Release of growth factors by neuronal precursor cells as a treatment for diseases with tau pathology

The intraneuronal accumulation of the microtubule associated protein tau in a hyperphosphorylated state and the extracellular deposit of ?amyloid protein constitute the defining neuropathological signature of Alzheimer's disease, the most common type of dementia in ageing Homo sapiens.There is accumulating evidence suggesting that transplantation of embryonic and adult derived neuronal precursor cells (NPCs) has a major role for cell based repair strategies in models of acute and chronic injury. In order to determine whether NPCs could rescue tau related neuronal cell death NPCs were transplanted into the transgenic mouse cortex of transgenic mice expressing human P301S tau protein at 2 month of age and the effect followed 2 and 3 months after transplantation. The results demonstrated that following transplantation mouse NPCs differentiated into astrocytes and exerted a neuroprotective effect. In particular, the expression of ciliary neurotrophic factor, nerve growth factor and glial cell derived neurotrophic factor was increased near the transplanted cells. A nonsignificant increase of brain derived neurotrophic factor expression was instead found in the area of the cortex where neuronal death was rescued.     

A transgenic mouse model that is useful for analyzing cellular and geographic differentiation of the intestine during fetal development

Regional as well as cell-specific differences in gene expression are established and maintained in the perpetually regenerating intestinal epithelium. We have recently linked regions of the 5'-nontranscribed domain of the rat "liver" fatty acid binding protein (L-FABP) gene which is normally expressed in both liver and intestine, to a reporter, the human growth hormone (hGH) gene, and examined hGH expression in adult transgenic mice (Sweetser, D. A., Birkenmeier, E. H., Hoppe, P. C., McKeel, D. W., and Gordon, J. I. (1988) Genes Dev. 2, 1318-1332). Our results indicated that cis-acting elements, including an orientation-independent suppressor, could produce a pattern of cellular and geographic expression of hGH which mimics that of the intact, endogenous murine Fabpl gene in both organs. We now show that nucleotides -4000 to +21 of the rat L-FABP gene can direct "appropriate" cell-specific, regional, and temporal expression of the hGH reporter during a period of remarkable cellular expansion, cytodifferentiation, and morphologic transformation of the fetal gut epithelium. These studies also indicate that the polyclonal stem cell population located in the intervillous regions of the late fetal intestine exhibits a different pattern of transgene regulation than does the monoclonally derived crypt stem cell population in adult transgenic mice. Nucleotides -4000 to +21 are not sufficient to reproduce the normal temporal pattern of L-FABP gene activation in liver. Precocious expression of growth hormone in this pedigree of transgenic mice results in early induction of insulin-like growth factor I mRNA accumulation in liver but has no effect on insulin-like growth factor II mRNA levels. In contrast, local synthesis of growth hormone in the small intestine does not influence its insulin-like growth factor I or II mRNA levels.     

A biologically active rhIGF-1 fusion accumulated in transgenic rice seeds can reduce blood glucose in diabetic mice via oral delivery

Human insulin-like growth factor 1(hIGF-1) is essential for cell proliferation and used therapeutically in treating various diseases including diabetes mellitus. Here, we present that a recombinant hIGF-1(rhIGF-1) was expressed fused with the C-terminus of a rice luminal binding protein and accumulated highly in rice seeds, reaching 6.8+/-0.5% of total seed protein. The rhIGF-1 fusion was demonstrated to possess biological activity to stimulate cell proliferation. Importantly, the unprocessed transgenic seeds could significantly increase plasma rhIGF-1 level and reduce blood glucose of diabetic mice via oral delivery. Further studies suggested that transgenic seeds reduced blood glucose of diabetic mice by enhancing islet cells survival and increasing insulin secretion rather than increasing insulin sensitivity. These results indicated the potential of the novel fusion expression system in production and oral delivery of biologically active small peptides for diseases.     

Male germ cell-specific RNA binding protein RBMY: a new oncogene explaining male predominance in liver cancer

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC.     

Insulin-like growth factor II plays a central role in atherosclerosis in a mouse model.

