Gonadal hormone deprivation regulates response to tibolone in neurodegenerative pathways

Gonadal hormone deprivation (GHD) and decline such as menopause and bilateral oophorectomy are associated with an increased risk of neurodegeneration. Yet, hormone therapies (HTs) show varying efficacy, influenced by factors such as sex, drug type, and timing of treatment relative to hormone decline. We hypothesize that the molecular environment of the brain undergoes a transition following GHD, impacting the effectiveness of HTs. Using a GHD model in mice treated with Tibolone, we conducted proteomic analysis and identified a reprogrammed response to Tibolone, a compound that stimulates estrogenic, progestogenic, and androgenic pathways. Through a comprehensive network pharmacological workflow, we identified a reprogrammed response to Tibolone, particularly within "Pathways of Neurodegeneration", as well as interconnected pathways including "cellular respiration", "carbon metabolism", and "cellular homeostasis". Analysis revealed 23 proteins whose Tibolone response depended on GHD and/or sex, implicating critical processes like oxidative phosphorylation and calcium signalling. Our findings suggest the therapeutic efficacy of HTs may depend on these variables, suggesting a need for greater precision medicine considerations whilst highlighting the need to uncover underlying mechanisms.


Introduction
Understanding the implications of menopause on health, particularly concerning sex-specific differences and precision medicine, remains a pivotal area of research.Menopause signifies a significant biological transition characterized by the decline in ovarian function, leading to reduced levels of circulating sex hormones like estrogen and progesterone, parallel to the male andropause which is a slower decline of androgens.Menopause is a well-known risk factor for numerous diseases, including osteoporosis, heart disease, and obesity [1][2][3], and emerging evidence suggests involvement in neurological changes such as shifts towards glucose metabolism [4], increased oxidative stress [5], and disruptions in various homeostatic processes [1] in the brain during the menopausal transition.Perhaps these alterations create an environment conducive to the development of neurological pathologies [6,7] such as cognitive decline, dementia, and mental disorders, with structural changes observed in the brain [8].Early onset menopause or induced menopause through procedures like bilateral oophorectomy has been associated with a heightened risk of dementia and other neurological conditions, underscoring its potential contribution to the higher prevalence of Alzheimer's disease (AD) among women compared to men [6,7,9].
The neuroprotective role of sex hormones prompted exploration into hormone therapy (HT) as a promising intervention for mitigating the effects of hormone decline during menopause.However, clinical studies investigating HT's efficacy, particularly in AD, yielded unexpected and different results.For example, a large population study revealed an increased prevalence of dementia and AD among individuals undergoing estrogen-progestin-specific HT for menopause, suggesting potential adverse effects or complex interactions between HT and AD risk factors [10].Still, HTs such as Tibolone emerge as promising candidates, with growing evidence of their potential to mitigate neuroinflammation and neurotoxicity [11][12][13].As an FDA-approved selective tissue estrogenic activity regulator, Tibolone offers the potential for repurposing in various conditions, including AD, making it suitable for addressing hormonal imbalances in the brain characteristic of the menopausal transition [11][12][13].
It is increasingly evident that the therapeutic potential of HT in reducing AD risk is influenced by several factors such as sex, timing of treatment, and the menopausal transition itself [8].This phenomenon underscores the importance of timing and individual characteristics in post-menopausal HT administration, to potentially elucidate the inconclusive findings of previous studies and trials [14].However, little is known of the underpinning mechanism behind the neuropharmacological plasticity of HTs, and their relationship to neurodegeneration and associated pathways.Addressing this knowledge gap could inform our understanding of the influence of HTs in the brain, and how gonadal activity perturbances may alter molecular functions and HT response.
We hypothesize that the therapeutic action of HTs is reprogrammed in neural homeostatic pathways following a loss of gonadal hormone production.Using a GHD mouse model subjected to acute Tibolone treatment, we aim to elucidate the impact of HT on gonadal hormonedeprived mice, considering the influence of sex and GHD on therapeutic response and identifying key functional pathways involved.Gonadectomy was performed under halothane anaesthesia at 2 months of age.250 µg Tibolone was administered to the mice at 3 months of age, and sacrifice was 24 hours later.Animal sacrifice was by cervical dislocation, and 1 g of pre-frontal cortex samples were collected before being delivered to the Centro Nacional de Biotecnología (CNB-CSIC, Madrid, Spain) for proteome analysis.This produced 8 groups (n=3 for each): our control groups were made up of male and female mice which received a saline injection, male and female mice which received a Tibolone injection, male and female gonadectomised which received a saline injection, and gonadectomised male and female mice which received the Tibolone injection.

