Progesterone receptor distribution in the human hypothalamus and its association with suicide.

The human hypothalamus modulates mental health by balancing interactions between hormonal fluctuations and stress responses. Stress-induced progesterone release activates progesterone receptors (PR) in the human brain and triggers alterations in neuropeptides/neurotransmitters. As recent epidemiological studies have associated peripheral progesterone levels with suicide risks in humans, we mapped PR distribution in the human hypothalamus in relation to age and sex and characterized its (co-) expression in specific cell types. The infundibular nucleus (INF) appeared to be the primary hypothalamic structure via which progesterone modulates stress-related neural circuitry. An elevation of the number of pro-opiomelanocortin+ (POMC, an endogenous opioid precursor) neurons in the INF, which was due to a high proportion of POMC+ neurons that co-expressed PR, was related to suicide in patients with mood disorders (MD). MD donors who died of legal euthanasia were for the first time enrolled in a postmortem study to investigate the molecular signatures related to fatal suicidal ideations. They had a higher proportion of PR co-expressing POMC+ neurons than MD patients who died naturally. This indicates that the onset of endogenous opioid activation in MD with suicide tendency may be progesterone-associated. Our findings may have implications for users of progesterone-enriched contraceptives who also have MD and suicidal tendencies.

Increased pituitary adenylate cyclase-activating peptide genes expression in the prefrontal cortex in schizophrenia in relation to suicide.

Introduction: Pituitary adenylate cyclase-activating peptide (PACAP) is a stress-related neuropeptide that is produced in several brain areas. It acts by 3 receptors: PACAP type-1 (PAC1), vasoactive intestinal peptide (VIP) -1 and -2 (VPAC1 and 2). Data on polymorphisms in PACAP and PAC1 indicate a relationship of the PACAP system with schizophrenia (SCZ). Methods: The prefrontal cortex was chosen to measure PACAP-gene related expression changes, since this is a central structure in the symptoms of schizophrenia (SCZ). We investigated alterations in the expression of the PACAP-related genes by qPCR in the human dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of 35 SCZ patients and 34 matched controls in relation to SCZ, suicide, gender and medication. Results: The ACC revealed an upregulation in PACAP, PAC1, VPAC1 and VPAC2 in SCZ suicide (S) completers compared to controls. An increase in PACAP, VPAC1 and VPAC2 expression was also present in the ACC in SCZ-S compared to SCZ patients who died naturally (SCZ-N). In the DLPFC, an increase in PAC1 was found in SCZ-N patients compared to SCZ-S and controls. Moreover, an increase in all PACAP-related genes was present in SCZ-N male patients compared to SCZ-N females. Concluding, expression changes were found in PACAP-related genes in relation to SCZ, suicide and gender. In particular, there was a higher PACAP-related gene expression in SCZ patients in the ACC in relation to suicide and in DLPFC in relation to SCZ. Discussion: These findings suggest a potential link between PACAP and the pathophysiology of SCZ and suicide. Further research is needed to understand the functional significance and potential clinical applications of these changes.

Alterations in pituitary adenylate cyclase-activating polypeptide in major depressive disorder, bipolar disorder, and comorbid depression in Alzheimer's disease in the human hypothalamus and prefrontal cortex.

BACKGROUND: Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is involved in the stress response and may play a key role in mood disorders, but no information is available on PACAP for the human brain in relation to mood disorders. METHODS: PACAP-peptide levels were determined in a major stress-response site, the hypothalamic paraventricular nucleus (PVN), of people with major depressive disorder (MDD), bipolar disorder (BD) and of a unique cohort of Alzheimer's disease (AD) patients with and without depression, all with matched controls. The expression of PACAP-(Adcyap1mRNA) and PACAP-receptors was determined in the MDD and BD patients by qPCR in presumed target sites of PACAP in stress-related disorders, the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). RESULTS: PACAP cell bodies and/or fibres were localised throughout the hypothalamus with differences between immunocytochemistry and in situ hybridisation. In the controls, PACAP-immunoreactivity-(ir) in the PVN was higher in women than in men. PVN-PACAP-ir was higher in male BD compared to the matched male controls. In all AD patients, the PVN-PACAP-ir was lower compared to the controls, but higher in AD depressed patients compared to those without depression. There was a significant positive correlation between the Cornell depression score and PVN-PACAP-ir in all AD patients combined. In the ACC and DLPFC, alterations in mRNA expression of PACAP and its receptors were associated with mood disorders in a differential way depending on the type of mood disorder, suicide, and psychotic features. CONCLUSION: The results support the possibility that PACAP plays a role in mood disorder pathophysiology.

Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease.

