Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders

Belsham, Denise D.; Cai, Fei; Cui, Hong; Smukler, Simon R.; Salapatek, Anne Marie; Shkreta, Lulzim

doi:10.1210/en.2003-0946

Cited by 184 publications

(189 citation statements)

References 21 publications

Supporting

Mentioning

182

Contrasting

Unclassified

Order By: Relevance

“…These cell lines have been thoroughly characterized, show neurosecretory properties, express neuron specific markers and have classical neuronal morphology. 17,19 In this study, we show that 17b-estradiol (E 2 ) directly decreases NPY secretion in both the mHypoE-42 and mHypoA-2/12 neurons. Using pharmacological estrogen receptor (ER)-a and (ER)-b receptor specific agonists/antagonists, we were able to determine that the E 2 -mediated decrease occurred through ER-a in both of the hypothalamic cell lines.…”

Section: Introductionmentioning

confidence: 60%

“…To examine the mechanisms by which E 2 regulates NPY within an individual NPY-expressing neuron, we used hypothalamic neuronal cell models derived from both embryonic (e17) and adult (2 month-old) primary cell culture, the mHypoE-42 17 and mHypoA-2/12, 18 respectively. These cell lines have been thoroughly characterized, show neurosecretory properties, express neuron specific markers and have classical neuronal morphology.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons

Dhillon

Belsham

2010

Int J Obes

View full text Add to dashboard Cite

Objective: Estrogen (E 2 ) has an inhibitory effect on food intake by acting centrally in the hypothalamus, although it is not clear which hypothalamic neurons are involved in this process. Earlier studies from our lab and others have implicated neuropeptide Y (NPY) as an important central anorexigenic target of E 2 . This study was designed to investigate whether E 2 can directly regulate NPY secretion and examine the cellular mechanisms and receptors responsible for this anorexigenic action of E 2 . Design: Clonal, murine, hypothalamic neuronal cell models, mHypoE-42 and mHypoA-2/12, were investigated for NPY secretory responses to 17b-estradiol (E 2 ) in the presence or absence of pharmacological inhibitors directed against the phosphatidylinositol-3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and AMP-activated kinase (AMPK) pathways or to estrogen receptor (ER) specific agonists/antagonists. Measurements: The presence of hypothalamic markers and characterization of neuronal cell lines was completed with polymerase chain reaction. NPY levels were measured using an enzyme immunoassay (EIA). The expression of ER-a and caveolin-1 was analyzed using immunocytochemistry. Results: E 2 significantly decreased NPY secretion in both the mHypoE-42 and mHypoA-2/12 neurons. The E 2 -mediated repression of NPY secretion in the mHypoE-42 and mHypoA-2/12 neurons required ER-a, but not ER-b, as shown by studies using an ER-specific agonist/antagonists. Additionally, using immunocytochemistry we detected colocalization of ER-a and the membrane-associated signaling protein caveolin-1. Importantly, using E 2 -conjugated bovine serum albumin (E 2 -BSA) and ER antagonists, we were able to show that the E 2 -mediated decrease in NPY secretion occurred through membrane-bound ER-a. Finally, using a combination of pharmacological inhibitors, we found that inhibition of the PI3K or AMPK pathway blocked the E 2 -mediated decrease in NPY secretion. Conclusion: These findings indicate that the central anorexigenic action of E 2 occurs at least partially through hypothalamic NPY-synthesizing neurons. This regulation of NPY secretion occurs through rapid signaling mechanisms through membrane bound ER-a.

show abstract

Section: Introductionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons

Dhillon

Belsham

2010

Int J Obes

View full text Add to dashboard Cite

show abstract

“…Having confirmed that the physical properties of the CD vector were suitable for siRNA delivery, in vitro testing was carried out in a mouse embryonic hypothalamic neuronal cell line, mHypoE N41, 36 and in primary rat embryonic (E18) hippocampal neurons. )) and zeta potential (charge (mV)) of CD-siRNA complexes at increasing mass ratios (MR; μg CD/μg siRNA) were measured in DIW (final siRNA concentration 3 μg/mL) using dynamic light scattering (Malvern ZetaSizer Nano).…”

Section: ■ Results and Discussionmentioning

confidence: 98%

Click-Modified Cyclodextrins as Nonviral Vectors for Neuronal siRNA Delivery

O’Mahony

Godinho

Ogier

et al. 2012

ACS Chem. Neurosci.

View full text Add to dashboard Cite

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date, they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesized, which complexed siRNA to form cationic nanoparticles of less than 200 nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalized hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labeled siRNA, while maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

show abstract

“…Belsham et al have managed to develop cell lines that are representative of the enormous range of hypothalamic cell types [2,14]. N39, developed from primary mouse fetal hypothalamic cultures, is one such homologous neuronal cell line.…”

Section: Introductionmentioning

confidence: 99%

“…N39, developed from primary mouse fetal hypothalamic cultures, is one such homologous neuronal cell line. As they express both CRF 1 and CRF 2 receptors and Ob-R [2,32] these hypothalamic N39 cells have been used to further understand endogenous receptor signal transduction, the possible function of leptin, and the regulation of CRF and Ucns by leptin in the hypothalamus. Cui et al used these cells to demonstrate that leptin signaling involves the JAK-STAT3 pathway [13].…”

Section: Introductionmentioning

confidence: 99%

Regulation of corticotropin-releasing factor and urocortin 2/3 mRNA by leptin in hypothalamic N39 cells

et al. 2013

View full text Add to dashboard Cite

Corticotropin-releasing factor (CRF) activates the pituitary-adrenal axis during stress, and shows anorectic effects via CRF type 1 receptors in the hypothalamus.Both urocortin (Ucn) 2 and Ucn3 also act as anorectic neuropeptides via CRF type 2 receptors. Leptin, a product of the obesity gene secreted mainly from adipose tissue, reduces food intake and increases energy expenditure. A possible interaction between leptin and CRF/Ucns has been suggested, as leptin can regulate expression and activation of CRF and Ucns in the hypothalamus. This study aimed to explore the possible function of leptin in the hypothalamus, and its effects in regulating CRF and Ucns. The study identified mRNA expression of the leptin receptor (Ob-R) and its subtypes, CRF, and Ucn2/3 in mouse hypothalamic N39 cells. Leptin stimulated signal transducer and activators of transcription type 3 (STAT3) phosphorylation, directly increased the mRNA levels of both CRF and Ucn2/3 in hypothalamic cells, and increased Ob-Rb mRNA levels. A Janus kinase inhibitor inhibited the leptin-mediated increase in STAT3 phosphorylation, and then the increases in CRF and Ucn2/3 mRNA levels. Leptin may contribute to a stress response or anorectic effect via the regulation 2 of CRF and Ucn2/3 in the hypothalamus.

show abstract

Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders

Cited by 184 publications

References 21 publications

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons

Click-Modified Cyclodextrins as Nonviral Vectors for Neuronal siRNA Delivery

Regulation of corticotropin-releasing factor and urocortin 2/3 mRNA by leptin in hypothalamic N39 cells

Contact Info

Product

Resources

About