Proteomic data generation
The prefrontal cortex was lysed with lysis buffer (500 µl of 5% SDS, 125 mM TEAB, 5 mM TCEP, 10 mM CAA, and protease/phosphatase inhibitors).The lysate was incubated at 60 • C for 30 minutes with agitation, then centrifuged at 16,000 x g for 15 minutes.The supernatant was then collected, and its protein concentration was determined using PIERCE 660 nm protein concentration assay.Protein samples were digested with trypsin at a 1:20 protease-to-protein ratio at 37ºC.After digestion, samples were dried, and peptide concentration was determined by fluorimetry (QuBit). 1 µg of peptide from each digest was analysed by liquid chromatography coupled with Triple-TOF mass spectrometry (1 technical replicate).A 250-minute gradient on a reversed-phase C-18 column was used for peptide separation.Eluted peptides were then fragmented and analysed on a TRIPLE-TOF mass spectrometer (in nano LC-ESI-MS/MS mode).MaxQuant [15] software (v1.10.43) was used to identify and quantify peptides against the Mus musculus Uniprot database.False-Discovery Rates (FDRs) for peptide and protein identifications were set to 1%.The Perseus computational platform (v1.10.43) was used for differential analysis, following these steps: (i) Data Transformation: Abundance data were log2-transformed to approximate a normal distribution; (ii) Protein Filtering: Proteins with fewer than 2 valid values in at least one group (for each comparison) were filtered out; (iii) Imputation: Missing values were replaced using simulated data generated from a normal distribution that was shrunk by a factor of 0.3 (width) and shifted down by 1.8 standard deviations; (iv) Statistical Testing: To identify differentially expressed proteins between two groups, a two-sample t-test was conducted.A permutation-based FDR was calculated with a significance threshold of q-value ≤ 0.05.

Network pharmacology analysis
Utilizing metascape [16], we performed a PPI enrichment analysis of our proteins of interest.Mus musculus specific PPI was analysed utilizing the following databases: STRING, BioGrid, OmniPath, InWeb_IM.STRING (physical score > 0.132) and BioGrid are used to identify physical interactions.The PPI networks generated include proteins with at least 1 interaction.A molecular complex detection (MCODE) algorithm then groups associated interactors which share functionality.We also utilized STRING (version 12.0, 2023) [17] for protein-protein association, interactions, functional enrichment, and co-expression analysis.

Functional enrichment meta-analysis
We utilized Metascape [16] to perform a functional enrichment meta-analysis which uses multiple online enrichment databases (PANTHER Pathway, KEGG, TRRUST, PaGenBase, Reactome, CORUM, WikiPathways and GO Biological Processes.Metascape determines similar pathways across these databases based on a Kappa-test score which results in functionally enriched interconnected clusters assigned a name by the most statistically significantly enriched function.This is limited to a maximum of 20 clusters, with less than 16 similar pathways, and less than 251 enriched pathways in total.Network visualisation is done utilizing Cytoscape [18,19], performed within metascape.

Functional analysis
Utilizing the insights from the enriched pathways and network pharmacology, the 23 proteins of interest were examined.A literature search was performed on the function of each protein, followed by determining their interconnected nature, to produce a clear picture of the specific functions which may be impacted by interactions between GHD, sex, and Tibolone.

Statistical analysis
Sex differences are generally subtle and require a large number of test subjects to uncover [22].This raises ethical issues of best practiceparticularly around the reduction of the three Rs.As such, with our 8 groups at an n=3, we elected for a p < 0.15 to identify trends before reducing to a p < 0.05 in our focused analysis.The proteomic data was tested via a three-way ANOVA in GraphPad.We utilized a power cut-off of p < 0.05 for protein-protein interactions (PPI), and q < 0.05 when identifying functionally enriched pathways impacted by sex-associated factors.

