Herman Depypere
email: herman.depypere@ugent.be

Andrea Vergallo

Pablo Lemercier

Simone Lista

Andrea Benedet

Nicholas Ashton

Enrica Cavedo

Henrik Zetterberg

Kaj Blennow

Eugeen Vanmechelen

Harald Hampel

Menopause hormone therapy significantly alters pathophysiological biomarkers of Alzheimer's disease

Introduction:

This increasing body of literature indicates that menopause hormonal replacement therapy (MHT) may substantially mitigate the risk of developing late-life cognitive decline due to progressive Alzheimer's disease (AD) pathophysiology. For the first time, we investigated the question whether MHT impacts AD biomarker-informed pathophysiological dynamics in de-novo diagnosed menopausal women.

Methods:

We analyzed baseline and longitudinal differences between MHT-taking and -not women in terms of concentrations of core pathophysiological AD plasma biomarkers, validated in symptomatic and cognitively healthy individuals, including biomarkers of (1) the amyloid-β (Aβ) pathway, (2) tau pathophysiology, (3) neuronal loss, and (4) axonal damage and neurodegeneration.

Results:

We report a prominent and significant treatment response at the Aβ pathway biomarker level. Women at genetic risk for AD (APOE e4 allele carriers) have particularly shown favorable results from treatment.

Discussion: To our knowledge, we present first prospective clinical evidence on effects of MHT on AD pathophysiology during menopause.

INTRODUCTION

Decades of failing late-stage neuropharmacological trials and highly variable therapeutic outcomes in current healthcare suggest that merely adjusting statistical regression models for sex may not be sufficient when it comes to an appropriate understanding of the question why sex-based biological differences may account for substantially different risk rates of disease. [START_REF] Klein | Sex differences in immune responses[END_REF][START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF][START_REF] Miller | Family matters: sexual dimorphism in cardiovascular disease[END_REF] Evidence clearly indicates that there is an increased age-independent prevalence of Alzheimer's disease (AD) in women compared to men (average odds ratio is 1.6) and that it is consistent with sexual dimorphism in AD pathophysiology, spanning molecular pathways of brain proteinopathies, neurodegeneration, neuroinflammation, and large-scale high-order organization of the brain. [START_REF] Hampel | Precision medicine and drug development in Alzheimer's disease: the importance of sexual dimorphism and patient stratification[END_REF][START_REF] Maki | The timing of estrogen therapy after ovariectomyimplications for neurocognitive function[END_REF][START_REF] Mosconi | Increased Alzheimer's risk during the menopause transition: a 3-year longitudinal brain imaging study[END_REF][START_REF] Wang | Interactive rather than independent effect of APOE and sex potentiates tau deposition in women[END_REF][START_REF] Li | Sex difference in network topology and education correlated with sex difference in cognition during the disease process of Alzheimer[END_REF][START_REF] Cavedo | Sex differences in functional and molecular neuroimaging biomarkers of Alzheimer's disease in cognitively normal older adults with subjective memory complaints[END_REF][START_REF] Damoiseaux | Gender modulates the APOE ε4 effect in healthy older adults: convergent evidence from functional brain connectivity and spinal fluid tau levels[END_REF][START_REF] Buckley | Sex differences in the association of global amyloid and regional tau deposition measured by positron emission tomography in clinically normal older adults[END_REF][START_REF] Vergallo | Brain Aβ load association and sexual dimorphism of plasma BACE1 concentrations in cognitively normal individuals at risk for AD[END_REF] In addition, women who experience early menopause, either naturally or iatrogenic, represent the highest risk cluster within the menopause population of late-life cognitive decline and dementia. [START_REF] Ferretti | Account for sex in brain research for precision medicine[END_REF][START_REF] Nebel | Understanding the impact of sex and gender in Alzheimer's disease: a call to action[END_REF] The higher vulnerability of perimenopausal (transition phase) and early-menopause women than men has been established by large-scale neuropsychological studies, post-mortem investigations, biomarkerbased studies, data-driven multi-variate analysis, and experimental models of aging and AD. Multi-modal imaging studies corroborate the evidence of sexual dimorphism in AD biology and specifically indicate a positive association of (peri)menopause status with AD proteinopathies, neuronal loss and neurodegeneration, and cognitive outcomes. [START_REF] Mosconi | Increased Alzheimer's risk during the menopause transition: a 3-year longitudinal brain imaging study[END_REF][START_REF] Mosconi | Perimenopause and emergence of an Alzheimer's bioenergetic phenotype in brain and periphery[END_REF][START_REF] Mosconi | Sex differences in Alzheimer risk: brain imaging of endocrine vs chronologic aging[END_REF][START_REF] Schelbaum | Association of reproductive history with brain MRI biomarkers of dementia risk in midlife[END_REF] In all these inter-sex comparison studies, the reported sexual dimorphism related to AD risk is clearly not significantly impacted by age, generating and supporting the hypothesis that midlife endocrinological, hormonal change patterns-rather than agingrelated metabolic pathways-may drive/trigger/facilitate AD related pathophysiological events. This evidence-driven model is consistent with the established effects of sexual steroids on physiological neurodevelopment, which also account for the sexual dimorphism of global brain volume, regional gray-and white-matter density, shape, and volume of regional anatomical structures. [START_REF] Hampel | Precision medicine and drug development in Alzheimer's disease: the importance of sexual dimorphism and patient stratification[END_REF][START_REF] Jäncke | Brain size, sex, and the aging brain[END_REF][START_REF] Williams | Neuroanatomical norms in the UK Biobank: the impact of allometric scaling, sex, and age[END_REF] Systematic and single-center studies provide indirect evidence of an endocrine-mediated AD risk in menopause since the chronic use of menopausal hormone therapy (MHT)-a therapeutic approach to mitigate menopause associated signs, symptoms, and complicationsreduces the incidence of AD. [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF][START_REF] Maki | The timing of estrogen therapy after ovariectomyimplications for neurocognitive function[END_REF][START_REF] Depypere | Alzheimer's disease, apolipoprotein E and hormone replacement therapy[END_REF] The timing of MHT commencement is an additional significant factor concerning the AD-risk and long-term clinical outcomes. [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF][START_REF] Maki | The timing of estrogen therapy after ovariectomyimplications for neurocognitive function[END_REF][START_REF] Depypere | Alzheimer's disease, apolipoprotein E and hormone replacement therapy[END_REF] Longer exposure to MHT (estrogen/progestogen) may trigger and facilitate neuroprotective pathways, as suggested by the calculated risk reduction of AD equal to 0.5% for every extra-month of steroids intake. [START_REF] Rahman | Sex-driven modifiers of Alzheimer risk: a multimodality brain imaging study[END_REF][START_REF] Brinton | Perimenopause as a neurological transition state[END_REF] While initiating MHT early during menopause seems significantly protective against development of AD, a delayed therapeutic program may less likely be effective. [START_REF] Maki | The timing of estrogen therapy after ovariectomyimplications for neurocognitive function[END_REF][START_REF] Brinton | Perimenopause as a neurological transition state[END_REF][START_REF] Craig | The Women's Health Initiative Memory Study: findings and implications for treatment[END_REF] Moreover, sub-analysis of the largescale randomized Women's Health Initiative (WHI) clinical trial show that delayed MHT may even enhance the risk of vascular incidents and worsen an established dementia syndrome. [START_REF] Craig | The Women's Health Initiative Memory Study: findings and implications for treatment[END_REF][START_REF] Shumaker | Conjugated equine estrogens and incidence of probable dementia and mild cognitive impairment in postmenopausal women: women's Health Initiative Memory Study[END_REF][START_REF] Manson | Menopausal hormone therapy and health outcomes during the intervention and extended poststopping phases of the Women's Health Initiative randomized trials[END_REF] In the present university-based expert outpatient menopause clinic prospective study, we recruited cognitively healthy women who had recently transitioned to menopause and met the indication criteria and were prescribed MHT (i.e., natural estrogens and progesterone).

