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Originally published In Press as doi:10.1074/jbc.M311993200 on February 9, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20539-20545, May 7, 2004
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Luteinizing Hormone, a Reproductive Regulator That Modulates the Processing of Amyloid-{beta} Precursor Protein and Amyloid-{beta} Deposition*

Richard L. Bowen,ab Giuseppe Verdile,bcd Tianbing Liu,e Albert F. Parlow,f George Perry,g Mark A. Smith,g Ralph N. Martins,cd and Craig S. Atwoodceghi

From the aVoyager Pharmaceutical Corporation, Raleigh, North Carolina 27615, the cCentre for Aging and Alzheimer's Disease, School of Biomedical and Sports Science, Edith Cowan University, Joondalup, Western Australia 6027, Australia, the dDepartment of Psychiatry and Clinical Neurosciences, The University of Western Australia and The Sir James McCusker Alzheimer's Disease Research Unit, Hollywood Private Hospital, Perth, Western Australia 6009, Australia, the eDepartment of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53705, the fNational Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, California 90509, the gInstitute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, and the hDepartment of Medicine, University of Wisconsin, Madison, Wisconsin 53705

Received for publication, November 3, 2003 , and in revised form, February 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hormonal changes associated with the dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis following menopause/andropause have been implicated in the pathogenesis of Alzheimer's disease (AD). Experimental support for this has come from studies demonstrating an increase in amyloid-{beta} (A{beta}) deposition following ovariectomy/castration. Because sex steroids and gonadotropins are both part of the HPG feedback loop, any loss in sex steroids results in a proportionate increase in gonadotropins. To assess whether A{beta} generation was due to the loss of serum 17{beta}-estradiol or to the up-regulation of serum gonadotropins, we treated C57Bl/6J mice with the anti-gonadotropin leuprolide acetate, which suppresses both sex steroids and gonadotropins. Leuprolide acetate treatment resulted in a 3.5-fold (p < 0.0001) and a 1.5-fold (p < 0.024) reduction in total brain A{beta}1-42 and A{beta}1-40 concentrations, respectively, after 8 weeks of treatment. To further explore the role of gonadotropins in promoting amyloidogenesis, M17 neuroblastoma cells were treated with the gonadotropin luteinizing hormone (LH) at concentrations equivalent to early adulthood (10 mIU/ml) or post-menopause/andropause (30 mIU/ml). LH did not alter amyloid-{beta} precursor protein (A{beta}PP) expression but did alter A{beta}PP processing toward the amyloidogenic pathway as evidenced by increased secretion and insolubility of A{beta}, decreased {alpha}A{beta}PP secretion, and increased A{beta}PP-C99 levels. These results suggest the marked increases in serum LH following menopause/andropause as a physiologically relevant signal that could promote A{beta} secretion and deposition in the aging brain. Suppression of the age-related increase in serum gonadotropins using anti-gonadotropin agents may represent a novel therapeutic strategy for AD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer's disease (AD)1 is a neurodegenerative disorder of the elderly that leads to progressive memory loss, impairments in behavior, language, visuo-spatial skills, and ultimately death. The one or more underlying biochemical mechanisms leading to AD are unknown. Genetic studies have shown that mutations in A{beta}PP and the presenilin genes lead to early onset (<=65 years) AD, which accounts for ~5% of all AD cases. The vast majority of these mutations promote the overproduction and deposition of amyloid-{beta} (A{beta}) (1-7), the major component of the extracellular amyloid plaques, in the hippocampus and frontal cortex (8, 9). Amyloid deposition also is a hallmark of the late-onset or "sporadic" form of AD, which accounts for ~95% of AD cases. The primary factors responsible for A{beta} deposition and disease progression in late onset AD remain to be elucidated.

