Gabor Szalai

Obesity in Aging Exacerbates Blood–Brain Barrier Disruption, Neuroinflammation, and Oxidative Stress in the Mouse Hippocampus: Effects on Expression of Genes Involved in Beta-Amyloid Generation and Alzheimer’s Disease

There is growing evidence that obesity has deleterious effects on the brain and cognitive function in the elderly population. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesity-induced cerebromicrovascular damage and neuroinflammation, we compared young (7 months) and aged (24 months) high fat diet-fed obese C57BL/6 mice. Aging exacerbated obesity-induced systemic inflammation and blood-brain barrier disruption, as indicated by the increased circulating levels of proinflammatory cytokines and increased presence of extravasated immunoglobulin G in the hippocampus, respectively. Obesity-induced blood-brain barrier damage was associated with microglia activation, upregulation of activating Fc-gamma receptors and proinflammatory cytokines, and increased oxidative stress. Treatment of cultured primary microglia with sera derived from aged obese mice resulted in significantly more pronounced microglia activation and oxidative stress, as compared with treatment with young sera. Serum-induced activation and oxidative stress were also exacerbated in primary microglia derived from aged animals. Hippocampal expression of genes involved in regulation of the cellular amyloid precursor protein-dependent signaling pathways, beta-amyloid generation, and the pathogenesis of tauopathy were largely unaffected by obesity in aged mice. Collectively, obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates blood-brain barrier disruption. The resulting neuroinflammation and oxidative stress in the mouse hippocampus likely contribute to the significant cognitive decline observed in aged obese animals.

Endothelial Fli1 Deficiency Impairs Vascular Homeostasis : A Role in Scleroderma Vasculopathy

Systemic sclerosis or scleroderma (SSc) is a complex autoimmune connective tissue disease characterized by obliterative vasculopathy and tissue fibrosis. The molecular mechanisms underlying SSc vasculopathy are largely unknown. Friend leukemia integration factor 1 (Fli1), an important regulator of immune function and collagen fibrillogenesis, is expressed at reduced levels in endothelial cells in affected skin of patients with SSc. To develop a disease model and to investigate the function of Fli1 in the vasculature, we generated mice with a conditional deletion of Fli1 in endothelial cells (Fli1 CKO). Fli1 CKO mice showed a disorganized dermal vascular network with greatly compromised vessel integrity and markedly increased vessel permeability. We show that Fli1 regulates expression of genes involved in maintaining vascular homeostasis including VE-cadherin, platelet endothelial cell adhesion molecule 1, type IV collagen, matrix metalloproteinase 9, platelet-derived growth factor B, and S1P(1) receptor. Accordingly, Fli1 CKO mice are characterized by down-regulation of VE-cadherin and platelet endothelial cell adhesion molecule 1, impaired development of basement membrane, and a decreased presence of alpha-smooth muscle actin-positive cells in dermal microvessels. This phenotype is consistent with a role of Fli1 as a regulator of vessel maturation and stabilization. Importantly, vascular characteristics of Fli1 CKO mice are recapitulated by SSc microvasculature. Thus, persistently reduced levels of Fli1 in endothelial cells may play a critical role in the development of SSc vasculopathy.

Molecular mechanisms of megakaryopoiesis

Abstract.One function of bone marrow megakaryocytes (MKs) is the controlled release of platelets into the circulation. Over the past few years, molecular mechanisms that contribute to MK development and differentiation have begun to be elucidated. This review provides a brief overview of megakaryopoiesis and platelet function, and the importance of selected hematopoietic transcription factors (including GATA-1, FOG, Fli-1, AML1, and NF-E2) and target genes in this biological process. In addition, a discussion of human diseases affecting megakaryopoiesis and mouse models of thrombocytopenia are presented with emphasis on how these systems have and will continue to provide further insights into mechanisms that control the biological functions of the megakaryocytic cell lineage. Ultimately, such knowledge may provide the basis for novel therapeutic approaches for modulation of platelet number and function.

Transcription Factor Fli1 Regulates Collagen Fibrillogenesis in Mouse Skin

ABSTRACT Biosynthesis of fibrillar collagen in the skin is precisely regulated to maintain proper tissue homeostasis; however, the molecular mechanisms involved in this process remain largely unknown. Transcription factor Fli1 has been shown to repress collagen synthesis in cultured dermal fibroblasts. This study investigated the role of Fli1 in regulation of collagen biosynthesis in mice skin in vivo using mice with the homozygous deletion of the C-terminal transcriptional activation (CTA) domain of the Fli1 gene (Fli1ΔCTA/ΔCTA). Skin analyses of the Fli1 mutant mice revealed a significant upregulation of fibrillar collagen genes at mRNA level, as well as increased collagen content as measured by acetic acid extraction and hydroxyproline assays. In addition, collagen fibrils contained ultrastructural abnormalities including immature thin fibrils and very thick irregularly shaped fibrils, which correlated with the reduced levels of decorin, fibromodulin, and lumican. Fibroblasts cultured from the skin of Fli1ΔCTA/ΔCTA mice maintained elevated synthesis of collagen mRNA and protein. Additional experiments in cultured fibroblasts have revealed that although Fli1 ΔCTA retains the ability to bind to the collagen promoter in vitro and in vivo, it no longer functions as transcriptional repressor. Together, these results establish Fli1 as a key regulator of the collagen homeostasis in the skin in vivo.