We assessed the baseline and longitudinal concentrations of validated core pathophysiological AD plasma biomarkers, including biomarkers of (1) the amyloid-β (Aβ) pathway, (2) tau pathophysiology,

(3) neuronal loss, and (4) axonal damage and neurodegeneration.

The Aβ pathway, currently known as the earliest biochemical and pathophysiological cycle occurring in the AD biological continuum by [START_REF] Hampel | The amyloid-β pathway in Alzheimer's disease[END_REF] analyzing Aβ peptide 42 over Aβ peptide 40 (Aβ-42/Aβ-40 ratio) [START_REF] Janelidze | Head-to-head comparison of 8 plasma amyloid-β 42/40 assays in Alzheimer disease[END_REF] and protein concentrations of the β-site amyloid precursor protein cleaving enzyme 1 (also known as β-secretase 1 or BACE1). [START_REF] Vergallo | Plasma β-secretase1 concentrations correlate with basal forebrain atrophy and neurodegeneration in cognitively healthy individuals at risk for AD[END_REF][START_REF] Hampel | The β-Secretase BACE1 in Alzheimer's disease[END_REF] Plasma Aβ1-42/Aβ1-40 ratio has close concordance with amyloid-PET status. [START_REF] Janelidze | Head-to-head comparison of 8 plasma amyloid-β 42/40 assays in Alzheimer disease[END_REF] BACE1 plasma concentration is hypothesized to reflect levels of gene expression of BACE1, and are associated with brain Αβ concentrations, axonal damage, and neurodegeneration biomarkers, volumetric loss in the cholinergic circuitry, and cognitive scores. [START_REF] Vergallo | Brain Aβ load association and sexual dimorphism of plasma BACE1 concentrations in cognitively normal individuals at risk for AD[END_REF][START_REF] Vergallo | Plasma β-secretase1 concentrations correlate with basal forebrain atrophy and neurodegeneration in cognitively healthy individuals at risk for AD[END_REF][START_REF] Cervellati | BACE1: from biomarker to Alzheimer's disease therapeutical target[END_REF][START_REF] Shen | Increased plasma beta-secretase 1 may predict conversion to Alzheimer's disease dementia in individuals with mild cognitive impairment[END_REF] We further implemented tau protein phosphorylated at threonine 231 (p-tau 231 ), a particularly early indicator of AD related tau protein phosphorylation and neurofibrillary pathology, [START_REF] Buerger | CSF phosphorylated tau protein correlates with neocortical neurofibrillary pathology in Alzheimer's disease[END_REF] rate of hippocampal atrophy [START_REF] Hampel | Correlation of cerebrospinal fluid levels of tau protein phosphorylated at threonine 231 with rates of hippocampal atrophy in Alzheimer disease[END_REF] and rate of regional brain atrophy, longitudinal patterns of brain networks functional decline, and indirectly reflects Aβ plaque rates of deposition when combined with Aβ1-42 in a composite value (Aβ1-42/p-tau 231 ratio). [START_REF] Ashton | Plasma p-tau231: a new biomarker for incipient Alzheimer's disease pathology[END_REF][START_REF] Suárez-Calvet | Novel tau biomarkers phosphorylated at T181, T217 or T231 rise in the initial stages of the preclinical Alzheimer's continuum when only subtle changes in Aβ pathology are detected[END_REF] Plasma NfL and to a lesser extent total tau (t-tau) are surrogate biomarkers of axonal damage, neurodegeneration, and neuronal loss, respectively. [START_REF] Vergallo | Plasma β-secretase1 concentrations correlate with basal forebrain atrophy and neurodegeneration in cognitively healthy individuals at risk for AD[END_REF][START_REF] Ashton | A multicentre validation study of the diagnostic value of plasma neurofilament light[END_REF] The primary objective of the present study was to investigate whether women receiving MHT show significantly different longitudinal changes of core pathophysiological AD plasma biomarkers compared with women who opted out of MHT. The second objective was to address the question whether age and the presence of at least one APOE ε4 allele may influence biomarker expression and trajectories and partially explain potential significant inter-group differences (treated vs. untreated menopausal women). and adherence to the Mediterranean diet were also quantified and recorded.