Aging, the strongest risk factor for late-onset AD, is associated with major changes in serum concentrations of all hormones that comprise the hypothalamic-pituitary-gonadal (HPG) axis, including declines in the serum concentrations of the sex steroids, 17{beta}-estradiol and testosterone. Such changes have been correlated with the prevalence of the disease (e.g. Refs. 10-14), and it has been shown that there is a decreased incidence (15) and delay in the onset (16) of AD among women on hormone replacement therapies following menopause (17). There also is biochemical evidence suggesting that sex steroids modulate A{beta} deposition in animal models (18, 19). Taken together, these studies have driven the idea that the loss of sex steroids following menopause/andropause is primarily responsible for the cognitive and neuropathological changes observed in the disease. Sex steroid production is, however, under the control of complex feedback loops within the HPG axis, such that during menopause the loss of negative feedback by estrogen on gonadotropin production (20) results in 3-4-fold and 4-18-fold increases in the concentrations of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), respectively (21). Likewise, during andropause, men experience increases of more than 2- and 3-fold in LH and FSH, respectively (22). Thus, the neurological and biochemical changes previously ascribed to sex steroids could just as easily be explained by the large increases in the circulating concentrations of LH and/or FSH or other hormones of the HPG axis that are dysregulated by menopause/andropause. Evidence for a role of gonadotropins in the disease process is indicated by the 2-fold increase in the concentration of circulating gonadotropins in individuals with AD, above that of the already elevated concentrations of serum gonadotropins in age-matched control individuals (13, 23). Further support for a role of LH in AD is given by the findings that the highest density of LH receptors in the brain is found within the hippocampus (24-26), that LH crosses the blood-brain barrier (27), and that LH accumulates intracellularly in the pyramidal neurons of AD compared with age-matched control brains (28). These findings have led us to investigate whether the expression and processing of A{beta}PP, a molecular pathway relevant to AD neuropathology, also is regulated by gonadotropins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The human-specific PS1 monoclonal antibody, NT1, directed against residues 41-49 of PS1 and the A{beta}PP monoclonal antibody C1/1.6, directed against the last 20 residues of A{beta}PP, have been described previously (29, 30). Antibody WO2, raised against amino acid residues 5-8 (31) of the A{beta} domain of A{beta}PP, was kindly provided by Prof. C. L. Masters (University of Melbourne, Victoria, Australia). Antibody 6E10 (amino acid residues 1-17 of A{beta}) was from Senetek (Maryland Heights, MO). Rabbit antisera R208 (specific for A{beta}40) was kindly provided by Prof. P. D. Mehta (Institute for Basic Research in Developmental Disabilities, New York, NY). Rabbit anti-rat LH receptor polyclonal antibodies (raised against the N-terminal peptide sequence (15-38) of the rat LH/CG receptor) were kindly provided by Dr. P. C. Roche (Mayo Clinic, Rochester, MN). Anti-human LH receptor monoclonal antibody 3B5 was kindly provided by Dr. J. Wimalasena (The University of Tennessee, Knoxville, TN). Horseradish peroxidase linked goat anti-mouse and goat anti-rabbit IgG were from Amersham Biosciences (Buckinghamshire, UK).

Radioimmunoassay—Pooled fetal calf serum (FCS) (from HyClone, Logan, UT) was analyzed for LH, FSH, and prolactin by radioimmunoassay using reagents from The National Hormone and Peptide Program, NIDDK, National Institutes of Health. Table I summarizes the concentration ranges of pituitary hormones in FCS. The concentration of bovine LH in media containing 0.5% FCS was ~80-fold lower than that of the lowest concentration (1 mIU/ml or 0.25 ng/ml) of human LH added to media.


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  		TABLE I
Concentration of pituitary hormones in fetal calf serum

 
Cell Culture—The M17 human neuroblastoma cell line was maintained in Opti-MEM media supplemented with 5% (v/v) FCS, 2 mM L-glutamine, 10 mM D-glucose, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 0.7 mM {beta}-mercaptoethanol. All cells were cultured in a humidified atmosphere containing 5% CO2 in air, at 37 °C. Cells were plated at a density of 1.0 x 106 cells/ml 1 day prior to experiments in Opti-MEM media containing dialyzed 0.5% FCS (to remove endogenous sex steroids and to minimize the effects of endogenous gonadotropins). The next day, M17 cells were treated with human LH (0-30 mIU/ml; National Hormone and Peptide Program, Harbor-UCLA, Torrance, CA) or 17{beta}-estradiol (0-100 nM) at 37 °C for various time periods as indicated in the results section. Medium was changed with fresh medium containing the appropriate hormone concentration every 2 days.

Cell Lysis and Western Immunoblotting—Following incubation with LH the M17 cells were scraped in ice-cold PBS using a rubber policeman and centrifuged at 200 x g for 10 min to obtain a cell pellet. The pellet was washed with ice-cold PBS prior to lysing the cells with lysis buffer (50 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride). Total protein (25 µg) was resuspended in sample buffer (70 mM Tris-HCl, pH 6.8, containing 3.2% (w/v) SDS, 0.4 mM glycine, 6 M urea, 0.1 M dithiothreitol, and 0.01% (w/v) phenol red) and separated using polyacrylamide gel electrophoresis. Following electrophoresis and electrophoretic transfer, the membranes were immunoblotted using the antibodies against full-length A{beta}PP (WO2), PS1 (NT1), A{beta}PP-C99 (C1/1.6), and/or LH receptor (rabbit polyclonal and 3B5) using standard techniques as previously described (29, 32).

