Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

Kisspeptin Stimulates Growth Hormone Release by Utilizing Neuropeptide Y Pathways and Is Dependent on the Presence of Ghrelin in the Ewe.

Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.

Reproduction and beyond, kisspeptin in ruminants.

Kisspeptin (Kp) is synthesized in the arcuate nucleus and preoptic area of the hypothalamus and is a regulator of gonadotropin releasing hormone in the hypothalamus. In addition, Kp may regulate additional functions such as increased neuropeptide Y gene expression and reduced proopiomelanocortin (POMC) gene expression in sheep. Other studies have found a role for Kp to release growth hormone (GH), prolactin and luteinizing hormone (LH) from cattle, rat and monkey pituitary cells. Intravenous injection of Kp stimulated release LH, GH, prolactin and follicle stimulating hormone in some experiments in cattle and sheep, but other studies have failed to find an effect of peripheral injection of Kp on GH release. Recent studies indicate that Kp can stimulate GH release after intracerebroventricular injection in sheep at doses that do not release GH after intravenous injection. These studies suggest that Kp may have a role in regulation of both reproduction and metabolism in sheep. Since GH plays a role in luteal development, it is tempting to speculate that the ability of Kp to release GH and LH is related to normal control of reproduction.

Interaction of estrogen and progesterone on kisspeptin-10-stimulated luteinizing hormone and growth hormone in ovariectomized cows.

BACKGROUND/AIMS: Growth hormone (GH) is necessary for optimal reproductive efficiency and its secretion is influenced by sex steroids. This study was designed to determine whether kisspeptin-10 (Kp10) could stimulate GH and if gonadal steroids enhance the GH response to Kp10 in cows. METHODS AND RESULTS: Intravenous injection of Kp10 at 100 or 200 pmol/kg body weight with or without treatment with estradiol cypionate and/or progesterone increased luteinizing hormone (p < 0.01) plasma concentrations. Plasma concentrations of GH were increased following Kp10 in cows treated with estradiol cypionate and/or progesterone (p < 0.05) but not in cows treated with Kp10 without gonadal steroids. CONCLUSIONS: These data suggest that reproductive steroids enhance the sensitivity of the somatotropic axis to physiologically relevant doses of Kp10, and support the possibility that Kp10 is an integrator of luteinizing hormone and GH release.

Pharmacological analyses of two naturally occurring porcine melanocortin-4 receptor mutations in domestic pigs.

The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Numerous mutations in the MC4R gene have been identified from obese humans. So far two naturally occurring porcine MC4R (pMC4R) mutations, D298N and R236H, have been identified from various strains of pigs and D298N is being utilized as a genetic marker to screen performance traits of pigs. In this study, we performed functional analyses of pMC4R D298N and R236H, including their ligand binding and signaling properties in transiently transfected HEK293T cells. Ligand binding assays showed that both D298N and R236H pMC4Rs had similar binding capacities and affinities for the natural agonist alpha-MSH and the natural antagonist Agouti-related protein as wild-type pMC4R. In signaling assays, both mutants had normal EC50 and maximal signaling to alpha-MSH. In summary, pMC4R mutants D298N and R236H do not have any overt functional defects; therefore we suggest caution using these mutations as selection markers in breeding programs.

Molecular cloning and pharmacological characterization of porcine melanocortin-3 receptor.

Mouse gene targeting studies revealed that the melanocortin-3 receptor (MC3R) affected feeding efficiency and fat storage in mice. The functions of the MC3R in other mammalian species remain to be investigated. We are interested in exploring the functions of the porcine MC3R (pMC3R) in regulating fat storage because of the economical importance of swine industry. Although nucleotide sequences of MC3Rs from several species have been reported, pMC3R had not been cloned and sequenced. We reported herein the molecular cloning and pharmacological analysis of the pMC3R. Sequence analysis revealed that pMC3R was highly homologous (>80%) at nucleotide and amino acid sequences to human, rat, and mouse MC3Rs. With human MC3R (hMC3R) as a control, the binding and signaling properties of pMC3R were investigated using several agonists including alpha- and gamma-melanocyte-stimulating hormone (alpha- and gamma-MSH), D-Trp(8)-gamma-MSH, and [Nle(4)-D-Phe(7)]-MSH (NDP-MSH) and the natural antagonist agouti-related protein (AgRP). The results showed that pMC3R bound NDP-MSH with the highest affinity followed by D-Trp(8)-gammaMSH, gamma-, and alpha-MSH. The same ranking was also found for hMC3R, although pMC3R had two- to ninefold higher affinities for these ligands. Both pMC3R and hMC3R bound AgRP with high affinity. D-Trp(8)-gamma-MSH was the most potent agonist to stimulate cAMP generation followed by NDP-, alpha-, and gamma-MSH. This ranking was the same as that of hMC3R. The availability of pMC3R and its pharmacological characteristics will facilitate the investigation of pMC3R in regulating food intake and fat storage.

