C. Dominique Toran Allerand, M.D., Sc.D.

Research Summary
Estrogen, estrogen receptors, and development of the brain and neural progenitor cells.

We are investigating the cellular and molecular mechanisms underlying our discovery of estrogen enhancement of neuronal growth and differentiation. We have documented the ability of estrogen to elicit rapid and sustained activation of the MAPK cascade, a pathway usually associated with the actions of growth factors such as the neurotrophins. We have shown that the estrogen receptor responsible for mediating estrogen activation of the MAPK cascade is neither the classical receptor ERalpha nor ERbeta. My recent work on the intersection between estrogen action and neurotrophin signaling challenges conventional mainstream thinking and led to the hypothesis that this non-ERalpha/non-ERbeta receptor is associated with caveolar-like microdomains of the plasma membrane .These findings led directly to the identification of a novel putative membrane ER that I have designated ER-X. We have provided compelling evidence that ER-X is a novel and unique, plasma membrane-associated, developmentally regulated estrogen-binding protein which is functionally distinct from the classical intranuclear receptors ERalpha and ERbeta and is re-expressed following postnatal and adult ischemic brain injury and in Alzheimer’s disease. Current studies focus on the roles and mechanisms of 17alpha- and 17beta-estradiol action in cultures of the postnatal neocortex and in neural progenitor cells of the adult rat hippocampal dentate gyrus. We are also comparing the therapeutic value of the physiological ovarian hormone 17 beta-estradiol and 17alpha-estradiol, the inactive stereoisomer and preferred ligand of ER-X, in the proliferation and differentiation of progenitor cells of the adult hippocampal dentate gyrus.

1. Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly Jr ES, Nethrapalli IS, Tinnikov A. (2002) “ER-X”: A novel, plasma-membrane-associated, putative estrogen receptor that is regulated during development and following ischemic brain Injury. J. Neurosci., 22 8391-8401.

2. Toran-Allerand, CD (2001) Interactions of estrogen with the neurotrophins and their receptors. In Neurobiology of the Neurotrophins, I. Mochetti, ed., F.P Graham Publishing Co, Johnson City TN, 607-3.

3. Toran-Allerand, CD (2000) Novel sites and mechanisms of oestrogen action in the brain. In: Neuronal and Cognitive Effects of Oestrogens, Wiley, Chichester (Novartis Foundation Symposium 230), pp 56-73.

4. Singh M, Sétáló Jr. G, Guan X, Frail DF, Toran-Allerand CD (2000). Estrogen-induced activation of the MAP kinase cascade in the cerebral cortex of estrogen receptor-a knock-out mice. J. Neurosci. 20 1694-1700.

5. Singh M, Sétáló Jr., G, Guan X, Warren M, Toran-Allerand CD (1999) Estrogen-induced activation of MAP Kinase (ERK) in cerebral cortical explants: Convergence of estrogen and neurotrophin signaling pathways. J. Neurosci. 19 1179-1188.

6. Toran-Allerand CD, Singh M, and Sétáló Jr. Gy. (1999) Novel mechanisms of estrogen action in the developing brain: New players in an old story. Frontiers in Neuroendocrinology, 20 97-121.

1. Cloning a Membrane Estrogen Receptor in Developing Brain
The proposed studies are designed to obtain preliminary data concerning the molecular structure and chromosome localization of a novel and unique plasma membrane-associated, estrogen receptor (ER) that is neither ER-alpha nor ER-beta, which we have designated "ER-X". (1) Using a cDNA-based approach, we will characterize the "ER-X" protein indirectly by first determining the mRNA, using rapid amplification of cDNA 5'ends (5'RACE) and comparing wild-type and ERKO neocortex. (2) Using a protein-based approach, we will determine the protein structure directly, using Maldi-Mass Spectrometry and protein data base searching. Chromosomal assignment will be determined by karyotyping and mapping its subchromosomal position on wild-type, embryonic mouse metaphase chromosomes using fluorescent in situ hybridization (FISH) on DAPI banded chromosomes.
National Institute of Mental Health
8/1/2002-7/31/2004

2. Developmental Action of Estrogen on Cognitive Substrates
We have compelling evidence in both wild-type (+/+) and estrogen receptor-alpha (ER-alpha) gene-disrupted (-/-) (ERKO) postnatal day- (P7) 7 mouse neocortex and uterus that points strongly to the existence of a novel, plasma-membrane-associated, putative ER that is neither ER-alpha (67 kDa) nor ER-beta (54-64 kDa). The proposed studies are designed to further characterize this putative ER, which I have designated "ER-X." "ER-X" is an about 62-63 kDa protein that: (a) is expressed and enriched in purified caveolar-like microdomains of P7, but not adult, neocortical and uterine plasma-membranes; (b) binds 3H-estradiol with high affinity but with ligand specificities radically different from ER-alpha and ER-beta; (c) exhibits some homology with the ligand binding domain of ER-alpha; (d) appears to mediate estrogen activation of the Mitogen-Activated Protein Kinase (MAPK) cascade; (e) is developmentally regulated; and (f) is up-regulated in adult neocortical tissue surrounding an ischemic stroke.
National Institute of Mental Health
7/01/1993-8/31/2004

1. Estrogen signaling in brain during development and aging
Estrogen enhances the growth and differentiation of axons and dendrites (neurites) in cultured slices of the developing forebrain and estrogen and neurotrophin receptors co-localize in developing forebrain neurons. This proposal addresses the intracellular pathways estrogen may use in mediating its growth-or neurite-promoting signals to the nucleus. The hypothesis underlying this proposal is that the estrogen receptor (ER) in the developing brain may be more than a ligand-induced transcriptional enhancer and may also act as a receptor with tyrosine kinase activity. Characterizing estrogen signaling pathways that may or may not be brain-specific could lead to the synthesis of estrogen- like drugs, which could duplicate estrogen's positive attributes for the brain, while eliminating many of its undesirable peripheral effects on non-neural tissues.
National Institute on Aging
1/1/1998-12/31/2003

Keywords: estradiol, estrogen receptor, molecular cloning, protein structure, receptor expression, cell membrane, developmental neurobiology, organotypic cultures neural progenitor cells, genetic mapping, membrane protein, molecular genetics, structural biology, laboratory mouse