Sub-cellular Fractionation and Quantitation of A{beta}—Following incubation with LH, the cells were washed twice with ice-cold PBS. After washing, the cells were lysed with 50 mM Tris-HCl (pH 7.6), containing 1% Triton X-100 and 150 mM NaCl. The cell lysate was centrifuged at 60,000 x g for 20 min to obtain a supernatant and cell pellet. The supernatant (Triton-soluble fraction) was analyzed using ELISA. The cell pellet was washed once with 50 mM Tris-HCl (pH 7.6), containing 1% Triton X-100 and 150 mM NaCl, to remove remaining Triton-soluble proteins. The pellet was sonicated in 50 mM Tris-HCl (pH 7.6) containing 6 M guanidine hydrochloride (33-35). Following centrifugation at 250,000 x g for 20 min, the supernatant was diluted 1:12 to give a final concentration of guanidine hydrochloride of 0.5 M (which has been shown not to affect protein-antibody binding (34)). The samples were then subjected to a double sandwich ELISA as previously described (36, 37). Briefly, ELISA plates were coated with the antibody 6E10, and the A{beta} was captured with the rabbit polyclonal R208 antibody conjugated with biotin. The optical density was measured at 450 nm using a Bio-Rad Model 3550 microplate reader. All samples were treated similarly to allow comparisons between treatments. The levels of A{beta} are expressed as picograms per milligram of total protein.

Leuprolide Acetate Treatment of Mice and Quantitation of Brain A{beta}—Female C57Bl/6J mice (3 months of age) received either leuprolide acetate (1.5 mg/kg, slow release (depot) formulation) or vehicle for 0, 4, or 8 weeks and were then sacrificed. Brains were removed and dissected in half, and one hemisphere was frozen (-80 °C) for biochemical analysis. This hemisphere (to maximize yield and minimize inter-animal variability) was homogenized and used to determine the concentrations of A{beta}1-40 and A{beta}1-42 using an ELISA assay as described above or previously (38) with a modification for rodent brain tissues (Techno-Synapse Inc., Quebec, Canada) (39). Each sample was run in duplicate or triplicate and normalized with {alpha}-tubulin content for each sample. As in other species, leuprolide acetate has been shown to lower serum gonadotropin and sex steroid levels in previous studies (40, 41) and in this study.2

Data Analysis—Exposed films from Western blots were scanned (transmission scanning) using a visual light scanner (UMAX) at a resolution of 600 dpi. Scanned images were quantified using the public domain program Image (National Institutes of Health; version 1.61) to measure the density of the protein bands. Data collation and analysis were performed using Microsoft® Excel 2000. This program was also used for statistical analysis of data via the Students t test (independent, two-tailed, assuming unequal variances).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Gonadotropin Lowering Hormone Decreases the Concentration of Brain A{beta} in Mice—To determine whether gonadotropins alter A{beta}PP processing and A{beta} generation, we administered a GnRH analogue (leuprolide acetate, 1.5 mg/kg) to depress serum LH concentrations in 3-month-old C57Bl/6J female mice (n = 6) and then analyzed A{beta}1-40 and A{beta}1-42 levels. Leuprolide is a GnRH agonist that down-regulates GnRH receptors and acts as a potent antagonist, suppressing LH and FSH secretion (reviewed in Ref. 42). The actions of GnRH and its analogues are mediated by high affinity receptors for GnRH found on the membranes of the pituitary gonadotrophs (43). An acute administration of agonists of GnRH induces a marked initial release of LH and FSH. However, continuous stimulation of the pituitary by chronic administration of GnRH agonists produces an inhibition of the hypophyseal-gonadal axis through the process of "down-regulation" of pituitary receptors for GnRH, desensitization of the pituitary gonadotrophs, and a suppression of circulating levels of LH and sex steroids (42). This down-regulation of GnRH receptors, produced by sustained administration of GnRH agonists, prevents GnRH signaling for the secretion of LH and FSH and provides the basis for clinical applications in numerous reproductive hormone-related disorders (42).