Treatment assignment

Given the multi-dimensional (medical, psychological, and social) burden of menopause on the individual experiencing it, [START_REF] Monteleone | Symptoms of menopause -global prevalence, physiology and implications[END_REF] we opted to give the women the possibility to decide which treatment/control arm to be allocated in, that is, whether starting an MHT program.

MHT was prescribed in accordance with the guidelines of international societies IMS, EMAS, NAMS, and NICE. [START_REF] Baber | IMS recommendations on women's midlife health and menopause hormone therapy[END_REF] Women with a history of hysterectomy or in women with a levonorgestrel-releasing intrauterine system (LNG-IUS), [START_REF] Depypere | A 60-month non-comparative study on bleeding profiles with the levonorgestrel intrauterine system from the late transition period to estrogen supplemented menopause[END_REF] natural oral or transdermal estrogen was prescribed. All other women were prescribed a combination of estrogen and natural progesterone. Natural progesterone was used, since it offers a well-documented protection for the endometrium, [START_REF] Furness | Hormone therapy in postmenopausal women and risk of endometrial hyperplasia[END_REF] is safe for the breast, [START_REF] Fournier | Unequal risks for breast cancer associated with different hormone replacement therapies: results from the E3N cohort study[END_REF] and does not interfere with the beneficial effects of estrogens on the serum lipid profile. [START_REF]Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. The Writing Group for the PEPI Trial[END_REF] Oral or transdermal route of estrogen supplementation was prescribed according to the woman's preference. If a woman opted for an oral estrogen, estradiol valerate 1 mg (Progynova 1 mg once daily) was prescribed. If a woman opted for a transdermal intake of hormones, an estrogen gel (Oestrogel two doses per day) or spray (Lenzetto two spray applications per day) was used. If, women had menopausal complaints, but after extensive counselling about the advantages and risks of MHT, opted not to take hormonal therapy, they were asked to participate in this trial as controls.

Women without menopausal complaints, who met inclusion criteria for the study, were equally asked to participate in this trial as controls.

Hereafter, women who underwent a therapeutic MHT program are identified in the "MHT" arm, whereas women who opted out for any MHT treatment are identified in the "control" arm.

Global cognition

Medical exclusion criteria were a diagnosis of mild cognitive impairment/dementia, any type of disease potentially affecting cognition in the short-or long-term, including severe cardiac comorbidities (severe congestive heart failure -NYHA III-IV-; severe arrhythmias), [START_REF] Yancy | ACCF/AHA guideline for the management of heart failure: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines[END_REF] severe psychiatric comorbidities, alcohol and/or substances abuse (excluding nicotine) according to the Diagnostic and Statistical Manual of Mental Disorders-fifth edition (DSM-V), [START_REF] Weinstein | Diagnostic precision in the detection of mild cognitive impairment: a comparison of two approaches[END_REF] or more cognitive domains if tested with an extensive psychometric battery. [START_REF] Arevalo-Rodriguez | Mini-Mental State Examination (MMSE) for the early detection of dementia in people with mild cognitive impairment (MCI)[END_REF] We did not assess MMSE at V2 since we do not expect significant changes in the global cognition status over the 6-month follow-up.

Blood collection, pre-analytical processing, and AD plasma biomarkers assessment: Analytical protocols

Blood withdrawal was performed at V1 and V2 following the same pre-analytical protocol. BD vacutainers n#368861 (K2 EDTA as anticoagulant) were used for blood collection. All blood samples were handled in a standardized way and centrifuged at 1200 × g during 15 min. After that, the plasma was extracted, aliquoted per 1 ml, and frozen at -80 • C within 1 h. Aliquoting was done in Sarstedt PP tube 1.5 ml (Cat No 72.694.105) using PP tips Greiner Cat No 741050. Six months later, participating women had another blood draw. Plasma and whole blood were subsequently stored at -80 • C. The subsequent neurological plasma parameters were all determined in one run to exclude inter-assay variation.

Plasma t-tau and NfL concentrations were measured using the Single molecule array (Simoa) Tau2.0 and the NF-Light Advantage assays, respectively (Quanterix, Billerica, MA). The p-tau231 concentration was measured using an in-house Simoa assay on an HD-X Analyzer (Quanterix, Billerica, MA), as previously described. [START_REF] Ashton | Plasma p-tau231: a new biomarker for incipient Alzheimer's disease pathology[END_REF] Plasma BACE1, Aβ1-42, and Aβ1-40 were measured at ADx Neuro-Sciences, Ghent, Belgium. The previously described amyblood assay [START_REF] Thijssen | Highly specific and ultrasensitive plasma test detects Abeta(1-42) and Abeta(1-40) in Alzheimer's disease[END_REF] was used to measure plasma Aβ1-42 and Aβ1-40 was used to measure plasma Aβ1-42 and Aβ1-40.

F I G U R E 1 Study design overview of the study inclusion / exclusion flowchart

See Supplementary Methods for more details about the AD plasma biomarkers assessment.

