Evaluation of the cerebrospinal fluid amyloid-ß1-42/amyloid-ß1-40 ratio measured by alpha-LISA to distinguish Alzheimer's disease from other dementia disorders.

BACKGROUND: The well-established core biomarkers used to identify Alzheimer's disease (AD) overlap with other dementia disorders such as dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). This study aimed to evaluate whether the cerebrospinal fluid (CSF) amyloid-ß (Aß)1-42/Aß1-40 ratio, measured by a novel method, could improve the differential diagnosis of AD, DLB and PDD. METHOD: CSF levels of Aß1-40 and Aß1-42 in patients with PDD, DLB, AD, Parkinson's disease and controls were analyzed using an amplified luminescent proximity homogenous immunoassay along with conventional immunoassays. RESULTS: The CSF Aß1-42/Aß1-40 ratio increased discrimination of AD from PDD and DLB compared with either of the two Aß biomarkers individually. CONCLUSION: The use of the Aß1-42/Aß1-40 ratio could improve the differentiation of AD from PDD and DLB.

Soluble amyloid precursor protein α and ß in CSF in Alzheimer's disease.

OBJECTIVE: Cerebral accumulation of amyloid ß (Aß) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or ß-secretase results in two soluble metabolites, sAPPα and sAPPß, respectively. However, previous data have shown that both α- and ß-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPß in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. METHODS: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPß from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. RESULTS: Four different C-terminal forms of sAPP were identified, sAPPß-M671, sAPPß-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPß. CONCLUSION: Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPß levels are unaltered in AD.

Aß1-15/16 as a potential diagnostic marker in neurodegenerative diseases.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aß1-15) is produced by concerted ß- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aß1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aß1-15 and Aß1-16. Here, we investigate a novel immunoassay for Aß1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aß1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aß1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aß15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aß1-16 to Aß1-15, which is then detected by the Aß1-15/16 assay. Significantly, lower levels of Aß1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aß1-15/16 assay, a reliable quantification of Aß1-15 or Aß1-15/16 in CSF samples is obtained. We found reduced levels of Aß1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.

Amyloid-ß(1-15/16) as a marker for γ-secretase inhibition in Alzheimer's disease.

Amyloid-ß (Aß) producing enzymes are key targets for disease-modifying Alzheimer's disease (AD) therapies since Aß trafficking is at the core of AD pathogenesis. Development of such drugs might benefit from the identification of markers indicating in vivo drug effects in the central nervous system. We have previously shown that Aß(1-15) is produced by concerted ß-and α-secretase cleavage of amyloid-ß protein precursor (AßPP). Here, we test the hypothesis that this pathway is more engaged upon γ-secretase inhibition in humans, and cerebrospinal fluid (CSF) levels of Aß(1-15/16) represent a biomarker for this effect. Twenty healthy men were treated with placebo (n = 5) or the γ-secretase inhibitor semagacestat (100 mg [n = 5], 140 mg [n = 5], or 280 mg [n = 5]). CSF samples were collected hourly over 36 hours and 10 time points were analyzed by immunoassay for Aß(1-15/16), Aß(x-38), Aß(x-40), Aß(x-42), sAßPPα, and sAßPPß. The CSF concentration of Aß(1-15/16) showed a dose-dependent response over 36 hours. In the 280 mg treatment group, a transient increase was seen with a maximum of 180% relative to baseline at 9 hours post administration of semagacestat. The concentrations of Aß(x-38), Aß(x-40), and Aß(x-42) decreased the first 9 hours followed by increased concentrations after 36 hours relative to baseline. No significant changes were detected for CSF sAßPPα and sAßPPß. Our data shows that CSF levels of Aß(1-15/16) increase during treatment with semagacestat supporting its feasibility as a pharmacodynamic biomarker for drug candidates aimed at inhibiting γ-secretase-mediated AßPP-processing.

Stromal cell-specific apoptotic and antiestrogenic mechanisms may explain uterine defects in humans after clomiphene citrate therapy.