Leuprolide treatment dramatically reduced the concentration (picograms/mg of protein) of brain A{beta}1-42, by 40% after 4 weeks (24.1 ± 2.0 versus 14.5 ± 2.6, p < 0.015) and further decreased the concentration to 71% (24.1 ± 2.0 versus 7.1 ± 1.0, p < 0.0001; mean ± S.E.) that of controls after 8 weeks (Fig. 1). Leuprolide treatment also reduced brain A{beta}1-40 levels by 23% after 4 weeks (5.8 ± 0.3 versus 4.5 ± 1.1, ns) and further reduced the concentration to 35% (5.8 ± 0.3 versus 3.8 ± 0.4, p < 0.024) after 8 weeks compared with control mice. Given that leuprolide acetate suppresses serum gonadotropin concentrations, these results suggest that gonadotropin hormones may modulate A{beta} generation (44).



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  		FIG. 1.
Leuprolide, a gonadotropin-lowering drug, decreases brain A{beta} levels in mice. C57Bl/6J mice (3 months old) were administered either vehicle or a slow release leuprolide acetate (1.5 mg/kg; intraperitoneal monthly) mixture at 0 and 4 weeks. Mice were euthanized at 0, 4, and 8 weeks, the brains were dissected, the frontal cortex tissues were homogenized and centrifuged, and the supernatant analyzed for A{beta}1-40 and A{beta}1-42 levels via an A{beta} ELISA assay. Results are expressed as picograms/mg of total protein (mean ± S.D., n = 6 mice at each time point). *, p < 0.05; **, p < 0.0001 for differences between vehicle and treated animals at the same time point.

 
Secreted, Triton-soluble, and Triton-insoluble Amyloid-{beta} Levels in Cells Treated with LH—To determine how gonadotropins may be acting to modulate the concentration of A{beta} in the brain, we next assessed the effects of gonadotropins on the expression and processing of A{beta}PP in a neuroblastoma cell line. Because FSH receptors are primarily located in reproductive tissues, and others and we have not detected FSH receptors in neuronal cell types, we focused our efforts on biochemical changes modulated by LH. To test whether LH impacts A{beta} generation we used the M17 neuroblastoma cell line (widely used for studying A{beta}PP processing (1, 3, 45, 46)) to examine whether LH altered A{beta}PP expression and/or processing. Immunoblot analysis using polyclonal (Fig. 2D) and monoclonal (Fig. 2E) antibodies against LH receptor indicated that M17 neuroblastoma cells contain a number of LH receptor variants, including the mature glycosylated and phosphorylated LH receptor protein (~92 kDa), the immature full-length LH receptor (59 kDa isoform (47, 48)) and a number of minor variants migrating at 36, 40, 48, 68, and 110 kDa (Fig. 2, D and E). These results indicate the presence of the immature, mature, and truncated forms of the LH receptor on M17 neuroblastoma cells by which LH might mediate signaling.



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  		FIG. 2.
LH induces A{beta} secretion and insolubility in neuroblastoma cells. Human M17 neuroblastoma cells were cultured in Opti-MEM medium with 0.5% of dialyzed donor calf serum and 1% of penicillin/streptomycin and treated with 0, 10, and 30 mIU/ml of LH for 5 days. Media with corresponding LH concentrations were replaced every 2 days. The medium from each experiment was used to measure secreted A{beta}1-40 (A). Cell pellets were solubilized in Triton X-100 and centrifuged to generate soluble (B) and insoluble fractions (C). A{beta} concentration is expressed as picograms/mg of total protein (mean ± S.D.). Experiments were performed three times in duplicate (i.e. n = 6, p < 0.01). LH receptor expression pattern in human M17 neuroblastoma cells was determined by immunoblot analysis with (D) a rabbit polyclonal antibody against residues 15-38 and (E) a mouse monoclonal antibody (3B5). Arrows indicate the immature (~59 kDa) full-length LH receptor.

 
We first treated M17 neuroblastoma cells with concentrations of LH representative of serum concentrations during early adulthood (1-10 mIU/ml) and during menopause/andropause (15-30 mIU/ml) for 5 d. We then measured secreted, Triton X-100-soluble (cytoplasmic and membranous components) and Triton X-100-insoluble cell fractions for A{beta}. LH treatment at both early adult and post-menopausal concentrations significantly increased the concentration of secreted A{beta} (Fig. 2A), indicating that LH may promote secretion of A{beta} from neurons. No change in the concentration of Triton X-100-soluble A{beta} was detected with either LH concentration (Fig. 2B). Together these results indicate that LH is involved in either the processing of A{beta}PP or the trafficking of A{beta}PP/A{beta} (see below). Interestingly, there was a doubling in the concentration of Triton X-100-insoluble A{beta} with 30 mIU/ml but not with 10 mIU/ml LH (Fig. 2C), suggesting higher concentrations of LH might drive the aggregation of the protein. In summary, these results indicate that LH promotes the secretion of A{beta} from neuroblastoma cells and that high concentrations of LH might promote A{beta} insolubility, a cardinal feature of AD.

