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Like other members of the superfamily of nuclear receptors, the peroxisome proliferator-activated receptor γ (PPARγ), is a ligand-activated transcription factor known for its insulin-sensitizing actions in the periphery. Despite only sparse evidence for PPARγ in the CNS, many reports suggest direct PPARγ-mediated actions in the brain. This study aimed to (i) map PPARγ expression in rodent brain areas, involved in the regulation of cognitive, motivational, and emotional functions, (ii) examine the regulation of central PPARγ by physiological variables (age, sex, obesity); (iii) chemotypically identify PPARγ-expressing cells in the frontal cortex (FC) and hippocampus (HP); (iv) study whether activation of PPARγ by pioglitazone (Pio) in FC and HP cells can induce target gene expression; and (v) demonstrate the impact of activated PPARγ on learning behavior and motivation. Immunoreactive PPARγ was detectable in specific sub-nuclei/subfields of the FC, HP, nucleus accumbens, amygdala, hypothalamus, thalamus, and granular layers of the cerebellum. PPARγ protein levels were upregulated during aging and in high fat diet-induced obesity. PPARγ mRNA expression was upregulated in the amygdala of females (but not males) that were made obese. Neural precursor cells, mature neurons, and astrocytes in primary FC and HP cultures were shown to express PPARγ. Pioglitazone dose-dependently upregulated PPARγ target genes in manner that was specific to the origin (FC or HP) of the cultures. Lastly, administration of Pio impaired motivation and associative learning. Collectively, we provide evidence for the presence of regulatable PPARγ in the brain and demonstrate their participation the regulation of key behaviors.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with a complex physiopathology whose initiators are poorly defined. Accumulating clinical and experimental evidence suggests a causal role of lifetime stress in AD. This chapter summarizes current knowledge about how chronic stress and its accompanying high levels of glucocorticoid (GC) secretion, trigger the two main pathomechanisms of AD: (i) misprocessing of amyloid precursor protein (APP) and the generation of amyloid beta (Aβ) and (ii) Tau hyperphosphorylation and aggregation. Given that depression is a well-known stress-related illness, and the evidence that depression may precede AD, this chapter also explores neurobiological mechanisms that may be common to depressive and AD pathologies. This review also discusses emerging insights into the role of Tau and its malfunction in disrupting neuronal cascades and neuroplasticity and, thus triggering brain pathology.

In the ram, changes in daylength influence testicular activity by modifying the release of luteinizing hormone-releasing hormone (LH-RH) and thus the gonadotrophins. To investigate the nature of this response the hourly fluctuations in the circulating levels of prolactin, luteinizing hormone (LH) and melatonin were measured in rams kept under various artificial lighting conditions. In Exp. 1, 8 Soay rams (4 control and 4 from which the superior cervical ganglia had been removed) were exposed to alternating 16-week periods of short days (8L: 16D) and long days (16L: 8D) for over 2 years, and blood samples were collected hourly for 25 h on two occasions. The lighting regimen resulted in marked testicular and endocrine changes in the controls but not in the ganglionectomized rams which had low or undetectable levels of melatonin (less than or equal to 33 pg/ml) and an unusual diurnal rhythm in prolactin. In Exp. 2, 8 intact Soay rams were exposed to an ahemeral lighting regimen of 8L: 28D for 16 weeks; at the end of this period blood samples were collected hourly for 52 h and assayed for prolactin. During the pretreatment period of long days (16L: 8D), the testes became fully regressed. During the 16 weeks of 8L: 28D, redevelopment occurred, but the growth of the testes was slow compared to that normally occurring under short days of 8L: 16D. The prolactin profiles showed evidence of circadian rhythm in hormone secretion, with a correlation between the timing and duration of the rhythm and the degree of testicular development. These combined results support the idea that the photoperiodic response in the ram involves an interplay between the secretory activity of the pineal gland, and a light/dark entrained circadian mechanism in the brain.

