wild-type mice. • Influence of MNPSO 2 milk levels by Abcg2 may have important consequences for the undesirable presence of residues in milk. [ABSTRACT FROM AUTHOR]

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD), but the pathogenic mechanism underlying LRRK2 mutations remains unresolved. In this study, we investigate the consequence of inactivation of LRRK2 and its functional homolog LRRK1 in male and female mice up to 25 months of age using behavioral, neurochemical, neuropathological, and ultrastructural analyses. We report that LRRK1 and LRRK2 double knock-out (LRRK DKO) mice exhibit impaired motor coordination at 12 months of age before the onset of dopaminergic neuron loss in the substantia nigra (SNpc). Moreover, LRRK DKO mice develop age-dependent, progressive loss of dopaminergic terminals in the striatum. Evoked dopamine (DA) release measured by fast-scan cyclic voltammetry in the dorsal striatum is also reduced in the absence of LRRK. Furthermore, LRRK DKO mice at 20-25 months of age show substantial loss of dopaminergic neurons in the SNpc. The surviving SNpc neurons in LRRK DKO mice at 25 months of age accumulate large numbers of autophagic and autolysosomal vacuoles and are accompanied with microgliosis. Surprisingly, the cerebral cortex is unaffected, as shown by normal cortical volume and neuron number as well as unchanged number of apoptotic cells and microglia in LRRK DKO mice at 25 months. These findings show that loss of LRRK function causes impairments in motor coordination, degeneration of dopaminergic terminals, reduction of evoked DA release, and selective loss of dopaminergic neurons in the SNpc, indicating that LRRK DKO mice are unique models for better understanding dopaminergic neurodegeneration in PD. [ABSTRACT FROM AUTHOR]

Acid sphingomyelinase (ASM) regulates a variety of physiological processes and plays an important role in emotional behavior. The role of ASM in fear-related behavior has not been investigated so far. Using transgenic mice overexpressing ASM (ASMtg) and ASM deficient mice, we studied whether ASM regulates fear learning and expression of cued and contextual fear in a classical fear conditioning paradigm, a model used to investigate specific attributes of post-traumatic stress disorder (PTSD). We show that ASM does not affect fear learning as both ASMtg and ASM deficient mice display unaltered fear conditioning when compared to wild-type littermates. However, ASM regulates the expression of contextual fear in a sex-specific manner. While ASM overexpression enhances the expression of contextual fear in both male and female mice, ASM deficiency reduces the expression of contextual fear specifically in male mice. The expression of cued fear, however, is not regulated by ASM as ASMtg and ASM deficient mice display similar tone-elicited freezing levels. This study shows that ASM modulates the expression of contextual fear but not of cued fear in a sex-specific manner and adds a novel piece of information regarding the involvement of ASM in hippocampal-dependent aversive memory. [ABSTRACT FROM AUTHOR]

Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation by PKA and Cdk5 in the modulation of this process is unknown. We expressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression of WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5-dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival. Significance statement: Synapsin III is an atypical member of the synapsin family of synaptic vesicle-associated phosphoproteins that is precociously expressed in neurons and is downregulated afterward. Although experimental evidence suggests a specific role for Synapsin III in neuronal development, the molecular mechanisms are still largely unknown. We found that Synapsin III plays a central role in early stages of neuronal development involving neuronal survival, polarization, and neuritic growth and that these effects are dependent on phosphorylation by cAMP-dependent protein kinase and cyclin-dependent protein kinase-5. These results explain the recently described neurodevelopmental defects in the migration and orientation of Synapsin III-depleted cortical neurons and support the potential association of Synapsin III with neurodevelopmental disorders such as schizophrenia.

Chronic cholestatic liver diseases including primary sclerosing cholangitis (PSC) present a complex spectrum with regards to the cause, age of manifestation and histopathological features. Current treatment options are severely limited primarily due to a paucity of model systems mirroring the disease. Here, we describe the Keratin 5 (K5)-Cre; Klf5fl/fl mouse that spontaneously develops severe liver disease during the postnatal period with features resembling PSC including a prominent ductular reaction, fibrotic obliteration of the bile ducts and secondary degeneration/necrosis of liver parenchyma. Over time, there is an expansion of Sox9+ hepatocytes in the damaged livers suggestive of a hepatocyte-mediated regenerative response. We conclude that Klf5 is required for the normal function of the hepatobiliary system and that the K5-Cre; Klf5fl/fl mouse is an excellent model to probe the molecular events interlinking damage and regenerative response in the liver. [ABSTRACT FROM AUTHOR]

We have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function. [ABSTRACT FROM AUTHOR]