APOE genotyping

APOE variants were genotyped by sequencing at the Genetic Service Facility (GSF, www.vibgeneticservicefacility.be) of the VIB Department of Molecular Genetics, in line with previous works carried out by the same group. [START_REF] Adamczuk | Polymorphism of brain derived neurotrophic factor influences β amyloid load in cognitively intact apolipoprotein E ε4 carriers[END_REF]

STATISTICAL ANALYSIS

The statistical analyses were conducted on the 224 participants who had no missing data on plasma biomarkers collected at the two timepoints, education, MMSE, and APOE genotype (see study flow-chart illustrated in Figure 1). The Gaussian distribution and homoskedasticity were checked visually through histograms and density plots with normal probability density function curve overlaid as well as Q-Q plots. To perform group-wise comparisons for categorical and continuous variable, we used Pearson's chi-squared test and Student's t-test, respectively.

Outliers were visually inspected for each single biomarker and not for the composite ratio values.

We used linear models (LM) to assess the potential difference of plasma biomarkers concentrations at V1 (baseline) between the two study groups (treatment vs. non-treatment). Age and APOE ε4 carrier status (presence vs. absence) were included as covariates, to rule out potential confounding effect and to better weight in on treatment effect size in case of significant results. For longitudinal analysis, we calculated the annual rate of change (ARC) as the difference in plasma concentration between the two visits (V1 and V2) divided by the delay in years. [START_REF] Garcia | Statistical approaches to longitudinal data analysis in neurodegenerative diseases: Huntington's disease as a model[END_REF] ARC = Concentration at V1 -Concentration at V2 Delay in year (1)

We used LM to test whether the two study groups may have a different ARC. Age and APOE ε4 status were set as covariates.

To follow, we performed an additional subgroup analysis to examine whether APOE ε4 status impacts the treatment effect on plasma biomarkers. We used a LM with an interaction term between the group (MHT arm or control arm) and APOE ε4 status (APOE ε4 status*group).

Eventually, we performed post-hoc analysis to breakdown significant results and capture the difference between each pair of subgroups created by the interaction term.

For all models, we reported regression coefficients (β) and standard error (SE). In addition, effect sizes were estimated using partial Cohen's f2, which represent the amount of variance of the response variables (outcome) that is explained by an explanatory variable (predictor) after accounting for other predictors in the regression model. [START_REF] Cohen | Statistical Power Analysis for the Behavioral Sciences[END_REF] All tests were two-sided and p-values, p < .05 were considered significant in all statistical elaboration. Influent data points on model outputs were inspected through Cook's distance. When the distance is equal or higher than 1, models were refitted with a robust regression to consider the presence of influent data point. [START_REF] Faraway | Extending the Linear Model with R : Generalized Linear, Mixed Effects and Nonparametric Regression Models[END_REF] Since the plasma biomarker investigated track partially independent molecular pathways, we ran separate LM for each biomarker and applied no correction of p.

Statistical analyses were performed using R software, version 4.0.5.

Robust linear models, post-hoc analyses, and Cohen's f2 were carried with the libraries "MASS", "emmeans", and "effectsize", respectively, all available at http://cran.r-project.org/web/packages. different between the control and the MHT study arms (Table 1).

RESULTS

Group-wise comparison for socio-demographic and clinical features

There was no group-wise difference in terms of MMSE scores. Women enrolled in the control group were significantly older than participants enrolled in the MHT arm (p = .033). No significant differences but a trend was present in education levels with the control group having lower levels (p = .055). The mean delay between V1 and V2 was 7.2 ± 2.4 months in the control group and 6.7 ± 1.6 months in the MHT group, with no significant between-group difference.

Baseline and longitudinal comparison between MHT and control arms for AD biomarkers

Description of plasma biomarkers at baseline and follow-up, and the ARC are reported in Table S1. We report no significant differences

Longitudinal APOE-wise comparison between MHT and control arms for AD biomarkers

The MHT-control groups difference for Aβ1-42/p-tau 231 ratio is significant at the longitudinal analysis. At the 6-month follow-up evaluation with the pairwise subgroups comparison indicating that controls with APOE ε4 showed greater reduction in Aβ1-42/p-tau 231 ratio levels than both MHT group individuals APOE ε4 allele carriers and noncarriers (p = .007 and p = .008 respectively, see Figure 3 and Table S2)).

Although Aβ1-42/p-tau 231 ratio does not differ between the two MHT subgroups split by APOE ε4 status, the MHT APOE ε4-positive individuals exhibit a significantly greater reduction of Aβ1-42 levels than the MHT APOE ε4-negative individuals (p = .03, see Figure 4 and Table S2).

Moreover, APOE ε4 carrier control individuals have significantly different levels of Aβ1-42/p-tau 231 ratio than non-carriers (p = .02, Cohen f2 = 2.65e-3, see Figure 3 and Table S2). MHT group individuals carrying at least one APOE ε4 allele have smaller reduction of Aβ1-42 than APOE ε4-negative treated women (see also Figure 4). Of note, HT APOE ε4-positive women have a mild within-group increment of plasma Aβ1-42 over time (2.41, 95% confidence interval [CI] -1.83 to 6.65; p = .26), while HT APOE ε4-negative women show a significant longitudinal reduction of the same biomarker (-3.17, 95% CI -5.89 to 0.46, p = .02). The two MHT subgroups show opposite directions, albeit with slight changes, in p-tau 231 levels with either an increase or a decrease in APOE ε4-positive and ε4-negative women, respectively. Notably, control APOE ε4-positive individuals display an average higher increment rate of p-tau 231 levels than each MHT subgroups (and the control APOE ε4-negative individuals as well). Hence, it is conceivable to infer that the effect of MHT in APOE ε4-positive women is on the Aβ pathway.