Since foods with high hedonic value are often consumed in excess of energetic needs, this study was designed to identify the mechanisms that may counter anorexigenic signalling in the presence of hedonic foods in lean animals.Mice, in different states of satiety (fed/fasted, or fed/fasted and treated with ghrelin or leptin, respectively), were allowed to choose between high-fat/high-sucrose and standard foods. Intake of each food type and the activity of hypothalamic neuropetidergic neurons that regulate appetite were monitored. In some cases, food choice was monitored in leptin-injected fasted mice that received microinjections of galanin receptor agonists into the lateral hypothalamus.Appetite-stimulating orexin neurons in the lateral hypothalamus are rapidly activated when lean, satiated mice consume a highly palatable food (PF); such activation (upregulated c-Fos expression) occurred even after administration of the anorexigenic hormone leptin and despite intact leptin signalling in the hypothalamus. The ability of leptin to restrain PF eating is restored when a galanin receptor 2 (Gal2R) agonist is injected into the lateral hypothalamus.Hedonically-loaded foods interrupt the inhibitory actions of leptin on orexin neurons and interfere with the homeostatic control of feeding. Overeating of palatable foods can be curtailed in lean animals by activating Gal2R in the lateral hypothalamus. This article is protected by copyright. All rights reserved.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with a complex physiopathology whose initiators are poorly defined. Accumulating clinical and experimental evidence suggests a causal role of lifetime stress in AD. This chapter summarizes current knowledge about how chronic stress and its accompanying high levels of glucocorticoid (GC) secretion, trigger the two main pathomechanisms of AD: (i) misprocessing of amyloid precursor protein (APP) and the generation of amyloid beta (Aβ) and (ii) Tau hyperphosphorylation and aggregation. Given that depression is a well-known stress-related illness, and the evidence that depression may precede AD, this chapter also explores neurobiological mechanisms that may be common to depressive and AD pathologies. This review also discusses emerging insights into the role of Tau and its malfunction in disrupting neuronal cascades and neuroplasticity and, thus triggering brain pathology.

Synaptic dysfunction during early stages of Alzheimer's disease (AD) is triggered by soluble amyloid-β (Aβ) oligomers that interact with NMDA receptors (NMDARs). We previously showed that Aβ induces synaptic protein loss through NMDARs, albeit through undefined mechanisms. Accordingly, we here examined the contribution of individual NMDAR subunits to synaptotoxicity and demonstrate that Aβ exerts differential effects on the levels and distribution of GluN2A and GluN2B subunits of NMDAR in dendrites. Treatment of cultured hippocampal neurons with Aβ1-40 (10 μM, 1 h) induced a significant increase of dendritic and synaptic GluN2B puncta densities with parallel decreases in the puncta densities of denritic and synaptic pTyr1472-GluN2B. Conversely, Aβ significantly decreased dendritic and synaptic GluN2A and dendritic pTyr1325-GluN2A puncta densities and increased synaptic pTyr1325-GluN2A puncta densities. Unexpectedly, Aβ treatment resulted in a significant reduction of GluN2B and pTyr1472-GluN2B protein levels but did not influence GluN2A and pTyr1325-GluN2A levels. These results show that Aβ exerts complex and distinct regulatory effects on the trafficking and phosphorylation of GluN2A and GluN2B, as well as on their localization within synaptic and non-synaptic sites. Increased understanding of these early events in Aβ-induced synaptic dysfunction is likely to be important for the development of timely preventive and therapeutic interventions.

In Press, Corrected Proof, Environmental enrichment (EE), comprising positive physical (exercise) and cognitive stimuli, influences neuronal structure and usually improves brain function. The promise of EE as a preventative strategy against neuropsychiatric disease is especially high during early postnatal development when the brain is still amenable to reorganization. Despite the fact that male and female brains differ in terms of connectivity and function that may reflect early life experiences, knowledge of the neural substrates and mechanisms by which such changes arise remains limited. This study compared the impact of EE combined with physical activity on neuroplasticity and its functional consequences in adult male and female rats; EE was provided during the first 3 months of life and our analysis focused on the hippocampus, an area implicated in cognitive behavior as well as the neuroendocrine response to stress. Both male and female rats reared in EE displayed better object recognition memory than their control counterparts. Interestingly, sex differences were revealed in the effects of EE on time spent exploring the objects during this test. Independently of sex, EE increased hippocampal turnover rates of dopamine and serotonin and reduced expression of 5-HT1A receptors; in addition, EE upregulated expression of synaptophysin, a presynaptic protein, in the hippocampus. As compared to their respective controls, EE-exposed males exhibited parallel increases in phosphorylated Tau and the GluN2B receptor, whereas females responded to EE with reduced hippocampal levels of glutamate and GluN2B. Together, these observations provide further evidence on the differential effects of EE on markers of hippocampal neuroplasticity in males and females., This work was funded by an ``Education and Lifelong Learning, Supporting Postdoctoral Researchers”, co-financed by the European Social Fund (ESF) and the General Secretariat for Research and Technology, Greece. This work was also supported by the Portuguese North Regional Operational Program (ON.2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER), the Project Estratégico co-funded by FCT (PEst-C/SAU/LA0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01-0124-FEDER-037,298) as well as the project NORTE-01-0145-FEDER-000,013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), info:eu-repo/semantics/acceptedVersion

