Downregulation of peripheral luteinizing hormone rescues ovariectomy-associated cognitive deficits in APP/PS1 mice.

Alzheimer's disease (AD) is more prevalent in women than men, supposing due to the decline of estrogens in menopause, accompanied by increased gonadotropins such as luteinizing hormone (LH). We and others found that the transcription factor early growth response-1 (EGR1) regulates cholinergic function including the expression of acetylcholinesterase (AChE) and plays a significant role in cognitive decline of AD. Here we investigated in APP/PS1 mice by ovariectomy (OVX) and estradiol (E2) supplementation or inhibition of LH the effect on hippocampus-related cognition and related molecular changes. We found that OVX-associated cognitive impairment was accompanied by increased dorsal hippocampal EGR1 expression, which was rescued by downregulating peripheral LH rather than by supplementing E2. We also found in postmortem AD brains a higher expression of pituitary LH-mRNA and higher EGR1 expression in the posterior hippocampus. Both, in human and mice, there was a significant positive correlation between respectively posterior/dorsal hippocampal EGR1 and peripheral LH expression. We conclude that peripheral increased LH and increased posterior hippocampal EGR1 plays a significant role in AD pathology.

Sex differences in hippocampal ß-amyloid accumulation in the triple-transgenic mouse model of Alzheimer's disease and the potential role of local estrogens.

Introduction: Epidemiological studies show that women have a higher prevalence of Alzheimer's disease (AD) than men. Peripheral estrogen reduction during aging in women is proposed to play a key role in this sex-associated prevalence, however, the underlying mechanism remains elusive. We previously found that transcription factor early growth response-1 (EGR1) significantly regulates cholinergic function. EGR1 stimulates acetylcholinesterase (AChE) gene expression and is involved in AD pathogenesis. We aimed to investigate whether the triple-transgenic AD (3xTg-AD) mice harboring PS1 M146V , APP Swe , and Tau P301L show sex differences in ß-amyloid (Aß) and hyperphosphorylated tau (p-Tau), the two primary AD hallmarks, and how local 17ß-estradiol (E2) may regulate the expression of EGR1 and AChE. Methods: We first sacrificed male and female 3xTg-AD mice at 3-4, 7-8, and 11-12 months and measured the levels of Aß, p-Tau, EGR1, and AChE in the hippocampal complex. Second, we infected SH-SY5Y cells with lentivirus containing the amyloid precursor protein construct C99, cultured with or without E2 administration we measured the levels of extracellular Aß and intracellular EGR1 and AChE. Results: Female 3xTg-AD mice had higher levels of Aß compared to males, while no p-Tau was found in either group. In SH-SY5Y cells infected with lentivirus containing the amyloid precursor protein construct C99, we observed significantly increased extracellular Aß and decreased expression of intracellular EGR1 and AChE. By adding E2 to the culture medium, extracellular Aß(l-42) was significantly decreased while intracellular EGR1 and AChE expression were elevated. Discussion: This data shows that the 3xTg-AD mouse model can be useful for studying the human sex differences of AD, but only in regards to Ap. Furthermore, in vitro data shows local E2 may be protective for EGR1 and cholinergic functions in AD while suppressing soluble Aß(1-42) levels. Altogether, this study provides further in vivo and in vitro data supporting the human epidemiological data indicating a higher prevalence of AD in women is related to changes in brain estrogen levels.

Sexually dimorphic age-related molecular differences in the entorhinal cortex of cognitively intact elderly: Relation to early Alzheimer's changes.

INTRODUCTION: Women are more vulnerable to Alzheimer's disease (AD) than men. The entorhinal cortex (EC) is one of the earliest structures affected in AD. We identified in cognitively intact elderly different molecular changes in the EC in relation to age. METHODS: Changes in 12 characteristic molecules in relation to age were determined by quantitative immunohistochemistry or in situ hybridization in the EC. They were arbitrarily grouped into sex steroid-related molecules, markers of neuronal activity, neurotransmitter-related molecules, and cholinergic activity-related molecules. RESULTS: The changes in molecules indicated increasing local estrogenic and neuronal activity accompanied by a higher and faster hyperphosphorylated tau accumulation in women's EC in relation to age, versus a mainly stable local estrogenic/androgenic and neuronal activity in men's EC. DISCUSSION: EC employs a different neurobiological strategy in women and men to maintain cognitive function, which seems to be accompanied by an earlier start of AD in women. HIGHLIGHTS: Local estrogen system is activated with age only in women's entorhinal cortex (EC). EC neuronal activity increased with age only in elderly women with intact cognition. Men and women have different molecular strategies to retain cognition with aging. P-tau accumulation in the EC was higher and faster in cognitively intact elderly women.