γ‐Aminobutyric acid (GABA) acting through heteropentameric GABAA receptors plays a pivotal role in the sleep‐promoting circuitry. Whereas the role of the different GABAA receptor α‐subunits in sleep regulation and in mediating the effect of benzodiazepines for treatment of insomnia is well‐described, the β‐subunits are less studied. Here we report the first study characterizing sleep in mice lacking the GABAA receptor β1‐subunit (β1−/− mice). We show that β1−/− mice have a distinct and abnormal sleep phenotype characterized by increased delta power in non‐rapid eye movement (NREM) sleep and decreased theta activity in rapid eye movement (REM) sleep compared to β1+/+ mice, without any change in the overall sleep‐wake architecture. From GABAA receptor‐specific autoradiography, it is further demonstrated that functional β1‐subunit‐containing GABAA receptors display the highest binding levels in the hippocampus and frontal cortex. In conclusion, this study suggests that the GABAA receptor β1‐subunit does not play an important role in sleep initiation or maintenance but instead regulates the power spectrum and especially the expression of theta rhythm. This provides new knowledge on the complex role of GABAA receptor subunits in sleep regulation. In addition, β1−/− mice could provide a useful mouse model for future studies of the physiological role of delta and theta rhythms during sleep. [ABSTRACT FROM AUTHOR]

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock‐out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild‐type and TC10 knock‐out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron‐intrinsic. [ABSTRACT FROM AUTHOR]

Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: "lethality", "mice", "knock-out", "deficient", "embryonic", "perinatal", "rescue". Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human diseaserelevant homozygous KO mice lethal phenotypes were summarized. [ABSTRACT FROM AUTHOR]

BackgroundLipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models.MethodsLp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. β- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice.ResultsPAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released β-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice.ConclusionsWe conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge.

• Prrrt2 −/− mice exhibit paroxysmal dystonia with face validity. • Prrt2 transcripts reside in granule cells but not Purkinje cells or interneurons within cerebellar cortex. • In the cerebellar molecular layer, Prrt2 −/− mice have increased numbers of docked vesicles but decreased total vesicles. • In Prrt2 −/− mice, we found reduced parallel fiber facilitation at parallel fiber-Purkinje cell synapses. PRRT2 loss-of-function mutations have been associated with familial paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions and choreoathetosis, and benign familial infantile seizures. Dystonia is the foremost involuntary movement disorder manifest by patients with PKD. Using a lacZ reporter and quantitative reverse-transcriptase PCR, we mapped the temporal and spatial distribution of Prrt2 in mouse brain and showed the highest levels of expression in cerebellar cortex. Further investigation into PRRT2 localization within the cerebellar cortex revealed that Prrt2 transcripts reside in granule cells but not Purkinje cells or interneurons within cerebellar cortex, and PRRT2 is presynaptically localized in the molecular layer. Analysis of synapses in the cerebellar molecular layer via electron microscopy showed that Prrt2 −/− mice have increased numbers of docked vesicles but decreased vesicle numbers overall. In addition to impaired performance on several motor tasks, approximately 5% of Prrt2 −/− mice exhibited overt PKD with clear face validity manifest as dystonia. In Prrt2 mutants, we found reduced parallel fiber facilitation at parallel fiber-Purkinje cell synapses, reduced Purkinje cell excitability, and normal cerebellar nuclear excitability, establishing a potential mechanism by which altered cerebellar activity promotes disinhibition of the cerebellar nuclei, driving motor abnormalities in PKD. Overall, our findings replicate, refine, and expand upon previous work with PRRT2 mouse models, contribute to understanding of paroxysmal disorders of the nervous system, and provide mechanistic insight into the role of cerebellar cortical dysfunction in dystonia. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy (DCM) is a severe cardiovascular complication of diabetes mellitus (DM). Detecting DCM during the early stages of the disease remains a challenge, as the molecular mechanisms underlying early-stage DCM are not clearly understood. Circular RNA (circRNA), a type of non-coding RNA, has been confirmed to be associated with numerous diseases. However, it is still unclear how circRNAs are involved in early-stage DCM. In the present study, heart tissues harvested from BKS-db/db knock-out mice were identified through high-throughput RNA sequencing technology. A total of 58 significantly differentially expressed circRNAs were identified in the db/db sample. Among these, six upregulated circRNAs and seven downregulated circRNAs were detected by reverse transcription-quantitative PCR and analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Furthermore, based on the predicted binding site with microRNAs (miRNAs) involved in DCM, five circRNAs (mmu_circ_0000652, mmu_circ_0000547, mmu_circ_0001058, mmu_circ_0000680 and novel_circ_0004285) were shown to serve as competing endogenous (ce)RNAs. The corresponding miRNAs and mRNAs of the ceRNAs were also verified, and two promising circRNA-miRNA-mRNA regulatory networks were determined. Finally, internal ribosome entry site prediction combined with open reading frame prediction indicated that it was highly possible that mmu_circ_0001160 encoded a protein. In the present study, a comprehensive analysis of the circRNA expression profile during the early phase of DCM was performed, and two promising circRNA-miRNA-mRNA regulatory networks were identified. These results lay the foundation for unravelling the underlying pathogenesis of DCM, and highlight potential biomarkers and therapeutic targets for the treatment of DCM at an early stage. [ABSTRACT FROM AUTHOR]

