Proteomic identification of non-erythrocytic alpha-spectrin-1 down-regulation in the pre-optic area of neonatally estradiol-17β treated female adult rats

Vijayakumar Govindaraj; Addicam Jagannadha Rao

doi:10.1515/hmbci-2016-0008

Article

Proteomic identification of non-erythrocytic alpha-spectrin-1 down-regulation in the pre-optic area of neonatally estradiol-17β treated female adult rats

Vijayakumar Govindaraj and Addicam Jagannadha Rao

Published/Copyright: May 11, 2016

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information

From the journal Hormone Molecular Biology and Clinical Investigation Volume 26 Issue 3

Abstract

It is well established that sexually dimorphic brain regions, which are critical for reproductive physiology and behavior, are organized by steroid hormones during the first 2 weeks after birth in the rodents. In our recent observation, neonatal exposure to estradiol-17β (E2) in the female rat revealed increase in cyclooxygenase 2 (COX-2) level, sexually dimorphic nucleus (SDN)-pre-optic area (POA) size and down-regulation of synaptogenesis related genes in POA in the adult stage. In the present study, using the same animal model, the protein profile of control and neonatally E2-treated POA was compared by 1D-SDS-PAGE, and the protein that shows a change in abundance was identified by LC-MS/MS analysis. Results indicated that there was a single protein band, which was down-regulation in E2-treated POA and it was identified as spectrin alpha chain, non-erythrocytic 1 (SPTAN1). Consistently, the down-regulation of SPTAN1 expression was also confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The SPTAN1 was identified as a cytoskeletal protein that is involved in stabilization of the plasma membrane and organizes intracellular organelles, and it has been implicated in cellular functions including DNA repair and cell cycle regulation. The evidence shows that any mutation in spectrins causes impairment of synaptogenesis and other neurological disorders. Also, protein-protein interaction analysis of SPTAN1 revealed a strong association with proteins such as kirrel, actinin, alpha 4 (ACTN4) and vinculin (VCL) which are implicated in sexual behavior, masculinization and defeminization. Our results indicate that SPTAN1 expression in the developing rat brain is sexually dimorphic, and we suggest that this gene may mediate E2-17β-induced masculinization and defeminization, and disrupted reproductive function in the adult stage.

Keywords: COX-2; estradiol-17β; neonatal; pre-optic area; SPTAN1

Corresponding author: Dr. Vijayakumar Govindaraj, Research Associate, Department of Biochemistry, Indian Institute of Science, Bangalore – 560012, India, Phone: +91-80-22932308

Funding source: Science and Engineering Research Board

Award Identifier / Grant number: SR/SO/HS-0016/2012

Funding statement: Authors thank the Department of Biochemistry, Indian Institute of Science for the facilities. Author AJR thanks the INSA (Indian National Science Academy) for a senior Scientist Fellowship. This study was supported by the Department of Science and Technology (DST), Government of India, Science and Engineering Research Board (Grant No. SR/SO/HS-0016/2012).

Acknowledgments:

Authors thank the Department of Biochemistry, Indian Institute of Science for the facilities. Author AJR thanks the INSA (Indian National Science Academy) for a senior Scientist Fellowship. This study was supported by the Department of Science and Technology (DST), Government of India, Science and Engineering Research Board (Grant No. SR/SO/HS-0016/2012).

Conflict of interest statement: The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

References

1. McCarthy MM, Nugent BM. At the frontier of epigenetics of brain sex differences. Front Behav Neurosci 2015;9:221.10.3389/fnbeh.2015.00221Search in Google Scholar

2. McCarthy M. How it’s made: organisational effects of hormones on the developing brain. J Neuroendocrinol 2010;22:736–42.10.1111/j.1365-2826.2010.02021.xSearch in Google Scholar

3. McCarthy MM, Albrecht ED. Steroid regulation of sexual behavior. Trends Endocrinol Metab 1996;7:324–7.10.1016/S1043-2760(96)00153-1Search in Google Scholar

4. Frye C, Bo E, Calamandrei G, Calza L, Dessì-Fulgheri F, Fernández M, Fusani L, Kah O, Kajta M, Le Page Y. Endocrine disrupters: a review of some sources, effects, and mechanisms of actions on behaviour and neuroendocrine systems. J Neuroendocrinol 2012;24:144–59.10.1111/j.1365-2826.2011.02229.xSearch in Google Scholar

5. McCarthy M, Schwarz J, Wright C, Dean S. Mechanisms mediating oestradiol modulation of the developing brain. J Neuroendocrinol 2008;20:777–83.10.1111/j.1365-2826.2008.01723.xSearch in Google Scholar

6. Schwarz JM, McCarthy MM. Cellular mechanisms of estradiol-mediated masculinization of the brain. J Steroid Biochem Mol Biol 2008;109:300–6.10.1016/j.jsbmb.2008.03.012Search in Google Scholar

7. Tobet S, Knoll JG, Hartshorn C, Aurand E, Stratton M, Kumar P, Searcy B, McClellan K. Brain sex differences and hormone influences: a moving experience? J Neuroendocrinol 2009;21:387–92.10.1111/j.1365-2826.2009.01834.xSearch in Google Scholar

