Dissecting the involvement of tropomyosin-related kinase A and p75 neurotrophin receptor signaling in NGF deficit-induced neurodegeneration.

NGF, the principal neurotrophic factor for basal forebrain cholinergic neurons (BFCNs), has been correlated to Alzheimer's disease (AD) because of the selective vulnerability of BFCNs in AD. These correlative links do not substantiate a comprehensive cause-effect mechanism connecting NGF deficit to overall AD neurodegeneration. A demonstration that neutralizing NGF activity could have consequences beyond a direct interference with the cholinergic system came from studies in the AD11 mouse model, in which the expression of a highly specific anti-NGF antibody determines a neurodegeneration that encompasses several features of human AD. Because the transgenic antibody binds to mature NGF much more strongly than to proNGF and prevents binding of mature NGF to the tropomyosin-related kinase A (TrkA) receptor and to p75 neurotrophin receptor (p75NTR), we postulated that neurodegeneration in AD11 mice is provoked by an imbalance of proNGF/NGF signaling and, consequently, of TrkA/p75NTR signaling. To test this hypothesis, in this study we characterize the phenotype of two lines of transgenic mice, one in which TrkA signaling is inhibited by neutralizing anti-TrkA antibodies and a second one in which anti-NGF mice were crossed to p75NTR(exonIII(-/-)) mice to abrogate p75NTR signaling. TrkA neutralization determines a strong cholinergic deficit and the appearance of beta-amyloid peptide (Abeta) but no tau-related pathology. In contrast, abrogating p75NTR signaling determines a full rescue of the cholinergic and Abeta phenotype of anti-NGF mice, but tau hyperphosphorylation is exacerbated. Thus, we demonstrate that inhibiting TrkA signaling activates Abeta accumulation and that different streams of AD neurodegeneration are related in complex ways to TrkA versus p75NTR signaling.

A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.

Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington's disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington's disease-like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington's disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington's disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.

Maternal thyroid hormones are transcriptionally active during embryo-foetal development: results from a novel transgenic mouse model.

Even though several studies highlighted the role of maternal thyroid hormones (THs) during embryo-foetal development, direct evidence of their interaction with embryonic thyroid receptors (TRs) is still lacking. We generated a transgenic mouse model ubiquitously expressing a reporter gene tracing TH action during development. We engineered a construct (TRE2×) containing two TH-responsive elements controlling the expression of the LacZ reporter gene, which encodes ß-galactosidase (ß-gal). The specificity of the TRE2× activation by TH was evaluated in NIH3T3 cells by cotransfecting TRE2× along with TRs, retinoic or oestrogen receptors in the presence of their specific ligands. TRE2× transgene was microinjected into the zygotes, implanted in pseudopregnant BDF1 (a first-generation (F1) hybrid from a cross of C57BL/6 female and a DBA/2 male) mice and transgenic mouse models were developed. ß-gal expression was assayed in tissue sections of transgenic mouse embryos at different stages of development. In vitro, TRE2× transactivation was observed only following physiological T3 stimulation, mediated exclusively by TRs. In vivo, ß-gal staining, absent until embryonic day 9.5-10.5 (E9.5-E10.5), was observed as early as E11.5-E12.5 in different primordia (i.e. central nervous system, sense organs, intestine, etc.) of the TRE2× transgenic embryos, while the foetal thyroid function (FTF) was still inactive. Immunohistochemistry for TRs essentially colocalized with ß-gal staining. No ß-gal staining was detected in embryos of hypothyroid transgenic mice. Importantly, treatment with T3 in hypothyroid TRE2× transgenic mice rescued ß-gal expression. Our results provide in vivo direct evidence that during embryonic life and before the onset of FTF, maternal THs are transcriptionally active through the action of embryonic TRs. This model may have clinical relevance and may be employed to design end-point assays for new molecules affecting THs action.

A novel peroxisome proliferator-activated receptor responsive element-luciferase reporter mouse reveals gender specificity of peroxisome proliferator-activated receptor activity in liver.