Different oxytocin and corticotropin-releasing hormone system changes in bipolar disorder and major depressive disorder patients.

BACKGROUND: Oxytocin (OXT) and corticotropin-releasing hormone (CRH) are both produced in hypothalamic paraventricular nucleus (PVN). Central CRH may cause depression-like symptoms, while peripheral higher OXT plasma levels were proposed to be a trait marker for bipolar disorder (BD). We aimed to investigate differential OXT and CRH expression in the PVN and their receptors in prefrontal cortex of major depressive disorder (MDD) and BD patients. In addition, we investigated mood-related changes by stimulating PVN-OXT in mice. METHODS: Quantitative immunocytochemistry and in situ hybridization were performed in the PVN for OXT and CRH on 6 BD and 6 BD-controls, 9 MDD and 9 MDD-controls. mRNA expressions of their receptors (OXTR, CRHR1 and CRHR2) were determined in anterior cingulate cortex and dorsolateral prefrontal cortex (DLPFC) of 30 BD and 34 BD-controls, and 24 MDD and 12 MDD-controls. PVN of 41 OXT-cre mice was short- or long-term activated by chemogenetics, and mood-related behavior was compared with 26 controls. FINDINGS: Significantly increased OXT-immunoreactivity (ir), OXT-mRNA in PVN and increased OXTR-mRNA in DLPFC, together with increased ratios of OXT-ir/CRH-ir and OXTR-mRNA/CRHR-mRNA were observed in BD, at least in male BD patients, but not in MDD patients. PVN-OXT stimulation induced depression-like behaviors in male mice, and mixed depression/mania-like behaviors in female mice in a time-dependent way. INTERPRETATION: Increased PVN-OXT and DLPFC-OXTR expression are characteristic for BD, at least for male BD patients. Stimulation of PVN-OXT neurons induced mood changes in mice, in a pattern different from BD. FUNDING: National Natural Science Foundation of China (81971268, 82101592).

Distinct proteomic profiles in prefrontal subareas of elderly major depressive disorder and bipolar disorder patients.

We investigated for the first time the proteomic profiles both in the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of major depressive disorder (MDD) and bipolar disorder (BD) patients. Cryostat sections of DLPFC and ACC of MDD and BD patients with their respective well-matched controls were used for study. Proteins were quantified by tandem mass tag and high-performance liquid chromatography-mass spectrometry system. Gene Ontology terms and functional cluster alteration were analyzed through bioinformatic analysis. Over 3000 proteins were accurately quantified, with more than 100 protein expressions identified as significantly changed in these two brain areas of MDD and BD patients as compared to their respective controls. These include OGDH, SDHA and COX5B in the DLPFC in MDD patients; PFN1, HSP90AA1 and PDCD6IP in the ACC of MDD patients; DBN1, DBNL and MYH9 in the DLPFC in BD patients. Impressively, depending on brain area and distinct diseases, the most notable change we found in the DLPFC of MDD was 'suppressed energy metabolism'; in the ACC of MDD it was 'suppressed tissue remodeling and suppressed immune response'; and in the DLPFC of BD it was differentiated 'suppressed tissue remodeling and suppressed neuronal projection'. In summary, there are distinct proteomic changes in different brain areas of the same mood disorder, and in the same brain area between MDD and BD patients, which strengthens the distinct pathogeneses and thus treatment targets.

Sexual differentiation of the human hypothalamus: Relationship to gender identity and sexual orientation.