8. McCarthy MM. Estradiol and the developing brain. Physiol Rev 2008;88:91–124.10.1152/physrev.00010.2007Search in Google Scholar

9. Bakker J, De Mees C, Douhard Q, Balthazart J, Gabant P, Szpirer J, Szpirer C. Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens. Nat Neurosci 2006;9:220–6.10.1038/nn1624Search in Google Scholar

10. Gorski RA, Gordon JH, Shryne JE, Southam AM. Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain Res 1978;148:333–46.10.1016/0006-8993(78)90723-0Search in Google Scholar

11. Tsukahara S. Sex differences and the roles of sex steroids in apoptosis of sexually dimorphic nuclei of the preoptic area in postnatal rats. J Neuroendocrinol 2009;21:370–6.10.1111/j.1365-2826.2009.01855.xSearch in Google Scholar

12. Radhika NS, Govindaraj V, Sarangi SK, Rao AJ. Neonatal exposure to 17beta-estradiol down-regulates the expression of synaptogenesis related genes in selected brain regions of adult female rats. Life Sci 2015;141:1–7.10.1016/j.lfs.2015.09.013Search in Google Scholar

13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.10.1016/S0021-9258(19)52451-6Search in Google Scholar

14. Govindaraj V, Rao AJ. Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats. Syst Biol Reprod Med 2015;61:367–75.10.3109/19396368.2015.1077903Search in Google Scholar PubMed

15. Gupta K, Kumar P, Chatterji D. Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis. PLoS One 2010;5:e15072.10.1371/journal.pone.0015072Search in Google Scholar PubMed PubMed Central

16. Govindaraj V, Keralapura Basavaraju R, Rao AJ. Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats. Reprod Biomed Online 2015;30:303–10.10.1016/j.rbmo.2014.11.010Search in Google Scholar PubMed

17. Von Mering C, Huynen M, Jaeggi D, Schmidt S, Bork P, Snel B. STRING: a database of predicted functional associations between proteins. Nucleic Acids Res 2003;31:258–61.10.1093/nar/gkg034Search in Google Scholar PubMed PubMed Central

18. Shridharan RN, Krishnagiri H, Govindaraj V, Sarangi S, Rao AJ. Neonatal exposure to estradiol-17β modulates tumour necrosis factor alpha and cyclooxygenase-2 expression in brain and also in ovaries of adult female rats. Horm Mol Biol Clin Investig 2016;25:149–56.10.1515/hmbci-2015-0072Search in Google Scholar PubMed

19. Rasband MN. The axon initial segment and the maintenance of neuronal polarity. Nat Rev Neurosci 2010;11:552–62.10.1038/nrn2852Search in Google Scholar PubMed

20. Xu K, Zhong G, Zhuang X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 2013;339:452–6.10.1126/science.1232251Search in Google Scholar PubMed PubMed Central

21. Zhang R, Zhang C, Zhao Q, Li D. Spectrin: structure, function and disease. Sci China Life Sci 2013;56:1076–85.10.1007/s11427-013-4575-0Search in Google Scholar

22. Zagon I, Mclaughlin PJ, Goodman SR. Localization of spectrin in mammalian brain. J Neurosci 1984;4:3089–100.10.1523/JNEUROSCI.04-12-03089.1984Search in Google Scholar

23. Goodman SR, Zimmer WE, Clark MB, Zagon IS, Barker JE, Bloom ML. Brain spectrin: of mice and men. Brain Res Bull 1995;36:593–606.10.1016/0361-9230(94)00264-2Search in Google Scholar

24. Riederer BM, Zagon IS, Goodman SR. Brain spectrin (240/235) and brain spectrin (240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells. J Cell Biol 1986;102:2088–97.10.1083/jcb.102.6.2088Search in Google Scholar

25. Zagon IS, Higbee R, Riederer BM, Goodman S. Spectrin subtypes in mammalian brain: an immunoelectron microscopic study. J Neurosci 1986;6:2977–86.10.1523/JNEUROSCI.06-10-02977.1986Search in Google Scholar

26. Chi P, Greengard P, Ryan TA. Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 2003;38:69–78.10.1016/S0896-6273(03)00151-XSearch in Google Scholar

27. Hilfiker S, Pieribone VA, Czernik AJ, Kao H-T, Augustine GJ, Greengard P. Synapsins as regulators of neurotransmitter release. Philos Trans R Soc Lon B Biol Sci 1999;354:269–79.10.1098/rstb.1999.0378Search in Google Scholar PubMed PubMed Central

28. Hamdan FF, Saitsu H, Nishiyama K, Gauthier J, Dobrzeniecka S, Spiegelman D, Lacaille J-C, Décarie J-C, Matsumoto N, Rouleau GA. Identification of a novel in-frame de novo mutation in SPTAN1 in intellectual disability and pontocerebellar atrophy. Eur J Hum Genet 2012;20:796–800.10.1038/ejhg.2011.271Search in Google Scholar PubMed PubMed Central