LH Does Not Modulate A{beta}PP or PS1 Expression in Neuroblastoma Cells—To determine how LH was promoting the increased generation of A{beta}, we examined whether LH was increasing the expression of A{beta}PP. M17 neuroblastoma cells were treated with increasing concentrations of LH (0-30 mIU/ml) for 2 days, and changes in the expression of A{beta}PP and PS1 were determined (Fig. 3A). Subsequent experiments were preformed at 0, 10, and 30 mIU/ml LH for quantitation. No significant change in the expression of A{beta}PP was observed following quantitation at 5 days (or at 2 and 10 days, not shown), indicating that LH had no effect on modulating the expression of A{beta}PP (Fig. 3, B and C).



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  		FIG. 3.
LH does not alter A{beta}PP or PS1 expression. Human M17 neuroblastoma cells were treated with 0-30 mIU/ml LH for 2 days (A) or 5 days (B-E) as described in Fig. 2. The cell lysates (B-E are same as used in Fig. 2) also underwent Western immunoblotting with antibodies against full-length A{beta}PP (WO2), and full-length-PS1 and N-terminal PS1 (NT1). The full-length A{beta}PP (B), PS1 holoprotein, and N-terminal fragment (D) signals were quantitated and were expressed as a percentage of untreated cells (C and E). +ve = HEK-293 cells overexpressing PS1wt and A{beta}PP695. Experiments were performed three times in duplicate (i.e. n = 6, p < 0.05). Mol. weight markers are shown on the left hand-side.

 
Because evidence suggests PS1 is a component of the {gamma}-secretase complex, we also tested whether LH altered the expression of cellular full-length PS1 and its N-terminal fragment. No change in PS1 expression was detected, indicating that LH does not promote {gamma}-secretase cleavage by increasing PS1 expression (Fig. 3, D and E). LH also did not alter the processing of PS1 to its N-terminal fragment (Fig. 3, D and E). These results suggest that the mechanism by which LH increases A{beta} generation is not via an increase in A{beta}PP expression nor an increase in PS1 expression or processing.

LH Modulation of Secreted {alpha}-A{beta}PPs Levels and A{beta}PP-C99 —Because LH did not affect the expression of A{beta}PP or PS1 but did increase secreted A{beta} levels, we next investigated if LH modulated A{beta}PP processing through the amyloidogenic pathway (as reflected by a decrease in {alpha}-secretase cleavage of A{beta}PP to soluble {alpha}-A{beta}PP). The same media collected for A{beta} analysis (Fig. 2) was used for immunoblotting of {alpha}-A{beta}PPs using the mouse monoclonal antibody WO2 (recognizes {alpha}-A{beta}PPs). Consistent with the increased concentration of soluble A{beta} in the media of M17 neuroblastoma cells, there was a significant decrease in the concentration of {alpha}-A{beta}PPs (Fig. 4). These results indicate that LH drives A{beta}PP processing toward the amyloidogenic pathway.



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  		FIG. 4.
LH decreases {alpha}-A{beta}PPs and promotes A{beta}PP-C99 generation. Human M17 neuroblastoma cells were treated as described in Fig. 2, and the media were analyzed for revised {alpha}-A{beta}PPs (A) and cell lysates analyzed for A{beta}PP-C99 generation (C) by immunoblot analysis. Immunoblotting with antibodies WO2 (residues 5-8 of the A{beta} domain) or C1/1.6 revealed a band of ~110 kDa representing {alpha}-A{beta}PPs (A) or a band of ~10 kDa representing A{beta}PP-C99 (C), respectively. Signals were quantitated and are expressed as a percentage of untreated cells (B and D). Experiments were performed three times in duplicate (i.e. n = 6, p < 0.05).