Gender identity (an individual's perception of being male or female) and sexual orientation (heterosexuality, homosexuality, or bisexuality) are programmed into our brain during early development. During the intrauterine period in the second half of pregnancy, a testosterone surge masculinizes the fetal male brain. If such a testosterone surge does not occur, this will result in a feminine brain. As sexual differentiation of the brain takes place at a much later stage in development than sexual differentiation of the genitals, these two processes can be influenced independently of each other and can result in gender dysphoria. Nature produces a great variability for all aspects of sexual differentiation of the brain. Mechanisms involved in sexual differentiation of the brain include hormones, genetics, epigenetics, endocrine disruptors, immune response, and self-organization. Furthermore, structural and functional differences in the hypothalamus relating to gender dysphoria and sexual orientation are described in this review. All the genetic, postmortem, and in vivo scanning observations support the neurobiological theory about the origin of gender dysphoria, i.e., it is the sizes of brain structures, the neuron numbers, the molecular composition, functions, and connectivity of brain structures that determine our gender identity or sexual orientation. There is no evidence that one's postnatal social environment plays a crucial role in the development of gender identity or sexual orientation.

Matching of the postmortem hypothalamus from patients and controls.

The quality of postmortem hypothalamus research depends strongly on a thorough clinical investigation and documentation of the patient's disorder and therapies. In addition, a systematic and professional neuropathological investigation of the entire brain of both the cases and the controls is absolutely crucial. In the experience of the Netherlands Brain Bank (NBB), about 20% of the clinical neurological diagnoses, despite being made in first rate clinics, have to be revised or require extra diagnoses after a complete and thorough neuropathologic review by the NBB. The neuropathology examination may reveal for instance that the elderly "controls" already have preclinical neurodegenerative alterations. In postmortem studies, the patient and control groups must be matched for as many as possible of the known confounding factors. This is necessary to make the groups as similar as possible, except for the topic being investigated. Confounding factors are present (i) before, (ii) during, and (iii) after death. They are, respectively: (i) genetic background, systemic diseases, duration and gravity of illness, medicines and addictive compounds used, age, sex, gender identity, sexual orientation, clock- and seasonal time of death, and lateralization; (ii) agonal state, stress of dying; and (iii) postmortem delay, freezing procedures, fixation, and storage time. Agonal state is generally estimated by measuring the pH of the brain. However, there are disorders in which pH is lower as a part of the disease process. Because of the large number of potentially confounding factors that differ according to, for instance, brain area and disease, a brain bank should have a large number of controls at its disposal for appropriate matching. If matching fails for some confounders, the influence of the confounders may be determined by statistical methods, such as analysis of variance or the regression models.

Sex differences in the neuropathological hallmarks of Alzheimer's disease: focus on cognitively intact elderly individuals.

AIMS: Women are more vulnerable to Alzheimer's disease (AD) than men. We investigated (i) whether and at what age the AD hallmarks, that is, ß-amyloid (Aß) and hyperphosphorylated Tau (p-Tau) show sex differences; and (ii) whether such sex differences may occur in cognitively intact elderly individuals. METHODS: We first analysed the entire post-mortem brain collection of all non-demented 'controls' and AD donors from our Brain Bank (245 men and 403 women), for the presence of sex differences in AD hallmarks. Second, we quantitatively studied possible sex differences in Aß, Aß42 and p-Tau in the entorhinal cortex of well-matched female (n = 31) and male (n = 21) clinically cognitively intact elderly individuals. RESULTS: Women had significantly higher Braak stages for tangles and amyloid scores than men, after 80 years. In the cognitively intact elderly, women showed higher levels of p-Tau, but not Aß or Aß42, in the entorhinal cortex than men, and a significant interaction of sex with age was found only for p-Tau but not Aß or Aß42. CONCLUSIONS: Enhanced p-Tau in the entorhinal cortex may play a major role in the vulnerability to AD in women.

Changes of Hypocretin (Orexin) System in Schizophrenia: From Plasma to Brain.