29. Saitsu H, Tohyama J, Kumada T, Egawa K, Hamada K, Okada I, Mizuguchi T, Osaka H, Miyata R, Furukawa T. Dominant-negative mutations in α-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay. Am J Hum Genet 2010;86:881–91.10.1016/j.ajhg.2010.04.013Search in Google Scholar PubMed PubMed Central

30. Stankewich MC, Gwynn B, Ardito T, Ji L, Kim J, Robledo RF, Lux SE, Peters LL, Morrow JS. Targeted deletion of βIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes. Proc Natl Acad Sci USA 2010;107:6022–7.10.1073/pnas.1001522107Search in Google Scholar PubMed PubMed Central

31. Dick KA, Ikeda Y, Day JW, Ranum L. Spinocerebellar ataxia type 5. Ataxic Disorders: Handbook of Clinical Neurology (Series Editors: Aminoff, Boller and Swaab) 2011;103:451.10.1016/B978-0-444-51892-7.00028-0Search in Google Scholar

32. Lise S, Clarkson Y, Perkins E, Kwasniewska A, Akha ES, Schnekenberg RP, Suminaite D, Hope J, Baker I, Gregory L. Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development. PLoS Genet 2012;8:e1003074.10.1371/journal.pgen.1003074Search in Google Scholar PubMed PubMed Central

33. Moorthy S, Chen L, Bennett V. Caenorhabditis elegans β-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function. J Cell Biol 2000;149:915–30.10.1083/jcb.149.4.915Search in Google Scholar PubMed PubMed Central

34. Wright CL, Schwarz JS, Dean SL, McCarthy MM. Cellular mechanisms of estradiol-mediated sexual differentiation of the brain. Trends Endocrinol Metab 2010;21:553–61.10.1016/j.tem.2010.05.004Search in Google Scholar PubMed PubMed Central

35. Wechsler A, Teichberg VI. Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin. EMBO J 1998;17:3931–9.10.1093/emboj/17.14.3931Search in Google Scholar PubMed PubMed Central

36. Barraclough CA, Gorski RA. Evidence that the hypothalamus is responsible for androgen-induced sterility in the female rat 1. Endocrinology 1961;68:68–79.10.1210/endo-68-1-68Search in Google Scholar PubMed

37. Marcondes RR, Carvalho KC, Duarte DC, Garcia N, Amaral VC, Simões MJ, Turco EG, Soares JM, Baracat EC, Maciel GA. Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels. Gen Comp Endocrinol 2015;212:28–33.10.1016/j.ygcen.2015.01.006Search in Google Scholar PubMed

38. Wong KK, Li W, An Y, Duan Y, Li Z, Kang Y, Yan Y. β-Spectrin regulates the Hippo signaling pathway and modulates the basal actin network. J Biol Chem 2015;290:6397–407.10.1074/jbc.M114.629493Search in Google Scholar PubMed PubMed Central

39. Prince JE, Brignall AC, Cutforth T, Shen K, Cloutier J-F. Kirrel3 is required for the coalescence of vomeronasal sensory neuron axons into glomeruli and for male-male aggression. Development 2013;140:2398–408.10.1242/dev.087262Search in Google Scholar PubMed PubMed Central

40. Schwarz JM, McCarthy MM. The role of neonatal NMDA receptor activation in defeminization and masculinization of sex behavior in the rat. Horm Behav 2008;54:662–8.10.1016/j.yhbeh.2008.07.004Search in Google Scholar PubMed PubMed Central

41. Clarkson J, Busby ER, Kirilov M, Schütz G, Sherwood NM, Herbison AE. Sexual differentiation of the brain requires perinatal kisspeptin-GnRH neuron signaling. J Neurosci 2014;34:15297–305.10.1523/JNEUROSCI.3061-14.2014Search in Google Scholar PubMed PubMed Central

42. Dobkin-Bekman M, Naidich M, Rahamim L, Przedecki F, Almog T, Lim S, Melamed P, Liu P, Wohland T, Yao Z. A preformed signaling complex mediates GnRH-activated ERK phosphorylation of paxillin and FAK at focal adhesions in LβT2 gonadotrope cells. Mol Endocrinol 2009;23:1850–64.10.1210/me.2008-0260Search in Google Scholar

43. Speert DB, Konkle AT, Zup SL, Schwarz JM, Shiroor C, Taylor ME, McCarthy MM. Focal adhesion kinase and paxillin: novel regulators of brain sexual differentiation? Endocrinology 2007;148:3391–401.10.1210/en.2006-0845Search in Google Scholar

44. Gore AC. Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle. Brain Res Rev 2001;37:235–48.10.1016/S0165-0173(01)00121-7Search in Google Scholar

Received: 2016-1-27

Accepted: 2016-3-21

Published Online: 2016-5-11

Published in Print: 2016-6-1

You are currently not able to access this content.

Articles in the same Issue

https://doi.org/10.1515/hmbci-2016-0008

Keywords for this article

COX-2; estradiol-17β; neonatal; pre-optic area; SPTAN1