Considering high drug attrition rates in clinical studies and the overall complexity and challenging environment of drug development, it is increasingly important to understand the therapeutic molecule and target and how they intersect with disease biology as fully as possible. This requires one to use numerous tools and investigative approaches in combination. Genetically engineered mouse models are a critical component to the drug development toolbox as they can provide key insights across multiple steps of the drug development process. While knock-out and knock-in mice can inform questions of basic biology, genetically engineered mice can also be applied to model diseases for efficacy studies, to discriminate on-target and off-target effects of novel therapeutics, and to inform an array of biologic and pharmacologic questions, including pharmacodynamics, pharmacokinetics, and biomarker discovery. However, use of these models requires not only an understanding of their strengths and limitations but also a careful consideration of the context in which they are being used and the hypotheses being addressed by them. Additionally, they should not be used in isolation, but instead in combination with other biochemical, in vitro, and clinical data to create a broad understanding of the drug, target, and disease biology. [ABSTRACT FROM AUTHOR]

Photoreceptors of the retina adapt to ambient light in a manner that allows them to detect changes in illumination over an enormous range of intensities. We have discovered a novel form of adaptation in mouse rods that persists long after the light has been extinguished and the rod's circulating dark current has returned. Electrophysiological recordings from individual rods showed that the time that a bright flash response remained in saturation was significantly shorter if the rod had been previously exposed to bright light. This persistent adaptation did not decrease the rate of rise of the response and therefore cannot be attributed to a decrease in the gain of transduction. Instead, this adaptation was accompanied by a marked speeding of the recovery of the response, suggesting that the step that rate-limits recovery had been accelerated. Experiments on knockout rods in which the identity of the rate-limiting step is known suggest that this adaptive acceleration results from a speeding of G protein/effector deactivation.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines. [ABSTRACT FROM AUTHOR]

The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knock-out mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knock-out mice (ADAPfl/fl PF4-Cretg). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even after stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and TGF-β1. In vitro, platelets from these mice revealed reduced P-selectin expression, lower TGF-β1 release, diminished integrin αIIbβ3 activation and decreased fibrinogen binding after stimulation with podoplanin, the ligand of the C-type lectin-like receptor-2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of PLCγ2 and Erk1/2. Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-ß1 early after T cell transfer improved EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo. [ABSTRACT FROM AUTHOR]

Abstract A clinically important and well-studied transporter of the blood-brain barrier (BBB) is P-glycoprotein (P-gp), the gene product of ABCB1. Animal studies have shown that brain concentrations of many antidepressants depend on P-gp. However, biochemical properties, which might allow the prediction of pharmacodynamical involvement of P-gp have not yet been identified, hence thorough experimental testing of each novel drug is needed to determine its P-gp substrate status. In the current study, we tested the P-gp substrate status for the antidepressant vortioxetine using double abcb1ab knock-out (KO) mice. Cerebral concentrations of vortioxetine were 2.3 times higher in P-gp deficient mice compared to wildtype (WT) controls. No significant difference was found regarding the concentration of the drug in the plasma and other organs (liver, kidney, spleen) between KO and WT mice. The results of our study provide conclusive in-vivo evidence that in mice vortioxetine's brain bioavailability is P-gp dependent, expanding previous findings on this topic. [ABSTRACT FROM AUTHOR]

It has been suggested that the DGCR2 gene plays a role in the pathogenesis of 22q11.2 deletion syndrome. To analyze its function, we used our Dgcr2-knock-out/EGFP-knock-in mice (Dgcr2-KO mice). At 20-26 weeks of age, approximately 20% of Dgcr2-KO mice showed gait abnormalities with trembling and difficulty in balancing. Footprint test revealed awkward movements in Dgcr2-KO mice soon after they were placed on the floor. Once they started walking, their stride lengths were not different from wild-type mice. In short-term open field test, Dgcr2-KO mice travelled a significantly shorter distance and walked more slowly than wild-type mice during the initial 5 min after being placed in a new environment. In long-term open field test, Dgcr2-KO mice exhibited reduced cage activity compared to wild-type mice on the first day, but not on later days. Dgcr2-KO mice showed reduced latency to fall in the rotarod test, and the latency was not improved in the 3-day test. Histology revealed sparseness of cerebellar Purkinje cells in Dgcr2-KO mice. Our results suggest that Dgcr2 plays a role in motor control related to Purkinje cell function and that the deficiency of DGCR2 contributes at least to some of the symptoms of patients of 22q11.2 deletion syndrome.

