Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (−776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (−101/−94 and −59/−52) and glucocorticoid response element (GRE) half-site (−210/−205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (−101/−94) but not to CRE (−59/−52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.
The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1–3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10–10–10–4 M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10–10M, an increase above control at 10–8–10–6M and a reduction at higher concentrations (10–5– 10–4M). The stimulatory effect of 10–8M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10–7M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10–3M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.
Background Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. While NSCs have been isolated from intestinal musclularis, their presence in mucosa has not been well described. Mucosa-derived NSCs are accessible endoscopically and could be used autologously. Brain-derived Nestin-positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa-derived NSCs, determine their relationship to Nestin-expressing cells and demonstrate capacity to produce neuroglial networks in vitro and in vivo. Methods Neurospheres were generated from periventricular brain, colonic muscularis (Musc), and mucosa-submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin-GFP). NSCs were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed by immunohistochemistry using glial and neuronal markers. Brain and gut derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. Results Musc- and MSM-derived neurospheres expressed Nestin and gave rise to cells of neuronal, glial and mesenchymal lineage. While Nestin expression in tissue was mostly limited to glia colabelled with glial fibrillary acid protein (GFAP), neurosphere-derived neurons and glia both expressed Nestin in vitro, suggesting Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover, following transplantation into aneural colon, brain- and gut-derived NSCs were able to differentiate into neurons. Conclusions Nestin-expressing intestinal NSCs cells give rise to neurospheres, differentiate into neuronal, glial and mesenchymal lineages in vitro, generate neurons in vivo and can be isolated from mucosa. Further studies are needed exploring their potential for treating neuropathies.
Knowledge of the importance of docosahexaenoic acid (DHA), arachidonic acid (AA), and long-chain polyunsaturated fatty acids (LCPUFAs) in neurodevelopment was originally obtained from animal studies. These fatty acids are rapidly accreted in brain during the first postnatal year in animal and human infants, and they are found in high concentrations in breast milk. Reports of enhanced intellectual development in breast-fed children, and reports linking LCPUFA deficiency with neurodevelopmental disorders have stressed the physiological importance of DHA in visual and neural systems. In addition to high concentrations of fatty acids in breast milk, they are also present in fish and algae oil and have recently been added to infant formulas. Esterified poplyunsaturated fatty acids act in cellular membranes, in signal transduction, in neurotransmission, and in the formation of lipid rafts. Nonesterified polyunsaturated fatty acids can modulate gene expression and ion channel activities, thus becoming neuroprotective agents. The conversion of linoleic acid and alpha-linolenic acid into ARA and DHA have led to randomized clinical trials that have studied whether infant formulas supplemented with DHA or both DHA and ARA would enhance visual and cognitive development. This review gives an overview of fatty acids and neurodevelopment, focusing on the findings from these studies.
Background Enteric neurospheres derived from postnatal intestine represent a promising avenue for cell replacement therapy to treat Hirschsprung disease and other neurointestinal diseases. We describe a simple method to improve the neuronal yield of spontaneously-formed gut-derived neurospheres. Materials and Methods Enteric neurospheres were formed from the small and large intestines of mouse and human subjects. Neurosphere size, neural crest cell content, cell migration, neuronal differentiation, and neuronal proliferation in culture were analyzed. The effect of supplemental neurotrophic factors, including glial-derived neurotrophic factor (GDNF) and endothelin-3 (ET3), was also assessed. Results Mouse small intestine-derived neurospheres contained significantly more P75-expressing neural crest-derived cells (49.9 ± 15.3 vs. 21.6 ± 11.9%, p<0.05) and gave rise to significantly more Tuj1-expressing neurons than colon-derived neurospheres (69.9 ± 8.6 vs. 46.2 ± 15.6%, p<0.05). A similar pattern was seen in neurospheres isolated from human small and large intestine (32.6 ± 17.5 vs. 10.2 ± 8.2% neural crest cells, p<0.05; 29.7 ± 16.4 vs. 16.0 ± 13.5% enteric neurons, p<0.05). The addition of GDNF to the culture media further improved the neurogenic potential of small intestinal neurospheres (75.9 ± 4.0 vs. 67.8 ± 5.8%, p<0.05) whereas ET3 had no effect. Conclusions Enteric neurospheres formed from small intestine and supplemented with GDNF yield an enriched population of neural crest-derived progenitor cells and give rise to a high density of enteric neurons.
Background: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3′,5-L-triiodothyronine (T3) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (–59/–52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (–101/–94). Methods: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (–242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T3. Results: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T3 while they increased by 1 mM 8Br-cAMP (± T3). Site-4 was protected from DNase I digestion with nuclear extracts from T3-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T3 + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Krüppel family. A TRβ2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T3. A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T3. Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T3. Discussion: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRβ2 is bound; pCREB and TR do not present mutual interference on their binding sites.
Aim The aim of this study was to determine whether a direct relationship existed between absolute telomere length (aTL), obesity and familial functionality in a group of Mexican children. Methods We recruited 134 children (52% boys) aged 8‐10 years during regular primary care check‐ups in 2016 and evaluated physical activity (PA), feeding practices, anthropometrics, body fat percentage (BF%) and family dysfunction. Optimised quantitative PCR determined aTL from genomic deoxyribonucleic acid isolated from saliva samples. Results Boys with a healthy BF% showed a higher aTL than their high BF% counterparts (P < .01). aTL was higher in children who performed PA than their sedentary counterparts (P < .05). Alarmingly, 90% of the children belonged to dysfunctional families and a dysfunctional family was correlated with a higher BF% (r = −.57). Negative correlations between the BF% and aTL (r = −.1765) and the BF% and time dedicated to PA (r = −.031) were observed in boys. On the contrary, we found a positive correlation between the aTL and weekly PA (r = .1938). These correlations were not observed in girls. Conclusion Telomere shortening was associated with a high BF% in boys, but not girls. Dysfunctional families were also a key factor. School PA programmes should be mandatory.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.