Preliminary pathway and process enrichment meta-analysis of proteins effected by sex, gonad, tibolone, and interactions
Functional enrichment meta-analysis of the 666 proteins found 'Pathways of neurodegeneration -multiple diseases' to be the most enriched term (q = 3.8×10 − 38 ) (Table 1).
All enriched functions with p < 0.01, ≥ 3 proteins, and at least a 1.5 enrichment factor were then grouped into < 20 clusters.Enriched functions were then assigned to a cluster based on sharing a similarity score of > 30%.From the 20 most significant clusters (Fig. 1A) a network plot is created where enriched functions are represented as nodes, and related nodes are connected by edges (> 30% similarity) with 15 or fewer nodes (enriched functions) per cluster, and less than 250 nodes total (Fig. 1B).
The functional enrichment meta-analysis revealed a network of enriched functions, with 6 mega-clusters of pathways and processes which share proteins.The mega-cluster containing the most enriched cluster 'Pathways of neurodegeneration -multiple diseases' has 40 nodes and 260 edges and is connected to 3 other clusters: 'Cellular Homeostasis' (q = 1.45×10 − 11 ), 'Cellular Respiration' (8.91×10 − 27 ), and 'Carbon Metabolism' (q = 3.89×10 − 15 ) (Fig. 2A).Of the 344 proteins involved, 184 are unique to its cluster, 119 are shared between two clusters, 39 are shared between three clusters, and 2 proteins (Dld, Vdac1) are shared between all four clusters (Fig. 2B).Protein-protein interactions (PPI) found seven functional clusters to be enriched, as described in Fig. 2C.

Focused process and pathway enrichment analysis
Given the phenomenon of HT resulting in often timing-specific results in studies of neurodegenerative diseases, we opted to explore how proteins implicated in the 'neurodegenerative pathways' super-cluster respond specifically to interactions of sex-gonad (SG), sex-Tibolone (ST), gonad-tibolone (GT), and sex-gonad-Tibolone (SGT).As such, the 344 proteins present within these four clusters were isolated for a higher power proteomic exploration, focused on the interactions of (Fig. 3A-D).Each list of proteins significantly affected interactions underwent functional enrichment analysis (Supp.File 1).
Of the 80 proteins, 7 overlapped between each list (Fig. 4A), but as expected many functions overlapped (Fig. 4B).The only functional pathway to be enriched by all interactions is 'pathways of neurodegenerationmultiple diseases' (Fig. 4C) (Table 2), the only gene ontology biological process to overlap all lists is 'metabolic process' (Fig. 4D).Enrichment analysis utilizing DisGeNET revealed no diseases were enriched across all protein lists.The diseases with a q < 0.01 were Respiratory Insufficiency, Leukoencephalopathy, Familial Alzheimer's Disease (FAD), and Tauopathies (Fig. 4E).STRING PPI analysis found SG interactions PPI enrichment to not be significant (p = 0.0801) (Fig. 4F), ST interactions PPI enrichment was also not significant (p = 0.527) (Fig. 4G), GT Interactions were found to be PPI enriched (p = 8.43 ×10 − 4 ) (Fig. 4H), and SGT PPI interactions were again found to not be significant (p = 0.357) (Fig. 4I).When the 80 proteins significantly impacted by interactions were clustered together in a PPI analysis, the PPI enrichment was very significant (p = 9.56 ×10 − 10 ) (Fig. 4J) with significant functional enrichments (Table 3) (Supp File 2).This 72-protein PPI network had 150 edges to an expected 80 edges, with only proteins disconnected from the network.

Pathway analysis and network pharmacology of sex-hormone interactions in pathways of neurodegeneration
We next drew our focus to the standout enriched function KEGG pathway 'Pathways of Neurodegeneration -Multiple Diseases'.proteins with sex, gonad, and Tibolone interactions, of which only two proteins overlap between two groups: NDUFS8 (SG+GT), and UBA1 (ST+GT) (Fig. 5A).There are 7 proteins in the pathway affected by SG interactions (Fig. 5B), 6 proteins affected by ST interactions (Fig. 5C), proteins affected by GT interactions (Fig. 5D), and 6 proteins affected by SGT interactions (Fig. 5E).These proteins contribute to multiple and diverse networks within the KEGG pathway (Supp Fig. 1).PPI enrichment analysis revealed significant interactions in the combined sexhormone interactions network (p = 1.97 ×10 − 10 ) (Fig. 5F) and enriched functions within the network (Table 4), as well as of the protein co-expression (Fig. 5G).