Longevity is associated with increased vascular resistance to high glucose-induced oxidative stress and inflammatory gene expression in Peromyscus leucopus

Vascular aging is characterized by increased oxidative stress and proinflammatory phenotypic alterations. Metabolic stress, such as hyperglycemia in diabetes, is known to increase the production of ROS and promote inflammatory gene expression, accelerating vascular aging. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower steady-state production of ROS and/or superior resistance to the prooxidant effects of metabolic stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), which show a more than twofold difference in maximum lifespan potential (8.2 and 3.5 yr, respectively). We compared interspecies differences in steady-state and high glucose (HG; 30 mmol/l)-induced production of O(2)(*-) and H(2)O(2), endothelial function, mitochondrial ROS generation, and inflammatory gene expression in cultured aortic segments. In P. leucopus aortas, steady-state endothelial O(2)(*-) and H(2)O(2) production and ROS generation by mitochondria were less than in M. musculus vessels. Furthermore, vessels of P. leucopus were more resistant to the prooxidant effects of HG. Primary fibroblasts from P. leucopus also exhibited less steady-state and HG-induced ROS production than M. musculus cells. In M. musculus arteries, HG elicited significant upregulation of inflammatory markers (TNF-alpha, IL-6, ICAM-1, VCAM, and monocyte chemoattractant protein-1). In contrast, the proinflammatory effects of HG were blunted in P. leucopus vessels. Thus, increased life span potential in P. leucopus is associated with decreased cellular ROS generation and increased resistance to prooxidant and proinflammatory effects of metabolic stress, which accord with predictions of the oxidative stress hypothesis of aging.

Peromyscus as a Mammalian epigenetic model.

Deer mice (Peromyscus) offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential.

Evolution of Monogamy, Paternal Investment, and Female Life History in Peromyscus

The timing of reproductive development and associated trade-offs in quantity versus quality of offspring produced across the life span are well documented in a wide range of species. The relation of these aspects of maternal life history to monogamy and paternal investment in offspring is not well studied in mammals, due in part to the rarity of the latter. By using five large, captive-bred populations of Peromyscus species that range from promiscuous mating with little paternal investment (P. maniculatus bairdii) to social and genetic monogamy with substantial paternal investment (P. californicus insignis), we modeled the interaction between monogamy and female life history. Monogamy and high paternal investment were associated with smaller litter size, delayed maternal reproduction that extended over a longer reproductive life span, and larger, higher quality offspring. The results suggest monogamy and paternal investment can alter the evolution of female life-history trajectories in mammals.

Peromyscus (deer mice) as developmental models

Deer mice (Peromyscus) are the most common native North American mammals, and exhibit great natural genetic variation. Wild‐derived stocks from a number of populations are available from the Peromyscus Genetic Stock Center (PGSC). The PGSC also houses a number of natural variants and mutants (many of which appear to differ from Mus). These include metabolic, coat‐color/pattern, neurological, and other morphological variants/mutants. Nearly all these mutants are on a common genetic background, the Peromyscus maniculatus BW stock. Peromyscus are also superior behavior models in areas such as repetitive behavior and pair‐bonding effects, as multiple species are monogamous. While Peromyscus development generally resembles that of Mus and Rattus, prenatal stages have not been as thoroughly studied, and there appear to be intriguing differences (e.g., longer time spent at the two‐cell stage). Development is greatly perturbed in crosses between P. maniculatus (BW) and Peromyscus polionotus (PO). BW females crossed to PO males produce growth‐restricted, but otherwise healthy, fertile offspring which allows for genetic analyses of the many traits that differ between these two species. PO females crossed to BW males produce overgrown but severely dysmorphic conceptuses that rarely survive to late gestation. There are likely many more uses for these animals as developmental models than we have described here. Peromyscus models can now be more fully exploited due to the emerging genetic (full linkage map), genomic (genomes of four stocks have been sequenced) and reproductive resources.

Prostate-Derived ETS Factor Regulates Epithelial-to-Mesenchymal Transition through Both SLUG-Dependent and Independent Mechanisms

The 5-year survival rate is very low when breast cancer becomes metastatic. The metastatic process is governed by a network of molecules of which SLUG is known to play a major role as a regulator of epithelial-to-mesenchymal transition (EMT). Prostate-derived ETS factor (PDEF) has been proposed as a tumor suppressor, possibly through inhibition of invasion and metastasis; therefore, understanding the mechanism of PDEF regulation may help to better understand its role in breast cancer progression. This study shows for the first time that the transcription factor SLUG is a direct target of PDEF in breast cancer. We show that the expression of PDEF is able to suppress/dampen EMT through the negative regulation of SLUG. In addition, we show that PDEF is also able to regulate downstream targets of SLUG, namely E-cadherin, in both SLUG-dependent and -independent manners, suggesting a critical role for PDEF in regulating EMT.

The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus-including deer mice-are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.