Gene expression profiling of the preclinical scrapie-infected hippocampus

The molecular events that underlie prion disease neuropathology remain poorly defined. Within the hippocampus of the ME7/CV mouse scrapie model, profound CA1 neuronal loss occurs between 160 and 180 days post-infection (dpi). To elucidate the molecular events that may contribute to this neuronal loss, we have applied Affymetrix high-density oligonucleotide probe arrays to the study of ME7-infected hippocampal gene expression at 170 dpi. The study has identified 78 genes that are differentially expressed greater than 1.5-fold within the preclinical ME7-infected hippocampus prior to the profound late stage glial cell activation. The results indicate oxidative and endoplasmic reticulum (ER) stress, activated ER and mitochondrial apoptosis pathways, and activated cholesterol biosynthesis within the scrapie-infected hippocampus, and offer insight into the molecular events which underlie the neuropathology.     

L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C > G mutation (CTT–GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.     

Cloning and expression analysis of androgen receptor gene in chicken embryogenesis

We cloned a full-length androgen receptor (AR) cDNA from chicken (Gallus gallus) gonads. The cDNA sequence has an open reading frame of 2109 bp encoding 703 amino acids. The chicken AR (cAR) shares high homology with ARs from other species in its amino acid sequences, in particular DNA binding domain (DBD) and ligand binding domain (LBD). RT-PCR analysis revealed that cAR mRNA is expressed in several embryonic tissues of both sexes, and relatively higher expression was observed in left ovary compared with testis. The immunoreactive signal of AR was co-localized within the ovarian cell nucleus, while such nuclear localization was not detected in those of testis. To get insight on the possible role of androgen-AR signaling during gonadal development, non-steroidal AR antagonist, flutamide, was administrated in ovo. The treatment induced the disorganization of sex cords in ovarian cortex at day 12 of incubation. The effect was restored by testosterone co-treatment, implying the possibility that AR mediated signaling may be involved in ovarian morphogenesis. Furthermore, co-treatment of flutamide with estradiol-17beta (E2) also restored the phenotype, suggesting androgen-AR signaling might activate aromatase expression that is necessary for estrogen synthesis. These findings suggest androgen-AR signaling might contribute to chicken embryonic ovarian development.     

Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression

Differences in gene expression patterns between adult and postnatal day 7 (P7) mouse cerebellum, at the peak of granule neuron migration, were analyzed by hybridization to the GLYCOv2 glycogene array. This custom designed oligonucleotide array focuses on glycosyl transferases, carbohydrate-binding proteins, proteoglycans and related genes, and 173 genes were identified as being differentially expressed with statistical confidence. Expression levels for 11 of these genes were compared by RT-PCR, and their differential expression between P7 and adult cerebellum confirmed. Within the group of genes showing differential expression, the sialyltransferases (SiaTs) and GalNAc-Ts that were elevated at P7 prefer glycoprotein substrates, whilst the SiaTs and GalNAc-Ts that were elevated in the adult preferentially modify glycolipids, consistent with a role for gangliosides in maintaining neuronal function in the adult. Also within this group, three proteoglycans--versican, bamacan and glypican-2--were elevated at P7, along with growth factor midkine, which is known to bind to multiple types of proteoglycans, and fibroblast growth factor receptor 1, whose activity is known to be influenced by heparan sulfate proteoglycans. Two sulfotransferases that can modify the extent of proteoglycan sulfation were also differentially regulated, and may modify the interaction of a subset of proteoglycans with their binding partners during cerebellar development. Bamacan, glypican-2 and midkine were shown to be expressed in different cell types, and their roles in cerebellar development during granule neuron migration and maturation are discussed.     

Role of androgens and the androgen receptor in remodeling of spine synapses in limbic brain areas

Accumulating evidence indicate that structural synaptic plasticity in limbic areas plays a vital role not only in normal brain functions, such as cognition and mood, but also in the development of neurological and mental disorders. We have learned from studies investigating neuronal remodeling that estrogens have an exceptional synaptogenic potential that seems to be specific to limbic areas of the adult female brain. On the other hand, structural synaptic plasticity in the adult male brain and the synaptogenic effect of androgens received relatively little attention. During the last five years, the Leranth laboratory provided conclusive evidence that the hippocampus and prefrontal cortex of adult male rodents and non-human primates retain considerable structural synaptic plasticity similar to the female, and that androgens are capable of inducing spine synapse growth in both the hippocampus and prefrontal cortex similar to estrogens. Our recent work also demonstrates that androgen-induced remodeling of spine synapses in the prefrontal cortex of adult male rats is dependent, at least to some extent, on functional androgen receptors, while being entirely independent of the androgen receptor in the hippocampus. Based on these findings and on their many beneficial effects, we believe that androgens hold a great and undeservingly neglected therapeutic potential that could be employed to reverse synaptic pathology in various neurocognitive and neuropsychiatric disorders.     

Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.     

Demonstration of a stage-specific expression of the ZFY protein in fetal mouse testis using anti-peptide antibodies.

The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno-staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development.     

Stage dependent and androgen inductive expression of orphan receptor TR4 in rat testis

In this study, we investigated the expression of TR4 in different stages of seminiferous tubules and the relationship between TR4 and androgen in rat testis. We found that TR4 was stage-dependently expressed in rat seminiferous tubules, T withdrawal induced by high doses of testosterone undecanoate and ethane dimethane sulfonate inhibit TR4 expression in rat testis, and testosterone induced TR4 expression in co-cultured primary germ/Sertoli cells. Furthermore, we demonstrated that androgen receptor could enhance TR4-mediated transactivation activity in testis cells in the presence of testosterone. Together, these data indicate that the expression of TR4 in rat testis is stage dependent and androgen inductive, and suggest the important role of orphan receptor TR4 in spermatogenesis.     

Protein kinase activity of the insulin receptor is essential for insulin-regulated gene expression

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven -galactosidase (pRSVLTR-gal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV₂HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-gal and pMLSV₂HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters.     

Pomegranate polyphenols down-regulate expression of androgen-synthesizing genes in human prostate cancer cells overexpressing the androgen receptor

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state in which they progress in the absence of circulating testosterone, leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis, which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In this study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes and the AR. We measured expression of the HSD3B2 (3beta-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5alpha reductase type 1) genes for the respective androgen-synthesizing enzymes in LNCaP, LNCaP-AR and DU-145 human prostate cancer cells. A twofold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P=.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is up-regulated.     

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control

VSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix^® Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNA levels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11b2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.