Exosomes derived from human induced pluripotent stem cell-derived neural progenitor cells protect neuronal function under ischemic conditions.

Compared with other stem cells, human induced pluripotent stem cells-derived neural progenitor cells (iPSC-NPCs) are more similar to cortical neurons in morphology and immunohistochemistry. Thus, they have greater potential for promoting the survival and growth of neurons and alleviating the proliferation of astrocytes. Transplantation of stem cell exosomes and stem cells themselves have both been shown to effectively repair nerve injury. However, there is no study on the protective effects of exosomes derived from iPSC-NPCs on oxygen and glucose deprived neurons. In this study, we established an oxygen-glucose deprivation model in embryonic cortical neurons of the rat by culturing the neurons in an atmosphere of 95% N2 and 5% CO2 for 1 hour and then treated them with iPSC-NPC-derived exosomes for 30 minutes. Our results showed that iPSC-NPC-derived exosomes increased the survival of oxygen- and glucose-deprived neurons and the level of brain-derived neurotrophic factor in the culture medium. Additionally, it attenuated oxygen and glucose deprivation-induced changes in the expression of the PTEN/AKT signaling pathway as well as synaptic plasticity-related proteins in the neurons. Further, it increased the length of the longest neurite in the oxygen- and glucose-deprived neurons. These findings validate the hypothesis that exosomes from iPSC-NPCs exhibit a neuroprotective effect on oxygen- and glucose-deprived neurons by regulating the PTEN/AKT signaling pathway and neurite outgrowth. This study was approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China (approval No. SRRSH20191010) on October 10, 2019.

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.

Early growth response-1 regulates acetylcholinesterase and its relation with the course of Alzheimer's disease.

Our previous studies showed that the transcription factor early growth response-1 (EGR1) may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer's disease (AD). In order to elucidate the mechanisms involved, we first performed data mining on our previous microarray study on postmortem human prefrontal cortex (PFC) for the changes in the expression of EGR1 and acetylcholinesterase (AChE) and the relationship between them during the course of AD. The study contained 49 patients, ranging from non-demented controls (Braak stage 0) to late AD patients (Braak stage VI). We found EGR1-mRNA was high in early AD and decreased in late AD stages, while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages. A significant positive correlation was found between the mRNA levels of these two molecules. In addition, we studied the relationship between EGR1 and AChE mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD (3xTg-AD) mice. EGR1- and AChE-mRNA were lower in 3xTg-AD mice compared with wild-type (WT) mice. A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice. Subsequently, AChE expression was determined following up- or down-regulating EGR1 in cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein levels. Dual-luciferase assay and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter. We conclude that the EGR1 can upregulate AChE expression by a direct effect on its gene promoter, which may contribute significantly to the changes in cholinergic function in the course of AD. The 3xTg-AD mouse model only reflects later stage AD.

Autophagy-related protein expression was associated with BRAF V600E mutation in epilepsy associated glioneuronal tumors.

PURPOSE: The aim of this study was to explore the expression level of autophagy-related proteins in epileptic patients with glioneuronal tumors (GNTs) and evaluate the association with clinicopathological features. MATERIALS AND METHODS: We obtained the brain specimens from 33 patients with GNTs, including 22 gangliogliomas (GGs) and 11 dysembryoplastic neuroepithelial tumors (DNTs). The expression of two autophagy-related proteins (LC3B and Beclin-1) was evaluated by immunohistochemistry, and BRAF V600E mutation was examined by DNA sequencing. RESULTS: Among 33 epileptic patients with GNTs, the frequency of high expression of LC3B was 36.4% (12/33), and that of Beclin-1 was 39.4% (13/33). High expression of LC3B and Beclin-1 proteins was significantly associated with BRAF V600E mutation in GNTs (P=0.008; P=0.018), and LC3B overexpression was also correlated with temporal location of GNTs (P=0.002). In GGs alone, high expression of LC3B revealed significant correlation with BRAF V600E mutation and temporal location (P=0.020; P=0.015), while Beclin-1 showed no correlation with them (P>0.05). Furthermore, autophagy-related proteins did not show any association with other studied clinicopathological features, such as gender, age at seizure onset, epilepsy duration and postoperative seizure outcome. CONCLUSIONS: Our observations demonstrated that impaired autophagy may be associated with BRAF V600E mutation. However, large sample studies with long-term follow-up were required.