G protein‐coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein‐coupled receptors (GPCRs). The short‐splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T‐lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure inGit2−/− mice. CD4+ T cells fromGit2−/− mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR‐stimulated activation. Our results suggested that GIT2 plays an important role in T‐cell functionin vivo andin vitro.

Background: Innate immune response components such as toll-like receptors (TLRs) and NLRP3-inflammasome act in concert to increase IL-1α/β secretion by synovial macrophages. Previous results suggest that IL-1α/β could be an important mediator involved in the pathogenesis of osteoarthritis (OA).Objectives: The aim of our study was to evaluate the role of NLRP3, IL-1β, and IL-1α in the menisectomy (MNX) model of murine OA.Methods: Murine chondrocytes (CHs) and bone marrow-derived machrophages (BMDM) were stimulated with hydroxyapatite (HA) crystals, a form of calcium-containing crystal found in human OA, and IL-1β and IL-6 secretion assayed by ELISA.Conversely, the ability of IL-1β and IL-6 to induce CHs calcification was assessed in vitro by Alizarin red staining. Knees from 8 to 10 weeks old C57Bl/6J wild-type (WT) (n = 7), NLRP3−/− (n = 9), IL-1α−/− (n = 5), and IL-1β−/− (n = 5) mice were menisectomized, using the sham-operated contralateral knee as control. 8 weeks later, knee cartilage degradation and synovial inflammation were evaluated by histology. In addition, apoptotic chondrocytes, metalloproteases activity, and collagen-type 2 expression were evaluated in all mice. Joint calcification and subchondral bone parameters were quantified by CT-scan in WT and IL-1β−/− menisectomized knees.Results:In vitro, HA crystals induced significant increased IL-6 secretion by CHs, while IL-1β remained undetectable.Conversely, both IL-6 and IL-1β were able to increase chondrocytes mineralization. In vivo, operated knees exhibited OA features compared to sham-operated knees as evidenced by increased cartilage degradation and synovial inflammation. In menisectomized KO mice, severity and extent of cartilage lesions were similar (IL-1α−/− mice) or exacerbated (IL-1β−/− and NLRP3−/− mice) compared to that of menisectomized WT mice. Metalloproteases activity, collagen-type 2 expression, chondrocytes apoptosis, and synovial inflammation were similar between KO and WT mice menisectomized knees. Moreover, the extent of joint calcification in osteoarthritic knees was comparable between IL-1β−/− and WT mice.Conclusions: MNX knees recapitulated features of OA, i.e, cartilage destruction, synovial inflammation, cell death, and joint calcification. Deficiency of IL-1α did not impact on the severity of these features, whereas deficiency of IL-1β or of NLRP3 led to increased cartilage erosion. Our results suggest that IL-1α and IL-1β are not key mediators in this murine OA model and may explain the inefficiency of IL-1 targeted therapies in OA.

Introduction R-Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell responses. Methods Here, we investigated the role of R-Ras in allergen-induced immune response (type 2 immune response) in Rras deficient (R-Ras KO) and wild type (WT) mice. Results Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R-Ras KO mice. The expression of co-stimulatory CD80 and CD86 molecules on these cells was also reduced on DC's from the R-Ras KO mice. However, there was no difference in papain-induced immune response between the R-Ras WT and KO as measured by serum IgE levels after the immunization. Interestingly, neither the DC number nor co-stimulatory molecule expression was different between WT and R-Ras KO animals after the immunization. Conclusions Taken together, despite having reduced number of conventional DC's in the R-Ras KO mice and low expression of CD80 on DC's, the R-Ras KO mice are capable of mounting papain-induced IgE responses comparable to that of the WT mice. To our knowledge, this is the first report addressing potential differences in in vivo allergen responses regulated by the R-Ras GTPase. [ABSTRACT FROM AUTHOR]

