Progesterone induction of tau phosphorylation during the differentiation of human embryonic stem cells into neuroectodermal rosettes

Abstract

Background:

Tau phosphorylation is associated with neuronal division and differentiation in the fetal brain, in neuroblastoma cells, in the hibernating brains of ground squirrels and black bears, and in post-mitotic neurons in the Alzheimer's disease (AD) brain. The disassembly of the rigid microtubule structure of neurons for neuronal division and neurite remodeling requires the removal of the microtubule stabilizing protein tau via its phosphorylation.

Objective:

To determine if tau phosphorylation is required during neural embryogenesis.

Methods:

Using an in vitro human model of early embryonic development, human embryonic stem cells (hESC) were differentiated into embryoid bodies (EBs; akin to an early blastocyst) and then into neuroectodermal rosettes (akin to a rudimentary neural tube containing neuroectodermal precursor cells) upon treatment with progesterone. The neuroectodermal rosettes were then treated with and without LiCl (Cdk5 inhibitor) or roscovitine (GSK-3β inhibitor) and assayed for the expression of tau, P-tau, nestin (an early marker of neurogenesis), Cdk5 and GSK-3β.

Results:

Tau was not expressed in hESC, but tau expression and its phosphorylation increase upon progesterone-induced differentiation of hESC into neuroectodermal rosettes. Both Cdk5 and GSK-3β, enzymes associated with tau phosphorylation, were expressed in hESCs, EBs, and neuroectodermal rosettes. The GSK-3β inhibitor LiCl, but not the Cdk-5 inhibitor roscovitine, prevented tau phosphorylation and nestin expression and the formation of neuroectodermal precursor cells.

Conclusions:

These preliminary results suggest that progesterone induces tau expression and its phosphorylation during the differentiation of neuroectodermal rosettes from hESC and suggest that tau and its phosphorylation is obligatory for neuronal precursor cell mitosis. The parallels between neural embryogenesis and neurodegeneration are discussed in the context of tau phosphorylation and the aberrant re-entry of neurons into the cell cycle in AD.

Keywords

Alzheimer's disease cell cycle embryoid body human embryonic stem cell microtubule neuroectodermal rosette neuronal differentiation progesterone tau tau phosphorylation

Introduction

Tau is a microtubule-associated protein normally expressed in neuronal and glial cytoplasm including cell bodies, neurites and axons, and that binds to and stabilizes microtubules.^1–3 Tau's major cellular role is thought to involve regulation of neuronal microtubule assembly and stabilization of microtubules against severing and depolymerization in vivo.^1–4 It has been shown that disassembly of the rigid microtubule structure of neurons for neuronal division is achieved by the removal of the microtubule stabilizing protein tau via its phosphorylation.^5–9 Tau phosphorylation also is required for the assembly and disassembly of MAPs during the extension or retraction of neurites.^5–7

Tau phosphorylation was been observed in dividing and differentiating neurons in the fetal brain,^2,10 in neuroblastoma cells,^{5,8,9,11–14} in the hibernating brains of European and arctic ground squirrels, Syrian hamsters and black bears, and with aging,^15–17 situations where neuronal division or remodeling occur. Alzheimer's disease (AD) is another situation where post-mitotic neurons are thought to be re-entering the cell cycle.^18–23 Goedert et al. experimentally showed that while Ser-202 is a normal phosphorylation site in development, in AD patients this key site undergoes abnormal phosphorylation.^24,25 In addition, among the 37 serine and threonine residues that have been found to be phosphorylated in PHF-tau, the C-terminal Ser-396 and Ser-404 represent one of the major AD epitopes.^26,27 Smith and colleagues elegantly demonstrated that activation of the cell cycle in post-mitotic primary cortical neurons via adenoviral-mediated expression of c-myc and ras oncogenes induced tau phosphorylation and conformational changes similar to that seen in the AD brain.²⁸

Similarities and differences in the phosphorylation sites of tau between the fetus and the adult brain have been reported.^10,29 Phosphorylation of tau normally occurs during metaphase of neuronal division, and during differentiation hyperphosphorylated tau is observed in neurons of the fetal brain, suggesting that it is also developmentally regulated.^2,10 In this respect, tau phosphorylation has been demonstrated to be reversible in the brains of hibernating squirrels (tau protein is highly phosphorylated in torpor states and phosphorylation levels decrease after arousal in all species¹⁷), supporting a role for tau phosphorylation as a physiological process involved in neuron mitosis and/or the remodeling of neurites.^16,30,31 Since tau phosphorylation is involved in developmental processes, we examined when tau expression and phosphorylation first occur during human embryogenesis. Using human embryonic stem cells (hESC) as a model of human embryogenesis, we find that tau is not expressed in hESC, but that tau phosphorylation increases upon progesterone-induced differentiation of hESC into embryoid bodies (akin to a blastocyst) and neuroectodermal rosettes (akin to a rudimentary neural tube). The GSK-3β inhibitor LiCl prevented tau phosphorylation and nestin expression, implicating GSK-3β as one mediator of tau phosphorylation and neurogenesis during early embryogenesis.