There is a growing interest in peroxisome proliferator-activated receptors (PPARs) as major players in the regulation of lipid and carbohydrate metabolism. Drugs targeting PPARs were in fact shown to have major relevance for the treatment of diseases associated with aging, such as arteriosclerosis and diabetes. However, a variety of toxic effects associated with PPAR ligand administration has been documented, including hepatocarcinogenesis, which may severely limit its therapeutic use. A better comprehension of the multiplicity of PPAR physiological functions is therefore mandatory for the development of novel, safer drugs. We here describe the generation of a novel transgenic mouse for the detection of the generalized activities of PPARs, the PPAR responsive element-Luc reporter mouse. In this model luciferase expression is under the control of a PPAR-inducible promoter in all target organs. By optical imaging and ex vivo analysis, we were able to demonstrate the remarkable gender specificity of the PPAR transcriptional activity in liver. In fact, in the liver of female PPAR responsive element-Luc, the PPAR reporter transgene is more than one order of magnitude less expressed, thus leading to the conclusion that the signaling in females is much less activated than in males. Diet or hormonal manipulations as demonstrated here by treatments with high-fat diet or gonad removal and hormone replacement do not influence this low activation. The extent of the gender difference in PPAR transcriptional activity and the ineffectiveness of hormone treatments or diet to significantly elevate liver PPAR activity in females led us to hypothesize that gender-specific epigenetic events occurring during development may affect PPAR signaling in the liver. This study sets the ground for understanding the differential susceptibility of the two genders to metabolic disorders; furthermore, the model generated provides a novel opportunity for the molecular characterization of PPAR activity in pathophysiological conditions.

Overexpression of the ped/pea-15 gene causes diabetes by impairing glucose-stimulated insulin secretion in addition to insulin action.

Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Calpha activation and block of insulin induction of protein kinase Czeta. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Calpha activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.

Tissue-specific sensitivity to AID expression in transgenic mouse models.

Activation-induced cytidine deaminase (AID), an enzyme with homology to members of the APOBEC family, is involved in somatic hypermutation (SHM) of immunoglobulin (Ig) genes, either by direct deamination of DNA or by an indirect action through its putative RNA editing activity. AID is able to mutate both Ig-like reporter constructs and selected non-Ig genes in normal B cells and in other cells when ectopically overexpressed in mammalian cells and transgenic mice. However, in spite of the fact that in these transgenic animals AID activity was driven by an ubiquitous promoter, only T lymphomas and lung adenomas occurred. In the present work, we constructed three sets of transgenic mice in which AID was under the control of lck, HTLV-I and MMTV promoters, respectively. The lck/AID mice developed thymic lymphomas with variable but high efficiency, while no tumor was detected in HTLV-I/AID mice after two years of monitoring. Four MMTV/AID founder mice died with an atypical clinical picture, although no mammary tumor was found. These findings suggest that additional factors, present in thymocytes but not in other tissues or in lymphoid cells at different stages of differentiation, are needed for AID to fully manifest its tumorigenic potential in mouse. Alternatively, the display of full AID mutagenic and transforming activity could be related to the existence of physiologic DSBs which occur in both thymocytes and switching B cells.

Insights from a transgenic mouse model on the role of SLC26A2 in health and disease.

Mutations in the SLC26A2 cause a family of recessive chondrodysplasias that includes in order of decreasing severity achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia and recessive multiple epiphyseal dysplasia. The gene encodes for a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate that is needed for proteoglycan sulfation. To investigate the mechanisms leading to skeletal dysplasia, we generated a transgenic mouse with a mutation in Slc26a2 causing a partial loss of function of the sulfate transporter. Homozygous mutant mice were characterized by skeletal dysplasia with chondrocytes of irregular size, delay in the formation of the secondary ossification centre and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts, but proteoglycan undersulfation was detected only in cartilage. The similarity with human diastrophic dysplasia makes this mouse a model to explore pathogenetic and therapeutic aspects of SLC26A2-related disorders.