OBJECTIVE: The purpose of this study was to investigate clomiphene citrate (CC)-induced modulation of uterine cell function in vivo. STUDY DESIGN: Prepubertal female Sprague-Dawley rats were treated intraperitoneally with CC for 6 or 24 hours or with a combination of CC and/or 17-beta-estradiol (E2) for 4 days. RESULTS: Chronic CC treatment induced apoptosis in a fraction of uterine stromal cells by activating the caspase-3-mediated apoptotic pathway. The damage was prevented by successive E2 treatment; however, pretreatment or concomitant treatment with E2 did not protect against CC-induced uterine apoptosis. CC decreased the protein expression of estrogen receptor alpha and increased its phosphorylation but did not affect estrogen receptor beta expression or phosphorylation. Furthermore, changes in Hoxa11, p27, and progesterone receptor protein levels and localization were associated with CC treatment. CONCLUSION: We provide novel mechanistic insights into cellular and molecular events by which CC regulates uterine stromal cell function and hence the implantation process and pregnancy outcome.

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube.

BACKGROUND: The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations. METHODS: A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone. RESULTS: The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls. CONCLUSIONS: The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

Distribution and hormonal regulation of membrane progesterone receptors beta and gamma in ciliated epithelial cells of mouse and human fallopian tubes.

BACKGROUND: The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma. METHODS: Western blot and immunohistochemistry with specific antibodies were used to characterize the expression and cellular localization of the mPRs in mouse and human tissues. Taqman (Quantitative Polymerase Chain Reaction) assays were used to quantify mRNA levels in the fallopian tubes of two different mouse models after injections with different hormones and specific antagonists. RESULTS: In the fallopian tubes of both mouse and human, the expression of mPR beta and mPR gamma proteins was exclusively found in the ciliated cells. Whereas mPR beta was found on the cilia, mPR gamma was localized at the base of the same ciliated cells, as previously reported. In gonadotropin-primed mice, both mPRs genes were down-regulated after an injection with progesterone. Treatment with estradiol rapidly down-regulated the level of mPR beta mRNA and protein in immature mice. The mPR gamma protein was down-regulated around the time of ovulation in cycling women, similar to the regulation observed in mice stimulated to ovulate via gonadotropin injections. CONCLUSION: Our findings show the presence and hormonal regulation of two distinct mPRs associated with the cilia of the fallopian tubes in both mice and women. It is hypothesized that these receptors are involved in the control of ciliary movement and, thus, gamete transport in the fallopian tubes of mammals.

Downregulation of cilia-localized Il-6R alpha by 17beta-estradiol in mouse and human fallopian tubes.

The action of interleukin-6 (IL-6) impacts female reproduction. Although IL-6 was recently shown to inhibit cilia activity in human fallopian tubes in vitro, the molecular mechanisms underlying IL-6 signaling to tubal function remain elusive. Here, we investigate the cellular localization, regulation, and possible function of two IL-6 receptors (IL-6R alpha and gp130) in mouse and human fallopian tubes in vivo. We show that IL-6R alpha is restricted to the cilia of epithelial cells in both mouse and human fallopian tubes. Exogenous 17beta-estradiol (E(2)), but not progesterone (P(4)), causes a time-dependent decrease in IL-6R alpha expression, which is blocked by the estrogen receptor (ER) antagonist ICI-182,780. Exposure of different ER-selective agonists propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol or 2,3-bis-(4-hydroxyphenyl)-propionitrile demonstrated an ER subtype-specific regulation of IL-6R alpha in mouse fallopian tubes. In contrast to IL-6R alpha, gp130 was detected in tubal epithelial cells in mice but not in humans. In humans, gp130 was found in the muscle cells and was decreased in the periovulatory and luteal phases during the reproductive cycles, indicating a species-specific expression and regulation of gp130 in the fallopian tube. Expression of tubal IL-6R alpha and gp130 in IL-6 knockout mice was found to be normal; however, E(2) treatment increased IL-6R alpha, but not gp130, in IL-6 knockout mice when compared with wild-type mice. Furthermore, expression levels of IL-6R alpha, but not gp130, decreased in parallel with estrogenic accelerated oocyte-cumulus complex (OCC) transport in mouse fallopian tubes. Our findings open the possibility that cilia-specific IL-6R alpha may play a role in the regulation of OCC transport and suggest an estrogen-regulatory pathway of IL-6R alpha in the fallopian tube.

Clomiphene citrate causes aberrant tubal apoptosis and estrogen receptor activation in rat fallopian tube: implications for tubal ectopic pregnancy.