Methods

Human embryonic stem cell culture

Pluripotent H9 hESCs (passage 22 to 32; XX karyotype; also known as WA09, a National Institute of Health registered line) were obtained from the WiCell Research Institute (Madison, WI). The research was approved by the UW-Madison Department of Medicine. Propagation of hESC, and their differentiation into EBs and into neural precursor cells is extensively described in Porayette et al.,^32,33 and Gallego et al.,.^34,35 Essentially, SD1 cultures were generated by culturing hESCs on a mouse embryonic fibroblast (MEF) feeder layer for 10 d; SD2 cultures were generated by culturing hESCs on Matrigel supplemented with defined media (mTeSR1) for 10 d; EB generation – hESC colonies were removed intact from the MEF feeder layer and grown in serum-containing medium with constant gentle rocking for 10 d to produce EBs; Rosettes generation – hESC colonies were detached intact from the MEF feeder layer using dispase. The ESC aggregates after 4–6 d in suspension culture were adhered to the culture surface where they form monolayer colonies in defined neural induction medium (containing N2 supplement). Under this culture condition, columnar neuroectodermal cells appear in the center of each colony and organize into neural tube-like rosettes after 14 d of differentiation culture. The neuroectodermal cells in the rosettes were isolated through differential enzymatic and adhesion treatment.

Inhibition of GSK-3β and Cdk-5 in hESC

Undifferentiated H9 hESCs were evenly plated in 6-well plates coated with Matrigel^TM in 2.5 mL of mTeSR1 media per well. The cells in each well of one plate were trypsinized after 24 h using 400 μL of TrypLE^TM Express (Invitrogen) per well. TrypLE was neutralized after 5 min. by adding 1 mL of MEF culture media. To confirm even plating, cells were counted using a hemocytometer after mixing equal volumes of neutralized sample and trypan blue stain (0.4%, Thermo Fisher Scientific, MA, USA). After confirmation of even plating, cells cultured in mTeSR1 media were treated with either doubly deionized H₂O, NaCl (5 mM; Fisher Scientific, PA, USA), the GSK-3β inhibitor LiCl (5 mM; Sigma-Aldrich Co., MO, USA), the Cdk-5 inhibitor roscovitine (300 nM dissolved in DMSO; Calbiochem, CA, USA) or a corresponding concentration of DMSO (Thermo Fisher Scientific, MA, USA). After 5 days of treatment, the cells were trypsinized and cell proliferation was assessed using the trypan blue assay. Cells were also collected in Dulbecco's phosphate-buffered saline and stored at −80°C for examination of protein expression.

Inhibition of GSK-3β and Cdk-5 in neuroectodermal cells

H9 hESC cultured in neural induction medium were treated as described above with H₂O, NaCl, LiCl, roscovitine or DMSO for 10 days. Cells were then trypsinized and cell proliferation, and GSK-3β and Cdk-5 expression, assessed.

Immunoblotting

Following collection of the lineages, these samples, M17 neuroblastoma cells and MEFs were quantitated for protein, equal amounts of protein run on SDS-PAGE and the immunoblot probed using well characterized monoclonal antibodies against Oct3/4 (against C-terminal 10 amino acids, Santa Cruz Biotechnology, TX, USA), Nestin (Millipore Sigma, MA, USA), tau (tau-1; clone PC1C6; recognizes most forms of tau and some bias towards dephosphorylated serine sites at 195, 198, 199 and 202; Millipore Sigma, MA, USA), P-tau (AT8; Thermo Fisher Scientific, MA, USA; recognizes tau phosphorylated at both serine 202 and threonine 205) and PHF-1 (which recognizes phosphorylated ser 396 and/or ser 404, kindly provided by Dr Peter Davies).³⁶ Polyclonal antibodies were used to detect human Cdk5 (Stressgen Bioreagent Corp., BC, Canada) and GSK-3β (Biolegend, CA, USA). Results are representative of 2–3 experiments.

Results

To examine when tau expression and its phosphorylation commences during human embryogenesis, we differentiated hESC towards embryoid bodies and neuroectodermal rosettes as previously described.^32–35 Non-phosphorylated tau expression was not detected in pluripotent hESC or EBs but was observed in neuroectodermal rosettes (53 kDa; Figure 1A) indicating that tau expression is functionally important during the development of the neural tube. Immunoblot analysis of the hESC and differentiated lineages using the monoclonal antibody AT8 which recognizes tau phosphorylated at both serine 202 and threonine 205²⁵ indicated two phosphorylated tau (43–61 kDa) isoforms in rosettes (Figure 1B, third panel). In a separate experiment, but using an antibody against tau phosphorylated at serine 396 and/or serine 404 (PHF-1³⁶), we detected a single phosphorylated tau species at ∼56 kDa in both EBs and rosettes, but not pluripotent hESC or hESC maintained in MEF conditioned media for 50 days (CM; Figure 1C). These results indicate that tau becomes expressed and is phosphorylated at different residues along the path of differentiation toward neural stem cells, and that microtubule disassembly is required during cell division associated with neuroectodermal differentiation. Moreover, the progesterone present in the neural induction media, which is obligatory for the formation of neuroectodermal rosettes,^34,35 is inducing the expression of tau and its phosphorylation during NPC differentiation from hESC.