Insulin-like growth factor II is a fetal promoter of cell proliferation that is involved in some forms of cancer and overgrowth syndromes in humans. Here, we provide two sources of genetic evidence for a novel, pivotal role of locally produced insulin-like growth factor II in the development of atherosclerosis. First, we show that homozygosity for a disrupted insulin-like growth factor II allele in mice lacking apolipoprotein E, a widely used animal model of atherosclerosis, results in aortic lesions that are approximately 80% smaller and contain approximately 50% less proliferating cells compared with mice lacking only apolipoprotein E. Second, targeted expression of an insulin-like growth factor II transgene in smooth muscle cells, but not the mere elevation of circulating levels of the peptide, causes per se aortic focal intimal thickenings. The insulin-like growth factor II transgenics presented here are the first viable mutant mice spontaneously developing intimal masses. These observations provide the first direct evidence for an atherogenic activity of insulin-like growth factor II in vivo.     

Human apolipoprotein C-I expression in mice impairs learning and memory functions

The H2 allele of APOC1, giving rise to increased gene expression of apolipoprotein C-I (apoC-I), is in genetic disequilibrium with the APOE4 allele and may provide a major risk factor for Alzheimer's disease (AD). We found that apoC-I protein is present in astrocytes and endothelial cells within hippocampal regions in both human control and AD brains. Interestingly, apoC-I colocalized with beta-amyloid (Abeta) in plaques in AD brains, and in vitro experiments revealed that aggregation of Abeta was delayed in the presence of apoC-I. Moreover, apoC-I was found to exacerbate the soluble Abeta oligomer-induced neuronal death. To establish a potential role for apoC-I in cognitive functions, we used human (h) APOC1(+/0) transgenic mice that express APOC1 mRNA throughout their brains and apoC-I protein in astrocytes and endothelial cells. The hAPOC1(+/0) mice displayed impaired hippocampal-dependent learning and memory functions compared with their wild-type littermates, as judged from their performance in the object recognition task (P = 0.012) and in the Morris water maze task (P = 0.010). ApoC-I may affect learning as a result of its inhibitory properties toward apoE-dependent lipid metabolism. However, no differences in brain mRNA or protein levels of endogenous apoE were detected between transgenic and wild-type mice. In conclusion, human apoC-I expression impairs cognitive functions in mice independent of apoE expression, which supports the potential of a modulatory role for apoC-I during the development of AD.     

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5′- and 3′-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.     

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30 °C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30 °C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30 °C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.     

Expression of epidermal growth factor in transgenic mice causes growth retardation

The epidermal growth factor (EGF) family of peptides signals through the erbB family of receptor tyrosine kinases and plays important roles in development and tumorigenesis. Both EGF and transforming growth factor (TGF)-alpha only bind to erbB1 and activate it. The precursor of EGF is distinct from that of TGF-alpha in having eight additional EGF-like repeats. We have recently shown that the EGF precursor without these repeats is biologically active and leads to hypospermatogenesis in transgenic mice. Here we present evidence that the growth of transgenic mice widely expressing this engineered EGF precursor is also stunted. These mice were consistently born at half the normal weight and reached almost 80% of normal weight at adulthood. The mechanism involved a reduction of serum insulin-like growth factor-binding protein-3. Chondrocyte development in the growth plate was affected, and osteoblasts accumulated in the endosteum and periosteum. Besides these novel findings on the in vivo effects of EGF on bone development, we observed no sign of tumor formation in our transgenic animals. In contrast to previous reports on TGF-alpha transgenic mice, we show that the biological functions of EGF and TGF-alpha are clearly distinct.     

Mild calorie restriction does not affect testosterone levels and testicular gene expression in mutant mice

The hypothalamic-pituitary-gonadal (HPG) axis and the somatotropic axis are influenced by nutritional factors. Calorie restriction (CR) extends lifespan but suppresses both the HPG and the somatotropic axes. Since most CR studies use a fairly severe (40%-60%) reduction of calorie intake, we hypothesized that a milder CR (20%) might not be deleterious to reproduction in male mice. To test this hypothesis, we evaluated the effects of 20% CR on testicular testosterone content and on testicular expression of genes that are relevant to testicular function and reproductive competence, including insulin-like growth factor-I, cytochrome P450 aromatase (Cyp19a1), androgen receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, cytochrome P450c17 and 3-beta-hydroxysteroid dehydrogenase/isomerase. To relate CR effects to the activity of the somatotropic axis, we have used growth hormone-resistant GHR knockout mice as well as transgenic mice overexpressing GH. Mild CR did not affect testosterone levels in testis homogenates and had little effect on expression of the examined genes in the reproductive organs. Altered activity of the GH/insulin-like growth factor-1 axis had a major impact on the parameters analyzed. The results also suggest that expression of several key genes involved in the control of testicular function is preserved under conditions of mild CR and encourage speculation that mild regimens of CR can produce longevity benefits without impairing reproduction.     