Table 1
Neurodegeneration pathways are enriched following wide-scope proteomic analysis of sex-gonad-Tibolone interactions.

Discussion
To explore the phenomenon where HT action depends on gonadal hormone activity, we investigated the action of acute Tibolone therapy in GHD male and female mice.The results indicate that Tibolone action can be altered by GHD.Interestingly, neurodegeneration-associated pathways are most significantly influenced by interactions between Tibolone, GHD, and sex.A detailed functional analysis reveals the functional pathways include oxidative phosphorylation, calcium transport, protein degradation, and intracellular transport.

Neurodegenerative pathways are impacted by sex, gonadal hormone deprivation, HT, and interactions
We performed an exploratory study to identify functions enriched by sex, GHD, Tibolone, and interactions.Fascinatingly, most enriched pathway across each variable was a KEGG pathway 'Pathways of Neurodegeneration', indicating that pathways associated with neurodegeneration are differently regulated in males and females, by sex-hormone action, and interactions.This analysis found this pathway to be clustered with similar processes and pathways.The linked clusters were 'cellular respiration', 'cellular homeostasis', and 'carbon metabolism'.Each of these pathway clusters are implicated within neurodegeneration, but it is only recently emerging that they are differently regulated by sex, sex-hormone activity, and interactions [1].Our PPI analysis bolstered our functional enrichment with overlapping results.We then performed a focused proteomic analysis of the proteins within these linked pathways focusing on the interactions between sex, GHD, and Tibolone to investigate functions whose response to Tibolone reprogrammed following GHD and/or sex-specific to understand this reprogramming phenomenon.

Functional analysis reveals the functions differently regulated by HT action in gonadal hormone deprivation in males and females
To investigate the functions influenced by this reprogramming phenomenon, we performed a functional and PPI analysis of the 23 proteins within the most enriched KEGG pathway 'Pathways of Neurodegeneration'.Functional enrichment and PPI analysis of these proteins reveal networks associated with oxidative phosphorylation, calcium signalling, autophagy, and microtubule-based transport.

Oxidative phosphorylation
Interactions between sex, hormonal status changes such as the MT, HT, or GHD, and mitochondrial metabolism have been identified in multiple tissues such as adipocytes [24], prostate and ovarian/testicular cancer cells [25], and metabolism in cardiac muscle [26].The work revealing these interactions in neural tissue has identified these interactions [27], and given these proteins each contribute to oxidative phosphorylation, which is associated with cellular stress and neurodegeneration [28], taking steps to reveal the underlying mechanism is paramount.The expression of 6 subunits of respiratory complex 1 (ND4, NDUFA2, NDUFA7, NDUFS5, NDUFS6, NDUFS8), 1 subunit of complex 3 (UQCR10), 2 subunits of complex 4 (COX2, COX6A1), and ADP/ATP translocase 2 (SLC25A5) is dependent on interactions between Tibolone, GHD, and sex.The regulation of the respiratory complex subunits shares the transcription factor NRF-1, either directly or by the downstream expression of TFAM which is the transcription factor for mitochondrial encoded subunits [29].However, there is a disparity in how each factor of sex, gonadectomy, and HT impacts each subunit (even within a complex, or shared gene regulation pathway).For example, in complex 1, 4 subunits (ND4, NDUFA2, NDUFA7, and NDUFS5) are impacted by sex, and 2 (NDUFS6 and NDUFS8) are not.5 (ND4, NDUFA2, NDUFA7, NDUFS6, and NDUFS8) are impacted by GHD, and 1 (NDUFS5) is not.5 (NDUFA2, NDUFA7, NDUFS5, NDUFS6, and NDUFS8) are impacted by Tibolone HT, and 1 (ND4) is not.It is difficult to identify a pattern to explain these mechanisms as the way that these factors affect each subunit is different.However, we note that ND4 is the only subunit not impacted by Tibolone, and it is the only subunit of mitochondrial DNA origin which may reveal a route for further research.For UQCR10 of complex 3, there is no sex difference at baseline but responses to GHD and Tibolone are fascinatingly contrary between the sexes.In complex 4, each protein is impacted by GHD but only COX2 (encoded by mitochondrial DNA) by Tibolone, and COX6A1 by sex differences.Lastly, ANT2 is a critical transporter in maintaining ADP/ATP homeostasis for mitochondrial metabolism.It is regulated by interactions of sex and Tibolone.Interestingly it is linked to the X-chromosome.