Uncorrected proof, The hippocampus and prefrontal cortex (PFC) are connected in a reciprocal manner: whereas the hippocampus projects directly to the PFC, a polysynaptic pathway that passes through the nucleus reuniens (RE) of the thalamus relays inputs from the PFC to the hippocampus. The present study demonstrates that lesioning and/or inactivation of the RE reduces coherence in the PFC-hippocampal pathway, provokes an antidepressant-like behavioral response in the forced swim test and prevents, but does not ameliorate, anhedonia in the chronic mild stress (CMS) model of depression. Additionally, RE lesioning before CMS abrogates the well-known neuromorphological and endocrine correlates of CMS. In summary, this work highlights the importance of the reciprocal connectivity between the hippocampus and PFC in the establishment of stress-induced brain pathology and suggests a role for the RE in promoting resilience to depressive illness., Greece for providing sertraline. This work was supported by an ‘Education and Lifelong Learning, Supporting Postdoctoral Researchers’, co-financed by the European Social Fund (ESF) and the General Secretariat for Research and Technology, Greece, the Life and Health Sciences Research Institute (ICVS), ON.2—O NOVO NORTE—North Portugal Regional Operational Program 2007/2013 of the National Strategic Reference Framework (NSRF) 2007/2013 through the European Regional Development Fund (ERDF), the Portuguese Foundation for Science and Technology (FCT; grant no. NMC-113934) and an InEurope program funded by International Brain Research Organization, info:eu-repo/semantics/publishedVersion

The FK506-binding protein 51 (FKBP51, encoded by the FKBP5 gene) is an established risk factor for stress-related psychiatric disorders such as major depression. Drug discovery for FKBP51 has been hampered by the inability to pharmacologically differentiate against the structurally similar but functional opposing homolog FKBP52, and all known FKBP ligands are unselective. Here, we report the discovery of the potent and highly selective inhibitors of FKBP51, SAFit1 and SAFit2. This new class of ligands achieves selectivity for FKBP51 by an induced-fit mechanism that is much less favorable for FKBP52. By using these ligands, we demonstrate that selective inhibition of FKBP51 enhances neurite elongation in neuronal cultures and improves neuroendocrine feedback and stress-coping behavior in mice. Our findings provide the structural and functional basis for the development of mechanistically new antidepressants.

Early-life obesity predisposes to obesity in adulthood, a condition with broad medical implications including sleep disorders, which can exacerbate metabolic disturbances and disrupt cognitive and affective behaviors. In this study, we examined the long-term impact of transient peripubertal diet-induced obesity (ppDIO, induced between 4 and 10 weeks of age) on sleep–wake behavior in male mice. EEG and EMG recordings revealed that ppDIO increases sleep during the active phase but reduces resting-phase sleep quality. This impaired sleep phenotype persisted for up to 1 year, although animals were returned to a non-obesiogenic diet from postnatal week 11 onwards. To better understand the mechanisms responsible for the ppDIO-induced alterations in sleep, we focused on the lateral hypothalamus (LH). Mice exposed to ppDIO did not show altered mRNA expression levels of orexin and melanin-concentrating hormone, two peptides that are important for sleep–wake behavior and food intake. Conversely, the LH of ppDIO-exposed mice had reduced contents of serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter involved in both sleep–wake and satiety regulation. Interestingly, an acute peripheral injection of the satiety-signaling peptide YY 3–36 increased 5-HT turnover in the LH and ameliorated the ppDIO-induced sleep disturbances, suggesting the therapeutic potential of this peptide. These findings provide new insights into how sleep–wake behavior is programmed during early life and how peripheral and central signals are integrated to coordinate sleep.SIGNIFICANCE STATEMENTAdult physiology and behavior are strongly influenced by dynamic reorganization of the brain during puberty. The present work shows that obesity during puberty leads to persistently dysregulated patterns of sleep and wakefulness by blunting serotonergic signaling in the lateral hypothalamus. It also shows that pharmacological mimicry of satiety with peptide YY3–36can reverse this neurochemical imbalance and acutely restore sleep composition. These findings add insight into how innate behaviors such as feeding and sleep are integrated and suggest a novel mechanism through which diet-induced obesity during puberty imposes its long-lasting effects on sleep–wake behavior.