Sexually Dimorphic Changes of Hypocretin (Orexin) in Depression.

BACKGROUND: Neurophysiological and behavioral processes regulated by hypocretin (orexin) are severely affected in depression. However, alterations in hypocretin have so far not been studied in the human brain. We explored the hypocretin system changes in the hypothalamus and cortex in depression from male and female subjects. METHODS: We quantified the differences between depression patients and well-matched controls, in terms of hypothalamic hypocretin-1 immunoreactivity (ir) and hypocretin receptors (Hcrtr-receptors)-mRNA in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex. In addition, we determined the alterations in the hypocretin system in a frequently used model for depression, the chronic unpredictable mild stress (CUMS) rat. RESULTS: i) Compared to control subjects, the amount of hypocretin-immunoreactivity (ir) was significantly increased in female but not in male depression patients; ii) hypothalamic hypocretin-ir showed a clear diurnal fluctuation, which was absent in depression; iii) male depressive patients who had committed suicide showed significantly increased ACC Hcrt-receptor-2-mRNA expression compared to male controls; and iv) female but not male CUMS rats showed a highly significant positive correlation between the mRNA levels of corticotropin-releasing hormone and prepro-hypocretin in the hypothalamus, and a significantly increased Hcrt-receptor-1-mRNA expression in the frontal cortex compared to female control rats. CONCLUSIONS: The clear sex-related change found in the hypothalamic hypocretin-1-ir in depression should be taken into account in the development of hypocretin-targeted therapeutic strategies.

Direct Involvement of Androgen Receptor in Oxytocin Gene Expression: Possible Relevance for Mood Disorders.

Oxytocin (OXT), synthesized in the hypothalamic paraventricular nucleus (PVN) and then released into different brain areas, may play a crucial role in various behaviors and neuropsychiatric disorders, including depression. Testosterone has been proposed by clinical studies to have the opposite effect of oxytocin in these disorders. We began by studying, in the postmortem hypothalamus of fifteen patients with mood disorders and fifteen matched controls, the expression of OXT in the PVN by means of immunocytochemistry (ICC) and the co-localization of OXT and androgen receptor (AR) by means of double labeling ICC. Subsequently, the regulatory effect of AR on OXT gene expression was studied in vitro. We found a higher expression of PVN OXT in the mood disorder patients than in the control subjects, and observed a clear co-localization of AR in OXT-expressing neurons, both in the cytoplasm and in the nucleus. In addition, a significant decrease in OXT-mRNA levels was observed after pre-incubation of the SK-N-SH cells with testosterone. A further potential androgen-responsive element in the human OXT gene promotor was revealed by electrophoretic mobility shift assays and co-transfections in neuroblastoma cells. Finally, in vitro studies demonstrated that AR mediated the down-regulation of OXT gene expression. These results suggest that the fact that OXT and testosterone appear to have opposite effects in neuropsychiatric disorders might be based upon a direct inhibition of AR on OXT transcription, which may provide a novel target for therapeutic strategies in depression.

Aromatase changes in depression: A postmortem and animal experimental study.

