Elsevier

Neurobiology of Aging

Volume 28, Issue 11, November 2007, Pages 1670-1681
Neurobiology of Aging

Estrogen receptor α and its splice variants in the hippocampus in aging and Alzheimer's disease

https://doi.org/10.1016/j.neurobiolaging.2006.07.024Get rights and content

Abstract

Clinical and experimental studies show that estrogens can have beneficial effects on hippocampus-dependent cognitive functions that may be mediated by estrogen receptor (ER)α. We investigated whether menopause and Alzheimer's disease (AD) cause changes in this ER subtype. Immunocytochemical staining of ERα, aromatase and Golgi complex (GC) was performed on paraffin embedded hippocampal tissue from women of the pre-, peri- and postmenopausal age. Canonical ERα mRNA amplicons, ERα splice variants (del.2, del.4, del.7, MB1) and aromatase transcripts were measured by Q-PCR in frozen hippocampal samples of AD and matched control cases. Nuclear ERα, aromatase and the GC enhanced during aging in women indicating availability of locally synthesized estrogens that may up-regulate ERα by which neuronal metabolism can be augmented in the hippocampus after the menopause. In AD cases canonical and alternatively spliced ERα mRNA, and aromatase gene expression were down-regulated suggesting a deficit in local estrogen levels and diminished signalling through ERα. The major ERα splice variants in the hippocampus were found to be MB1 and del.4. A novel ERα isoform TADDI was isolated and sequenced from two female patients. It lacks 31 bp at the junction between exons 3 and 4 with an insertion of 13 nucleotides from the middle of the exon 2.

Introduction

The human hippocampus is particularly vulnerable to aging and Alzheimer's disease (AD) as shown, e.g. by neuronal loss [56], increased neurofilament protein accumulation [54] and changes in plasticity [13]. In early AD impaired verbal, visual and episodic memory correlates with a reduced volume of the hippocampus and entorhinal cortex and with the occurrence of neurofibrillary tangles in these brain areas (reviewed in [36]). Episodic memory is defined as an ability to recollect unique personal experiences [10] and represents one of the major components of declarative memory, in which the hippocampal formation plays a critical role [10]. The hippocampus is also important for the acquisition of semantic or factual knowledge and spatial learning [2], [10]. In addition to memory consolidation, this brain area is involved in minute-to-minute cognitive processing of space, time and relationships between objects in the environment [47]. Finally, the hippocampus was identified as a “neurogenic” region in the adult CNS [11], [49], [50].

Although questions remain [20], estrogen therapy (ET) only if initiated early within a critical window from the onset of menopause, was reported to be associated with a reduced risk for AD and to protect against age-related decline in specific cognitive functions [6], [20], [32]. Clinical and experimental studies show that beneficial effects of ET on cognition are likely to be mediated at the level of the hippocampus. Indeed, women receiving ET performed better on verbal and visual memory tests reflective of the involvement of the hippocampus and revealed over 2 years greater relative blood flow increase for the hippocampus and other medial temporal lobe structures [31], [32]. Moreover, women taking ET had larger right anterior hippocampal volumes than women not taking ET [9]. Cognitive studies in rodents also point to favorable effects of ET on hippocampus-associated memory functions, i.e. improvement of spatial memory in aged females [33]. In the hippocampus estrogens induce synaptogenesis [44], increase pre- and postsynaptic proteins (synaptophysin, syntaxin, spinofilin) [7], regulate BDNF expression [4], stimulate cell proliferation [49] and diminish apoptosis [45]. Ovariectomy in the female rat resulted in: (i) a decrease in the spine density of CA1 pyramidal cells [44], (ii) diminishment in the number of BrdU-labelled cells in the dentate gyrus [50] and (iii) reduction in neuronal and endothelial NO synthase expression [17], while estrogen replacement reversed all of these effects. The changes observed in ovariectomized animals suggest the presence of menopause-associated alterations in the human hippocampus. Since estrogen effects on synaptic plasticity in this brain area seem to be mediated via estrogen receptor (ER)α [1], and since the number of ERα-positive CA1–CA2 neurons was reported to be decreased in AD patients [23], in the present study we examined for the first time whether hippocampal ERα changes in relation to menopause in women and whether its canonical or splice variants mRNAs are affected in the elderly and AD cases. In addition, we investigated the possibility of the altered local estrogen biosynthesis in the hippocampus in relation to the age of menopause and AD by an immunocytochemical and Q-PCR detection of aromatase. We also addressed the question whether estrogens could influence the hippocampal cellular metabolism in women in the pre-, peri- and postmenopausal periods by examination of the Golgi complex (GC) immunoreactivity, that is a marker of neuronal metabolic activity [25].

Section snippets

Human brain tissue samples

Frozen hippocampi (stored at −80 °C) were obtained at autopsy in the framework of the Netherlands Brain Bank from five AD and five matched control patients (Table 1). Three control women of 46, 52 and 64 years of age were included in a separate study of age-related changes in ERα splice variants expression in women (Table 1). Since the frozen brain material from younger cases was not available, we performed additional immunocytochemical stainings of ERα, aromatase and Golgi complex proteins in

ERα

ERα immunolabelling in the hippocampus was mainly confined to the neuronal nuclei and nucleoli. A clear age-related increase in nuclear ERα immunocytochemical expression was already apparent at light microscopical examination in control women (Fig. 2, 15 cases in Table 2). Following computer-assisted measurements, the %area covered by ERα immunoreactivity (ERα-ir) was significantly enhanced during aging in women in CA4 (r = 0.589, p = 0.021), hilus (r = 0.532, p = 0.041) and dentate granular cell layer

Menopause and AD-related changes in the human hippocampus

In the present study we found for the first time that menopause in women is accompanied by a profound up-regulation of ERα, aromatase and neuronal metabolic activity in the hippocampus. It is plausible that the drop in ovarian hormonal levels triggers a rise in de novo hippocampal estrogen production as judged from the enhanced aromatase immunoreactivity. Consequently, elevated local estrogen levels may cause an up-regulation of the nuclear ERα via a para/auto/intracrine feedback loop, a

Acknowledgements

Brain material was obtained in the framework of the Netherlands Brain Bank, Amsterdam (co-ordinator Dr. R. Ravid). In all cases written consent was received for the brain autopsy and the use of the material and clinical information for research purposes. We also wish to thank Dr. E. Hol, Mr. E. Ehlert, Mr. M. Kooreman, Ms. A. van den Berg, Ms. J. Wouda, Mrs. A. Sluiter, Mr. B. Fisser and Mr. J.J. van Heerikhuize. This study was supported by the Netherlands Institute for Brain Research.

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    Present address: BioFocus, A Galapagos Company, Leiden, The Netherlands.

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