Figure 1.

Differentiation of hESC towards a neural phenotype promotes tau expression and phosphorylation. Pluripotent H9 hESC were cultured and differentiated into neural lineages as described in the methods. Following collection of the lineages, these samples, together with M17 neuroblastoma cells, MEFs, and control and AD brain were quantitated for protein, and equal amounts of protein run on SDS-PAGE and the immunoblots probed using well characterized antibodies against (A) anti-tau-1 (clone PC1C6), (B) Oct3/4, nestin, P-tau (AT8), Cdk5, and (C) PHF-1. Molecular weight markers are shown on the left-hand side.

Although tau is expressed in aged control brain, multiple tau species are phosphorylated in the AD brain as can be seen by the smear in Figure 1C. Thus, like differentiation-induced changes in AβPP processing,^32,33 the tau results suggest a developmental component to the neurodegeneration associated with AD.

Tau is phosphorylated by a number of kinases, of which most evidence for a role in vivo has been reported for GSK-3β and Cdk5.^37–39 To examine the expression of these kinases during hESC differentiation into rosettes, we probed the immunoblot described above with anti-GSK-3β and anti-Cdk5 polyclonal antibodies. GSK-3β (64 kDa) expression was detected in hESCs, increased in SD1 and SD2 differentiated cells and decreased in embryoid bodies to low levels in rosettes (data not shown). Cdk5 expression increased with differentiation of hESCs and was maximal at the rosette stage (Figure 1B). Despite the early expression of Cdk-5 in hESC, the Cdk-5 inhibitor roscovitine did not decrease hESC proliferation or tau phosphorylation (data not shown). However, treatment with LiCl, an inhibitor of GSK-3β did result in a decrease in hESC and neural precursor cell numbers. LiCl also decreased the expression of phosphorylated tau (Figure 2), and the expression of nestin, an early neural precursor cell marker (Figure 2), suggesting that LiCl inhibits the differentiation of hESC into neuroectodermal cells.

Figure 2.

LiCl prevents neuroectodermal differentiation and tau phosphorylation. hESC were cultured in neural induction media treated with H₂O, NaCl (5 mM) or LiCl (5 mM) for 10 d, collected and then analyzed by immunoblot with antibodies against nestin, P-tau (AT8), tau-1, and β-actin. Molecular weight markers are shown on the left-hand side.

Discussion

We have previously shown that progesterone is the hormonal factor that induces the differentiation of hESC into neuroectodermal rosettes.^34,35 We now show that GSK-3β-induced tau phosphorylation is necessary for the formation of neuroectodermal precursor cells during the differentiation of hESCs into neuroectodermal rosettes. Progesterone induced an increase in the expression of tau, and tau phosphorylation at both serine 202 and threonine 205 (AT8) and at ser 396 and/or ser 404 (PHF-1) (Figure 1) during differentiation of hESC into neuroectodermal rosettes. This is consistent with studies reporting that long-term progesterone treatment increases cortical tau and MAP2 expression in ovariectomized rats,⁴⁰ and induces the phosphorylation of tau (PHF-1) via the upregulation of GSK-3β activity.⁴¹ Blocking tau phosphorylation with LiCl, an inhibitor of GSK-3β, inhibited neural precursor cell differentiation as indicated by the suppression of nestin expression (Figure 2). This is consistent with previous findings that neurite elongation is dependent upon tau and microtubule stabilization.⁴² Taken together, the dramatic hCG-induced synthesis and secretion of progesterone from hESC of the pre-implantation embryo^34,35 and also from the corpus luteum at this time,⁴³ indicates progesterone may be the physiological signal that upregulates tau and its phosphorylation during early embryonic neurogenesis.

Our data indicate tau expression and its phosphorylation commences with the division and differentiation of hESC into a neural phenotype (Figures 1 and 2). This data is consistent with tau phosphorylation reported during neuronal division and/or differentiation in (1) the fetal brain,^2,10 (2) neuroblastoma cells,^{5,8,9,11–14} and (3) in the hibernating brains of European and arctic ground squirrels, Syrian hamsters and black bears, with aging,^15–17 situations where neuronal division, differentiation or remodeling occur. Interestingly, tau phosphorylation is a hallmark of neurofibrillary tangles in the AD cortex and hippocampus, suggesting post-mitotic pyramidal neurons in the AD brain that display tau phosphorylation are receiving a signal to divide, differentiate and/or remodel. Post-mitotic neurons in the AD brain have been demonstrated to re-enter the cell cycle,^18–23 while activation of the cell cycle in post-mitotic primary cortical neurons via adenoviral-mediated expression of c-myc and ras oncogenes induces tau phosphorylation.²⁸ Together, this data illustrates the parallels that exist between neural embryogenesis and neurodegeneration and how neural precursor cells can be a useful model for examining microtubule assembly and disassembly.^44,45 Future complimentary in vivo neuroimaging studies of tau and thymidine labeling (of DNA) in control versus AD subjects would allow spatial correlation of tau expression with (attempted) neuronal proliferation of post-mitotic, terminally differentiated neurons.