Calcium signalling
Given that calcium signalling dysregulation is heavily implicated in neurodegeneration [30][31][32], it is interesting that we found 6 proteins associated with calcium signalling presented with sex-related regulation.GRIA1 and ITPR1 are both calcium ion channels which are regulated by sex-gonad-Tibolone interactions.,In the control mice which were treated with Tibolone the expression of GRIA1, which increases intracellular calcium concentration from the extracellular matrix, was increased in females, and decreased in males.However, in a GHD state, Tibolone HT produced different outcomes on the expression of GRIA1 in males and females.Similarly, ITPR1 which increases the concentration of intracellular calcium by releasing stores from the endoplasmic reticulum [33] is also found to have sex-gonad-Tibolone interactions.Tibolone HT did not affect ITPR1 expression in male mice but decreased expression in female mice.When Tibolone was given to GHD mice it decreased expression in males and increased expression in females.4 proteins identified are involved in downstream calcium signalling: Calcineurin, CaMKII, β-Catenin, and MAPK.Intracellular calcium is transported by Calmodulin (CaM), which can then activate these proteins directly or indirectly [34].When the Calcium-CaM complex activates Calcineurin it can regulate a range of transcription activities regulating neurotransmission, apoptosis, plasticity, and homeostasis [35].Tibolone HT decreases Calcineurin expression, but in hormonally deprived mice it increases expression.Calcium-CaM can activate CaMKII which is a phosphorylation-capable enzyme heavily involved in regulating neurotransmitter homeostasis, and neuronal morphology and function [34].Importantly, CaMKII is essential for maintaining calcium and glutamate homeostasis [36].We identified that it is more expressed in males, and in a state of GHD, it is increased in males and decreased in females.CaMKII also inhibits β-Catenin, which in combination with Wnt, is involved in regulating neuroprotective functions [37].We found that GHD downregulated β-Catenin in males and females.Tibolone HT reduced expression in control males, but increased expression in GHD males.Conversely, Tibolone HT increased expression in control females, but decreased expression in GHD females.Lastly, the Calcium-CaM complex can combine with PKC which can trigger the MAPK/ERK signalling pathway which is heavily involved in regulating inflammatory pathways which contribute to neurodegenerative pathologies [38].Male mice were found to have increased expression of MAPK, but following Tibolone HT MAPK expression was increased in females, and decreased in males.