A hyperactive hypothalamo-pituitary-adrenal (HPA) axis is a prominent feature in depression. It has been shown that androgens inhibit HPA activity and that estrogens stimulate it. We have therefore investigated, in human postmortem hypothalamus, whether depression features an increase in aromatase, which is the rate-limiting enzyme for the conversion of androgens to estrogens. In addition, we have tested the effect of an aromatase inhibitor on depression-like symptoms in a frequently used animal model for depression. At first, aromatase immunoreactivity (ir) was quantified in the central part of the hypothalamic paraventricular nucleus (PVN) of 10 major depressive disorder (MDD) patients and 10 well-matched control subjects. Subsequently an animal experimental study was performed using the chronic unpredictable mild stress (CUMS) rats as depression model. The effect of administration of 1,4,6-androstatriene-3,17-dione (ATD), an aromatase inhibitor, was investigated by silastic capsule implantation. In the postmortem study, the amount of PVN aromatase-ir decreased significantly in the MDD group compared to the controls (P=0.029). In the animal study, ATD was found to cause significantly increased testosterone (T) levels, both in plasma and in the hypothalamus. However, ATD administration did not show significant effects on the depression-like behaviors or plasma corticosterone levels in CUMS rats. Based on our observations in human postmortem material and the animal experiment, we have to conclude that alterations in aromatase in adulthood do not seem to play a major role in the pathogenesis of the symptoms of depression.

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.

Effect of pentobarbital and isoflurane on acute stress response in rat.

BACKGROUND: Anesthesia administration before sacrificing animals is a common practice in stress-related studies, but the effect of anesthesia on the results remains understudied. We aimed to reveal the interference of different anesthetics, i.e. intraperitoneal (i.p.) sodium-pentobarbital injection or isoflurane inhalation, with the acute stress responses in rats. METHODS: Rats were randomly divided into foot shock (FS) and non-stressed control groups, and further grouped according to the sacrificing procedure: direct decapitation, decapitation after i.p. sodium-pentobarbital injection, or isoflurane inhalation. There was also a non-stressed group sacrificed by decapitation following i.p. saline injection. Plasma levels of corticosterone (CORT), testosterone and estradiol, hypothalamic stress-related molecule mRNA expression of corticotropin-releasing hormone, arginine vasopressin and oxytocin, and frontal lobe stress-related molecule mRNA expression of NMDA receptor subunit NR2B, GABAA receptor and the neuronal-type nicotinic acetylcholine receptor were measured. RESULTS: FS significantly increased plasma CORT levels in direct decapitation and isoflurane groups, while this stress response 'disappeared' following i.p. sodium-pentobarbital injection. In control animals, both the injection of saline and pentobarbital caused a significant increase of plasma CORT. Neither the sex hormone levels nor the mRNA expression of stress-related molecules in the brain showed significant differences among the groups. CONCLUSION: The injection of the anesthetic compound rather than the compound itself may cause extra stress which interferes with the plasma CORT levels, but not with plasma sex hormone levels nor with the brain mRNA expression. Isoflurane inhalation leaves the stress response intact and is also optimal from an ethical point of view.

Sex differences in the stress response in SD rats.

Sex differences play an important role in depression, the basis of which is an excessive stress response. We aimed at revealing the neurobiological sex differences in the same study in acute- and chronically-stressed rats. Female Sprague-Dawley (SD) rats were randomly divided into 6 groups: chronic unpredictable mild stress (CUMS), acute foot shock (FS) and controls, animals in all 3 groups were sacrificed in proestrus or diestrus. Male SD rats were randomly divided into 3 groups: CUMS, FS and controls. Comparisons were made of behavioral changes in CUMS and control rats, plasma levels of corticosterone (CORT), testosterone (T) and estradiol (E2), and of the hypothalamic mRNA-expression of stress-related molecules, i.e. estrogen receptor α and ß, androgen receptor, aromatase, mineralocorticoid receptor, glucocorticoid receptor, corticotropin-releasing hormone, arginine vasopressin and oxytocin. CUMS resulted in disordered estrus cycles, more behavioral and hypothalamic stress-related molecules changes and a stronger CORT response in female rats compared with male rats. Female rats also showed decreased E2 and T levels after FS and CUMS, while male FS rats showed increased E2 and male CUMS rats showed decreased T levels. Stress affects the behavioral, endocrine and the molecular response of the stress systems in the hypothalamus of SD rats in a clear sexual dimorphic way, which has parallels in human data on stress and depression.