DISCUSSION

To our knowledge, this is the first-ever prospective longitudinal study performed with healthy, MHT-naïve women during menopause who carriers. [START_REF] Hampel | Precision medicine and drug development in Alzheimer's disease: the importance of sexual dimorphism and patient stratification[END_REF][START_REF] Maki | The timing of estrogen therapy after ovariectomyimplications for neurocognitive function[END_REF][START_REF] Mosconi | Increased Alzheimer's risk during the menopause transition: a 3-year longitudinal brain imaging study[END_REF][START_REF] Wang | Interactive rather than independent effect of APOE and sex potentiates tau deposition in women[END_REF][START_REF] Li | Sex difference in network topology and education correlated with sex difference in cognition during the disease process of Alzheimer[END_REF][START_REF] Cavedo | Sex differences in functional and molecular neuroimaging biomarkers of Alzheimer's disease in cognitively normal older adults with subjective memory complaints[END_REF][START_REF] Damoiseaux | Gender modulates the APOE ε4 effect in healthy older adults: convergent evidence from functional brain connectivity and spinal fluid tau levels[END_REF][START_REF] Buckley | Sex differences in the association of global amyloid and regional tau deposition measured by positron emission tomography in clinically normal older adults[END_REF][START_REF] Vergallo | Brain Aβ load association and sexual dimorphism of plasma BACE1 concentrations in cognitively normal individuals at risk for AD[END_REF] Our findings are also in agreement with the large-scale, randomized WHI study showing that MHT can reduce the risk of late-life dementia and indicated that the timing of MHT influences this outcome. In this sense, our study provides preliminary biological context for the WHI clinical results. [START_REF] Shumaker | Conjugated equine estrogens and incidence of probable dementia and mild cognitive impairment in postmenopausal women: women's Health Initiative Memory Study[END_REF][START_REF] Manson | Menopausal hormone therapy and health outcomes during the intervention and extended poststopping phases of the Women's Health Initiative randomized trials[END_REF] volumetric reduction (a surrogate marker of regional neuronal loss and neurodegeneration) over time. [START_REF] Schelbaum | Association of reproductive history with brain MRI biomarkers of dementia risk in midlife[END_REF][START_REF] Than | Interactions between age, sex, menopause, and brain structure at midlife: a UK Biobank Study[END_REF] We provide preliminary evidence of an MHT-associated effect on core biomarkers of AD pathophysiology, especially the central Aβ pathway (currently the target of late-stage anti-Aβ drug developments) and mostly in females carrying the APOE ε4 risk allele, the greatest genetic risk factor for late-onset AD. [START_REF] Zhao | Apolipoprotein E, receptors, and modulation of Alzheimer's disease[END_REF] A recent study investigating sexual dimorphism in AD brain endophenotypes showed for the first time that the accumulation of soluble Aβ aggregates was higher in women than men, especially in peri-menopausal and post-menopausal women carrying the APOE ε4 allele. [START_REF] Mosconi | Menopause impacts human brain structure, connectivity, energy metabolism, and amyloid-beta deposition[END_REF] The APOE ε4 allele-wise effect we found in the MHT arm is consistent with other therapeutic approaches for AD, either symptomatic or candidate disease-modifying drugs, reported to be associated with better cognitive-functional outcomes in APOE ε4 allele carriers. [START_REF] Tolar | Aducanumab, gantenerumab, BAN2401, and ALZ-801-the first wave of amyloid-targeting drugs for Alzheimer's disease with potential for near term approval[END_REF][START_REF] Wattmo | Predictors of longterm cognitive outcome in Alzheimer's disease[END_REF][START_REF] Cummings | Alzheimer's disease drug development pipeline: 2021[END_REF] Clinical and animal models indicate the menopause-related risk of AD in women is non-linearly increased by the presence of the APOE ε4 allele. [START_REF] Zhao | Apolipoprotein E, receptors, and modulation of Alzheimer's disease[END_REF][START_REF] Zhao | Estrogen deficiency induces mitochondrial damage prior to emergence of cognitive deficits in a postmenopausal mouse model[END_REF][START_REF] Ratnakumar | Estrogen activates Alzheimer's disease genes[END_REF][START_REF] Hou | Differential contributions of ApoE4 and female sex to BACE1 activity and expression mediate Aβ deposition and learning and memory in mouse models of Alzheimer's disease[END_REF] Mouse models suggest that estrogen upregulates the APOE gene, whereas progesterone acts antagonistically to estrogen at the gene expression level. It is hypothesized that such balanced cross-talk is impaired during menopause. [START_REF] Ratnakumar | Estrogen activates Alzheimer's disease genes[END_REF] Therefore, all these clinical-biological findings are backed by mouse models of aging and AD showing that induced estradiol deficiency is associated with cognitive worsening, synaptic decline, and AD biological signatures. [START_REF] Zhao | Estrogen deficiency induces mitochondrial damage prior to emergence of cognitive deficits in a postmenopausal mouse model[END_REF][START_REF] Hao | Interactive effects of age and estrogen on cognition and pyramidal neurons in monkey prefrontal cortex[END_REF][START_REF] Qin | Estradiol replacement at the critical period protects hippocampal neural stem cells to improve cognition in APP/PS1 mice[END_REF][START_REF] Bailey | Interactive effects of age and estrogen on cortical neurons: implications for cognitive aging[END_REF][START_REF] Candeias | Middle-aged diabetic females and males present distinct susceptibility to Alzheimer diseaselike pathology[END_REF] Stratifying pharmacological clinical trial outcomes for female sex alongside concomitant intake of MHT may uncover specific population clusters with selective response rates to pathways-targeting drugs. [START_REF] Martinkova | Proportion of women and reporting of outcomes by sex in clinical trials for Alzheimer disease: a systematic review and meta-analysis[END_REF] This reasoning may apply to late stage-development/already approved anti-Aβ treatments and developing anti-tau compounds, given the experimental and clinical evidence of a sex-APOE ε4 allele interaction effect on tau-mediated pathways related trajectories. [START_REF] Wang | Interactive rather than independent effect of APOE and sex potentiates tau deposition in women[END_REF][START_REF] Buckley | Sex differences in the association of global amyloid and regional tau deposition measured by positron emission tomography in clinically normal older adults[END_REF][START_REF] Tolar | Aducanumab, gantenerumab, BAN2401, and ALZ-801-the first wave of amyloid-targeting drugs for Alzheimer's disease with potential for near term approval[END_REF] We believe that an extensive and thorough elaboration of the limitation of this study is useful to facilitate future replication studies.