Caveolae nitration of Janus kinase-2 at the 1007Y-1008Y site: coordinating inflammatory response and metabolic hormone readjustment within the somatotropic axis.

Life-threatening proinflammatory response (PR) induces severe GH resistance. Although low-level PR is much more commonly encountered clinically, relatively few studies have investigated the accompanying change in GH signal transduction progression and, in particular, the impact of low-level PR on Janus kinase (JAK)-2. Using a low-level, in vivo endotoxin [lipopolysaccharide (LPS)] challenge protocol, we demonstrated that the liver tissue content of JAK2 declined 24 h (62%, P < 0.02) after LPS and that tyrosine-nitrated JAK2 could be immunoprecipitated from post-LPS liver biopsy homogenates. With antibodies developed to probe specifically for nitration at the (1007)Y-(1008)Y phosphorylation epitope of JAK2, we demonstrated that the nitrated (1007)Y-(1008)Y-JAK-2 (nitro-JAK2) coimmunoprecipitated with caveolin-1 and (1177)phospho-SER-endothelial nitric oxide synthase when post-LPS liver homogenates were treated with anticaveolin-1 and protein A/G. The magnitude of increase in nitro-JAK2 was attenuated in animals treated with vitamin E prior to LPS. The increase in nitro-JAK2 after LPS was greater in a line of experimental animals with a genetic propensity for higher PR at the given LPS dose than responses measured in their normal counterparts. The development and remission of nitro-JAK2 was temporally concordant with changes in plasma concentrations of IGF-I; hepatocellular IGF-I mRNA content was inversely proportional to nitro-JAK2 content. Localized changes in the state of nitration of regulatory phosphorylation domains of JAK2 in caveolar microenvironments and tissue content of JAK2 during PR suggest a unique mechanism through which discrete signal transduction switching might occur in the liver to fine tune cellular responses to the endocrine-immune signals that develop during low-level, transient proinflammatory stress.

Amplification of proopiomelanocortin mRNA in canine skin: preliminary results.

Skin and pituitary specimens were obtained from a normal chow-chow cross-bred dog. Total messenger RNA (mRNA) was extracted, reversed transcribed and the proopiomelanocortin (POMC) mRNA was amplified by polymerase chain reaction using canine specific primers. The expected 391 bp amplification product was detected in both canine skin and pituitary samples. Sequencing of this product showed 100% homology to the GenBank sequence for canine PMOC cDNA, and confirmed its remarkable homology to sequences of human, pigtailed macaque, mink, pig, mouse, rat and cow POMC. These preliminary results suggest that transcription of POMC mRNA occurs in canine skin. The nature of resident or nonresident cells transcribing the POMC gene in canine skin remains unknown at this time.

A role for agouti-related protein in appetite regulation in a species with continuous nutrient delivery.

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Mechanisms underlying growth hormone effects in augmenting nitric oxide production and protein tyrosine nitration during endotoxin challenge.

The present study defined the effects of GH administration on components of the nitric oxide (NO)-generating cascade to account for observed increases in NO production and protein nitration after an immune challenge. Calves were assigned to groups with or without GH treatment (100 microg GH/kg body weight or placebo im, daily for 12 d) and with or without low-level endotoxin [lipopolysaccharide (LPS), 2.5 microg/kg, or placebo, iv]. Plasma was obtained for estimation of NO changes as [NO(2)(-) + NO(3)(-)] (NO(x)). Transcutaneous liver biopsies were collected for measurement of protein tyrosine nitration, cationic amino acid transporter (CAT)-2 mRNA transporter, and constitutive NO synthase (cNOS), inducible NOS (iNOS), and arginase activity. Liver protein nitration increased more than 10-fold 24 h after LPS and an additional 2-fold in animals treated with GH before LPS. GH increased plasma NO(x) after LPS to levels 27% greater than those measured in non-GH-treated calves. LPS increased CAT-2 mRNA after LPS; GH was associated with a 24% reduction in CAT-2 mRNA content at the peak time response. cNOS activity was 3-fold greater than iNOS after LPS. NOS activities were increased 140% (cNOS) at 3 h and 169% (iNOS) at 6 h, respectively, after LPS; GH treatment increased cNOS activity and the phosphorylation of endothelial NOS after LPS more than 2-fold over that measured in non-GH-treated calves. The data suggest that an increased production of nitrated protein develops in the liver during low-level, proinflammatory stress, and nitration is increased by GH administration through a direct effect on the competing activities of NOS and arginase, modulatable critical control points in the proinflammatory cascade.