 
Given that secreted A{beta} levels increased and {alpha}-A{beta}PPs levels decreased, we tested to confirm that there was a corresponding increase in the levels of A{beta}PP-C99 (i.e. increased {beta}-secretase activity) in the cell lysates treated with LH. The same cell lysate that was used for intracellular A{beta} levels was therefore used for Western immunoblotting analysis of A{beta}PP-C99 levels. Consistent with the decrease in {alpha}-A{beta}PPs levels, there was an increase in A{beta}PP-C99 levels at both 10 and 30 mIU/ml LH, supportive of the increased processing of A{beta}PP to A{beta}. These results further support the idea that LH modulates {beta}-secretase cleavage of A{beta}PP in the secretory pathway. These in vitro results indicate that LH drives A{beta}PP processing toward the amyloidogenic pathway by decreasing {alpha}-secretase cleavage and promoting {beta}-secretase (and {gamma}-secretase) cleavage of A{beta}PP to yield A{beta}.

LH Receptor Expression and Hormonal Regulation—Because LH receptor expression is modulated in reproductive tissues by 17{beta}-estradiol (49) and LH (50), we tested to determine whether 17{beta}-estradiol (and LH) regulated LH receptor expression in neurons. Physiological concentrations of 17{beta}-estradiol (0.1 nM) promoted an increase in the 59-kDa band representative of the immature form of the full-length LH receptor (Fig. 5) when compared with untreated M17 cells. However, treatment with increasing concentrations of 17{beta}-estradiol, including physiologically relevant concentrations of 17{beta}-estradiol (1 nM), decreased the expression of immature LH receptor in a dose-dependent manner (Fig. 5). Interestingly, levels of the mature forms of the LH receptor (~68, 92, and 110 kDa) were increased at high concentrations of 17{beta}-estradiol (10 and 100 nM).



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  		FIG. 5.
Human M17 neuroblastoma cells were treated with 17{beta}-estradiol (0-100 nM) or LH (0-30 mIU/ml), and the cell lysates were analyzed by Western immunoblot for LH receptor isoforms using a rabbit polyclonal LH receptor antibody.

 
Treatment of M17 cells with LH (as little as 1 mIU/ml) resulted in a small dose-dependent increase in immature and mature LH receptor isoforms. Truncated forms of the LH receptor (<55 kDa) also increased in a dose-dependent manner with higher LH concentrations (10 and 30 mIU/ml). Together these results indicate that 17{beta}-estradiol and LH can modulate LH receptor expression and maturation and may therefore regulate signaling mediated via the LH receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that the gonadotropin LH modulates the processing of A{beta}PP leading to increased A{beta} generation. In vivo, the potent GnRH agonist, leuprolide acetate, which suppresses the concentrations of serum gonadotropin and sex steroids, decreased the concentration of total brain A{beta} (Fig. 1). In vitro, LH directed A{beta}PP processing toward the amyloidogenic pathway as evidenced by increased A{beta} generation and secretion, a concurrent decrease in {alpha}A{beta}PP secretion and increased A{beta}PP-C99 generation (Figs. 2, 3, 4). Because LH induces biochemical changes consistent with those observed in the AD brain, our results implicate the normal age-related increase in serum LH following menopause/andropause as a potential mechanism responsible for the amyloidosis associated with aging and in AD.

Contribution of Sex Steroids and Gonadotropins to Amyloidosis—The importance of sex steroids in providing important trophic/protective support for normal brain function has been well described (51). That the decrease in the serum concentrations of these neurotrophic steroids following menopause (and andropause) is associated with disease progression has been based on epidemiological studies and findings that estrogen deficiency in vitro and in vivo promotes biochemical and neuropathological changes consistent with those observed in the aging and AD brains. For example, 17{beta}-estradiol and testosterone have been shown to alter neuronal A{beta}PP processing toward the non-amyloidogenic pathway both in vitro and in vivo. 17{beta}-estradiol at concentrations between 2 and 2000 nM increases {alpha}-A{beta}PPs production and reduces the generation of A{beta} in both mouse and human cell lines and primary cultures of rat, mouse, and human embryonic cerebrocortical neurons (52-55). Likewise, testosterone treatment (200-2000 nM) of mouse neuroblastoma cells and rat primary cerebrocortical neurons increases secretion of {alpha}-A{beta}PPs and decreases the secretion of A{beta} (56). Ovariectomy, which suppresses serum estrogen levels, also has been shown to increase total A{beta} concentrations in guinea pigs (18) and A{beta}PP transgenic mice (19). Conversely, 17{beta}-estradiol treatment was shown to partially and totally reverse the effects of ovariectomy in guinea pigs (18) and A{beta}PP transgenic mice (Tg2576 and Tg2576 x mutant PS1 (19)), respectively. A more recent study using A{beta}PPSWE transgenic mice did not see a significant increase in brain A{beta} following ovariectomy (57). One difference that might explain this discrepancy is that the A{beta}PPSWE mice were ovariectomized prior to puberty (4 weeks), whereas the Tg2576 mice were ovariectomized after puberty, indicating that establishment of an intact HPG axis is important with regards to A{beta}PP processing and pathological deposition of A{beta}.