Hypocretin (also called orexin) regulates various functions, such as sleep-wake rhythms, attention, cognition, and energy balance, which show significant changes in schizophrenia (SCZ). We aimed to identify alterations in the hypocretin system in SCZ patients. We measured plasma hypocretin-1 levels in SCZ patients and healthy controls and found significantly decreased plasma hypocretin-1 levels in SCZ patients, which was mainly due to a significant decrease in female SCZ patients compared with female controls. In addition, we measured postmortem hypothalamic hypocretin-1-immunoreactivity (ir), ventricular cerebrospinal fluid (CSF) hypocretin-1 levels, and hypocretin receptor (Hcrt-R) mRNA expression in the superior frontal gyrus (SFG) in SCZ patients and controls We observed a significant decrease in the amount of hypothalamic hypocretin-1 ir in SCZ patients, which was due to decreased amounts in female but not male patients. Moreover, Hcrt-R2 mRNA in the SFG was decreased in female SCZ patients compared with female controls, while male SCZ patients showed a trend of increased Hcrt-R1 mRNA and Hcrt-R2 mRNA expression compared with male controls. We conclude that central hypocretin neurotransmission is decreased in SCZ patients, especially female patients, and this is reflected in the plasma.

Sex differences in stress-related disorders: Major depressive disorder, bipolar disorder, and posttraumatic stress disorder.

Stress-related disorders, such as mood disorders and posttraumatic stress disorder (PTSD), are more common in women than in men. This sex difference is at least partly due to the organizing effect of sex steroids during intrauterine development, while activating or inhibiting effects of circulating sex hormones in the postnatal period and adulthood also play a role. Such effects result in structural and functional changes in neuronal networks, neurotransmitters, and neuropeptides, which make the arousal- and stress-related brain systems more vulnerable to environmental stressful events in women. Certain brainstem nuclei, the amygdala, habenula, prefrontal cortex, and hypothalamus are important hubs in the stress-related neuronal network. Various hypothalamic nuclei play a central role in this sexually dimorphic network. This concerns not only the hypothalamus-pituitary-adrenal axis (HPA-axis), which integrates the neuro-endocrine-immune responses to stress, but also other hypothalamic nuclei and systems that play a key role in the symptoms of mood disorders, such as disordered day-night rhythm, lack of reward feelings, disturbed eating and sex, and disturbed cognitive functions. The present chapter focuses on the structural and functional sex differences that are present in the stress-related brain systems in mood disorders and PTSD, placing the HPA-axis in the center. The individual differences in the vulnerability of the discussed systems, caused by genetic and epigenetic developmental factors warrant further research to develop tailor-made therapeutic strategies.

Identifying Plasma Biomarkers with high specificity for major depressive disorder: A multi-level proteomics study.

BACKGROUND: There are currently no objective diagnostic biomarkers for major depressive disorder (MDD) due to the biological complexity of the disorder. The existence of blood-based biomarkers with high specificity would be convenient for the clinical diagnosis of MDD. METHODS: A comprehensive plasma proteomic analysis was conducted in a highly homogeneous cohort [7 drug-naïve MDD patients and 7 healthy controls (HCs)], with bioinformatics analysis combined with machine learning used to screen candidate proteins. Verification of reproducibility and specificity was conducted in independent cohorts [60 HCs and 74 MDD, 42 schizophrenia (SZ) and 39 bipolar I disorder (BD-I) drug-naïve patients]. Furthermore, verification of consistency was accomplished by proteomic analysis of postmortem brain tissue from 16 MDD patients and 16 HCs. RESULTS: Levels of C-reactive protein (CRP), antithrombin III (ATIII), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and vitamin D-binding protein (VDB) were significantly higher in MDD patients, both in the discovery cohort and independent replication cohort. In comparison with SZ or BD-I patients, two proteins (VDB and ITIH4) were significantly elevated only in MDD patients. In addition, increased VDB and ITIH4 were observed consistently in both plasma and postmortem dorsolateral prefrontal cortex tissues of MDD patients. Furthermore, a panel consisting of all four plasma proteins was able to distinguish MDD patients from HCs or SZ or BD-I patients with the highest accuracy. CONCLUSION: Plasma ITIH4 and VDB may be potential plasma biomarkers of MDD with high specificity. The four-protein panel is more suitable as a potential clinical diagnostic marker for MDD.

Rapid membrane effect of estrogens on stimulation of corticotropin-releasing hormone.