Along with being the main target of many antidepressant medications, the serotonin transporter (5-HTT) is known to be involved in the pathophysiology of depression and anxiety disorders. In line with this, mice with varying 5-HTT genotypes are invaluable tools to study depression- and anxiety-like behaviours as well as the mechanisms mediating potential therapeutics. There is clear evidence that both genetic and environmental factors play a role in the aetiology of psychiatric disorders. In that regard, housing paradigms which seek to enhance cognitive stimulation and physical activity have been shown to exert beneficial effects in animal models of neuropsychiatric disorders. In the present study, we examined the effects of environmental enrichment on affective-like behaviours and sensorimotor gating function of 5-HTT knock-out (KO) mice. Using the elevated-plus maze and the light-dark box, we found that environmental enrichment ameliorated the abnormal innate anxiety of 5-HTT KO mice on both tests. In contrast, environmental enrichment did not rescue the depression-like behaviour displayed by 5-HTT KO mice in the forced-swim test. Finally, measuring pre-pulse inhibition, we found no effect of genotype or treatment on sensorimotor gating. In conclusion, our data suggest that environmental enrichment specifically reduces innate anxiety of 5-HTT KO mice with no amelioration of the depression-like behaviour. This has implications for the current use of clinical interventions for patients with symptoms of both anxiety and depression. [ABSTRACT FROM AUTHOR]

Testicular androgens are the major endocrine factor promoting masculine phenotypes in vertebrates, but androgen signaling is complex and operates via multiple signaling pathways and sites of action. Recently, selective androgen receptor mutants have been engineered to study androgenic mechanisms of sexual differentiation of the nervous system and behavior. The focus of these studies has been to evaluate androgenic mechanisms within the nervous system by manipulating androgen receptor conditionally in neural tissues. Here we review both the effects of neural loss of AR function as well as the effects of neural overexpression of AR in relation to global AR mutants. Although some studies have conformed to our expectations, others have proved challenging to assumptions underlying the dominant hypotheses. Notably, these studies have called into question both the primacy of direct, neural mechanisms and also the linearity of the relationship between androgenic dose and sexual differentiation of brain and behavior. [ABSTRACT FROM AUTHOR]

Background: Innate immune response components such as toll-like receptors (TLRs) and NLRP3-inflammasome act in concert to increase IL-1α/β secretion by synovial macrophages. Previous results suggest that IL-1α/β could be an important mediator involved in the pathogenesis of osteoarthritis (OA). Objectives: The aim of our study was to evaluate the role of NLRP3, IL-1β, and IL-1α in the menisectomy (MNX) model of murine OA. Methods: Murine chondrocytes (CHs) and bone marrow-derived machrophages (BMDM) were stimulated with hydroxyapatite (HA) crystals, a form of calcium-containing crystal found in human OA, and IL-1β and IL-6 secretion assayed by ELISA.Conversely, the ability of IL-1β and IL-6 to induce CHs calcification was assessed in vitro by Alizarin red staining. Knees from 8 to 10 weeks old C57Bl/6J wild-type (WT) (n = 7), NLRP3-/- (n = 9), IL-1α-/- (n = 5), and IL-1β-/- (n = 5) mice were menisectomized, using the sham-operated contralateral knee as control. 8 weeks later, knee cartilage degradation and synovial inflammation were evaluated by histology. In addition, apoptotic chondrocytes, metalloproteases activity, and collagen-type 2 expression were evaluated in all mice. Joint calcification and subchondral bone parameters were quantified by CT-scan in WT and IL-1β-/- menisectomized knees. Results: In vitro, HA crystals induced significant increased IL-6 secretion by CHs, while IL-1β remained undetectable. Conversely, both IL-6 and IL-1β were able to increase chondrocytes mineralization. In vivo, operated knees exhibited OA features compared to sham-operated knees as evidenced by increased cartilage degradation and synovial inflammation. In menisectomized KO mice, severity and extent of cartilage lesions were similar (IL-1α-/- mice) or exacerbated (IL-1β-/- and NLRP3-/- mice) compared to that of menisectomized WT mice. Metalloproteases activity, collagen-type 2 expression, chondrocytes apoptosis, and synovial inflammation were similar between KO and WT mice menisectomized knees. Moreover, the extent of joint calcification in osteoarthritic knees was comparable between IL-1β-/- and WT mice. Conclusions: MNX knees recapitulated features of OA, i.e, cartilage destruction, synovial inflammation, cell death, and joint calcification. Deficiency of IL-1α did not impact on the severity of these features, whereas deficiency of IL-1β or of NLRP3 led to increased cartilage erosion. Our results suggest that IL-1α and IL-1β are not key mediators in this murine OA model and may explain the inefficiency of IL-1 targeted therapies in OA. [ABSTRACT FROM AUTHOR]

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB1 receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D3 receptor null (D3R(−/−)) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R(−/−) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R(−/−) mice also exhibited different endocannabinoid and TRPV1, but not CB1, levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB1 and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R(−/−) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.

