REST/NRSF-induced changes of ChAT protein expression in the neocortex and hippocampus of the 3xTg-AD mouse model for Alzheimer's disease.

AIMS: The cholinergic system is one of the neurotransmitter systems altered in Alzheimer's disease (AD), the most common form of human dementia. The objective of this work was to determine the REST/NRSF involvement in altered ChAT expression in the neocortex and hippocampus of an AD transgenic mouse (homozygous 3xTg-AD) that over-expresses 3 proteins, amyloid-ß precursor protein, presenilin-1, and tau, all of which are associated with AD and cause cellular degeneration. MAIN METHODS: Two groups (WT and 3xTg-AD) of 11-month-old female mice were analyzed and compared. Half of the brains of each group were used for ChAT immunohistochemistry, and Western Blot analyses of ChAT and REST/NRSF were performed on the other half. KEY FINDINGS: We observed significant decreases in the number of ChAT-immunoreactive cells in the Meynert nucleus and of fibers in the frontal motor cortex and hippocampal CA1 area in transgenic mice compared with control mice. An increased level of REST/NRSF protein and a reduction of ChAT protein expression in the 3xTg-AD mice compared with their controls were also found in both in the latter two cerebral regions. SIGNIFICANCE: The increased REST/NRSF expression reported here and its effect on the regulatory region for ChAT transcription could explain the decreased expression of ChAT in the 3xTg-AD mouse; these findings may be associated with the degeneration observed in AD.

Chicken growth hormone: further characterization and ontogenic changes of an N-glycosylated isoform in the anterior pituitary gland.

Glycosylation is one of the post-translational modifications that growth hormone (GH) can undergo. This has been reported for human, rat, mouse, pig, chicken and buffalo GH. The nature and significance of GH glycosylation remains to be elucidated. This present study further characterizes glycosylated chicken GH (G-cGH) and examines changes in the pituitary concentration of G-cGH during embryonic development and post hatching growth. G-cGH was purified from chicken pituitaries by affinity chromatography (Concanavalin A-Sepharose and monoclonal antibody bound to Sepharose). Immunoreactive G-cGH has a MW of 26 kDa or 29 kDa as determined by SDS-PAGE, respectively, under non-reducing and reducing conditions. Evidence that it is N-glycosylated comes from its susceptibility to peptide N-glycosidase F, and its resistance to O-glycosidase. Based on the ability of G-cGH to bind Concanavalin A or wheat germ agglutinin but not other lectins and its susceptibility to peptide N-glycosidase F, a hybrid or biantennary type glycopeptide (GlcNac2, Man) structure is proposed. Some G-cGH can be observed in the pituitary at most ages examined (from 15-day embryo to adult). Moreover, electron microscopy revealed the presence of both immuno-reactive GH and Concanavalin A-reactive sites in the same secretory granules in the somatotrope. There were marked changes in the level and relative proportion of G-cGH in the pituitary gland during development and growth, the proportion of G-cGH rising during late embryonic development (e.g., between 15 and 18 days of development) and with further increases between 9 weeks and 15 weeks old. G-cGH was able to bind to chicken liver membrane preparations with less affinity than non-glycosylated monomer; on the other hand, however, G-cGH stimulated cell proliferation on Nb2 lymphoma bioassay whereas the non-glycosylated monomer was uncapable to do it.

Growth hormone in the male reproductive tract of the chicken: heterogeneity and changes during ontogeny and maturation.

Growth hormone (GH) gene expression is not confined to pituitary somatotrophs and occurs in many extrapituitary tissues. In this study, we describe the presence of GH moieties in the chicken testis. GH-immunoreactivity (GH-IR), determined by ELISA, was found in the testis of immature and mature chickens, but at concentrations <1% of those in the pituitary gland. The immunoassayable GH concentration in the testis was unchanged between 4 and 66 weeks of age, and approximately 10-fold higher than that at 1-week of age and 25-fold higher than that in 1-day-old chicks and perinatal (embryonic day 18) embryos. This immunoreactivity was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. However, while most of the GH-IR in the pituitary ( approximately 40 and 15%, respectively) is associated with monomer (26 kDa) or dimer (52 kDa) GH moieties GH-IR in the testis is primarily (30-50%) associated with a 17 kDa moiety. GH bands between 32 and 45 kDa are also relatively more abundant in the testis than in the pituitary. During ontogeny the relative abundance of a 14 kDa GH and 40 kDa GH moieties in the testis significantly declined, whereas the relative abundance of the 17 and 45 kDa moieties increased with advancing age. In adult birds, GH-IR was widespread and intense in the seminiferous tubules. Although the GH-IR was not present in the basal compartment of Sertoli cells, nor in spermatogonia and primary spermatocytes, it was abundantly present in secondary spermatocytes and spermatids in the luminal compartments of the tubules as well as in some surrounding myocytes and interstitial cells. In summary, immunoreactive GH moieties are present in the chicken testis but at concentrations far less than in the pituitary. Age-related changes in the relative abundance of testicular GH variants may be related to local (autocrine/paracrine) actions of testicular GH. The localization of GH in spermatocytes and spermatids suggests hitherto unsuspected roles in gamete development.

Glycerol-3-Phosphate dehydrogenase (E.C.1.1.1.8) is expressed in cultured chicken embryonic adipofibroblasts and upregulated by embryonic chicken serum.

Chicken embryonic adipofibroblasts (CEA) accumulate intracytoplasmic lipids when cultured in medium containing chicken serum (CS), but not in medium with fetal bovine serum (FBS). To characterize this process of lipid accumulation, we evaluated the expression of the enzyme glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8) (GPDH), first in chicken tissues and then in CEA cultured under diverse conditions. GPDH activity in adipose depots from 4-wk-old broiler chickens was similar or higher than that shown by liver, the main organ for fatty acid synthesis in chickens, while skeletal muscle had the lowest levels of GPDH. In vitro, GPDH activity increased in CEA cultured in the presence of CS but not in medium with FBS, paralleling the lipid accumulation by these cells. Both lipid accumulation and GPDH activity were further increased in CEA cultured in the presence of embryonic CS. Our results show that GPDH is highly expressed in avian tissues related to lipid metabolism and therefore can be a reliable marker for avian adipogenesis, and suggest that ECS is an optimum source for the purification of avian adipogenic factors.

Characterization of a bioactive 15 kDa fragment produced by proteolytic cleavage of chicken growth hormone.

There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.

Growth hormone size variants: changes in the pituitary during development of the chicken.

There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (>/=66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radioreceptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir-GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are under some differential control during growth and development of the chicken.