Nefiracetam activation of CaM kinase II and protein kinase C mediated by NMDA and metabotropic glutamate receptors in olfactory bulbectomized mice

Aberrant behaviors related to learning and memory in olfactory bulbectomized (OBX) mice have been documented in the previous studies. We reported that the impairment of long-term potentiation (LTP) of hippocampal CA1 regions from OBX mice was associated with down-regulation of CaM kinase II (CaMKII) and protein kinase C (PKC) activities. We now demonstrated that the nootropic drug, nefiracetam, significantly improved spatial reference memory-related behaviors as assessed by Y-maze and novel object recognition task in OBX mice. Nefiracetam also restored hippocampal LTP injured in OBX mice. Nefiracetam treatment restored LTP-induced PKCα (Ser657) and NR1 (Ser896) phosphorylation as well as increase in their basal phosphorylation in the hippocampal CA1 region of OBX mice. Likewise, nefiracetam improved LTP-induced CaMKIIα (Thr286) autophosphorylation and GluR1 (Ser831) phosphorylation and increased their basal phosphorylation. The enhancement of PKCα (Ser657) and CaMKIIα (Thr286) autophosphorylation by nefiracetam was inhibited by treatment with (±)-α-Methyl-(4-carboxyphenyl)glycine and DL-2-Amino-5-phosphonovaleric acid, respectively. The enhancement of LTP induced by nefiracetam is inhibited by treatment with 2-methyl-6-(phenylethynyl)-pyridine, but not by treatment with LY367385, suggesting that metabotropic glutamate receptor 5 (mGluR5) but not mGluR1 is involved in the nefiracetam-induced LTP enhancement. Taken together, nefiracetam ameliorates OBX-induced deficits in memory-related behaviors and impairment of LTP in the hippocampal CA1 region through activation of NMDAR and mGluR5, thereby leading to an increase in activities of CaMKIIα (Thr286) and PKCα (Ser657), respectively.     

Ca2+/calmodulin-dependent protein kinase IIalpha clusters are associated with stable lipid rafts and their formation traps PSD-95

Relatively large number of post-synaptic density (PSD) proteins, including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), have the potential to associate with lipid rafts. We in this study demonstrate that the CaMKIIα clusters induced by ionomycin in human embryonic kidney 293 cells, as well as unclustered CaMKIIα (Du F., Saitoh F., Tian Q. B., Miyazawa S., Endo S. and Suzuki T, 2006, Biochem. Biophys. Res. Commun 347, 814–820), were associated with lipid rafts. The CaMKIIα clusters associated with lipid raft fraction became resistant to treatment with methyl-β-cyclodextrin and subsequent cold Triton X-100, which suggests the stabilization of CaMKIIα cluster-associated lipid rafts. Next, we found that PSD-95, which is also a component of lipid raft fraction and does not interact directly with CaMKII, was trapped by stable CaMKIIα cluster-containing structure. Association of PSD-95 with CaMKIIα clusters was also observed in cultured neuronal cells. These results suggest the CaMKIIα clusters associated with the lipid rafts in the cytoplasmic region play a role in the assembly and stabilization of certain PSD proteins that have the potential to associate with lipid rafts.     

Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha

In the following study, we asked which steroid receptors regulate aggression and arginine vasopressin (AVP) immunoreactivity (– ir) in several limbic regions. Using spontaneous mutant and knockout mice, we generated a novel cross of mice whose offspring lacked estrogen receptor α (ERα), androgen receptor (AR) or both ERα and AR. The wild-type (WT) males and females were compared with ERα knockout (ERαKO) male, mutated AR (Tfm) male and ERαKO/Tfm (double knockout; DKO) male littermates. Animals were gonadectomized and treated with 17β-estradiol (E2) prior to resident-intruder aggression tests. WT and Tfm males showed aggression whereas WT females, ERαKO and DKO males did not. In the lateral septum, WT and Tfm male brains had significantly denser AVP-ir as compared with WT females and DKO males. ERαKO male brains were intermediate in the amount of AVP-ir present. In the medial amygdala, brains from all genotypes had equivalent AVP-ir, except DKO males, which had significantly less AVP-ir. Overall, the expression of aggressive behavior coincided with AVP-ir in WT, Tfm and DKO males. However, in ERαKO males and WT females, the amount of AVP-ir was not associated with resident-intruder aggression. In sum we have shown that E2 acts via ERα to regulate aggression in male mice. In contrast both ERα and AR contribute to AVP-ir in limbic brain regions.     