The physiological signals regulating tau phosphorylation/dephosphorylation for microtubule disassembly/assembly have been unclear, although it is clear that these signals fluctuate between seasons in hibernating animals, but are constant in those with AD. It had been suggested that temperature dependent mechanisms may drive the reversible phosphorylation of tau in hibernating animals.¹⁷ Indeed, early studies^46–49 demonstrated that heat shock of rats to 42°C induced a rapid dephosphorylation of tau and a concomitant increase in non-phosphorylated tau.⁴⁹ By 6 h after heat shock, there was progressive hyperphosphorylation of tau in female but not male rats. Since both low and high temperatures are stresses that modulate the reproductive axis (e.g., suppress progesterone and other sex steroid production), the temperature-induced changes in tau phosphorylation in normal and hibernating animals may be regulated by changes in HPG axis hormones.^{15–17,50,51} This is supported by our current findings, but also by the finding that tau rephosphorylation is estrogen-independent but is prevented by androgens.^46,48 Testosterone was subsequently shown to prevent the hyperphosphorylation of tau by inhibiting the heat shock-induced overactivation of GSK-3β.⁴⁷ Further evidence to support reproductive hormones in the phosphorylation of tau has been demonstrated in neuroblastoma cells where luteinizing hormone (LH) dose-dependently alters tau phosphorylation⁵² and promotes adult neurogenesis.⁵³ Likewise, intraperitoneal injections of FSH that cross the blood-brain barrier (BBB) upregulate both the expression and phosphorylation of brain tau in female 3xTg mice.⁵⁴ Similarly, gonadectomized male and female 3xTg-AD mice (elevated LH/FSH, suppressed sex steroids) also displayed increases in tau hyperphosphorylation compared with sham gonadectomized mice, and treatment with testosterone, progesterone and/or estradiol reduced tau hyperphosphorylation to levels equivalent to or lower than observed in sham animals).^55,56 Conversely, marked reductions in the elevated levels of phosphorylated tau were reported in APPsw⁺ mice lacking the LHCGR⁵⁷ or following FSH blockade.⁵⁴ Importantly, the phosphorylation of tau during torpor^15–17 correlates with increases in the gonadotropins LH and FSH during the winter and spring in hibernating golden-mantled ground squirrels.⁵⁸ Importantly, it remains to be determined from the above studies whether the change in tau phosphorylation is a direct effect of gonadotropins or an indirect effect of alterations in peripheral or brain sex steroids.

Since LH/FSH can cross the BBB,^54,59 the brain itself can synthesize gonadotropins⁶⁰ and sex steroids (neurosteroids),⁶¹ and hESC colonies also express LH/hCG and synthesize sex steroids,^34,35 it is likely that a higher ratio of brain sex steroids to gonadotropins regulate tau expression and the induction of specific tau phosphorylation during neurogenesis. We have previously discussed the importance of the sex steroid:gonadotropin ratio as dictating normal or dyotic signaling.⁶² A higher ratio (reproductive adult concentrations of sex steroids and gonadotropins) is associated with normal cell cycle signaling, while a lower ratio (menopausal/andropausal concentrations of sex steroids and gonadotropins) is associated with aberrant cell cycle dynamics as for example seen in the AD brain.⁶³ Mey and colleagues⁶⁴ and Meethal and colleagues⁶⁵ have indicated the complexity of this signaling as it refers to the brain following menopause in women and andropause in men. The loss of circulating gonadal hormones and concurrent elevations in circulating gonadotropins at this time may result in (1) elevated brain gonadotropins dependent upon BBB entry, (2) resulting in suppression or elevation of LHCGR and FSHR expression and/or signaling, and/or (3) lowered or elevated production of brain LH/FSH. Understanding these brain-related dynamics in LH/FSH signaling will dictate neurosteroid production under these scenarios, and ultimately the gonadotropin to sex steroid ratio and signaling. The respective contributions of gonadotropins and sex (neuro)steroids ratios in regulating tau phosphorylation can explain the neuroplasticity observed during development, but also the presence of excessive tau phosphorylation during gonadotropin-induced, aberrant, re-entry of post-mitotic neurons into the cell cycle during disease states such as AD.

