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Sex Steroid Regulation of Vasopressin Levels Secreted by hNT Neurons*

hNT Neurons used to imitate neurodegeneration


Rob D. Dickermana Thomas Fungweb N.Y. Zachariaha Walter J. McConathya,b
aDepartment of Medicine, bDepartment of Biomedical Sciences,
University of North Texas Health Science Center


This article describes an innovative technique that uses Stratagenes postmitotic human central nervious system cells (hNT Neurons) to imitate the aging human brain, neurodegeneration, and to investigate the role of sex steroids in neurotransmitter transcription and synthesis. Preliminary data show the potential of this model system.

The normal, physiological aging of the human brain has been associated with cellular atrophy and cell death. Sex differences in the aging brain have been documented by magnetic resonance imaging,1 suggesting that age-related changes in neuronal systems may be due in part to changes in the hormonal milieu. The age-related decline of circulating sex steroids is one of the most consistent findings in the aging literature.2,3 It has been demonstrated in the rat that vasopressinergic neurons are responsible for many of the behaviors and neurophysiological functions that are impaired in aging, including social memory, conditioned avoidance behavior and temperature regulation.4 A peptide hormone of interest, vasopressin, or antidiuretic hormone (ADH), is made in specialized neurons found in the hypothalamus and then transported to the pituitary gland. In neurons, both ADH content and ADH mRNA levels show a marked decline in senescence.5,6 Testosterone can reverse senescent decline in the levels of extrahypothalamic ADH mRNA in rats.7 In addition, the density of ADH nerve fibers is higher in male rats than in female rats.8 ADH nerve-fiber density can be reduced by castration and restored to normal density following testosterone replacement therapy.9

hNT Neurons and Alzheimers Disease

Another topic in neurogenerative research that could benefit from the hNT model system is Alzheimers disease. This disease is known to affect the female population more than the male population, and the females affected by Alzheimers disease suffer more severe forms of dementia.5 Dementia associated with Alzheimers disease is thought to be linked to the cholinergic system.6,7 Animal studies have demonstrated that the cholinergic system and overall brain activity can be enhanced by estrogen treatment.8,9 Recent studies have shown that estrogen stimulates mRNA for new growth of axons and dendrites and enhances gene expression for acetylcholine production.10,11 Several studies of menopausal women undergoing estrogen treatment demonstrate that estrogen directly affects memory.13-15 Interestingly, the male brain is thought to be less affected by Alzheimers due to the neural conversion of testosterone to estrogen by the enzyme aromatase.16 Thus, the normal hormonal milieu associated with aging may be directly responsible for age-related neurodegenerative changes and Alzheimers disease.

hNT Neurons and Sex Steroids

Steroid

Incubation Time
(hours)

Vasopressin
(pg/ml)

Control

6

1.8

24

3.6

48

6.3

Estrogen
(25 pg/ml)

6

3.6

24

3.6

48

6.3

Dihydrostestosterone

6

8.1

24

5.5

48

3.5

Testosterone
(3 ng/ml)

6

3.6

24

48

8.1

Testosterone
(9 ng/ml)

6

3.5

24

3.6

48

9.9

Testosterone
(18 ng/ml)

6

3.6

24

8.1

48

17.4

table 1

In order to demonstrate that sex steroids increase neuronal protein synthesis of the neuropeptide vasopressin, Stratagenes hNT Neurons were cultured with various physiological and supraphysiological levels of estrogen, testosterone and dihydrotestosterone (table 1). Cell culture media was collected at 6, 24 and 48 hours, and vasopressin was quantitated by radioimmunoassay (RIA). The more potent androgen, dihydrotestosterone, demonstrated the most immediate effect on vasopressin synthesis. High doses of testosterone had the greatest overall effect on vasopressin synthesis, while estrogen treatment mimicked control neurons. Neurons were also incubated with [35S]methionine and steroids to monitor sex steroid alterations in protein synthesis. Dihydrotestosterone appeared to be the most potent initiator of protein synthesis in the neurons.

These preliminary results demonstrate that hNT neurons synthesize the neuropeptide vasopressin. As previously demonstrated with animal models,2,5 hNT neuronal vasopressin synthesis responds to testosterone treatment. Incorporation of [35S]methionine indicates that hNT neurons appear to increase total neuronal protein synthesis in response to dihydrotestosterone.

Conclusions

Stratagenes hNT Neurons provide a viable model that may resemble the human aging brain more closely than previous animal models. Our results show that these neurons respond to various sex steroids and to varying concentrations of sex steroids. hNT neurons provide opportunities for research in the areas of steroid-responsive neuropeptides and age-related neurodegeneration.

Acknowledgments

The authors would like to thank the American Osteopathic Association and Glaxo-Wellcome for fellowship support.

REFERENCES

  1. Gur, R.C., et al. (1991) Proc. Natl. Acad. Sci. USA. 88: 2845-2849.

  2. Brenner, W.J., Vitiello, M.V., and Prinz, P.N. (1983) J. Clin. Endocrinol. Metab. 56: 1278-1287.

  3. Ravid, R., et al. (1987) Gerontology 33: 87-98.

  4. Dantzer, R., et al. (1988) Brain Res. 457: 143-147.

  5. Dorsa, D.M., and Bottemiller, L.A. (1982) Brain Res. 242: 3237-3242.

  6. Fliers, E., De Vries, G.J., and Swaab, D.F. (1985) Brain Res. 348: 1-8.

  7. Dobie, D.J., et al. (1992) Brain Res. 583: 247-252.

  8. Miller, M.A., Urban, J.H., and Dorsa, D.M. (1989) Endocrinology 125: 2335-2340.

  9. Devries, G.J., Duetz, W., and Buijs, R.M. (1984) Dev. Brain Res. 298: 141-145.

  10. Paganini-Hill, A., and Henderson, V.W. (1994) Am. J. Epidemiol. 140: 256-261.

  11. Rocca, W.A., et al. (1986) Ann. Neurol. 19: 415-424.

  12. Bartus, R.T., et al. (1982) Science 217: 408-414.

  13. Coyle, J.T., et al. (1983) Science 219: 1184-1190.

  14. Luine, V. (1985) Exp. Neurol. 89: 484-490.

  15. Luine, V., et al. (1980) Brain Res. 191: 273-277.

  16. Sohrabi, F., et al. (1994) J. Neurosci. 14: 459-471.


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