Although a significant effect of MHT on AD neurobiology at 6-months is potentially clinically meaningful, data need to be handled with caution and call for studies with longer follow-up periods. Replication studies may also indicate that menopause-related and biomarkerguided dose adjustments of MHT may be beneficial and needed to maintain a stable significant effect on biomarker indicators of AD pathophysiology.

Given the multi-dimensional (medical, psychological, and social) consequences of menopause on the affected individual, we chose to give the study participants the option to decide which treatment/control arm to be allocated to, that is, whether starting a MHT program. The consequence of giving priority to this matter is represented by the absence of randomized treatment assignment, which also translates into unbalanced numbers of subgroups. On the flip side, the study enrolled more women than the number reported in the longitudinal results.

Another potential caveat of the present study is of pharmacological nature and refers to the impossibility to differentiate whether oral or transdermal estrogen as well as whether the concomitant use of progesterone yielded different effects on AD biomarkers.

Mono-centric studies have the intrinsic limitations of not adequately addressing inter-ethnical differences that may exist due to genetic polymorphism variations accounting for downstream differences in disease vulnerability and drug metabolism.

Finally, several lines of cross-disciplinary evidence have been published between menopause and the dysregulation of neuroinflammatory and neuroimmune responses, [START_REF] Mishra | Inflammation: bridging age, menopause and APOEε4 genotype to Alzheimer's disease[END_REF][START_REF] Wang | Transitions in metabolic and immune systems from pre-menopause to post-menopause: implications for age-associated neurodegenerative diseases[END_REF][68] two critical pathophysiological mechanisms occurring early in AD and involved in complex dynamic cross-links with other AD-related pathobiological changes. [START_REF] Mishra | Inflammation: bridging age, menopause and APOEε4 genotype to Alzheimer's disease[END_REF][START_REF] Wang | Transitions in metabolic and immune systems from pre-menopause to post-menopause: implications for age-associated neurodegenerative diseases[END_REF][68]

CONCLUSIONS

Substantial progress in the analytical and clinical validation of bloodbased biomarkers, alongside the definition of their context-of-use, have recently accelerated AD clinical diagnostic and therapy research.

We provide the first prospective clinical evidence on the potential effect of MHT on core biomarkers associated to AD pathophysiology in menopause. In particular, we demonstrate a prominent response at the critical Aβ pathway biomarker level. Women at genetic risk for AD (carrying at least one APOE e4 allele) seem to be particularly benefiting from MHT.

  for plasma concentrations of AD biomarkers result from the V1 and V2 comparison of women enrolled in the MHT arm versus untreated women. As expected, LM shows that age is positively associated with a few AD biological pathways, as indicated by the Aβ1-42/p-tau 231 ratio, BACE1, and NfL (all p < .05). Such an age-wise significant association faded out at the follow-up (longitudinal changes of biomarkers). In addition, we observe an expected cross-sectional and longitudinal trend in the APOE ε4 positive group, exhibiting lower levels of plasma Aβ1-42 concentrations at baseline compared to the APOE ε4 negative group (baseline: Figure 2, β = -3.18, SE = 1.65, p = .055, Cohen f2 = 0.02, and longitudinal: β = 4.32, SE = 2.35, p = .067, Cohen f2 = 0.02).

F I G U R E 2

 2 Plasma Aβ1-42 concentrations at V1 according to APOE ε4 allele carrier status (positive or negative). Notes: The values represented refer to the estimated marginal means computed based on the linear model to account for the effect of age and treatment groups. Aβ, amyloid-β; APOE, apolipoprotein E; V1, baseline visit

F I G U R E 3

 3 Longitudinal changes of the Aβ1-42/p-tau 231 ratio according to treatment group and APOE ε4 allele carrier status (positive or negative). Notes: The values represented refer to the estimated marginal means computed based on the linear model to account for the effect of age. Post-hoc pairwise group comparison: Control APOE ε4-negative -MHT APOE ε4-negative: p = 0.76, Control APOE ε4-negative -Control APOE ε4-positive: p = 0.02, Control APOE ε4-negative -MHT APOE ε4-positive: p = 0.95, MHT APOE ε4-negative -Control APOE ε4-positive: p = 0.008, MHT APOE ε4-negative -MHT APOE ε4-positive: p = 0.71, Control APOE ε4-positive -MHT APOE ε4-positive: p = 0.007. Aβ, amyloid-β; APOE, apolipoprotein E; MHT, menopause hormone therapy; p-tau 231 , tau protein phosphorylated at site threonine 231 and biomarker assessment, the effect of treatment still differs according to APOE ε4 allele status (β = 4.18, 1.74, p = .02, Cohen f2 = 0.03),

  underwent two time point assessments of an extensive panel of blood-based biomarkers charting AD pathophysiological processes, including the Aβ and tau pathways, axonal damage, neurodegeneration. We report that MHT is associated with smaller changes toward AD pathophysiology than no-therapy (control groups) and that APOE ε4 allele carrying condition is associated with an amplified treatment outcome. Specifically, the most prominent effect of MHT on AD pathophysiological biomarkers focuses on the Aβ pathway, an early and central AD patho-biochemical cycle which defines and propagates progression in the disease continuum. Such a result is consistent with (a) the clinical features of the study cohort, cognitively healthy women with no familial history of AD; and (b) previous molecular imaging studies (Aβ-PET) reporting that women in menopause have higher rates of brain Aβ accumulation, especially in APOE ε4