Impaired NK cell development in an IFN-gamma transgenic mouse: aberrantly expressed IFN-gamma enhances hematopoietic stem cell apoptosis and affects NK cell differentiation

Aberrant expression of IFN-gamma has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-gamma in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1(+)CD3(-) cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-gamma transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-gamma transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-gamma inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-gammaRalpha subunit and undergo apoptosis in response to exogenous IFN-gamma. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-gamma transgenic mouse model. Furthermore, because exogenous IFN-gamma affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-gamma in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.     

Heterogeneity of Glia in the Retina and Optic Nerve of Birds and Mammals

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transitin. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100β, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.     

Rapid activation of astrocyte-specific expression of GFAP-lacZ transgene by focal injury

Astrocytes form a key cellular component of the central nervous system. They respond vigorously to diverse neurologic insults by undergoing hypertrophy and increasing expression of the glial fibrillary acidic protein (GFAP) gene, but their functions are largely unknown. To analyze astrocytes in vivo we constructed a transgenic vector from GFAP gene sequences and monitored its efficiency by fusing it to lacZ. Injection of the GFAP-lacZ hybrid gene into the germline of mice yielded six different lines of transgenic mice. In all lines the expression of lacZ was astrocyte-specific. In unmanipulated transgenic animals beta-galactosidase activity was much more prominent in astrocytes of the hippocampal formation, selected white matter tracts, and glial limitans than in astrocytes of other areas. This pattern of expression illustrates the physiologic heterogeneity of astrocytes and probably reflects differences in functional demands placed on these cells in different brain regions. Upmodulation of transgene expression was used to determine the time frame within which astroglial activation and increased GFAP gene expression occur following a neurologic insult. Induction of GFAP-lacZ expression was detectable within 1 hour after focal mechanical trauma. This demonstrates that the response of astrocytes to neurologic injury is very rapid and implies that these cells could fulfill important early functions in wound healing within the central nervous system.     

Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.     

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4(+) T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4(+) Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-gamma-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-gamma activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.     

Transforming growth factor-beta 1 potentiates amyloid-beta generation in astrocytes and in transgenic mice

Accumulation of the amyloid-beta peptide (Abeta) in the brain is crucial for development of Alzheimer's disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been correlated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia. Here, we demonstrate that TGF-beta1 drives the production of Abeta40/42 by astrocytes leading to Abeta production in TGF-beta1 transgenic mice. First, TGF-beta1 induces the overexpression of the amyloid precursor protein (APP) in astrocytes but not in neurons, involving a highly conserved TGF-beta1-responsive element in the 5'-untranslated region (+54/+74) of the APP promoter. Second, we demonstrated an increased release of soluble APP-beta which led to TGF-beta1-induced Abeta generation in both murine and human astrocytes. These results demonstrate that TGF-beta1 potentiates Abeta production in human astrocytes and may enhance the formation of plaques burden in the brain of Alzheimer's disease patients.     

Expression of granulosa cell-specific genes and induction of apoptosis in conditionally immortalized granulosa cell lines established from H-2Kb-tsA58 transgenic mice

Granulosa cell lines have been established from H-2Kb-tsA58 transgenic mice. Using immunocytochemistry, significant amounts of insulin-like growth factor-I (IGF-I) were found in all cell lines investigated, whereas estrogen and progesterone receptor expression could be detected in only some of the lines. All cell lines showed low basal production of the gonadal steroids estradiol and progesterone. The genes for the ovarian paracrine regulators IGF-I and basic fibroblast growth factor were expressed, as well as the genes for anti-Müllerian hormone and for the P450 side-chain cleavage enzyme (P450scc). Expression of P450scc could be shown to be up-regulated in the cell lines under conditions mimicking the hormonal environment of the luteinizing granulosa cells in vivo. Inactivation of the temperature-sensitive SV40 T antigen by a shift of the cell lines to the nonpermissive temperature of 39.5 degrees C led to massive induction of apoptosis in several cell lines. These cell lines will allow a detailed study of the mechanisms regulating the expression of granulosa cell-specific functions, as well as the induction of granulosa cell apoptosis.