The above observations suggest certain post-reproductive therapeutic strategies that involve maintaining or rebalancing of the HPG axis to produce a brain sex steroid:gonadotropin ratio found during reproductive life as a treatment for cognitive decline and AD. These strategies include hormone replacement therapy (HRT) to increase brain steroids, upregulation of neurosteroid production, or the suppression of circulating gonadotropin concentrations (that might elevate brain gonadotropin expression and signaling). There is evidence supporting each of these therapies in animal and/or human studies. HRT has been demonstrated to improve cognitive performance in ovariectomized mice,^66–69 and to halt or improve cognitive performance in post-reproductive men and women with and without AD (reviewed in⁷⁰). Recently, Mey and colleagues^71,72 have demonstrated that intracerebroventricular hCG, that likely upregulates neurosteroid production, increases hippocampal dendritic spine density and cognitive function in ovariectomized mice back to that of reproductively intact mice. Finally, suppression of circulating LH/FSH using GnRH agonists/antagonists has been demonstrated to improve AD neuropathology and cognitive performance in rodents.^63,73–76 Human studies have demonstrated that leuprolide-induced suppression of circulating gonadotropins together with an acetylcholinesterase inhibitor halts the progression of AD for 48 weeks in a Phase II trial of women with mild-moderate AD.⁷⁷ A second Phase II clinical trial testing leuprolide plus acetylcholinesterase inhibitors as a treatment for AD in women with mild to moderate AD is currently underway.⁷⁸

Our preliminary data indicates that progesterone upregulates the expression of tau and its phosphorylation by GSK-3β in the differentiation of hESC into neuroectodermal rosettes. The data adds support to the idea that reproductive hormones play a pivotal role in embryonic neurogenesis,^44,45 just as they do in adult neurogenesis, neuritogenesis and dendritic spine formation.^79–81 Further studies are required to determine which embryonic sex hormones regulate tau expression and specific tau post-translational modifications for neuronal differentiation and/or remodeling.

Footnotes

Acknowledgements

We thank the WiCell Research Institute (Madison, WI) for providing hESCs and technical expertise. Portions of this research were performed as part of a Senior Honors Thesis, University of Wisconsin-Madison (MMK). This material is the result of work supported with the use of facilities at the William S. Middleton Memorial Veterans Hospital, Madison, WI. The contents do not represent the views of the Department of Veterans Affairs or the United States Government. This is Geriatrics Research, Education and Clinical Center VA paper number 09-2025.

ORCID iDs

George Perry

Tracy Butler

Craig S Atwood

Ethical considerations

The research was approved by the UW-Madison Department of Medicine.

Author contributions

Prashab Porayette: Conceptualization; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft

Maria M Kaltcheva: Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft

George Perry: Conceptualization; Methodology; Writing – review & editing

Tracy Butler: Conceptualization; Methodology; Writing – review & editing

Sivan Vadakkadath Meethal: Formal analysis; Investigation; Methodology; Supervision; Validation; Writing – original draft

Craig Atwood: Conceptualization; Formal analysis; Funding acquisition; Project administration; Resources; Supervision; Writing – original draft; Writing – review & editing

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

Craig Atwood and George Perry are Editorial Board Members of this journal but were not involved in the peer-review process of this article nor had access to any information regarding its peer-review.

The remaining authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data supporting the findings of this study are available within the article.

References

Drubin

Kobayashi

Kirschner

. Association of tau protein with microtubules in living cells. Ann N Y Acad Sci 1986; 466: 257–268.

Drubin

Kirschner

. Tau protein function in living cells. J Cell Biol 1986; 103: 2739–2746.

Kanai

Takemura

Oshima

, et al. Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA. J Cell Biol 1989; 109: 1173–1184.

Drubin

Kobayashi

Kellogg

, et al. Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells. J Cell Biol 1988; 106: 1583–1591.

Diaz-Nido

Armas-Portela

Correas

, et al. Microtubule protein phosphorylation in neuroblastoma cells and neurite growth. J Cell Sci Suppl 1991; 15: 51–59.

Avila

Dominguez

Diaz-Nido

. Regulation of microtubule dynamics by microtubule-associated protein expression and phosphorylation during neuronal development. Int J Dev Biol 1994; 38: 13–25.

Avila

Ulloa

Gonzalez

, et al. Phosphorylation of microtubule-associated proteins by protein kinase CK2 in neuritogenesis. Cell Mol Biol Res 1994; 40: 573–579.

Pope

Lambert

Leypold

, et al. Microtubule-associated protein tau is hyperphosphorylated during mitosis in the human neuroblastoma cell line SH-SY5Y. Exp Neurol 1994; 126: 185–194.

Tanaka

Iqbal

Trenkner

, et al. Abnormally phosphorylated tau in SY5Y human neuroblastoma cells. FEBS Lett 1995; 360: 5–9.

10.

Pope

Enam

Bawa

, et al. Phosphorylated tau epitope of Alzheimer's disease is coupled to axon development in the avian central nervous system. Exp Neurol 1993; 120: 106–113.

11.

Sternberg

Mesco

Cole

, et al. Tau protein: its presence and metabolism in human neuroblastoma cells. Adv Exp Med Biol 1990; 265: 283–289.

