Generation of Pmel-dependent conditional and inducible Cre-driver mouse line for melanocytic-targeted gene manipulation

Conditional and inducible gene targeting using Cre/loxP-mediated recombination is a powerful reverse genetics approach used to study spatiotemporal gene functions in specified cell types. To enable temporal gene manipulation in the melanocyte lineage, we established a novel inducible Cre-driver mouse line by targeting an all-in-one tetracycline/doxycycline (Dox)-inducible Cre expression cassette into the Pmel locus (Pmel^{P2A-TetON3G-TRE3G-iCre}), a gene locus preferentially expressed in pigment cells. By crossing these Cre-driver mice with a strong Cre-reporter mouse line, Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}, we show the effectiveness of the Pmel^{P2A-TetON3G-TRE3G-iCre} mouse line in facilitating Dox-inducible Cre/loxP recombination in a wide variety of pigment cell lineages including hair follicle melanocytes and their stem cells. Furthermore, to demonstrate proof of concept, we ablated Notch signaling postnatally in the Pmel^{P2A-TetON3G-TRE3G-iCre} mice. In agreement with the previously reported phenotype, induced ablation of Notch signaling in the melanocyte lineage resulted in premature hair graying, demonstrating the utility of the Pmel^{P2A-TetON3G-TRE3G-iCre} allele. Therefore, the Pmel^{P2A-TetON3G-TRE3G-iCre} mouse line is suitable for assessing gene functions in melanocytes using an in vivo inducible reverse genetics approach. Furthermore, we unexpectedly identified previously unrecognized PMEL-expressing cells in non-pigmentary organs in the mice, suggesting unanticipated functions of PMEL other than melanosome formation.     

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a G_s-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection ¹. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.     

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.     

SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type 4 (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with the cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic L374 variant. Our data suggest that SLC45A2 maintains melanosome neutralization that is initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common allelic variant imparts reduced activity due to protein instability.     

Melanin: A Two Edged Sword?

Melanin is both photosensitizer and photoprotector. Skin cancer rates decrease with increasing constitutive pigmentation, yet the pigment has been shown to be photoreactive and capable of producing damaging reactive oxygen species. We utilized model systems of related cells or similar cell type that vary in constitutive and in induced pigment. Induction of eumelanin in Cloudman S91 mouse melanoma cells leads to less UV-induced killing and to less mutation induction at the ouabain locus (Na⁺, K⁺-ATPase). Pigmented mouse melanocytes, melan-b (brown) and melan-a (black) were slightly less sensitive than melan-c (albino) melanocytes to killing after UVC and UVA but were more sensitive to killing after UVB and UVB + UVA. Pigment had a small sensitizing effect on pyrimidine dimer DNA damage in both the melanoma cells and the melanocytes. The lack of consistency in these results suggests that intracellular pigment may disregulate the milieu intérieur resulting in end effects that are unrelated to the original genomic damage.     

Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice

The K14‐SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14‐Scf, tyrosinase‐mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14‐SCF Tyr^c2j/c2j animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14‐Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c‐kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.     

CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies

Background

Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo.

Methodology/Principal Findings

WT or CD40L^−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L^−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L^−/− mice. However, CD40L^−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L^−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels.

Conclusion

We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.     

Mutations in or near the transmembrane domain alter PMEL amyloid formation from functional to pathogenic

PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)--which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core--are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show that the DW and HoSi mutations alter PMEL TMD oligomerization and/or association with membranes, with consequent formation of aberrantly packed fibrils. The aberrant fibrils are associated with a loss of pigmentation in cultured melanocytes, suggesting that they inhibit melanin production and/or melanosome integrity. A secondary mutation in the Smoky chicken, which reverts the dominant DW phenotype, prevents the accumulation of PMEL in fibrillogenic compartments and thus averts DW-associated pigment loss; a secondary mutation found in the Dun chicken likely dampens a HoSi-like dominant mutation in a similar manner. We propose that the DW and HoSi mutations alter the normally benign amyloid to a pathogenic form that antagonizes melanosome function, and that the secondary mutations found in the Smoky and Dun chickens revert or dampen pathogenicity by functioning as null alleles, thus preventing the formation of aberrant fibrils. We speculate that PMEL mutations can model the conversion between physiological and pathological amyloid.     

Transport activity and presence of ClC‐7/Ostm1 complex account for different cellular functions

Loss of the lysosomal ClC-7/Ostm1 2Cl⁻/H⁺ exchanger causes lysosomal storage disease and osteopetrosis in humans and additionally changes fur colour in mice. Its conversion into a Cl⁻ conductance in Clcn7^unc/unc mice entails similarly severe lysosomal storage, but less severe osteopetrosis and no change in fur colour. To elucidate the basis for these phenotypical differences, we generated Clcn7^td/td mice expressing an ion transport-deficient mutant. Their osteopetrosis was as severe as in Clcn7^−/− mice, suggesting that the electric shunt provided by ClC-7^unc can partially rescue osteoclast function. The normal coat colour of Clcn7^td/td mice and their less severe neurodegeneration suggested that the ClC-7 protein, even when lacking measurable ion transport activity, is sufficient for hair pigmentation and that the conductance of ClC-7^unc is harmful for neurons. Our in vivo structure-function analysis of ClC-7 reveals that both protein-protein interactions and ion transport must be considered in the pathogenesis of ClC-7-related diseases.Subject Categories: Membrane & Intracellular Transport; Molecular Biology of Disease     