Loss of tyrosinase activity confers increased skin tumor susceptibility in mice.

The tyrosinase (Tyr) gene encodes the enzyme tyrosinase that catalyses the conversion of L-tyrosine into DOPA (3,4-dihydroxyphenylalanine)-quinone. The albino mutation abrogates functional activity of tyrosinase resulting in deficiency of melanin pigment production in skin and retina. Tyr maps to a region in the central position of Chromosome 7 that contains a skin tumor-modifier locus. We rescued the albino mutation in transgenic mice to assess a possible role of Tyr gene in two-stage skin carcinogenesis. Transgenic expression of the functional Tyr(Cys) allele in albino mice (Tyr(Ser)) caused a reduction in skin papilloma multiplicity, in four independent experiments and at three dose levels of DMBA (9,10-dimethyl-1,2-benzanthracene). In vitro mechanistic studies demonstrated that transfection of the Tyr(Cys) allele in a human squamous cell carcinoma cell line (NCI-H520) increases tyrosinase enzyme activity and confers resistance to hydrogen peroxide-induced oxidative DNA damage. These results provide direct evidence that the Tyr gene can act as a skin cancer-modifier gene, whose mechanism of action may involve modulation of oxidative DNA damage.

Impaired keratinocyte proliferative and clonogenic potential in transgenic mice overexpressing 14-3-3σ in the epidermis.

The 14-3-3 protein family controls diverse biochemical processes through interaction with phosphorylated consensus sequences in protein targets. Its epithelial specific member, 14-3-3σ, also known as stratifin, is highly expressed in differentiated keratinocytes, and in vitro evidence indicates that 14-3-3σ downregulation leads to keratinocyte immortalization. To define the role of 14-3-3σ in skin homeostasis in vivo, we generated transgenic mice overexpressing 14-3-3σ in proliferating keratinocytes of the epidermis and hair follicle. Transgenic animals show decreased epidermal thickness and hair follicle density associated with reduced number of proliferating keratinocytes and decreased levels of keratins 14, 5, and 15. Primary keratinocytes isolated from transgenic mice manifest reduced proliferation and migration. Moreover, clonogenicity assessment and label-retaining analysis reveal a reduction in keratinocyte progenitor cell number in transgenic mice. Response to IGF-1 is strongly impaired in cultured transgenic keratinocytes compared with wild-type cells. Consistently, activation of phosphoinositol 3-kinase (PI3K), AKT, and Rac1, all IGF-1 downstream mediators, is reduced. Our results demonstrate that 14-3-3σ controls the in vivo epidermal proliferation-differentiation switch by reducing proliferative potential and forcing keratinocytes to exit the cell cycle, and that this effect associates with inhibition of the IGF-1 pathway.

Peripheral neutralization of nerve growth factor induces immunosympathectomy and central neurodegeneration in transgenic mice.

We previously showed that anti-nerve growth factor (NGF) antibodies expressed in transgenic mice (AD11) elicit a progressive neurodegeneration, comprising the triad of Alzheimer's disease (AD) hallmarks: cholinergic loss, tau hyperphosphorylation, and amyloid-beta peptide formation. However, since anti-NGF antibodies are produced both in the brain and in peripheral tissues of AD11 mice, the contribution of peripheral neutralization of NGF to the onset of brain neurodegeneration was still unexplored. To address this question, we characterized a line of transgenic mice (AD10) in which anti-NGF antibodies are obligatorily produced only in lymphocytes, being initially found in blood. In AD10 mice, peripheral NGF neutralization elicits shrinkage of superior cervical ganglia (immunosympathectomy) and, as a consequence of this, peripheral anti-NGF antibodies cross the blood brain barrier (BBB) and reach the brain, generating an NGF-dependent neurodegeneration, largely superimposable to that observed in AD11 mice. This demonstrates that peripherally originated anti-NGF antibodies can generate a neurodegeneration in the central nervous system of an animal model. Consistently, peripherally-delivered NGF is effective in preventing the onset of the central cholinergic deficit. These findings could have a direct relevance for some human sporadic AD cases, highlighting the role of the BBB disruption and suggesting a causally relevant role of circulating antibodies in AD pathology.