Tau is a major component of the neurofibrillary tangles (NFT) that represent a pathological hallmark of Alzheimer's disease (AD). Although generally considered an axonal protein, Tau is found in the somato-dendritic compartment of degenerating neurons and this redistribution is thought to be a trigger of neurodegeneration in AD. Here, we show the presence of tau mRNA in a dendritic ribonucleoprotein (RNP) complex that includes Ca2+-calmodulin dependent protein kinase (CaMK)IIα mRNA and that is translated locally in response to glutamate stimulation. Further, we show that Tau mRNA is a component of mRNP granules that contain RNA-binding proteins, and that it interacts with Myosin Va, a postsynaptic motor protein; these findings suggest that tau mRNA is transported into dendritic spines. We also report that tau mRNA localized in the somato-dendritic component of primary hippocampal cells and that a sub-toxic concentration of glutamate enhances local translation and hyperphosphorylation of tau, effects that are blocked by the gluatamatergic antagonists MK801 and NBQX. These data thus demonstrate that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-d-aspartate (NMDA) stimulation redistributes tau to the somato-dendritic region of neurons where it may trigger neurodegeneration., Graphical Abstract Image 2, Highlights • Tau mRNA binds to Staufen1- and FMRP-containing ribonucleoprotein (RNP) particles in dendrites and synapse. • AMPA and NMDA receptor-mediated glutamatergic stimulation triggers local translation of tau protein. • Over stimulation of AMPA and NMDA receptors is a cause of somatodendritic localization of tau found in degenerating neurons. We show here that glutamate-activated AMPA and NMDA receptors regulate local translation of tau protein and recruit mechanisms that lead to the accumulation of hyperphosphorylated tau and enhance the neurofibrillary lesions. Glutamate stimulation is thought to cause neurodegeneration in AD; memantine, which relieves the aberrant excitation of glutamate on NMDA receptor, has efficacy in AD therapy. Aβ, stroke or head trauma, which are known as higher risk of AD in etiological studies, evoke an exaggerate NMDA receptor stimulation. Therefore, our conclusion possibly provides a common mechanism for causing neurodegeneration in AD.

The state-of-the-art regarding the important role that endogenous opioid peptides and their respective receptors play in the regulation of a variety of physiological and behavioral functions under normal and stressful conditions is presented. This chapter covers the biology, pharmacology, and molecular biology of opioids, as well as the implication of opioids in stress-related pathology.

Tau protein in dendrites and synapses has been recently implicated in synaptic degeneration and neuronal malfunction. Chronic stress, awell-known inducer of neuronal/synaptic atrophy, triggers hyperphosphorylation of Tau protein and cognitive deficits. However, the cause–effect relationship between these events remains to be established. To test the involvement of Tau in stress-induced impairments of cognition,we investigated the impact of stress on cognitive behavior, neuronal structure, and the synaptic proteome in the prefrontal cortex (PFC) of Tau knock-out (Tau-KO) and wild-type (WT) mice. Whereas exposure to chronic stress resulted in atrophy of apical dendrites and spine loss in PFC neurons as well as significant impairments in working memory in WT mice, such changes were absent in Tau-KO animals. Quantitative proteomic analysis of PFC synaptosomal fractions, combined with transmission electron microscopy analysis, suggested a prominent role for mitochondria in the regulation of the effects of stress. Specifically, chronically stressed animals exhibit Tau-dependent alterations in the levels of proteins involved in mitochondrial transport and oxidative phosphorylation aswell as in the synaptic localization of mitochondria in PFC. These findings provide evidence for a causal role of Tau in mediating stress-elicited neuronal atrophy and cognitive impairment and indicate that Tau may exert its effects through synaptic mitochondria., This work was funded by the Portuguese Foundation for Science & Technology (FCT, grant number NMC-113934 to I.S.), the EU Consortium Switchbox (grant number Health-FP7-2010-259772 to O.F.X.A. and N.S.), the Deutsche Forschungsgemeinschaft (grant number FI 1895/1-1 to M.D.F.) and the Max Planck Society (M.D.F., G.M., C.W.T., and O.F.X.A.). In addition, this workwas also co-financed by the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER) (N.S.). S.L. and I.S. are holders of FCT Fellowships. J.V-S. is a recipient of a PhD fellowship (PD/ BD/105938/2014) of the University of Minho MD/PhD Program funded by FCT., info:eu-repo/semantics/publishedVersion