Intracellular transport and protein degradation
Intracellular transport dysfunction is well understood to create protein aggregation and cellular stress which are neurodegenerative in nature [39].For example, mutations to the important CAP-Gly domain of Dynactin, which binds to both microtubules and dynein [40], can prevent intracellular transport in neurons, leading to neurodegenerative phenotypes [41].We identified two subunits of Dynactin to be contrarily regulated by GHD differently in males and females.ACTR1B is more abundant in males but following GHD it becomes more expressed in females than males.Conversely, DCTN1 (which contains the important CAP-Gly) is more abundant in females, but following GHD DCTN1 abundance increases in males, and decreases in females.DCTN1 mutations can occur which is associated with neurodegenerative diseases such as G59S mutations which leads to dysfunction and accumulation [42].When this occurs, the proteosome-ubiquitin pathway is the primary pathway involved in clearing out the misfolded protein.We identified 3 proteins involved in this system regulated by sex-associated interactions.From the 26 S proteasome, PSMA4 is more expressed in males, but following GHD drops in males, and increases in females.Whilst another subunit, PSMD13, is more expressed in females but following Tibolone treatment this increases again in females and decreases in males.We also identified an E1 ubiquitinating enzyme, UBA1, to be more expressed in females, but following Tibolone treatment it decreases in females, and increases in males.Together this reveals that there may be a sex-hormone interaction which is regulating these systems which insists on further study.Mutations of RAB1a are associated with neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's [43].As a GTPase, it plays a crucial role in intracellular membrane trafficking, which can clear waste and contribute to maintaining homeostasis which has made it a promising therapeutic target for neurodegeneration [43].We identified that, whilst it appears to be more expressed in females, Tibolone HT decreased expression in females, and increased expression in males.Highlighting that research targeting RAB1a may need to take a sex-informed precise research approach.Lastly, we identified APP, better known as the precursor to amyloid-β whose dysfunctional aggregation is argued to be the pathological source of Alzheimer's disease.Fascinatingly, we identified that Tibolone HT or GHD increases APP abundance, but when Tibolone is introduced in hormonally deprived mice APP abundance is reduced.Fig. 4. A Bolstered and Focused Meta-Enrichment Analysis Within the Super-Cluster.(A) We narrowed our investigation to focus on the proteins present within the supercluster, with a p<0.05 revealing 80 proteins of interest impacted by at least 1 interaction between sex, gonads, and Tibolone.(B) The prevalence of these proteins is presented in a circus plot where the inner arcs are made up of dark orange which represents proteins shared by multiple enriched pathways, with light orange representing genes which are unique to the interaction.Purple lines link proteins present in multiple interactions, and blue links proteins share presence in an enriched pathway.(C) A heatmap of enriched pathways with the human equivalent of 'pathways of neurodegeneration' being the most significantly enriched.(D) A heatmap representing gene ontology biological processes found 'metabolic processes' as the most enriched term with significance across all proteins impacted by interactions, followed by 'response to stimulus' present in GT, SGT, and SG.(E) A heatmap of the enrichment analysis from the DisGeNET database.Next, we performed a protein-protein interaction analysis utilizing STRING for (F) SG, (G) ST, (H) GT, and (I) SGT.(J) We performed a protein-protein meta-analysis of all 80 proteins revealing major connections across the network.S: sex, G: gonads, T: Tibolone.

Considerations and future perspectives
A fascinating outcome of this study is considering the results in the context of age (3 months old) of the mice where we observe gonadal hormone deprivation having such influence on neurodegenerative pathways in the prefrontal cortexpathways we would suspect to be associated with older age groups [44].Whilst this study can't determine that the same effects would be observed in older mice, or with chronic Tibolone treatment, it is insightful to observe how loss of gonads can be so influential in these pathways, and how response to acute Tibolone treatment outcome depends on sex and gonadal hormone status.This phenomenon warrants further exploration to understand the effect across ages, and to explore the potential biological mechanism underlying the new response to Tibolone.Our hypothesis of this mechanism is that loss of gonads reorganizes the expression of sex-steroid receptors in the prefrontal cortex, therefore changing the outcome of Tibolone treatment.However, studies must explore which of the tibolone metabolites that act across sex-steroid receptors are regulating neurodegenerative processes, and which receptors this is being mediated by.There are no sex-steroid receptors in the KEGG "pathways of neurodegeneration" we explored, but in Fig. 3D, which is a heatmap of proteins identified by sex, gonad, and Tibolone interactions, we can see PGRMC1, the membrane-bound progesterone receptor, is significantly regulated which concurs which our hypothesis and bolsters the need for further exploration.Concurrently, we must explore if this phenomenon is specific to the prefrontal cortex, and which neural cells may be most influential and responsive to GHD and HT perhaps via single cell omics, transcriptomics which provide better gene counts for robust cell labelling, or in vitro exploration.

Conclusions
Our study delves into the intricate interplay between sex hormones, gonadal hormone deprivation, and acute Tibolone treatment in modulating neurodegenerative pathways.Through comprehensive pathway enrichment and functional analyses, we have gained novel insights into the mechanisms underlying the hormone therapy effects on neurodegeneration.The identification of key pathways highlights the multifaceted nature of neurodegenerative processes and their regulation by hormonal status.Importantly, our findings emphasize the need for sexspecific preclinical research and analysis to inform therapeutic strategies to achieve precision medicine approaches in the treatment of neurodegenerative diseases.Moving forward, further research should focus on understanding degrees of GHD according to varying levels of gonadal atrophy associated with menopause and andropause with the molecular intricacies of hormone therapy interactions, and their implications for neurodegeneration.By elucidating these mechanisms, we can pave the way for the development of targeted interventions tailored to individual hormonal profiles, ultimately advancing the field of neurodegenerative disease treatment with HTs.