Role of the molybdoflavoenzyme aldehyde oxidase homolog 2 in the biosynthesis of retinoic acid: generation and characterization of a knockout mouse.

The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2(-/-) mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian gland's transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses.

Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy.

Ethylmalonic encephalopathy is an autosomal recessive, invariably fatal disorder characterized by early-onset encephalopathy, microangiopathy, chronic diarrhea, defective cytochrome c oxidase (COX) in muscle and brain, high concentrations of C4 and C5 acylcarnitines in blood and high excretion of ethylmalonic acid in urine. ETHE1, a gene encoding a beta-lactamase-like, iron-coordinating metalloprotein, is mutated in ethylmalonic encephalopathy. In bacteria, ETHE1-like sequences are in the same operon of, or fused with, orthologs of TST, the gene encoding rhodanese, a sulfurtransferase. In eukaryotes, both ETHE1 and rhodanese are located within the mitochondrial matrix. We created a Ethe1(-/-) mouse that showed the cardinal features of ethylmalonic encephalopathy. We found that thiosulfate was excreted in massive amounts in urine of both Ethe1(-/-) mice and humans with ethylmalonic encephalopathy. High thiosulfate and sulfide concentrations were present in Ethe1(-/-) mouse tissues. Sulfide is a powerful inhibitor of COX and short-chain fatty acid oxidation, with vasoactive and vasotoxic effects that explain the microangiopathy in ethylmalonic encephalopathy patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase. Sulfur dioxygenase activity was absent in Ethe1(-/-) mice, whereas it was markedly increased by ETHE1 overexpression in HeLa cells and Escherichia coli. Therefore, ETHE1 is a mitochondrial sulfur dioxygenase involved in catabolism of sulfide that accumulates to toxic levels in ethylmalonic encephalopathy.

Increased longevity and refractoriness to Ca(2+)-dependent neurodegeneration in Surf1 knockout mice.

Leigh syndrome associated with cytochrome c oxidase (COX) deficiency is a mitochondrial disorder usually caused by mutations of SURF1, a gene encoding a putative COX assembly factor. We present here a Surf1-/- recombinant mouse obtained by inserting a loxP sequence in the open reading frame of the gene. The frequency of -/-, +/+ and +/- genotypes in newborn mice followed a mendelian distribution, indicating that the ablation of Surf1 is compatible with postnatal survival. The biochemical and assembly COX defect was present in Surf1(loxP)-/- mice, but milder than in humans. Surprisingly, not only these animals failed to show spontaneous neurodegeneration at any age, but they also displayed markedly prolonged lifespan, and complete protection from Ca(2+)-dependent neurotoxicity induced by kainic acid. Experiments on primary neuronal cultures showed markedly reduced rise of cytosolic and mitochondrial Ca(2+) in Surf1(loxP)-/- neurons, and reduced mortality, compared to controls. The mitochondrial membrane potential was unchanged in KO versus wild-type neurons, suggesting that the effects of the ablation of Surf1 on Ca(2+) homeostasis, and possibly on longevity, may be independent, at least in part, from those on COX assembly and mitochondrial bioenergetics.

Increased Ca2+ storage capacity of the skeletal muscle sarcoplasmic reticulum of transgenic mice over-expressing membrane bound calcium binding protein junctate.

Junctate is an integral sarco(endo)plasmic reticulum protein expressed in many tissues including heart and skeletal muscle. Because of its localization and biochemical characteristics, junctate is deemed to participate in the regulation of the intracellular Ca2+ concentration. However, its physiological function in muscle cells has not been investigated yet. In this study we examined the effects of junctate over-expression by generating a transgenic mouse model which over-expresses junctate in skeletal muscle. Our results demonstrate that junctate over-expression induced a significant increase in SR Ca2+ storage capacity which was paralleled by an increased 4-chloro-m-cresol and caffeine-induced Ca2+ release, whereas it did not affect SR Ca2+-dependent ATPase activity and SR Ca2+ loading rates. In addition, junctate over-expression did not affect the expression levels of SR Ca2+ binding proteins such as calsequestrin, calreticulin and sarcalumenin. These findings suggest that junctate over-expression is associated with an increase in the SR Ca2+ storage capacity and releasable Ca2+ content and support a physiological role for junctate in intracellular Ca2+ homeostasis.