Although the above results indicate that a reduction in serum estrogen increases A{beta}PP processing toward the amyloidogenic pathway in vivo, the fact that leuprolide acetate suppresses both gonadotropins and sex steroids suggests that the effect could well be mediated via LH. Physiological concentrations of serum estrogen are no greater than 1 nM in women, and serum testosterone concentrations are no greater than 35 nM in men (58). Given that physiologically relevant concentrations of 17{beta}-estradiol (1 nM) decreased the expression of immature LH receptor in neuroblastoma cells (Fig. 5), it is likely that the high concentrations of sex steroids used in previous in vitro studies (~2-2000 nM 17{beta}-estradiol and ~200-1000 nM testosterone) down-regulated neuronal gonadotropin receptors (and perhaps other HPG axis-relevant receptors). Thus, suppression of LH receptors by high concentrations of estrogen in vitro would have the same effect as elevated serum estrogen in vivo, suppressing gonadotropin signaling, diverting A{beta}PP processing to the non-amyloidogenic pathway with a resultant decrease in A{beta} generation. Although it is possible that high concentrations of LH suppress estrogen receptor expression, thereby modulating A{beta}PP processing, the concentrations of LH used in this study would be unlikely to exert such an affect. In support of the affect of estrogen being dependent upon other hormones of the HPG axis such as LH, Manthey and colleagues have reported for mouse hippocampal HT22 and human neuroblastoma SK-N-MC cells that the enhancement of cellular {alpha}-A{beta}PPs release was independent of estrogen receptor expression (55).

Our results clearly indicate that 17{beta}-estradiol modulates immature LH receptor expression. At physiological concentrations of 17{beta}-estradiol (0.1 nM), expression of the 59-kDa LH receptor was maximal (greater than 0 nM 17{beta}-estradiol) but subsequently declined in a dose-dependent manner to low levels by 10 nM. Thus, following menopause when 17{beta}-estradiol levels are low (<0.1 nM), a moderate expression of tissue (brain) LH receptor would still be expected. Interestingly, the levels of higher molecular weight (mature) LH receptor isoforms increased at the higher concentrations of 17{beta}-estradiol (10-100 nM). Although the physiological relevance of this finding is unclear, high concentrations of 17{beta}-estradiol might drive LH receptor maturation at such times when pituitary LH secretion is decreased in order to mediate lowered LH signaling.

In rat ovaries (and testis), the preovulatory LH surge causes a marked down-regulation of cell surface LH receptors and its cognate mRNA, but both of these recover upon luteinization (reviewed in Ref. 50). At typical adult and post-reproductive concentrations of LH (10 and 30 mIU/ml, respectively) used in this study, we found that LH receptor expression increased slightly (Fig. 5). It will be interesting in future studies to determine the affects of LH concentrations normally detected during the LH surge (80 mIU/ml) on LH receptor numbers. Although down-regulation of receptor number by LH is associated with decreased responsiveness to subsequent LH administration, loss of receptor number alone may not completely explain the desensitization, because cAMP was shown to be ineffective in reversing desensitization, suggesting LH also inhibits some post-receptor signaling events (59).

Our results suggest that LH regulates A{beta} generation via modulation of A{beta}PP processing rather than via its clearance. It is unclear whether LH is mediating its affects via the LH receptor, although the modulation of LH receptor expression by both 17{beta}-estradiol and LH (Fig. 5) supports this possibility. Full-length LH receptor consists of 335 residues containing six N-linked glycosylation sites (60, 61). We have identified mature, immature, and truncated forms of the LH receptor in M17 neuroblastoma cells (Fig. 2D). Similar findings have recently been reported in numerous mouse, rat, and human tissues, including brain (62). Post-translational changes in glycosylation and phosphorylation of the immature LH receptor (~59 kDa) result in several LHR variants that migrate between ~93 and 44 kDa (Ref. 62 and references therein). Lower molecular mass species, below 48 kDa, are thought to represent the glycosylated extracellular domain (truncated receptor) and retain hormone-binding specificity (63). Whether mature, immature, or other variants of LH receptor identified in neuroblastoma cells play a role in regulating amyloidogenesis remains to be determined. It also is becoming clear that LH can mediate its affects not only via LH receptors but via mechanisms independent of the LH receptor and traditional downstream signaling mechanisms (59, 64). These mechanisms by which LH exerts its LH receptor-independent affects are yet to be defined. Whether LH acts via one of these traditional or non-traditional pathways, or both, in regulating A{beta}PP processing, remains to be determined. Given the complex feedback loops present within the HPG axis, it will be prudent in future studies to examine other HPG axis hormones and their receptors in mediating AD-related neurodegenerative changes.