PPARγ is one of the three isoforms of the Peroxisome Proliferator-Activated Receptors (PPARs). PPARγ is activated by thiazolidinediones such as pioglitazone and is targeted to treat insulin resistance. PPARγ is densely expressed in brain areas involved in regulation of motivational and emotional processes. Here, we investigated the role of PPARγ in the brain and explored its role in anxiety and stress responses in mice. The results show that stimulation of PPARγ by pioglitazone did not affect basal anxiety, but fully prevented the anxiogenic effect of acute stress. Using mice with genetic ablation of neuronal PPARγ (PPARγNestinCre), we demonstrated that a lack of receptors, specifically in neurons, exacerbated basal anxiety and enhanced stress sensitivity. The administration of GW9662, a selective PPARγ antagonist, elicited a marked anxiogenic response in PPARγ wild-type (WT), but not in PPARγNestinCre knock-out (KO) mice. Using c-Fos immunohistochemistry, we observed that acute stress exposure resulted in a different pattern of neuronal activation in the amygdala (AMY) and the hippocampus (HIPP) of PPARγNestinCre KO mice compared with WT mice. No differences were found between WT and KO mice in hypothalamic regions responsible for hormonal response to stress or in blood corticosterone levels. Microinjection of pioglitazone into the AMY, but not into the HIPP, abolished the anxiogenic response elicited by acute stress. Results also showed that, in both regions, PPARγ colocalizes with GABAergic cells. These findings demonstrate that neuronal PPARγ is involved the regulation of the stress response and that the AMY is a key substrate for the anxiolytic effect of PPARγ. [ABSTRACT FROM AUTHOR]

This study investigates the functional role of calsequestrin 2 (CASQ2) in both fast-twitch and slow-twitch skeletal muscles by using CASQ2−/− mice; CASQ2 is expressed throughout life in slow-twitch muscles, but only in the developmental and neonatal stages in fast-twitch muscles. CASQ2−/− causes increase in calsequestrin 1 (CASQ1) expression, but without functional changes in both muscle types. CASQ2−/− mice have ultrastructural changes in fast-twitch muscles only, i.e., formation of pentads and stacks in the sarcoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Neural α7 nicotinic acetylcholine receptors (α7 nAChRs) emerged as a potential pharmacologic target for treating cognitive deficits in schizophrenia and Alzheimer’s disease. Experiments modeling these dysfunctions, as well as clinical evidence, demonstrate the relatively consistent procognitive effects of α7 nAChR agonists. One preclinical observation supporting the procognitive role of α7 nAChRs is their ability to modulate neuronal network oscillations closely associated with learning and memory, especially hippocampal oscillations. Due to the high degree of structural similarity between α7 nACh and 5-HT receptors, the majority of α7 nAChR agonists to date also act as 5-HT 3 antagonists. To address this confounding property and determine the relevance of α7 nAChR agonist binding to 5-HT 3 receptors in modulating hippocampal activity, we tested two well-described α7 nAChR agonists, PNU-282987 and FRM-17874, in mice lacking α7 nAChRs (α7 knock-out, α7KO) using the brainstem simulation-elicited hippocampal theta oscillation assay. Under urethane anesthesia both agonists at equivalent doses demonstrated efficacy in wild-type (WT) mice, significantly enhancing theta power and theta phase-gamma amplitude coupling as compared to saline treated control mice. These effects are comparable to those seen with drugs clinically used to treat Alzheimer’s disease. Although α7KO mice showed no alterations in elicited hippocampal oscillations, both α7 nAChR agonists failed to enhance theta power or theta phase – gamma amplitude coupling in these mice. Our findings demonstrate that selective activation of α7 nAChRs can modulate hippocampal oscillation, and these receptors are the primary targets of the tested agonists, PNU-282987 and FRM-17874 and likely underlies their observed procognitive activity. [ABSTRACT FROM AUTHOR]