Neuroactive steroids are known neuroprotective agents and neurotransmitter regulators. We previously found that expression of the enzymes synthesizing 5α-dihydroprogesterone (5α-DHP), allopregnanolone (ALLO), and dehydroepiandrosterone sulfate (DHEAS) were reduced in the substantia nigra (SN) of Parkinson's Disease (PD) brain. Here, concentrations of a comprehensive panel of steroids were measured in human post-mortem brains of PD patients and controls. Gas chromatography-mass spectrometry (GC/MS) was used to measure steroid levels in SN (involved in early symptoms) and prefrontal cortex (PFC) (involved later in the disease) of five control (CTR) and nine PD donors, divided into two groups: PD4 (PD-Braak stages 1-4) and PD6 (PD-Braak stages 5-6). In SN, ALLO was increased in PD4 compared to CTR and 5α-DHP and ALLO levels were diminished in PD6 compared to PD4. The ALLO metabolite 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was higher in PD4 compared to CTR. In PFC, 3α5α20α-HHP was higher in PD4 compared to both CTR and PD6. The effects of 5α-DHP, ALLO and DHEAS were tested on human post-mortem brain slices of patients and controls in culture. RNA expression of genes involved in neuroprotection, neuroinflammation and neurotransmission was analysed after 5 days of incubation with each steroid. In PD6 slices, both 5α-DHP and ALLO induced an increase of the glutamate reuptake effector GLAST1, while 5α-DHP also increased gene expression of the neuroprotective TGFB. In CTR slices, ALLO caused reduced expression of IGF1 and GLS, while DHEAS reduced the expression of p75 and the anti-apoptotic molecule APAF1. Together these data suggest that a potentially protective upregulation of ALLO occurs at early stages of PD, followed by a downregulation of progesterone metabolites at later stages that may exacerbate the pathological changes, especially in SN. Neuroprotective effects of neurosteroids are thus dependent on the neuropathological stage of the disease.

Changes in glial gene expression in the prefrontal cortex in relation to major depressive disorder, suicide and psychotic features.

BACKGROUND: To establish whether major depressive disorder (MDD), suicidal behaviors and psychotic features contribute to glial alterations in the human prefrontal cortex. MATERIALS AND METHODS: We compared mRNA expression using real-time qPCR of 17 glia related genes in the dorsolateral prefrontal cortex (DLPFC) and the anterior cingulate cortex (ACC) between 24 patients with MDD and 12 well-matched controls without psychiatric or neurological diseases. The MDD group was subdivided into i) MDD who died of suicide (MDD-S) or natural causes (MDD-NS) and ii) MDD with or without psychotic features (MDD-P and MDD-NP). The results were followed up with confounder factor analysis. RESULTS: Astrocyte gene aldehyde dehydrogenase-1 L1 (ALDH1L1) showed an increased expression in the DLPFC of MDD-NS and the ACC of MDD-NP. S100 calcium-binding protein B (S100B) was upregulated in the DLPFC of MDD compared to the controls. Microglial markers CD11B and purinergic receptor 12 (P2RY12) both showed decreased expression in the ACC of MDD-NS. CD68 was increased in the DLPFC of MDD in both, MDD-S and MDD-P, compared to the controls. In addition, there was increased translocator protein (TSPO) expression in the DLPFC of MDD, especially MDD-NS. In the ACC, this gene had a lower expression in MDD-P than in MDD-NP. Myelin basic protein (MBP) mRNA in the DLPFC increased in MDD, in relation to psychotic features, but not to suicide. LIMITATIONS: Sample volumes are relatively small. CONCLUSIONS: Different glial functions in MDD were related to specific brain area, suicide or psychotic features.

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.

Sex difference in glia gene expression in the dorsolateral prefrontal cortex in bipolar disorder: Relation to psychotic features.

BACKGROUND: Suicide, psychotic features and gender influence the epidemiology and clinical prognosis of bipolar disorder (BD). Differences in glial function between the genders might contribute to these clinical variables. Here we studied expression of glial genes in human post-mortem prefrontal cortex of BD and control subjects in relation to suicide, psychotic features and sex. METHODS: Real time PCR was used to detect transcriptional alterations of 16 glia-related genes in two brain areas, the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC), from 30 patients with BD subdivided by suicide and psychotic features, and from 34 well-matched control cases. RESULTS: We found no evidence of immune activation in BD. Instead, we found three microglial genes to be downregulated in the DLPFC of non-suicidal individuals with BD, i.e. CD68, triggering receptor expressed on myeloid cells 2 (TREM2) and purinergic receptor 12 (P2RY12). A remarkable sex difference was observed in the DLPFC of patients with BD: 14 glia-related genes were expressed at significantly higher levels in males, including all three glial cell types. A subset analysis showed that the sex differences were closely associated with the presence of psychotic features. CONCLUSIONS: No evidence of immune activation was found in these two brain regions in BD. The sex-specific differences in glial gene expression in BD, found particularly in patients with psychotic features, may be associated with the potential co-existence of mania and psychotic features and could potentially contribute to the gender-biased characteristics in BD.