Menopause/Andropause-induced Dysregulation of the HPG Axis—The decline in reproductive function late in life leads to the complete dysregulation in the concentrations of all hormones of the hypothalamic-pituitary-gonadal (HPG) axis. In particular, the loss of negative feedback of 17{beta}-estradiol and testosterone on the hypothalamus/pituitary leads to a large increase in the circulating concentrations of the gonadotropins, LH and FSH. Ovariectomy also results in a well-documented increase in serum LH and FSH concentrations in the mouse and other species (66-69). Therefore, during menopause/andropause or following castration, our results suggest that elevated LH could drive A{beta}PP processing toward the amyloidogenic pathway. It is interesting to speculate that the increased deposition of A{beta} in female compared with male A{beta}PP-transgenic mice (70, 71) might be due to the more rapid dysregulation of the HPG axis in females compared with males.

LH, FSH, and human chorionic gonadotropin have been shown to play a critical role in brain development and neuron differentiation (72). During fetal development human chorionic gonadotropin, which binds to LH receptors, is present at concentrations 5000 times higher than during young adulthood. With the onset of menopause/andropause, LH concentrations increase to consistently higher levels (73, 74). Interestingly, the increase in LH is higher and occurs earlier in women, who are more susceptible to AD than men. The increase in gonadotropins following menopause/andropause suggests that the neuron hormonal environment reverts back to one more akin to that of the fetal brain, which has been shown to display many similarities to AD brains, namely the presence of A{beta} (75), hyperphosphorylated tau (76), and presenilin expression (77). Because the expression of these biochemical parameters is normally associated with neurogenesis, it is possible that the post-reproductive change in HPG hormones might promote abberant re-entry of neurons into the cell cycle.

Our results indicate that the role of HPG axis hormones, other than the sex steroids, in the etiology of AD warrants further investigation. Because leuprolide acetate suppressed A{beta} generation in our in vivo study, blockage of the age-related increase in serum gonadotropins using gonadotropin-releasing hormone agonists/antagonists may represent a novel treatment strategy for AD.


   FOOTNOTES
 
* This work was supported by National Institute of Health Grant RO1-AG19356 and Voyager Pharmaceutical Corporation (to C. S. A.) and by The McCusker Foundation for Alzheimer's Disease Research (to R. N. M.). Drs. Perry, Smith, and Atwood are consultants of Voyager Pharmaceutical Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

b Both authors contributed equally to this work. Back

i To whom correspondence should be addressed: Dept. of Medicine, University of Wisconsin-Madison and William S. Middleton Memorial VA Hospital (GRECC 11G), 2500 Overlook Terrace, Madison, WI 53705. Tel.: 608-256-1901 (ext. 11664); Fax: 608-280-7291; E-mail: csa{at}medicine.wisc.edu.

1 The abbreviations used are: AD, Alzheimer's disease; A{beta}, amyloid-{beta}; HPG, hypothalamic-pituitary-gonadal; LH, luteinizing hormone; FSH, follicle-stimulating hormone; A{beta}PP, amyloid-{beta} precursor protein; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; PBS, phosphate-buffered saline; GnRH, gonadotropin-releasing hormone; PS1, presenilin-1; {alpha}-A{beta}PPs, {alpha}-A{beta}PP soluble. Back

2 LH: control mice (8 months old = 0.5 ng/ml versus ovariectomized mice (8 months old) = 2.1 ng/ml; FSH: control mice (8 months old) = 10.2 ng/ml versus ovariectomized mice (8 months old) = 74.5 ng/ml (65). Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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A. C Wilson, M S. Salamat, R. J Haasl, K. M Roche, A. Karande, S. V. Meethal, E. Terasawa, R. L Bowen, and C. S Atwood
Human neurons express type I GnRH receptor and respond to GnRH I by increasing luteinizing hormone expression
J. Endocrinol., December 1, 2006; 191(3): 651 - 663.
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R. L. Bowen, R. N. Martins, C. W. Gregory, M. A. Smith, and C. S. Atwood
Dementia and Testosterone Levels in Men
JAMA, February 2, 2005; 293(5): 551 - 551.
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