Background: Brinps 1-3, and Astrotactins (Astn) 1 and 2, are members of the Membrane Attack Complex/Perforin (MACPF) superfamily that are predominantly expressed in the mammalian brain during development. Genetic variation at the human BRINP2/ASTN1 and BRINP1/ASTN2 loci has been implicated in neurodevelopmental disorders. We, and others, have previously shown that Brinp1-/- mice exhibit behavior reminiscent of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Method: We created Brinp2-/- mice and Brinp3-/- mice via the Cre-mediated LoxP system to investigate the effect of gene deletion on anatomy and behavior. Additionally, Brinp2-/-Brinp3-/- double knock-out mice were generated by interbreeding Brinp2-/- and Brinp3-/- mice. Genomic validation was carried out for each knock-out line, followed by histological, weight and behavioral examination. Brinp1-/-Brinp2-/-Brinp3-/- triple knock-out mice were also generated by crossing Brinp2/3 double knock-out mice with previously generated Brinp1-/- mice, and examined by weight and histological analysis. Results: Brinp2-/- and Brinp3-/- mice differ in their behavior: Brinp2-/- mice are hyperactive, whereas Brinp3-/- mice exhibit marked changes in anxiety-response on the elevated plus maze. Brinp3-/- mice also show evidence of altered sociability. Both Brinp2-/- and Brinp3-/- mice have normal short-term memory, olfactory responses, pre-pulse inhibition, and motor learning. The double knock-out mice show behaviors of Brinp2-/- and Brinp3-/- mice, without evidence of new or exacerbated phenotypes. Conclusion: Brinp3 is important in moderation of anxiety, with potential relevance to anxiety disorders. Brinp2 dysfunction resulting in hyperactivity may be relevant to the association of ADHD with chromosome locus 1q25.2. Brinp2-/- and Brinp3-/- genes do not compensate in the mammalian brain and likely have distinct molecular or cell-type specific functions. [ABSTRACT FROM AUTHOR]

Rationale: Melanin-concentrating hormone (MCH) is involved in the regulation of food intake and has recently been associated with alcohol-related behaviors. Blockade of MCH-1 receptors (MCH1-Rs) attenuates operant alcohol self-administration and decreases cue-induced reinstatement, but the mechanism through which the MCH1-R influences these behaviors remains unknown. MCH1-Rs are highly expressed in the nucleus accumbens shell (NAcSh) where they are co-expressed with dopamine (DA) receptors. MCH has been shown to potentiate responses to dopamine and to increase phosphorylation of DARPP-32, an intracellular marker of DA receptor activation, in the NAcSh. Methods: In the present study, we investigated the role of the MCH1-R in alcohol reward using the conditioned place preference (CPP) paradigm. We then used immunohistochemistry (IHC) to assess activation of downstream signaling after administration of a rewarding dose of alcohol. Results: We found that alcohol-induced CPP was markedly decreased in mice with a genetic deletion of the MCH1-R as well as after pharmacological treatment with an MCH1-R antagonist, GW803430. In contrast, an isocaloric dose of dextrose did not produce CPP. The increase in DARPP-32 phosphorylation seen in wildtype (WT) mice after acute alcohol administration in the NAcSh was markedly reduced in MCH1-R knock-out (KO) mice. Conclusion: Our results suggest that MCH1-Rs regulate the rewarding properties of alcohol through interactions with signaling cascades downstream of DA receptors in the NAcSh. [ABSTRACT FROM AUTHOR]

Extracellular pyrimidines activate P2Y receptors on both smooth muscle cells and endothelial cells, leading to vasoconstriction and relaxation respectively. The aim of this study was to utilize P2Y knock-out (KO) mice to determine which P2Y receptor subtype are responsible for the contraction and relaxation in the coronary circulation and to establish whether P2Y receptors have different functions along the mouse coronary vascular tree. We tested stable pyrimidine analogues on isolated coronary arteries from P2Y2 and P2Y6 receptor KO mice in a myograph setup. In larger diameter segments of the left descending coronary artery (LAD) (lumen diameter ~ 150 μm) P2Y6 is the predominant contractile receptor for both UTP (uridine triphosphate) and UDP (uridine diphosphate) induced contraction. In contrast, P2Y2 receptors mediate endothelial-dependent relaxation. However, in smaller diameter LAD segments (lumen diameter ~ 50 μm), the situation is opposite, with P2Y2 being the contractile receptor and P2Y6 functioning as a relaxant receptor along with P2Y2. Immunohistochemistry was used to confirm smooth muscle and endothelial localization of the receptors. In vivo measurements of blood pressure in WT mice revealed a biphasic response to the stable analogue UDPβS. Based on the changes in P2Y receptor functionality along the mouse coronary arterial vasculature, we propose that UTP can act as a vasodilator downstream of its release, after being degraded to UDP, without affecting the contractile pyrimidine receptors. We also propose a model, showing physiological relevance for the changes in purinergic receptor functionality along the mouse coronary vascular tree. [ABSTRACT FROM AUTHOR]

