Yongbum Koo

Kalirin is Under-Expressed in Alzheimer's Disease Hippocampus

To identify genes aberrantly expressed in the brain of individuals with Alzheimers Disease (AD), we analyzed RNA extracts from the hippocampus and cerebellum from 19 AD patients and 15 age- and sex-matched control subjects. Our analysis identified a number of genes that were over-expressed or under-expressed specifically in AD hippocampus. Among these genes, kalirin was the most consistently under-expressed in AD hippocampus, which was verified by semi-quantitative RT-PCR and real time PCR. Kalirin is predominantly expressed in the brain, particularly in the hippocampus, and plays crucial roles in neuronal stability and growth. Our observation is the first to relate kalirin to AD and a human disease. In addition to kalirin, the genes for voltage-gated Ca++ channel gamma subunit 3 and visinin-like protein 1 (a Ca++ sensor protein) were under-expressed, whereas inositol 1,4,5-triphosphate 3-kinase B was over-expressed in AD hippocampus. Collectively, these differential expressions could severely impair calcium homeostasis. Remarkably, these aberrant gene expressions in AD hippocampus were not observed in AD cerebellum. Furthermore, housekeeping genes such as ribosomal protein genes are not affected by AD. These results provide new insights into the biochemistry of AD.

Generation of Cyp17iCre transgenic mice and their application to conditionally delete estrogen receptor alpha (Esr1) from the ovary and testis

A transgenic mouse line that expresses iCre under regulation of the Cytochrome P450 17α‐hydroxylase/17, 20‐lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report, we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell‐specific manner. genesis 46:499‐505, 2008.

Disrupted kisspeptin signaling in GnRH neurons leads to hypogonadotrophic hypogonadism.

Landmark studies have shown that mutations in kisspeptin and the kisspeptin receptor (Kiss1r) result in reproductive dysfunction in humans and genetically altered mouse models. However, because kisspeptin and its receptor are present in target cells of the central and peripheral reproductive axis, the precise location(s) for the pathogenic signal is unknown. The study described herein shows that the kisspeptin-Kiss1r signaling pathway in the GnRH neuron is singularly critical for both the onset of puberty as well as the attainment of normal reproductive function. In this study, we directly test the hypothesis that kisspeptin neurons regulate GnRH secretion through the activation of Kiss1r on the plasma membrane of GnRH neurons. A GnRH neuron-specific Kiss1r knockout mouse model (GKirKO) was generated, and reproductive development and phenotype were assessed. Both female and male GKirKO mice were infertile, having low serum LH and FSH levels. External abnormalities such as microphallus and decreased anogenital distance associated with failure of preputial gland separation were present in GKirKO males. A delay in pubertal onset and abnormal estrous cyclicity were observed in female GKirKO mice. Taken together, these data provide in vivo evidence that Kiss1r in GnRH neurons is critical for reproductive development and fertility.

Orientation of Follicle-stimulating Hormone (FSH) Subunits Complexed with the FSH Receptor β SUBUNIT TOWARD THE N TERMINUS OF EXODOMAIN AND α SUBUNIT TO EXOLOOP 3

Follicle-stimulating hormone (FSH) comprises an α subunit and a β subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH β subunit is specifically labeled with the N-terminal peptide, whereas the α subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH β subunit is close to the N-terminal region and that the α subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the α subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.

Glucocorticoids modulate NF-κB-dependent gene expression by up-regulating FKBP51 expression in Newcastle disease virus-infected chickens

FK506-binding protein 51(FKBP51, coded by FKBP5) is a co-chaperone molecule that interacts with the chaperone HSP90 and the glucocorticoid receptor (GR) in an inactive GR complex. It is a negative regulator of glucocorticoid action and is replaced by the positive regulator, FK506-binding protein 52 (FKBP52, coded by FKBP4) when hormone binds to GR, which renders the GR complex active. In this study, we found that the expression of FKBP51 mRNA in 12 organs of Newcastle disease virus (NDV)-infected chickens was robustly induced. The level of corticosterone in NDV-infected chickens was also elevated, approximately 2- to 6.5-fold in the organs compared to non-infected control chickens. The induction of FKBP51 mRNA expression was reproduced by dexamethasone treatment, indicating a role for glucocorticoids in the systemic induction of FKBP51 mRNA expression. In chicken UMNSAH/DF-1 cells, nuclear factor kappaB (NF-kappaB) was activated in an FKBP51-dependent manner. Regulation of the three NF-kappaB-dependent, anti-apoptotic genes, bcl-2, bcl-x and bfl-1/A1 was investigated in UMNSAH/DF-1 cells. Dexamethasone treatment of UMNSAH/DF-1 cells resulted in up-regulation of bcl-2, and down-regulation of bcl-x and bfl-1/A1. Expression of FKBP51 also resulted in down-regulation of bfl-1/A1, but had no effect on bcl-2 and bcl-x, suggesting the involvement of glucocorticoid-FKBP51-NF-kappaB signaling in the regulation of expression of bfl-1/A1 in UMNSAH/DF-1 cells. We observed organ-specific up- or down-regulation of expression of, bcl-2, bcl-x and bfl-1/A1 in NDV-infected and dexamethasone-treated chickens. Differential regulation of bfl-1/A1, bcl-2 and bcl-x upon NDV-infection and dexamethasone treatment suggests that additional factors are involved in the regulation of these genes. These results suggest that systemic elevation of FKBP51 in NDV-infected chickens activates NF-kappaB, which cooperates with other factors to regulate the expression of NF-kappaB-dependent genes.