BACKGROUND: Classic nuclear-initiated estrogen signaling stimulates corticotropin-releasing hormone (CRH) gene expression as a transcription factor. However, the possible mechanism by which membrane-initiated estrogen signaling (MIES) influences CRH expression remains unclear. There are indications that MIES may upregulate nitric oxide (NO) production through the phosphatidylinositol 3-hydroxy kinase (PI3K) and potentially through the mitogen-activated protein kinase (MAPK) pathway. OBJECTIVES: We investigated the effect of MIES-mediated kinase pathways on CRH expression with or without NO synthesis. METHOD: In SK-N-SH cell culture, estradiol-bovine serum albumin (E2-BSA) was used as the specific membrane estrogen receptor activator, with a specific NO donor, and/or inhibitors for NO synthase (NOS), PI3K, MAPK, protein kinase A (PKA), and protein kinase C (PKC). RESULTS: E2-BSA significantly increased NO and CRH levels in the medium and NOS1-mRNA levels in the cells. In addition, NO donor up-regulated CRH expression, while NOS-inhibitor down-regulated it. When the inhibitor of MAPK and/or the inhibitor of PI3K was added to the medium, only the latter appeared to significantly block the stimulating effect of E2-BSA on NO synthesis, and this was accompanied by an increased CRH expression in the medium. We further studied the effect of the MIES-PKC-mediated pathway on CRH expression, with or without NOS-inhibitor, while the MIES-PKA(-PI3K) pathway served as a control. We found that MIES-PKC upregulated CRH expression independent of NO synthesis. CONCLUSION: MIES can efficiently upregulate CRH expression via various intracellular kinase pathways and may thus be a crucial component in the stress response.

The human hypothalamus in mood disorders: The HPA axis in the center.

There are no specific structural neuropathological hallmarks found in the brain of mood disorders. Instead, there are molecular, functional and structural alterations reported in many brain areas. The neurodevelopmental underpinning indicated the presence of various genetic and developmental risk factors. The effect of genetic polymorphisms and developmental sequalae, some of which may start in the womb, result in functional changes in a network mediated by neurotransmitters and neuropeptides, which make the emotion- and stress-related brain systems more vulnerable to stressful events. This network of stress-related neurocircuits consists of, for instance, brainstem nuclei, the amygdala, habenula, prefrontal cortex and hypothalamus. Various nuclei of the hypothalamus form indeed one of the crucial hubs in this network. This structure concerns not only the hypothalamo-pituitary-adrenal (HPA) axis that integrate the neuro-endocrine-immune responses to stress, but also other hypothalamic nuclei and systems that play a key role in the symptoms of depression, such as disordered day-night rhythm, lack of reward feelings, disturbed eating, sex, and disturbed cognitive functions. The present review will focus on the changes in the human hypothalamus in depression, with the HPA axis in the center. We will discuss the inordinate network of neurotransmitters and neuropeptides involved, with the hope to find the most vulnerable neurobiological systems and the possible development of tailor-made treatments for mood disorders in the future.

Activation of the Brain to Postpone Dementia: A Concept Originating from Postmortem Human Brain Studies.

Alzheimer's disease (AD) is characterized by decreased neuronal activity and atrophy, while hyperactivity of neurons seems to make them resistant to aging and neurodegeneration, a phenomenon which we have paraphrased as 'use it or lose it'. Our hypothesis proposes that (1) during their functioning, neurons are damaged; (2) accumulation of damage that is not repaired is the basis of aging; (3) the vulnerability to AD is determined by the genetic background and the balance between the amount of damage and the efficiency of repair, and (4) by stimulating the brain, repair mechanisms are stimulated and cognitive reserve is increased, resulting in a decreased rate of aging and risk for AD. Environmental stimulating factors such as bilingualism/multilingualism, education, occupation, musical experience, physical exercise, and leisure activities have been reported to reduce the risk of dementia and decrease the rate of cognitive decline, although methodological problems are present.

Quantification of Tyrosine Hydroxylase and ErbB4 in the Locus Coeruleus of Mood Disorder Patients Using a Multispectral Method to Prevent Interference with Immunocytochemical Signals by Neuromelanin.

The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients-thus validating the method-but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.

Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds.

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.