Prefrontal cortex alterations in glia gene expression in schizophrenia with and without suicide.

BACKGROUND: Patients with schizophrenia (SCZ) run a lifelong risk of suicide. Alterations in glia activities in the prefrontal cortex (PFC) have been reported in relation to suicide in patients with SCZ. While immune processes in the CNS have been related to the susceptibility and course of SCZ, there are hardly any direct comparisons between individuals with SCZ, both those who died of natural causes and those that committed suicide, and healthy controls. MATERIALS AND METHODS: We compared mRNA expression using real time qPCR of 16 glia-related genes in the dorsal lateral prefrontal cortex (DLPFC) and the anterior cingulate cortex (ACC) between 35 patients with SCZ (7 suicide completers and 28 patients who died of natural causes) and 34 well-matched controls without psychiatric or neurological diseases. RESULTS: We found an increased expression of the astrocytic gene aldehyde dehydrogenase-1 family member L1 (ALDH1L1) mRNA, a marker involved in dopaminergic activity, in SCZ versus controls. Excluding individuals with SCZ that committed suicide resulted in an elevated expression in the DLPFC of both ALDH1L1 and glutamine synthetase (GS) genes in patients with SCZ, compared to suicide completers and non-psychiatric controls. Regarding microglia genes: in the ACC, homeostatic markers such as chemokine (C-X3-C motif) ligand 1 (CX3CR1) mRNA expression was increased in SCZ without suicide as compared to suicide completers, while no change was found when compared to controls. Another, purinergic receptor 12 (P2RY12) mRNA was exclusively elevated in the ACC of suicide completers, compared to either other group. Triggering receptor expressed on myeloid cells 2 (TREM2) expression, which maintains microglial metabolism, was reduced in non-suicide patients with SCZ, compared to suicide victims and control subjects. CONCLUSIONS: Differential changes are found in astrocyte and microglia genes in the PFC subregions in relation to SCZ and suicide, indicating possible disturbances of glia homeostasis in these conditions.

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.

Alterations in the steroid biosynthetic pathways in the human prefrontal cortex in mood disorders: A post-mortem study.

Altered levels of steroids have been reported in the brain, cerebral spinal fluid and plasma of patients with mood disorders. Neuroimaging studies have reported both functional and structural alterations in mood disorders, for instance in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC). In order to determine whether the endogenous production of steroids is altered in the ACC and DLPFC of patients with major depressive disorder (MDD) or bipolar disorder (BPD), quantitative real-time PCR was performed to detect mRNA expression level of key enzymes in the steroid biosynthetic pathways. In MDD, a significant decrease in mRNA level of cytochrome P450 17A1 (CYP17A1, synthesizing C19 ketosteroids) in the ACC and a significant increase in mRNA levels of hydroxysteroid sulfotransferase 2A1 [SULT2A1, catalyzing the sulfate conjugation of dehydroepiandrosterone (DHEA)] were observed in the DLPFC, suggesting alterations in DHEA and its sulfate metabolite DHEAS levels. Decreased intensity and distribution of CYP17A1 immunohistochemical staining was found in the ACC of MDD patients. Interestingly, there was a significant positive correlation between the mRNA levels of CYP17A1 and tyrosine-related kinase B (TrkB) full length isoform. In a unique post-mortem human brain slice culture paradigm, BDNF mRNA expression was found to be significantly increased following incubation with DHEA. Together, these data indicate a close relationship between DHEA and BDNF-TrkB pathways in depression. Furthermore, in the DLPFC, higher mRNA levels of 11ß-hydroxysteroid dehydrogenase-1 (HSD11B1, reducing cortisone to the active hormone cortisol) and steroidogenic acute regulatory protein (STAR, facilitating the shuttle of cholesterol through the intermembrane space) were found in the MDD patients and BPD patients, respectively. In conclusion, this study suggests the presence of a disturbance in the endogenous synthesis of DHEA and DHEAS in mood disorders, which has a close relationship with BDNF-TrkB signaling.

Altered Loyalties of Neuronal Markers in Cultured Slices of Resected Human Brain Tissue.

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic markers. Surprisingly, the reactive cells also began to express neuronal markers Tubulin ßIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin ßIII expression disappeared from neurons. While NeuN expression decreased significantly, it did not entirely disappear from many neurons. Moreover, it was not observed in reactive cells, showing that NeuN is a reliable marker of neurons.

MicroRNA-132 and early growth response-1 in nucleus basalis of Meynert during the course of Alzheimer's disease.