Brain damaging insults cause alterations in neuronal networks that trigger epileptogenesis, and eventually lead to the appearance of spontaneous seizures. The present experiments were designed to study the cellular expression and functions of a cysteine proteinase inhibitor, cystatin C, whose gene expression is previously shown to be upregulated in the rat hippocampus during status epilepticus (SE)-induced epileptogenesis. The present data showed that the expression of cystatin C protein increased in the mouse hippocampus 7 days following SE and localized mainly to astrocytes and microglia. Acute neuronal death in the hippocampus at 24 h after SE was reduced in cystatin C−/− mice. Also, the basal level of neurogenesis in the subgranular layer of dentate gyrus was decreased in cystatin C−/− mice compared to wildtype littermates. Interestingly, migration of newly born neurons within the granule cell layer was attenuated in cystatin C−/− mice. These data demonstrate that cystatin C has a role in neuronal death and neurogenesis during SE-induced network reorganization.

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene ''targeting''). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.A capacidade de introduzir modificações genéticas na linhagem germinal de organismos complexos tem sido, por muito tempo, uma meta dos estudiosos da biologia do desenvolvimento. Neste aspecto, o camundongo, um modelo favorito de estudo dos mamíferos, é singular. Assim, desde o final dos anos setenta, tem sido possível não só adicionar genes ao genoma do camundongo, como em vários outros organismos complexos, mas, também realizar a substituição e modificação de seus genes. Isto tem sido possível graças a dois rasgos tecnológicos: 1) o isolamento e cultura de células-tronco embrionárias, que têm a capacidade singular de colonizar todos os tecidos de um embrião hospedeiro, inclusive de sua linhagem germinal; 2) o desenvolvimento de métodos que permitem a recombinação homóloga entre um DNA que ''ingressa'' e sua seqüência cromossomal cognata (''mira'' genética - ''gene targetting'' Como resultado, tornou-se possível criar camundongos que carreiam mutações nulas em qualquer gene clonado (camundongos ''knock out''). Tal possibilidade revolucionou a abordagem genética de quase todos os aspectos da biologia do camundongo. Em anos recentes, o escopo da ''mira'' genética ampliou-se ainda mais devido ao refinamento da tecnologia de ''knock out'': outros tipos de modificações genéticas podem agora ser criadas, incluindo mutações sutis (mutações punctiformes, micro-deleções ou inserções, etc) e rearranjos cromossomais tais como grandes deleções, duplicações e translocações. Finalmente, têm sido implementados métodos que permitem a criação de mutações condicionais, permitindo o estudo da função gênica ao longo da vida do animal, quando a inativação do gene acarreta a letalidade embrionária. Neste artigo, apresentamos uma revisão geral dos métodos e situações usadas para a modificação programada do genoma do camundongo e acentuamos seu enorme interesse para o estudo da biologia dos mamíferos.

Eicosapentaenoic acid (EPA)-derived prostaglandin E 3 (PGE 3 ) possesses an anti-inflammatory effect; however, information for transporters that regulate its peri-cellular concentration is limited. The present study, therefore, aimed to clarify transporters involved in local disposition of PGE 3 . PGE 3 uptake was assessed in HEK293 cells transfected with OATP2A1/ SLCO2A1 , OATP1B1/ SLCO1B1 , OATP2B1/ SLCO2B1 , OAT1/ SLC22A6 , OCT1/ SLC22A1 or OCT2/ SLC22A2 genes, compared with HEK293 cells transfected with plasmid vector alone (Mock). PGE 3 uptake by OATP2A1-expressing HEK293 cells (HEK/2A1) was the highest and followed by HEK/1B1, while no significantly higher uptake of PGE 3 than Mock cells was detected by other transporters. Saturation kinetics in PGE 3 uptake by HEK/2A1 estimated the K m as 7.202 ± 0.595 μM, which was 22 times higher than that of PGE 2 ( K m = 0.331 ± 0.131 μM). Furthermore, tissue disposition of PGE 3 was examined in wild-type (WT) and Slco2a1 -deficient ( Slco2a1 −/− ) mice after oral administration of EPA ethyl ester (EPA-E) when they underwent intraperitoneal injection of endotoxin ( e.g. , lipopolysaccharide). PGE 3 concentration was significantly higher in the lung, and tended to increase in the colon, stomach, and kidney of Slco2a1 −/− , compared to WT mice. Ratio of PGE 2 metabolite 15-keto PGE 2 over PGE 2 concentration was significantly lower in the lung and colon of Slco2a1 −/− than that of WT mice, suggesting that PGE 3 metabolism is downregulated in Slco2a1 −/− mice. In conclusion, PGE 3 was found to be a substrate of OATP2A1, and local disposition of PGE 3 could be regulated by OATP2A1 at least in the lung. [ABSTRACT FROM AUTHOR]