F I G U R E 4

 4 Longitudinal changes of Aβ1-42 concentrations according to the treatment group and APOE ε4 allele carrier status (positive or negative). Notes: The values represented refer to the estimated marginal means computed based on the linear model to account for the effect of age. Post-hoc pairwise group comparison: Control APOE ε4-negative -MHT APOE ε4-negative: p = 0.64, Control APOE ε4-negative -Control APOE ε4-positive: p = 0.67, Control APOE ε4-negative -MHT APOE ε4-positive: p = 0.37, MHT APOE ε4-negative -Control APOE ε4-positive: p = 0.88, MHT APOE ε4-negative -MHT APOE ε4-positive: p = 0.03, Control APOE ε4-positive -MHT APOE ε4-positive: p = 0.23. Aβ, amyloid-β; APOE, apolipoprotein E; MHT, menopause hormone therapy

  V1 socio-demographic features, clinical measures and scores, APOE ε4 status are reported in Table 1. In the 224 women participating in this study 67 (29.9%) were genotyped as carriers of at least one APOE ε4 allele. The percentage of APOE ε4 carriers was not significantly 15525279, 0, Downloaded from https://alz-journals.onlinelibrary.wiley.com/doi/10.1002/alz.12759 by Eisai Co., Ltd., Wiley Online Library on [13/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

	TA B L E 1 Sociodemographic variables, MMSE, and APOE ε4 status at V1
		Control (n = 31)	MHT (n = 193)	Total (n = 224)	p value
	Age, in years
	Mean (SD)	55.69 (4.94)	53.76 (4.63)	54.03 (4.71)	.03 a
	APOE ε4 status
	ε4-negative	21 (67.7%)	136 (70.5%)	157 (70.1%)	.76 b
	ε4-positive	10 (32.3%)	57 (29.5%)	67 (29.9%)
	APOE genotype
	ε2/ε2	0 (0.0%)	1 (0.5%)	1 (0.4%)	.83 b
	ε2/ε3	6 (19.4%)	24 (12.4%)	30 (13.4%)
	ε2/ε4	0 (0.0%)	3 (1.6%)	3 (1.3%)
	ε3/ε3	15 (48.4%)	111 (57.5%)	126 (56.2%)
	ε3/ε4	8 (25.8%)	43 (22.3%)	51 (22.8%)
	ε4/ε4	2 (6.5%)	11 (5.7%)	13 (5.8%)
	MMSE score
	Mean (SD)	29.77 (0.56)	29.52 (1.07)	29.55 (1.01)	.19 a
	Familial AD
	No	24 (77.4%)	146 (75.6%)	170 (75.9%)	.83 b
	Yes	7 (22.6%)	47 (24.4%)	54 (24.1%)

Abbreviations: APOE , apolipoprotein E; Familial AD, familial history of Alzheimer's or other neurodegenerative diseases with prominent cognitive impairment, MHT, menopause hormone therapy, MMSE, Mini Mental State Examination. a Student's t-test. b Pearson's chi-squared test.

  Moreover, our results are in line with structural neuroimaging studies showing that perimenopausal women not treated with MHT have significantly greater AD-vulnerable regions 15525279, 0, Downloaded from https://alz-journals.onlinelibrary.wiley.com/doi/10.1002/alz.12759 by Eisai Co., Ltd., Wiley Online Library on [13/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

15525279, 0, Downloaded from https://alz-journals.onlinelibrary.wiley.com/doi/10.1002/alz.12759 by Eisai Co., Ltd., Wiley Online Library on [13/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

ACKNOWLEDGMENTS

Harald Hampel: the present study and related article have been initiated and prepared as part of an academic position at Sorbonne University, Paris, France. At Sorbonne University he was supported by the AXA Research Fund, the "Fondation partenariale Sorbonne Université" and the "Fondation pour la Recherche sur Alzheimer", Paris, France. Andrea Vergallo: the present study and related article have been initiated and prepared as part of an academic position at Sorbonne University, Paris, France. At Sorbonne University he was supported by the translational research program "PHOENIX", administered by the Sorbonne University Foundation and sponsored by la Fondation pour la Recherche sur Alzheimer. Henrik Zetterberg is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862), the AD Strategic Fund and the Alzheimer's Association (#ADSF-21-831376-C, #ADSF-21-831381-C and #ADSF-21-831377-C), the Olav Thon Foundation, the Erling-Persson Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2019-0228), the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 860197 (MIRIADE), European Union Joint Program for Neurodegenerative Disorders (JPND2021-00694), and the UK Dementia Research Institute at UCL. Kaj Blennow is supported by the Swedish Research Council (#2017-00915), the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615), the Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986), the European Union Joint Program for Neurodegenerative Disorders (JPND2019-466-236), the National Institute of Health (NIH), USA, (grant #1R01AG068398-01), and the Alzheimer's Association 2021 Zenith Award (ZEN-21-848495). Kaj Blennow is a co-founder of ADx NeuroSciences. Nele Dewit and Dirk Jacobs performed the amyloid and BACE1 assays.

CONFLICTS OF INTEREST

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« Anthropo-scènes »

« Y-a-t-il quelque part où je puisse parler de la nature, c'est-à-dire de nousmêmes? » E. Burtynsky, Manufactured landscapes, 2006. Antropo-scènes est un carnet de recherches consacré à l'anthropocène, à ses déclinaisons et ses réappropriations dans l'épistémologie des sciences sociales. Ce carnet explore la compréhension d'objets et de situations localisées, territorialisées, et de configurations qui mobilisent de nouvelles approches théoriques et conceptuelles dans la compréhension des changements environnementaux d'échelle globale.