Generation of an estrogen receptor beta-iCre knock-in mouse

A novel knock‐in mouse that expresses codon‐improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2‐iCre mice were bred with ROSA26‐lacZ and Ai9‐RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2‐iCre mice will serve as a novel line for conditionally ablating genes in Esr2‐expressing tissues, identifying novel Esr2‐expressing cells, and differentiating the functions of ESR2 and ESR1. genesis 54:38–52, 2016.

Generation and characterization of an endothelin-2 iCre mouse.

A novel transgenic mouse line that expresses codon‐improved Cre recombinase (iCre) under regulation of the Endothelin‐2 gene (edn2) promoter was developed for the conditional deletion of genes in Endothelin‐2 lineage cells and for the spatial and temporal localization of Endothelin‐2 expression. Endothelin‐2 (EDN2, ET‐2, previously VIC) is a transcriptionally regulated 21 amino acid peptide implicated in vascular homeostasis, and more recently in female reproduction, gastrointestinal function, immunology, and cancer pathogenesis that acts through membrane receptors and G‐protein signaling. A cassette (edn2‐iCre) was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker in front of the endogenous start codon of the edn2 gene in a mouse genome BAC clone. The cassette was introduced into the C57BL/6 genome by pronuclear injection, and two lines of edn2‐iCre positive mice were produced. The edn2‐iCre mice were bred with ROSA26‐lacZ and Ai9 reporter mice to visualize areas of functional iCre expression. Strong expression was seen in the periovulatory ovary, stomach and small intestine, and colon. Uniquely, we report punctate expression in the corneal epithelium, the liver, the lung, the pituitary, the uterus, and the heart. In the embryo, expression is localized in developing hair follicles and the dermis. Therefore, edn2‐iCre mice will serve as a novel line for conditional gene deletion in these tissues. genesis 53:245–256, 2015.

Generation and characterization of an estrogen receptor alpha‐iCre knock‐in mouse

Two estrogen receptors, ESR1 and ESR2, are responsible for the classical actions of estrogens in mammalian species. They display different spatiotemporal expression patterns and nonoverlapping functions in various tissues and physiological conditions. In this study, a novel knock‐in mouse line that expresses codon‐improved Cre recombinase (iCre) under regulation of the natural Esr1 promoter (Esr1–iCre) was developed. Functional characterization of iCre expression by crossing them with reporter lines (ROSA26‐lacZ or Ai9‐RFP) showed that iCre is faithfully expressed in Esr1‐lineage cells. This novel transgenic mouse line will be a useful animal model for lineage‐tracing Esr1‐expressing cells, selective gene ablation in the Esr1‐lineage cells and for generating global Esr1 knockout mice.

A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

Changes in ovarian constriction by endothelin-2/receptor system in the feline ovary

Endothelin-2 (ET-2) is transiently expressed in the granulosa cells of mammalian periovulatory follicles immediately prior to ovulation. Ex vivo experiments showed that, upon treatment with ET-2, the rodent ovary rapidly contracts. When the endothelin receptor pathway is antagonized in vivo, ovulation is inhibited in rodents. These findings led us to postulate that ET-2–induced contraction of smooth muscles in the ovarian cortex is a final trigger for follicle rupture at the time of ovulation. Similar to humans and other mammals, feline ovaries possess a layer of contractile smooth muscle-like cells around developing follicles, known as the theca externa. In addition, feline ovaries have been documented to spontaneously contract ex vivo; however, the function of this spontaneous contraction and whether ET-2 induces ovulation remains to be determined. Here, we investigated the characteristics of feline ovarian cortical contraction using myography in the absence or presence of physiological doses of ET-2. Whole fresh feline ovaries were collected after spay procedures through the Junior Surgery Program of the College of Veterinary Medicine at the University of Illinois. Of 13 ovaries tested, all demonstrated a period of strong and sustained contraction when washed with a 50 nM solution of feline ET-2 peptide for 30 minutes, with an average increase in base tensile force of 2.48 ± 0.40 mN. Additionally, when washed for 20 minutes with a 140 nM solution of the dual ET receptor antagonist tezosentan contraction was reduced in a dose-dependent manner. Of these ovaries, 4 demonstrated spontaneous contractions prior to ET-2 treatment, with average amplitude of 4.08 ± 2.45 mN, duration of 22.2 ± 6.4sec, and a time of 60.9 ± 20.2sec between contractions. These contractions continued after ET-2 treatment, but contraction amplitude was reduced (1.25 ± 0.95 mN) as was the time between contractions (13.8 ± 6.5 sec) for all ovaries. There was no change in the duration of these contractions (20.13 ± 4.66 sec). Measurement of mRNA expression by polymerase chain reaction showed that feline ovaries express mRNA for ET-2, both isotypes of endothelin receptors (ET-A and ET-B), and endothelin converting enzymes 1 and 2 (ECE-1 and ECE-2). This study demonstrates that ET-2 produces a feline ovarian cortical contraction. Future work will determine the impact of inhibiting ET-2 and the endothelin receptor pathways in vivo on follicle rupture in the feline ovary.