Decreased Expression of Calmodulin Kinase II and Calcineurin Messenger RNAs in the Mouse Hippocampus After Kainic Acid-Induced Seizures

Abstract: The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIα and CaMKIIβ being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aα is the predominant form in the brain. We studied the expression of CaMKIIα, CaMKIIβ, and CaN Aα subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIα and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid-treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid-treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down-regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.     

Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.     

Female oxytocin gene-knockout mice, in a semi-natural environment, display exaggerated aggressive behavior

Compared to results from a generation of neuropharmacological work, the phenotype of mice lacking the oxytocin (OT) peptide gene was remarkably normal. An important component of the current experiments was to assay OT-knockout (OTKO) and wild-type (WT) littermate control mice living under controlled stressful conditions designed to mimic more closely the environment for which the mouse genome evolved. Furthermore, our experimental group was comprised of an all-female population, in contrast to previous studies which have focused on all-male populations. Our data indicated that aggressive behaviors initiated by OTKO during a food deprivation feeding challenge were considerably more intense and diverse than aggressive behaviors initiated by WT. From the measures of continuous social interaction in the intruder paradigm, it emerged that OTKO mice were more offensively aggressive (attacking rumps and tails) than WT. In a test of parental behaviors, OTKO mice were 100% infanticidal while WT were 16% infanticidal and 50% maternal. Finally, 'alpha females' (always OTKO) were identified in each experiment. They were the most aggressive, the first to feed and the most dominant at nesting behaviors. Semi-natural environments are excellent testing environments for elucidating behavioral differences between transgenic mice and their WT littermates which may not be ordinarily discernible. Future studies of mouse group behavior should include examining female groupings in addition to the more usual all-male groups.     

Mice with podocyte-specific overexpression of wild type α-actinin-4 are healthy controls for K256E-α-actinin-4 mutant transgenic mice

Mutations in the gene ACTN4 encoding the actin bundling protein—α-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant α-actinin-4 (K256E-ACTN4 ^pod+). However, whether α-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype α-actinin-4 (wt-ACTN4 ^pod+). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype α-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged α-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 ^pod+ mice are similar to K256E-ACTN4 ^pod+ mice. In contrast to K256E-ACTN4 ^pod+ mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 ^pod+ mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of α-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.     

Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons

Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1-deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.     

Liver-specific inducible nitric-oxide synthase expression is sufficient to cause hepatic insulin resistance and mild hyperglycemia in mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.     

Aggregate formation and toxicity by wild-type and R621C synphilin-1 in the nigrostriatal system of mice using adenoviral vectors

Synphilin-1 was described as a protein interacting with α-synuclein and is commonly found in Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Our group has previously described and characterized in vitro a mutation in the synphilin-1 gene (R621C) in PD patients. Providing the first characterization of synphilin-1 expression in an animal model, we here used adenoviral gene transfer to study the effects of wild-type (WT) and R621C synphilin-1 in dopaminergic neurons in mouse brain. As synphilin-1 is commonly used to trigger aggregation of α-synuclein in cell culture, we investigated not only non-transgenic C57Bl/6 mice but also A30P-α-synuclein transgenic animals. Both WT synphilin-1 and R621C synphilin-1 led to the formation of Thioflavine-S positive inclusions in C57Bl/6 mice and degeneration of dopaminergic neurons in the substantia nigra. R621C synphilin-1 induced more aggregate formation than WT synphilin-1 in A30P-α-synuclein transgenic mice, consistent with the role of the R621C mutation as a susceptibility factor for PD. Synphilin-1 expression may be used to improve current mouse models of PD, as it induced both the formation of aggregates and degeneration of dopaminergic neurons, two core characteristics of PD that have not been well reproduced with expression of α-synuclein.     

Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays

Social behavior involves both the recognition and production of social cues. Mice with selective deletion (knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -α or -β display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knockouts (KO: OTKO, αERKO, βERKO) from the odors of their wildtype littermates (WT: OTWT, αERWT, βERWT). Females showed marked initial choices of the urine odors of OTWT and βERWT males over those of OTKO and βERKO males, and αERKO males over αERWT males. The odors of OTKO and βERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the αERWT and αERKO males induced aversive, analgesic responses, with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females displaying significantly greater responses to the odors of stressed OTKO and βERKO males than their WTs, and significantly lower analgesia to the odors of stressed αERKO than αERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners' modified responses to their odors.     

Pollen-specific expression of theArabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genespromoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genes

We have characterized the promoter specificity of theArabidopsis thaliana α₁-tubulin (α ₁-tub) gene by studying expression patterns of gene fusions between the 2.2 kbp 5′ upstream region of theα ₁-tub gene and each of three different reporters: chloramphenical acetyltransferase, β-glucuronidase or the diphtheria toxin chain A gene. Analysis of transgenic tobacco andArabidopsis plants carrying the transgene showed that the chloramphenicol acetyltransferase and β-glucuronidase activities were not detected in any vegetative or reproductive organs except mature pollen. Transgenic tobacco plants carrying the diphtheria toxin chain A gene under the control of theα ₁-tub promoter were of normal phenotype but seed fertility was drastically reduced. Furthermore, the transgene could not be transmitted to the next generation through pollen, supporting the observation that theα ₁-tub promoter is active only in pollen. It was observed that the promoter activity was most active in mature pollen and decreased significantly duringin vitro pollen germination, indicating that the promoter is inactive or subdued in germinating pollen. The promoter activity was not affected by various plant growth hormones during pollen maturation.     

Transgenic maize endosperm containing a milk protein has improved amino acid balance

In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein α-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of α-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29–47% greater in endosperm from transgene positive kernels. The content of some other amino acids was changed to a lesser extent. Taken together, these changes resulted in the transgenic endosperms having an improved amino acid balance relative to non-transgenic endosperms produced on the same ear. Kernel appearance, weight, density and zein content did not exhibit substantial differences in kernels expressing the transgene when compared to non-expressing siblings. Assessment of the antigenicity and impacts on animal health will be required in order to determine the overall value of this technology.     

Mice transgenic for the Huntington's disease mutation are resistant to chronic 3-nitropropionic acid-induced striatal toxicity

Neuronal loss in Huntington's disease (HD) is seen first in the neostriatum. It has been suggested that impaired metabolism underlies this degeneration, as striatal vulnerability to excitotoxicity is increased by metabolic compromise. At 12 weeks of age, a transgenic mouse carrying the HD mutation (R6/2 line) has been shown to have an increased vulnerability to the mitochondrial toxin 3-nitropropionic acid (3-NP). However, in contrast, younger R6/2 mice appear to be less vulnerable than wild-type (WT) mice to the excitotoxins kainic acid and quinolinic acid (QA). In this study, we examine the possibility that the sensitivity of R6/2 mice to 3-NP might be age dependent. We treated young, symptomatic R6/2 mice with 3-NP and found that despite their progressive neurological phenotype, they were not more susceptible to 3-NP intoxication than their WT littermates. Further, fewer R6/2 than WT mice developed striatal lesions. We suggest that compensatory mechanisms exist in the R6/2 mouse brain that protect it against the toxic effect of the transgene and coincidentally protect against exogenous toxins such as 3-NP, QA, and kainic acid. The existence of similar compensatory mechanisms may explain why, in humans, HD is a late-onset disorder, despite early expression of the genetic mutation.     

Urokinase-type Plasminogen Activator mRNA is Expressed in Normal Developing Teeth and Leads to Abnormal Incisor Enamel in αMUPA Transgenic Mice