Construire la critique de l'environnementalisme global

Posé comme une alerte environnementale ouvrant à une prise de conscience globale des enjeux écologiques à l'échelle planétaire, le terme d'anthropocène s'est rapidement propagé comme un discours des politiques publiques et comme un paradigme scientifique transdisciplinaire à l'oeuvre dans des domaines aussi variés que les sciences de l'environnement, les sciences sociales et les humanités. S'il résulte d'une longue gestation dans l'histoire des sciences du système terre, ses conclusionsune humanité générique dont l'agentivité est au coeur du récit de terra formation et de son désastre -, demeurent au coeur de nombreux débats et présentent une scène de controverses scientifiques et épistémologiques : tout d'abord , dans les domaines disciplinaires qui l'ont engendré, ensuite dans la recherche en sciences sociales désormais situées dans l'orbite de sciences du système terre. Alors que nombre de chercheurs y voient l'expression d'un a-cosmisme politique qui dilue la responsabilité des désastres environnementaux dans une humanité générique, l'anthropocène porte aussi les risques d'un « brouillage scientifique » induits par une transdisciplinarité souvent confuse, ou encore la construction paradoxale d'un rapport à l'histoire à partir d'une ère géologique définie comme une unité de classification de l'époque dans laquelle nous vivons. Pourtant, l'anthropocène s'est imposé comme un environnementalisme global, c'està-dire une forme de gouvernementalité des hommes et de la nature, dont l'élaboration est connexe à la fois à l'émergence d'un référentiel environnemental d'échelle global (les grandes conférences internationales sur le climat, la biodiversité, etc. ) et à l'ancrage de la réflexion écologique dans le domaine des sciences de la terre et de ses géosciences. Fondé sur les mésusages d'un monde fini découvrant sa finitude, ou un dérèglement du système terre, cette gouvernementalité prône la possibilité de maîtriser la trajectoire future du « vaisseau terre » par le recours à des socio-techniques inspirées de la géo ingénierie.

Les anthropo-scènes

Anthropo-scènes suggèrent une autre manière d'aborder les changements socio-écologiques, de manière localisée et empirique, dans un ensemble de paradigmes existants qui se proposent comme des dépassements de la dichotomie nature culture de la modernité tardive. S'il s'agit de documenter et de décrire par les multiples scènes (extractives, pollution, destruction de la biodiversité, etc.) où se joue la « destruction de l'image du globe », cette problématique n'épuise cependant pas les possibilités ouvertes par l'heuristique des « anthropo-scènes ». Les recherches présentées et discutées dans ce blog interrogent aussi un nouveau monde, autrement dit comment l'anthropocène comme forme de gouvernementalité reconfigure les frontières de l'anthropos, les figures de l'humain et les éléments de nature. En ce sens, il s'agit de prendre en compte la manière dont les solutions, l'exploration et la création de nouveaux types de ressources, se traduisent, non pas seulement par le constat d'une voracité destructrice, mais par l'émergence de propriétés, liées à la situation anthropocènique.

Ce blog dédié aux changements environnementaux s'appuie sur des projets collaboratifs, interdisciplinaires, mis en oeuvre dans différentes parties du monde (Afrique, Europe, Asie, Asie du Sud-Est). Différents travaux de terrain, d'ateliers et de symposiums dédiés à la recherche interdisciplinaire empirique sur la conservation, les moyens de subsistance, la durabilité, l'énergie, la gestion des forêts et de l'eau, nous mènent à la conviction que la façon dont nous définissons l'approche interdisciplinaire trace elle-même les frontières de l'interdisciplinarité, et, in fine, la façon dont nous considérons les relations entre l'homme et la nature. Anthropo-scènes suggère la nécessité d'ouvrir un espace de diffusion des connaissances et des recherches empiriques produites dans ce cadre interdisciplinaire tout en maintenant fermement le cap d'une épistémologie des sciences sociales.

Lignes éditoriales du blog

Anthropo-scènes se veut l'écho de recherches internationales sur les changements environnementaux qui mobilisent des descriptions interdisciplinaires permettant de comprendre les interactions, les relations et les connexions entre les socio-systèmes (ou anthropo-systèmes) et les écosystèmes, les activités anthropiques et ces changements, tout comme les divers paradigmes mobilisés. Le carnet de recherche rend compte des recherches menées sur les "anthropo-scènes", c'est-à-dire les marges, les lieux de tensions et de conflictualités, les configurations critiques de l'anthropocène.

Le blog comprend une veille portant à la fois sur les débats théoriques et les différents paradigmes, des approches scientifiques fondées sur des enquêtes en cours ou sur un ensemble de données empiriques et spatialisées. Anthropo-scènes publie des billets de veille scientifique (500 signes), de recherche (1500 signes), des articles courts ("positions paper" exposant une question dans un format court (3000-5000 signes), des notes et comptes rendus de lecture axés sur des recherches empiriques ayant pour objet les changements environnementaux mondiaux et locaux. Il accueille des contributions en français et en anglais qui se proposent d'aborder les changements environnementaux à partir d'échelles de temps et d'espaces spécifiques. L'interdisciplinaire y est entendu comme un ensemble d'approches allant de travaux connectant des ensembles de données/proxies hétérogènes, à la recherche collaborative, y compris dans ses formes de recherche participative ou citoyenne. La continuité du blog s'appuie sur de "l'atelier de l'anthropocène" (séminaire de recherche de l'EHESS, Paris) en proposant des ressources, des comptes-rendus de débats et de discussions issus de l'atelier. Le blog produit aussi des notes des lecture ( notes critiques, comptes rendus d'ouvrages et de réflexions) et des liens vers des ressources (bibliographie, bases de données, site web).

  Anthropocene versus anthropo-scenes, in RIHN Journal, Kyoto, 2017, 3 p. (version anglaise sur : ⟨halshs-01505329⟩).