The dynamic turnover of hippocampal neurons is implicated in the regulation of cognitive and affective behavior. Extending our previous demonstration that administration of dexamethasone (ND) to neonatal rats depletes the resident population of neural precursor cells (NPC) and restrains the size of the neurogenic regions, we now show that the adverse effects of ND persist into adulthood. Specifically, ND impairs repletion of the neurogenic pool and neurogenesis; ND also compromises cognitive performance, the ability to actively adapt to an acute stressor and, the efficacy of glucocorticoid (GC) negative feedback. Interestingly, although ND depletes the neurogenic pool, it does not permanently abolish the proliferative machinery of the residual NPC population; however, ND increases the susceptibility of hippocampal granule neurons to apoptosis. Although the antidepressant fluoxetine (FLX) reverses the latter phenomenon, it does not replenish the NPC pool. Treatment of ND-treated adult rats with FLX also improves GC negative feedback, albeit without rescuing the deleterious effects of ND on behavior. In summary, ND leads to protracted disruption of mental functions, some of which are resistant to antidepressant interventions. We conclude that manipulation of the NPC pool during early life may jeopardize the therapeutic potential of antidepressants in adulthood., We thank Albin Varga and his team for invaluable help with animal housing and care. This study represents a contribution from the SwitchBox Consortium, supported by the European FP7 (Contract 259772), with additional suuport from the National Key Research & Development Program of China (2016YFC1306600) to YS. The funders did not have any role in the design or execution of the study and had no influence over the interpretation of its results or the writing of the paper. The research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest., info:eu-repo/semantics/publishedVersion

Early-life stress (ELS) increases the vulnerability thresholds for stress-related diseases such as major depression and anxiety by inducing alterations in the structure and function of neural circuits and endocrine pathways. We previously demonstrated the contribution of epigenetic mechanisms to the long-term programming of the hypothalamo-pituitary-adrenal axis activity following ELS exposure in male mice. Here, ELS comprising daily separation of pups from their dams on postnatal days 1–10 was observed to up-regulate the expression of the pituitary proopiomelanocortin (Pomc) gene; POMC serves as a prohormone for ACTH, a key mediator of the adrenocortical response to stress. Detailed analysis revealed that the increase in Pomc mRNA levels results from a reduction in DNA methylation at a critical regulatory region of the Pomc gene; interestingly, this change occurs with some delay after ELS and persists for up to 1 year. Using a Pomc-expressing pituitary cell line (AtT20), we confirmed a role for DNA methylation in restraining Pomc expression under resting conditions: specifically, we show that CpG site-specific methylation of the Pomc promoter represses Pomc mRNA transcription. Further, we show high-affinity binding of methyl-CpG binding protein-2 to the distal promoter of Pomc, suggesting that methyl-CpG binding protein-2 acts in association with the chromatin modifiers histone deacetylase 2 and DNA methyltransferase 1 to repress Pomc gene expression. Collectively, these experiments contribute to our understanding of the mechanisms through which environmental cues are translated into stable changes (“cellular memory”) in neuroendocrine cells.

We would like to thank Antonio Pinheiro, Hugo Almeida, and José Miguel Soares for help with the illustrations. We also thank all past and present members of our laboratories for stimulating discussions., Maladaptive responses to stress and the associated hypersecretion of glucocorticoids cause psychopathologies ranging from hyperemotional states and mood dysfunction to cognitive impairments. Research in both humans and animal models has begun to identify morphological correlates of these functional changes. These include dendritic and synaptic reorganization, glial remodeling, and altered cell fate in cortical and subcortical structures. The emerging view is that stress induces a disconnection syndrome’ whereby the transmission and integration of information that are critical for orchestrating appropriate physiological and behavioral responses are perturbed. High-resolution spatiotemporal mapping of the complete neural circuitry and identification of the cellular processes impacted by stress will help to advance discovery of strategies to reduce or reverse the burden of stress-related neuropsychiatric disorders., Work in our laboratories was supported by the German Academic Exchange Service - Acções Luso-Alemãs, Fundação para a Ciência e Tecnologia (PTDC/SAUNSC/ 111814/2009), and European Union FP7 (SwitchBox Project, Contract 259772; Neuroendocrine Immune Networks in Ageing Scientific Network, Contract 238665).