The urokinase-type plasminogen activator (uPA) is a secreted, inducible serine protease implicated in extracellular proteolysis and tissue remodeling. Here we detected uPA mRNA through in situ hybridization in developing molar and incisor teeth of normal mice at multiple sites of the cap and bell developmental stages. The mRNA was confined to epithelial cells, however, was undetectable in ameloblasts or their progenitor preameloblasts and the inner enamel epithelium. Furthermore, mice of five lines of previously described αMUPA transgenic mice, carrying a transgene consisting of the uPA cDNA linked downstream from the αA-crystallin promoter, overexpressed uPA mRNA in the same epithelial sites. In addition, αMUPA mice showed remarkably high levels of uPA mRNA in ameloblasts, however, exclusively in two specific sites late in incisor development. First, at the late secretory stage, but only on sides of the ameloblast layer. Second, in a limited zone of ameloblasts near the incisal end, coinciding with a striking morphological change of the ameloblast layer and the enamel matrix. In adult αMUPA mice, the incisor teeth displayed discoloration and tip fragility, and reduction of the outer enamel as determined by scanning electron microscopy. These results suggest that balanced uPA activity could play a role in normal tooth development. The αMUPA tooth phenotype demonstrates a remarkable sensitivity to excessive extracellular proteolysis at the incisor maturation stage of amelogenesis.     

Steroid-independent male sexual behavior in B6D2F2 male mice

It is well established that male sexual behavior (MSB) is regulated by gonadal steroids; however, individual differences in MSB, independent of gonadal steroids, are prevalent across a wide range of species, and further investigation is necessary to advance our understanding of steroid-independent MSB. Studies utilizing B6D2F1 hybrid male mice in which a significant proportion retain MSB after long-term orchidectomy, identified as steroid-independent-maters (SI-maters), have begun to unravel the genetic underpinnings of steroid-independent MSB. A recent study demonstrated that steroid-independent MSB is a heritable behavioral phenotype that is mainly passed down from B6D2F1 hybrid SI-maters when crossed with C57BL6J female mice. To begin to uncover whether the strain of the dam plays a role in the inheritance of steroid-independent MSB, B6D2F1 hybrid females were crossed with B6D2F1 hybrid males. While the present study confirms the finding that steroid-independent MSB is a heritable behavioral phenotype and that SI-mater sires are more likely to pass down some components of MSB than SI-non-maters to their offspring, it also reveals that the B6D2F2 male offspring that were identified as SI-maters that displayed the full repertoire of steroid-independent MSB had the same probability of being sired from either a B6D2F1 SI-mater or SI-non-mater. These results, in conjunction with previous findings, indicate that the specific chromosomal loci pattern that codes for steroid-independent MSB in the B6D2F2 male offspring may result regardless of whether the father was a SI-mater or SI-non-mater, and that the maternal strain may be an important factor in the inheritance of steroid-independent MSB.     

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as “maters”), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB₁) were orchidectomized and identified as either maters or “non-maters”. A significant proportion of the BXB₁ maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB₁ maters relative to the B6D2F1 and BXB₁ non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior.     

Targeted overexpression of OGFr in epithelium of transgenic mice suppresses cell proliferation and impairs full-thickness wound closure

The opioid growth factor (OGF) and its receptor, OGFr, play a regulatory role in cell proliferation, and maintain homeostasis through a tonically active negative feedback mechanism. To directly evaluate the repercussion of increased OGFr expression and consequent gain-of-function in epithelium, bovine keratin 5 promoter elements were used to direct the expression of OGFr to skin in a tetracycline-regulated manner. Three founder lines overexpressing OGFr (OGFrTG/K5-tTA) were established. Evidence for increased OGFr in the epithelium included a three-fold increase in OGFr binding activity, as well as significant increases in OGFr protein, as monitored by semiquantitative immunohistochemistry. DNA synthesis in target epithelium, including cornea, tongue, and skin of transgenic mice was decreased 41% to 80% from wild-type littermates; the liver, a nonepithelial organ, was not altered. Decreased DNA synthesis in corneal epithelium induced by transgenic expression of OGFr was further reduced by treatment with exogenous OGF but reversed by exposure to the opioid antagonist, naloxone. The number of cell layers in both epidermis and cornea of OGFrTG/K5-tTA animals was reduced nearly 45% from wild-type mice. Full-thickness wounds in mice overexpressing OGFr healed 37% to 75% slower than wild-type littermates. These data demonstrate for the first time that stable genetic amplification of OGFr downregulates homeostatic cell proliferation, as well as pathophysiological processes with respect to wound repair. These mice also can serve as a valuable model to dissect the mechanism of OGF-OGFr action and may be important in understanding the etiology, pathogenesis, and treatment of epithelium-related diseases.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.