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH ^−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH ^−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH ^−/− iPS cell lines, we aggregated FAH ^−/−-iPS cells with tetraploid embryos and obtained entirely FAH ^−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH ^−/− mice. Then, we transduced FAH cDNA into the FAH ^−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

Cellular senescence and organismal ageing in the absence of p21CIP1/WAF1 in ku80−/− mice

Ku80 is important in the repair of DNA double-strand breaks by its essential function in non-homologous end-joining. The absence of Ku80 causes the accumulation of DNA damage and leads to premature ageing in mice. We showed that mouse embryonic fibroblasts (MEFs) from ku80^−/− mice senesced rapidly with elevated levels of p53 and p21. Deletion of p21 delayed the early senescence phenotype in ku80^−/− MEFs, despite an otherwise intact response of p53. In contrast to ku80^−/−p53^−/− mice, which die rapidly primarily from lymphomas, there was no significant increase in tumorigenesis in ku80^−/−p21^−/− mice. However, ku80^−/−p21^−/− mice showed no improvement with respect to rough fur coat or osteopaenia, and even showed a shortened lifespan compared with ku80^−/− mice. These results show that the increased lifespan of ku80^−/− MEFs owing to the loss of p21 is not associated with an improvement of the premature ageing phenotypes of ku80^−/− mice observed at the organismal level.     

Characterization of Photorhabdus luminescens Growth for the Rearing of the Beneficial Nematode Heterorhabditis bacteriophora

Culturing the bioluminescent bacterium Photorhabdus luminescens in nutrient broth (NB) is used to recover phase I cells. These phase I cells were highly luminescent for up to 7 h in this media and the luminosity could also be seen with the naked eye after a 15 min eye adjustment period in a dark room. Red pigmentation is a known trait of phase I cells and was visually distinct within the culture media. The color shade of the red pigment varied on nutrient agar and in NB suggesting that the concentration of the pigment produced is dependent upon density of phase I cells within a specified area. The specific growth rate (μ) and doubling time (g) was determined during the logarithmic growth phase to be 0.36 h⁻¹ and 2.1 h, respectively in NB medium. The nematode-bacterium suspension was injected into larvae of Galleria mellonella to test for entomopathogencity. Within 24 h post-injection insect mortality was seen along with dark red pigmentation and extremely high luminosity indicating infection with P. luminescens.     

Insufficient glucose supply is linked to hypothermia upon cold exposure in high-fat diet-fed mice lacking PEMT

Mice that lack phosphatidylethanolamine N-methyltransferase (Pemt^−/− mice) are protected from high-fat (HF) diet-induced obesity. HF-fed Pemt^−/− mice show higher oxygen consumption and heat production, indicating that more energy might be utilized for thermogenesis and might account for the resistance to diet-induced weight gain. To test this hypothesis, HF-fed Pemt^−/− and Pemt^+/+ mice were challenged with acute cold exposure at 4°C. Unexpectedly, HF-fed Pemt^−/− mice developed hypothermia within 3 h of cold exposure. In contrast, chow-fed Pemt^−/− mice, possessing similar body mass, maintained body temperature. Lack of PEMT did not impair the capacity for thermogenesis in skeletal muscle or brown adipose tissue. Plasma catecholamines were not altered by Pemt genotype, and stimulation of lipolysis was intact in brown and white adipose tissue of Pemt^−/− mice. HF-fed Pemt^−/− mice also developed higher systolic blood pressure, accompanied by reduced cardiac output. Choline supplementation reversed the cold-induced hypothermia in HF-fed Pemt^−/− mice with no effect on blood pressure. Plasma glucose levels were ∼50% lower in HF-fed Pemt^−/− mice compared with Pemt^+/+ mice. Choline supplementation normalized plasma hypoglycemia and the expression of proteins involved in gluconeogenesis. We propose that cold-induced hypothermia in HF-fed Pemt^−/− mice is linked to plasma hypoglycemia due to compromised hepatic glucose production.     

Genetic meta-analysis of 15,901 African Americans identifies variation in EXOC3L1 is associated with HDL concentration

Meta-analyses of European populations has successfully identified genetic variants in over 150 loci associated with lipid levels, but results from additional ethnicities remain limited. Previously, we reported two novel lipid loci identified in a sample of 7,657 African Americans using a gene-centric array including 50,000 SNPs in 2,100 candidate genes. Initial discovery and follow-up of signals with P < 10⁻⁵ in additional African American samples confirmed CD36 and ICAM1. Using an additional 8,244 African American female samples from the Women’s Health Initiative SNP Health Association Resource genome-wide association study dataset, we further examined the previous meta-analyses results by attempting to replicate 20 additional putative lipid signals with P < 10⁻⁴. Replication confirmed rs868213, located in a splice donor region of exocyst complex component 3-like 1 (EXOC3L1) as a novel signal for HDL (additive allelic effect β = 0.02; P = 1.4 × 10⁻⁸; meta-analyses of discovery and replication). EXOC3L1 is strongly expressed in vascular endothelium and forms part of the exocyst complex, a key facilitator of the trafficking of lipid receptors. Increasing sample sizes for genetic studies in nonEuropean populations will continue to improve our understanding of lipid metabolism.