Clomiphene citrate (CC) therapy for disorders of anovulatory infertility has been linked to an increased frequency of tubal ectopic pregnancy. Although CC enhances apoptotic processes in the ovaries, villi, and decidual tissues, its effect on apoptosis in the fallopian tube is unknown. Here, we show that chronic treatment with CC induces tubal apoptosis, but not necrosis, through an intrinsic mitochondria-dependent signaling pathway in vivo. The apoptosis was specific to epithelial cells in the isthmus, and the damage was reversed with 17beta-estradiol (E2); however, pretreatment or concomitant treatment with E2 did not protect against tubal apoptosis induced by chronic treatment with CC. Chronic treatment activated estrogen receptors (ESRs), particularly cilia-localized ESR2A (formerly ERbeta2). In contrast to E2, acute treatment of superovulating rats with a high dose of CC or the ESR2-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) significantly delayed the transport of oocyte-cumulus complexes through the fallopian tube. Our findings suggest that in response to chronic CC therapy, isthmus-specific apoptosis of epithelial cells and activation of cilia-ESR2A act in parallel to block gamete and embryo passage through the fallopian tube, eventually resulting in tubal ectopic pregnancy.

Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice.

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.

Membrane progesterone receptor gamma: tissue distribution and expression in ciliated cells in the fallopian tube.

Non-genomic, rapid actions of steroids have long been known, suggesting the possible presence of non-classical steroid receptors. A membrane receptor for progestins (mPR) was recently described in the spotted seatrout, and transcripts of three related receptors (alpha, beta, and gamma) were subsequently identified in other species including human and mouse. To begin exploring the roles of mPRgamma in mammals, we have generated an antibody against this receptor. The specificity of the antibody was demonstrated by both overexpression and RNA interference experiments. Using the antibody, we show that mPRgamma is expressed in female mouse reproductive tissues such as ovary and fallopian tube, and also in the lung and liver of both sexes. Immunohistochemical studies revealed that mPRgamma is associated with the apical membrane of ciliated cells facing the lumen of the fallopian tube. The presence of mPRgamma in ciliated cells of the fallopian tube was also demonstrated in human samples. Rapid effects of progesterone on ciliary beat frequency in the fallopian tube have recently been reported. Together, this suggests a common role for mPRgamma in the regulation of ciliary activity in the fallopian tube and thus gamete transport in mammals. The presence of mPRgamma in lung and liver of mice suggests that the receptor mediates the actions of progesterone outside the reproductive tract as well.

Changes in spontaneous behavior in rats exposed to experimental disc herniation are blocked by selective TNF-alpha inhibition.

STUDY DESIGN: Study of pain behavior in animals by observation of changes in spontaneous behavior. OBJECTIVES: To assess if selective inhibition of tumor necrosis factor alpha may reduce changes in spontaneous behavior induced by experimental disc herniation in the rat as previously reported. SUMMARY OF BACKGROUND DATA: It is known that the proinflammatory cytokine tumor necrosis factor alpha may play a key role for the nucleus pulposus-induced nerve dysfunction seen in experimental set ups. However, it is not known if tumor necrosis factor alpha is also involved in pain production induced by the same procedure. METHODS: Thirty-two rats were used for the study. Twenty-two rats had an L4-L5 disc incision combined with a displacement of the L4 dorsal root ganglion. Twelve of these rats received an intraperitoneal injection of 0.125 mL of 10 mg/mL Remicade, and the remaining 10 were left untreated. Ten rats only had the L4-L5 disc exposed and formed the control group. The day before surgery and days 1, 3, 7, 14, and 21 after surgery, the rats were videotaped from below during a 20-minute period. The duration of four specific behaviors were determined and compared between the three experimental groups at each time point. RESULTS: Similar to a previous study, the nontreated showed increased signs of focal pain behavior (rotation of the head towards the operated leg and lifting of the operated leg) during the first 7 postoperative days. Treatment with the tumor necrosis factor alpha-inhibitor infliximab significantly reduced this behavior. At day 14, there were no differences between the groups, and at day 21, the nontreated group displayed reduced locomotion and increased immobility, similar to previous observations. Tumor necrosis factor alpha inhibition also seemed to reduce these behaviors. CONCLUSIONS: The data of the study clearly indicate a role for tumor necrosis factor alpha in the studied behavior changes after experimental disc herniation in the rat. Clinical trials must be performed in order to assess if there may be a clinical use for tumor necrosis factor alpha inhibition in the treatment of sciatica due to disc herniation.