Table 4
Functional Enrichments in PPI network of proteins regulated by sex, gonad, and tibolone interactions in pathways of neurodegeneration.Max of top 10 per database, minimum strength 1.5, q < 0.05.

2
months-old C57BL/6 mice born in the Cajal Institute (Madrid, Spain) were kept in a 12 h light:12 h dark schedule.Mice were handled in line with the NIH 'Care and Use of Laboratory Animals guidelines', the 'Use of Animals in Neuroscience Research' by the Society for Neuroscience and following the European Union guidelines (Council Directive 86/609/EEC).Ethical approval was provided by the Comité de Ética de Experimentación Animal del Instituto Cajal and by the Consejería del Medio Ambiente y Territorio (Comunidad de Madrid, PROEX 134/17).

Fig. 1 .
Fig. 1.Wide-Scope Proteomic Functional Enrichment Meta-Analysis Reveals 6 Super-Clusters of Enriched Function Networks Regulated by Sex, Gonads, Tibolone, and Interactions.(A) The functional enrichment meta-analysis revealed 20 statistically significant enriched functional clusters.(B) Within each of these clusters are nodes which represent enrichment pathways which share a significant number of proteins.The mega-clusters are labelled by numbers match to (A).Darker nodes indicate lower p-values.

Fig. 2 .
Fig. 2. Functional Enrichment and Protein-Protein Interaction Analysis of the Most Significantly Enriched Mega-Cluster Implicates Novel Regulation of Neurodegenerative, Metabolic, and Homeostasis Pathways by Sex-Gonad-Tibolone Effects and Interaction.(A) Here the most statistically significant mega-cluster was identified to be made up of 4 clusters 'Pathways of Neurodegeneration', 'Cellular Respiration', 'Carbon Metabolism', and 'Cellular Homeostasis'.The table represents the 5 most statistically significant pathways within each of the 4 clusters.(B) Here we show the identified overlap of proteins present within the clusters.(C) We then took these proteins and performed a protein-protein interaction analysis with clustering to identify further enriched functions.

Fig. 3 .
Fig. 3.A high-power heatmap of Sex (S), Gonads (G), and Tibolone (T) Interactions of Proteins within the Supercluster.Here are the proteins with significant (A) SG interactions, (B) GT interactions, (C) ST interactions, and (D) SGT interactions.Row Z-Score represents the change in protein abundance.M: male, F: female, C: control, T: Tibolone, G: gonads, nG: no gonads.

Fig. 5 .
Fig. 5.In depth look at Pathways of Neurodegeneration and the Proteins of interest.(A) A map describing the presence of each protein within each list of sex, gonad, and Tibolone interactions where green in present, and red is not.Next, we show which proteins in each interaction list are related to pathways of neurodegeneration with (B) SG, (C) ST, (D) GT, and (E) SGT.(F) We performed a protein-protein interaction analysis of only these proteins which revealed two core molecular groups associated with respiration, and intracellular signalling.(G) This graph describes the likelihood of the genes for each protein being co-expressed where darker squares represent increased likelihood.

Fig. 6 .
Fig. 6.An overview of the functions each protein regulated by Sex, Gonads, and Tibolone Interactions identified within 'Pathways of Neurodegeneration -Multiple Diseases.Specifically, the proteins are a part of intracellular processes such as (A) oxidative phosphorylation, (B) APP, which is a central protein in Alzheimer's disease research, (C) intracellular trafficking, (D) calcium homeostasis and signalling, (E) protein degradation by the proteosome, and trafficking from the endoplasmic reticulum to the Golgi apparatus.(G) Each of the proteins in A-F are shown here connected to their functional complexes, as well as showing the direction of expression change with each biological state.S: sex, nG: no gonads, T: Tibolone.

Table 2
Most Enriched Functions in Sex, Gonad, and Tibolone Interactions.Protein % represents the percentage of proteins present in the data within the enriched pathway.