12.

Molenaar

Baker

Pleasure

, et al. The neuroendocrine and neural profiles of neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Am J Pathol 1990; 136: 375–382.

13.

Hessler

Lopes

Frankfurter

, et al. Cytoskeletal immunohistochemistry of central neurocytomas. Am J Surg Pathol 1992; 16: 1031–1038.

14.

Delobel

Flament

Hamdane

, et al. Abnormal tau phosphorylation of the Alzheimer-type also occurs during mitosis. J Neurochem 2002; 83: 412–420.

15.

Hartig

Oklejewicz

Strijkstra

, et al. Phosphorylation of the tau protein sequence 199–205 in the hippocampal CA3 region of Syrian hamsters in adulthood and during aging. Brain Res 2005; 1056: 100–104.

16.

Wang

Drew

, et al. Physiological regulation of tau phosphorylation during hibernation. J Neurochem 2008; 105: 2098–2108.

17.

Stieler

Bullmann

Kohl

, et al. The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation. PLoS One 2011; 6: e14530.

18.

Raina

Zhu

Rottkamp

, et al. Cyclin’ toward dementia: cell cycle abnormalities and abortive oncogenesis in Alzheimer disease. J Neurosci Res 2000; 61: 128–133.

19.

Zhu

Raina

Boux

, et al. Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int J Dev Neurosci 2000; 18: 433–437.

20.

Raina

Zhu

Monteiro

, et al. Abortive oncogeny and cell cycle-mediated events in Alzheimer disease. Prog Cell Cycle Res 2000; 4: 235–242.

21.

Gerst

Raina

Pirim

, et al. Altered cell-matrix associated ADAM proteins in Alzheimer disease. J Neurosci Res 2000; 59: 680–684.

22.

Raina

Monteiro

McShea

, et al. The role of cell cycle-mediated events in Alzheimer's disease. Int J Exp Pathol 1999; 80: 71–76.

23.

Zhu

Raina

Smith

. Cell cycle events in neurons. Proliferation or death? Am J Pathol 1999; 155: 327–329.

24.

Goedert

Jakes

Crowther

, et al. The abnormal phosphorylation of tau protein at ser-202 in Alzheimer disease recapitulates phosphorylation during development. Proc Natl Acad Sci U S A 1993; 90: 5066–5070.

25.

Goedert

Jakes

Vanmechelen

. Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett 1995; 189: 167–169.

26.

Gong

Liu

Grundke-Iqbal

, et al. Dysregulation of protein phosphorylation/dephosphorylation in Alzheimer's disease: a therapeutic target. J Biomed Biotechnol 2006; 2006: 31825.

27.

Wang

, et al. Levels of nonphosphorylated and phosphorylated tau in cerebrospinal fluid of Alzheimer's disease patients : an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay. Am J Pathol 2002; 160: 1269–1278.

28.

McShea

Lee

Petersen

, et al. Neuronal cell cycle re-entry mediates Alzheimer disease-type changes. Biochim Biophys Acta 2007; 1772: 467–472.

29.

Kanemaru

Takio

Miura

, et al. Fetal-type phosphorylation of the tau in paired helical filaments. J Neurochem 1992; 58: 1667–1675.

30.

Arendt

Stieler

Strijkstra

, et al. Reversible paired helical filament-like phosphorylation of tau is an adaptive process associated with neuronal plasticity in hibernating animals. J Neurosci 2003; 23: 6972–6981.

31.

Hartig

Stieler

Boerema

, et al. Hibernation model of tau phosphorylation in hamsters: selective vulnerability of cholinergic basal forebrain neurons - implications for Alzheimer's disease. Eur J Neurosci 2007; 25: 69–80.

32.

Porayette

Gallego

Kaltcheva

, et al. Differential processing of amyloid-beta precursor protein directs human embryonic stem cell proliferation and differentiation into neuronal precursor cells. J Biol Chem 2009; 284: 23806–23817.

33.

Porayette

Gallego

Kaltcheva

, et al. Amyloid-beta precursor protein expression and modulation in human embryonic stem cells: a novel role for human chorionic gonadotropin. Biochem Biophys Res Commun 2007; 364: 522–527.

34.

Gallego

Porayette

Kaltcheva

, et al. The pregnancy hormones hCG and progesterone induce human embryonic stem cell proliferation and differentiation into neuroectodermal rosettes. Stem Cell Res Ther 2010; 1: 28.

35.

Gallego

Porayette

Kaltcheva

, et al. Opioid and progesterone signaling is obligatory for early human embryogenesis. Stem Cells Dev 2009; 18: 737–740.

36.

Otvos

Jr. Feiner

Lang

, et al. Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404. J Neurosci Res 1994; 39: 669–673.

37.

Liu

Perry

Chan

, et al. Amyloid-beta-induced toxicity of primary neurons is dependent upon differentiation-associated increases in tau and cyclin-dependent kinase 5 expression. J Neurochem 2004; 88: 554–563.