Targeted expression of RALT in mouse skin inhibits epidermal growth factor receptor signalling and generates a Waved-like phenotype.

Although it has been clearly established that negative feedback loops have a fundamental role in the regulation of epidermal growth factor receptor (EGFR) signalling in flies, their role in the regulation of mammalian EGFR has been inferred only recently from in vitro studies. Here, we report on the forced expression of RALT/MIG-6, a negative feedback regulator of ErbB receptors, in mouse skin. A RALT transgene driven by the K14 promoter generated a dose-dependent phenotype resembling that caused by hypomorphic and antimorphic Egfr alleles-that is, wavy coat, curly whiskers and open eyes at birth. Ex vivo keratinocytes from K14-RALT mice showed reduced biochemical and biological responses when stimulated by ErbB ligands. Conversely, knockdown of RALT by RNA interference enhanced ErbB mitogenic signalling. Thus, RALT behaves as a suppressor of EGFR signalling in mouse skin.

A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype.

Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) cause a family of recessively inherited chondrodysplasias including, in order of decreasing severity, achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia. The gene encodes a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate, which is needed for proteoglycan sulfation. To provide new insights in the pathogenetic mechanisms leading to skeletal and connective tissue dysplasia and to obtain an in vivo model for therapeutic approaches to DTD, we generated a Dtdst knock-in mouse with a partial loss of function of the sulfate transporter. In addition, the intronic neomycine cassette in the mutant allele contributed to the hypomorphic phenotype by inducing abnormal splicing. Homozygous mutant mice were characterized by growth retardation, skeletal dysplasia and joint contractures, thereby recapitulating essential aspects of the DTD phenotype in man. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, delay in the formation of the secondary ossification center and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts. In spite of the generalized nature of the sulfate uptake defect, significant proteoglycan undersulfation was detected only in cartilage. Chondrocyte proliferation and apoptosis studies suggested that reduced proliferation and/or lack of terminal chondrocyte differentiation might contribute to reduced bone growth. The similarity with human DTD makes this mouse strain a useful model to explore pathogenetic and therapeutic aspects of DTDST-related disorders.

Griseofulvin induces mitotic delay and aneuploidy in bone marrow cells of orally treated mice.

Griseofulvin (GF) is a fungicide drug well characterized for its aneugenic activity in vitro. In vivo strong evidence of aneuploidy and polyploidy induction has been obtained in germ cells, especially in oocytes. More controversial are the data on the aneugenicity of griseofulvin in somatic cells. In this paper we provide evidence that GF induces non-disjunction and cell cycle delay in bone marrow cells of orally treated mice. Adult female mice were administered olive oil suspensions of 200, 666 or 2000 mg/kg GF by gavage and killed 18 or 24 h later. To minimize animal-to-animal variation in the absorption and distribution of GF, mice were fasted from the time of GF administration to the time of killing. Two hours before treatment the animals were s.c. implanted with a bromodeoxyuridine tablet to obtain differential chromatid staining and to determine the number of divisions after GF treatment for each metaphase. Mitostatic effects of GF were assessed by the relative proportions of first, second and third generation metaphases and the average generation time (AGT) method. A statistically significant increase with respect to the control AGT value was observed after treatment with 666 and 2000 mg/kg, suggesting that GF, as already shown in meiosis, interfered with cell cycle progression. Hyperploidy was scored in second generation metaphases. Eighteen hours after treatment, the frequencies of hyperploid cells were significantly (P < 0.05) higher in all GF-treated groups than in their matched control group. The effect was not dose-dependent. No further increase in aneuploidy was observed at 24 h, suggesting that cells overcoming mitotic arrest did not have a higher rate of non-disjunction. No induction of polyploidy was demonstrated. We conclude that GF induces mitotic delay and aneuploidy in mouse bone marrow and suggest that the protocol used to formulate the gavage suspensions and the after-treatment fasting of the animals enhanced the bioavailability of GF to bone marrow cells.

Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family, comprising at least five cytokines specifically involved in the regulation of vascular and/or lymphatic endothelium differentiation. Several lines of evidence indicate a role for PlGF in monocyte chemotaxis and in potentiating the activity of VEGF, but the exact function of this cytokine is not fully understood. To define the biological role of PlGF in vivo, we have produced a transgenic mouse model overexpressing this factor in the skin by using a keratin 14 promoter cassette. Our data indicate that PlGF has strong angiogenic properties in both fetal and adult life. PlGF overexpression results in a substantial increase in the number, branching and size of dermal blood vessels as well as in enhanced vascular permeability. Indeed, intradermally injected recombinant PlGF was able to induce vessel permeability in wild-type mice. The analysis of vascular endothelial growth factor receptor 1/flt-1 and vascular endothelial growth factor receptor 2/flk-1 indicates that the two receptors are induced in the skin endothelium of transgenic mice suggesting that both are involved in mediating the effect of overexpressed PlGF.

Constitutive knockout of Surf1 is associated with high embryonic lethality, mitochondrial disease and cytochrome c oxidase deficiency in mice.

We report here the creation of a constitutive knockout mouse for SURF1, a gene encoding one of the assembly proteins involved in the formation of cytochrome c oxidase (COX). Loss-of-function mutations of SURF1 cause Leigh syndrome associated with an isolated and generalized COX deficiency in humans. The murine phenotype is characterized by the following hallmarks: (1) high post-implantation embryonic lethality, affecting approximately 90% of the Surf1(-/-) individuals; (2) early-onset mortality of post-natal individuals; (3) highly significant deficit in muscle strength and motor performance; (4) profound and isolated defect of COX activity in skeletal muscle and liver, and, to a lesser extent, heart and brain; (5) morphological abnormalities of skeletal muscle, characterized by reduced histochemical reaction to COX and mitochondrial proliferation; (6) no obvious abnormalities in brain morphology, reflecting the virtual absence of overt neurological symptoms. These results indicate a function for murine Surf1 protein (Surf1p) specifically related to COX and recapitulate, at least in part, the human phenotype. This is the first mammalian model for a nuclear disease gene of a human mitochondrial disorder. Our model constitutes a useful tool to investigate the function of Surf1p, help understand the pathogenesis of Surf1p deficiency in vivo, and evaluate the efficacy of treatment.

Conserved POU binding DNA sites in the Sox2 upstream enhancer regulate gene expression in embryonic and neural stem cells.

The Sox2 transcription factor is expressed early in the stem cells of the blastocyst inner cell mass and, later, in neural stem cells. We previously identified a Sox2 5'-regulatory region directing transgene expression to the inner cell mass and, later, to neural stem cells and precursors of the forebrain. Here, we identify a core enhancer element able to specify transgene expression in forebrain neural precursors of mouse embryos, and we show that the same core element efficiently activates transcription in inner cell mass-derived embryonic stem (ES) cells. Mutation of POU factor binding sites, able to recognize the neural factors Brn1 and Brn2, shows that these sites contribute to transgene activity in neural cells. The same sites are also essential for activity in ES cells, where they bind different members of the POU family, including Oct4, as shown by gel shift assays and chromatin immunoprecipitation with anti-Oct4 antibodies. Our findings indicate a role for the same POU binding motifs in Sox2 transgene regulation in both ES and neural precursor cells. Oct4 might play a role in the regulation of Sox2 in ES (inner cell mass) cells and, possibly, at the transition between inner cell mass and neural cells, before recruitment of neural POU factors such as Brn1 and Brn2.