The cholinergic nucleus basalis of Meynert, which is important for memory functions, shows neuronal activation ('up-phase') during the early stages of Alzheimer's disease and neurodegeneration ('down-phase') in later stages of Alzheimer's disease. MicroRNA-132 (miR-132) and the transcription factor early growth response-1 (EGR1) were proposed as possible candidate molecules regulating such an up-down activity pattern of the nucleus basalis of Meynert during the course of Alzheimer's disease, as they both show this up-down pattern of expression in the prefrontal cortex during the course of Alzheimer's disease. Not only do these two molecules stimulate synaptic activity and plasticity, they are also involved in Alzheimer's disease pathology and might, in addition, affect cholinergic function. In the nucleus basalis of Meynert, we investigated the expression of miR-132 and EGR1 along the entire course of Alzheimer's disease. Forty-nine post-mortem nucleus basalis of Meynert samples were studied, ranging from non-demented controls (Braak stage = 0) to late Alzheimer's disease patients (Braak stage = VI), and from clinical Reisberg scale 1 to 7. Each Braak stage contained seven samples, that were all well matched for confounding factors, i.e. age (range 58-91), sex, post-mortem delay, cerebrospinal fluid pH (as a measure for agonal state), APOE genotype, clock time of death, tissue fixation time, and tissue storage time. The alterations of these two molecules were studied over the course of Alzheimer's disease in relation to the expression of 4G8-stained amyloid-ß, hyperphosphorylated tau stained by antibody AT8, neuronal fibrillary tangles and neuropil threads stained by silver, and in relation to alterations in choline acetyltransferase. We found that the expression of miR-132 and EGR1 in the nucleus basalis of Meynert was quite stable during the early stages of Alzheimer's disease and decreased significantly only during late Alzheimer's disease stages. In addition, miR-132 and EGR1 showed a significant positive correlation with choline acetyltransferase expression (r = 0.49, P < 0.001 and r = 0.61, P < 0.001), while choline acetyltransferase expression showed a significantly negative correlation with hyperphosphorylated tau (r = -0.33, P = 0.021) but no correlation with 4G8-stained amyloid-ß. From the functional changes of miR-132 and EGR1 along the course of Alzheimer's disease we conclude: (i) that these two molecules may play a role in keeping the cholinergic function intact in early Alzheimer's disease stages; and (ii) that these molecules may contribute to the late neurodegeneration of this cholinergic nucleus.

Injury Response of Resected Human Brain Tissue In Vitro.

Brain injury affects a significant number of people each year. Organotypic cultures from resected normal neocortical tissue provide unique opportunities to study the cellular and neuropathological consequences of severe injury of adult human brain tissue in vitro. The in vitro injuries caused by resection (interruption of the circulation) and aggravated by the preparation of slices (severed neuronal and glial processes and blood vessels) reflect the reaction of human brain tissue to severe injury. We investigated this process using immunocytochemical markers, reverse transcriptase quantitative polymerase chain reaction and Western blot analysis. Essential features were rapid shrinkage of neurons, loss of neuronal marker expression and proliferation of reactive cells that expressed Nestin and Vimentin. Also, microglia generally responded strongly, whereas the response of glial fibrillary acidic protein-positive astrocytes appeared to be more variable. Importantly, some reactive cells also expressed both microglia and astrocytic markers, thus confounding their origin. Comparison with post-mortem human brain tissue obtained at rapid autopsies suggested that the reactive process is not a consequence of epilepsy.

Increased metabolic activity in nucleus basalis of Meynert neurons in elderly individuals with mild cognitive impairment as indicated by the size of the Golgi apparatus.

In this study, we examined the metabolic activity of nucleus basalis of Meynert (NBM) neurons in individuals clinically diagnosed with no cognitive impairment (NCI, n = 8), mild cognitive impairment (MCI, n = 9), and subjects with moderate Alzheimer disease (AD, n = 7). We used Golgi apparatus (GA) size as a measure of neuronal metabolic activity. Subjects with MCI showed increased NBM metabolic activity; they had significantly more neurons with larger GA size as compared with NCI and AD subjects. In contrast, more NBM neurons with extremely small GA sizes, indicating reduced metabolic activity, were seen in AD. When these cases were classified according to their AD pathology (Braak I-II, III-IV, or V-VI), Braak III-IV subjects showed significantly increased GA sizes, comparable with the increase in clinically diagnosed MCI, whereas in Braak V-VI, GA sizes were dramatically reduced. Of all MCI and NCI subjects with similar Braak III-IV pathology, the MCI subjects again had significantly larger GA sizes. The larger NBM neuronal GA size seen in MCI suggests increased metabolic activity, associated with both the clinical progression from NCI to MCI, and with the early stages of AD pathology.

Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators.

Toll-like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro-inflammatory factors and enhances antigen-presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro-inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti-inflammatory cytokines including interleukin-9 (IL-9), IL-10, and IL-11 and downregulation of the p40 subunit of IL-12 and IL-23. The collective TLR3-induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro-inflammatory reaction.

Cells in human postmortem brain tissue slices remain alive for several weeks in culture.

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno-associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.