38.

Imahori

Uchida

. Physiology and pathology of tau protein kinases in relation to Alzheimer's disease. J Biochem (Tokyo) 1997; 121: 179–188.

39.

Mandelkow

. Tau in Alzheimer's disease. Trends Cell Biol 1998; 8: 425–427.

40.

Camacho-Arroyo

Gonzalez-Arenas

Espinosa-Raya

, et al. Short- and long-term treatment with estradiol or progesterone modifies the expression of GFAP, MAP2 and Tau in prefrontal cortex and hippocampus. Life Sci 2011; 89: 123–128.

41.

Guerra-Araiza

Amorim

Camacho-Arroyo

, et al. Effects of progesterone and its reduced metabolites, dihydroprogesterone and tetrahydroprogesterone, on the expression and phosphorylation of glycogen synthase kinase-3 and the microtubule-associated protein tau in the rat cerebellum. Dev Neurobiol 2007; 67: 510–520.

42.

Caceres

Mautino

Kosik

. Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation. Neuron 1992; 9: 607–618.

43.

Csapo

Pulkkinen

Wiest

. Effects of luteectomy and progesterone replacement therapy in early pregnant patients. Am J Obstet Gynecol 1973; 115: 759–765.

44.

Atwood

Vadakkadath Meethal

. Gonadotropins and progestogens: obligatory developmental functions during early embryogenesis and their role in adult neurogenesis, neuroregeneration, and neurodegeneration. In: Gravanis

Mellon

(eds) Hormones in neurodegeneration, neuroprotection and neurogenesis. Weinheim, Germany: WILEY-VCH Verlag GmbH & Co. KgaA, 2011, pp.305–319.

45.

Atwood

Vadakkadath Meethal

. Human embryonic stem cells as a model system for understanding early human embryogenesis and age-related diseases. In: Atwood

(eds) Embryonic stem cells: the hormonal regulation of pluripotency and embryogenesis. Rijeka, Croatia: InTech, 2011, pp.251–270.

46.

Papasozomenos

. Androgens prevent the heat shock-induced hyperphosphorylation but not dephosphorylation of t in female rats. implications for Alzheimer's disease. J Alzheimers Dis 1999; 1: 147–153.

47.

Papasozomenos

Shanavas

. Testosterone prevents the heat shock-induced overactivation of glycogen synthase kinase-3 beta but not of cyclin-dependent kinase 5 and c-Jun NH2-terminal kinase and concomitantly abolishes hyperphosphorylation of tau: implications for Alzheimer's disease. Proc Natl Acad Sci U S A 2002; 99: 1140–1145.

48.

Papasozomenos

. The heat shock-induced hyperphosphorylation of tau is estrogen-independent and prevented by androgens: implications for Alzheimer disease. Proc Natl Acad Sci U S A 1997; 94: 6612–6617.

49.

Papasozomenos

. Heat shock induces rapid dephosphorylation of tau in both female and male rats followed by hyperphosphorylation only in female rats: implications for Alzheimer's disease. J Neurochem 1996; 66: 1140–1149.

50.

Korneyev

Binder

Bernardis

. Rapid reversible phosphorylation of rat brain tau proteins in response to cold water stress. Neurosci Lett 1995; 191: 19–22.

51.

Okawa

Ishiguro

Fujita

. Stress-induced hyperphosphorylation of tau in the mouse brain. FEBS Lett 2003; 535: 183–189.

52.

Casadesus

Webber

Atwood

, et al. Luteinizing hormone mediates Alzheimer-type changes in neurons. In: 10th International Conference on Alzheimer’s Disease and Related Disorders, Madrid, Spain, 15–20 July 2006.

53.

Mak

Enwere

Gregg

, et al. Male pheromone-stimulated neurogenesis in the adult female brain: possible role in mating behavior. Nat Neurosci 2007; 10: 1003–1011.

54.

Xiong

Kang

Wang

, et al. FSH Blockade improves cognition in mice with Alzheimer's disease. Nature 2022; 603: 470–476.

55.

Carroll

Rosario

Chang

, et al. Progesterone and estrogen regulate Alzheimer-like neuropathology in female 3xTg-AD mice. J Neurosci 2007; 27: 13357–13365.

56.

Rosario

Carroll

Pike

. Testosterone regulation of Alzheimer-like neuropathology in male 3xTg-AD mice involves both estrogen and androgen pathways. Brain Res 2010; 1359: 281–290.

57.

Lin

Yuan

, et al. Genetic ablation of luteinizing hormone receptor improves the amyloid pathology in a mouse model of Alzheimer disease. J Neuropathol Exp Neurol 2010; 69: 253–261.

58.

Barnes

. Annual cycles of gonadotropins and androgens in the hibernating golden-mantled ground squirrel. Gen Comp Endocrinol 1986; 62: 13–22.

59.

Lukacs

Hiatt

Lei

, et al. Peripheral and intracerebroventricular administration of human chorionic gonadotropin alters several hippocampus-associated behaviors in cycling female rats. Horm Behav 1995; 29: 42–58.

60.

Wilson

Salamat

Haasl

, et al. Human neurons express type I GnRH receptor and respond to GnRH I by increasing luteinizing hormone expression. J Endocrinol 2006; 191: 651–663.

61.

Baulieu

Robel

Schumacher

. Neurosteroids: beginning of the story. Int Rev Neurobiol 2001; 46: 1–32.

62.

Atwood

Hayashi

Meethal

, et al. Does the degree of endocrine dyscrasia post-reproduction dictate post-reproductive lifespan? Lessons from semelparous and iteroparous species. Geroscience 2017; 39: 103–116.

63.

Bowen

Verdile

Liu

, et al. Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition. J Biol Chem 2004; 279: 20539–20545.

64.

Mey

Bhatta

Casadesus

. Luteinizing hormone and the aging brain. Vitam Horm 2021; 115: 89–104.

65.

Meethal

Liu

Chan

, et al. Identification of a regulatory loop for the synthesis of neurosteroids: a steroidogenic acute regulatory protein-dependent mechanism involving hypothalamic-pituitary-gonadal axis receptors. J Neurochem 2009; 110: 1014–1027.

66.

Takuma

Matsuo

Himeno

, et al. 17beta-estradiol Attenuates hippocampal neuronal loss and cognitive dysfunction induced by chronic restraint stress in ovariectomized rats. Neuroscience 2007; 146: 60–68.

67.

Frye

Walf

. Progesterone to ovariectomized mice enhances cognitive performance in the spontaneous alternation, object recognition, but not placement, water maze, and contextual and cued conditioned fear tasks. Neurobiol Learn Mem 2008; 90: 171–177.

68.

Frye

Walf

. Effects of progesterone administration and APPswe + PSEN1Deltae9 mutation for cognitive performance of mid-aged mice. Neurobiol Learn Mem 2008; 89: 17–26.

69.

Frye

Duffy

Walf

. Estrogens and progestins enhance spatial learning of intact and ovariectomized rats in the object placement task. Neurobiol Learn Mem 2007; 88: 208–216.

70.

Butler

Tey

Galvin

, et al. Endocrine dyscrasia in the etiology and therapy of Alzheimer's disease. J Alzheimers Dis 2024; 101: 705–713.

71.

Mey

Bhatta

Suresh

, et al. Therapeutic benefits of central LH receptor agonism in the APP/PS1 AD model involve trophic and immune regulation and are reproductive status dependent. Biochim Biophys Acta Mol Basis Dis 2024; 1870: 167165.

72.

Mey

Bhatta

Suresh

, et al. The LH receptor regulates hippocampal spatial memory and restores dendritic spine density in ovariectomized APP/PS1 AD mice. bioRxiv 2023. DOI: 10.1101/2023.12.22.573087. [Preprint]. Posted December 23, 2023.

73.

Casadesus

Webber

Atwood

, et al. Luteinizing hormone modulates cognition and amyloid-beta deposition in Alzheimer APP transgenic mice. Biochim Biophys Acta 2006; 1762: 447–452.

74.

Blair

Bhatta

Casadesus

. CNS Luteinizing hormone receptor activation rescues ovariectomy-related loss of spatial memory and neuronal plasticity. Neurobiol Aging 2019; 78: 111–120.

75.

Bryan

Mudd

Richardson

, et al. Down-regulation of serum gonadotropins is as effective as estrogen replacement at improving menopause-associated cognitive deficits. J Neurochem 2010; 112: 870–881.

76.

Palm

Chang

Blair

, et al. Down-regulation of serum gonadotropins but not estrogen replacement improves cognition in aged-ovariectomized 3xTg AD female mice. J Neurochem 2014; 130: 115–125.

77.

Bowen

Perry

Xiong

, et al. A clinical study of lupron depot in the treatment of women with Alzheimer's disease: preservation of cognitive function in patients taking an acetylcholinesterase inhibitor and treated with high dose lupron over 48 weeks. J Alzheimers Dis 2015; 44: 549–560.

78.

Butler

Goldberg

Galvin

, et al. Rationale, study design and implementation of the LUCINDA trial: leuprolide plus cholinesterase inhibition to reduce neurologic decline in Alzheimer's. Contemp Clin Trials 2021; 107: 106488.

79.

Liu

Wang

Zhao

, et al. Progesterone increases rat neural progenitor cell cycle gene expression and proliferation via extracellularly regulated kinase and progesterone receptor membrane components 1 and 2. Endocrinology 2009; 150: 3186–3196.

80.

Woolley

McEwen

. Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol 1993; 336: 293–306.

81.

Hojo

Higo

Kawato

, et al. Hippocampal synthesis of sex steroids and corticosteroids: essential for modulation of synaptic plasticity. Front Endocrinol (Lausanne) 2011; 2: 43.