Early-stage Alzheimer's disease (AD) is characterized by synaptic dysfunction, a phenomenon in which soluble oligomers of amyloid-beta (Aβ) and N-methyl-D-aspartate receptor (NMDAR) are implicated. Here, we demonstrated that astrocytes express NMDARs and therefore have the potential to modulate the synaptotoxic actions of Aβ. We found that specific pharmacological antagonism of two of the major NMDAR subunits, GluN2A and GluN2B, exacerbates Aβ-induced synaptotoxicity suggesting, for the first time, that astrocytic GluN2A and GluN2B mediate synaptoprotection. From the perspective of the pathogenic mechanisms of Alzheimer's disease, in which Aβ and NMDAR play significant roles, these observations are striking since neuronal GluN2A and GluN2B are well known modulators of neurodegeneration. We did initial studies to understand the basis for the differential effects of astrocytic and neuronal GluN2A and GluN2B in the promotion of synapse survival, and identified a neurotrophin produced by astrocytes, nerve growth factor β (β-NGF), as a likely mediator of the synaptoprotective effects of astrocytic GluN2A and GluN2B activation. The results presented suggest that astrocytes may be suitable druggable targets for the prevention and/or delay of the synaptic loss that occurs during early stages of AD.

Exposure to chronic stress is frequently accompanied by cognitive and affective disorders in association with neurostructural adaptations. Chronic stress was previously shown to trigger Alzheimer's-like neuropathology, which is characterized by Tau hyper-phosphorylation and missorting into dendritic spines followed by memory deficits. Here, we demonstrate that stress-driven hippocampal deficits in wild-type mice are accompanied by synaptic missorting of Tau and enhanced Fyn/GluN2B-driven synaptic signaling. In contrast, mice lacking Tau [Tau knockout (Tau-KO) mice] do not exhibit stress-induced pathological behaviors and atrophy of hippocampal dendrites or deficits of hippocampal connectivity. These findings implicate Tau as an essential mediator of the adverse effects of stress on brain structure and function., We thank Dr. Peter Davies (Albert Einstein College) for the PHF1 antibody. This work was funded by Portuguese Foundation for Science & Technology (FCT) Grants PTDC/SAU-NMC/113934/2009 (to I.S.); the European Union FP7 Project SwitchBox (N.S. and O.F.X.A.); the Portuguese North Regional Operational Program (ON.2-O Novo Norte) under the National Strategic Reference Framework (QREN) through the European Regional Development Fund (FEDER); and the Education and Lifelong Learning, Supporting Postdoctoral Researchers and Large Scale Cooperative Project, cofinanced by the European Social Fund and the Greek General Secretariat for Research and Technology. J.V.-S. is a recipient of a PhD fellowship (PD/BD/105938/2014) of the University of Minho MD/PhD Program funded by the FCT.

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies., Aggregation of microtubule associated protein tau is one of cause of neuronal loss in tauopathies including Alzheimer's disease. Here, the authors show that compounds with a 1,2-dihydroxybenzene skeleton can modify cysteine residues in tau and block toxic tau aggregation.

Alternative splicing serves to increase biological diversity and adaptation. Many genes, including the glucocorticoid receptor (GR), contain multiple 5'-untranslated exons in their promoter regions that can give rise to various mRNA isoforms encoding the same protein. To date, information on the mouse GR promoter remains sparse. Here, we extensively characterize alternative first exons of the mouse GR to reveal homology to the rat and human. We further find that, although most promoters are broadly expressed in various tissues, transcription of individual promoters can be differentially regulated by growth factor- and depolarization-induced signaling. Moreover, in addition to selective promoter usage, the alternative first exon transcripts differentially control RNA stability and translation efficiency, indicative of their role in GR expression. In conclusion, the composite GR promoter enables multilayered adjustments in gene expression through transcriptional and posttranscriptional mechanisms that may serve varying physiological demands.

Soluble oligomeric amyloid-β (Aβ) is thought to induce synaptic dysfunction during early stages of Alzheimer's disease (AD). In this report, we show that soluble Aβ downregulates the levels of two synaptic proteins, PSD-95 and synaptophysin, and that this effect can be blocked by MK-801 (NMDAR antagonist) and ifenprodil (NR2B antagonist). Low (1 μM) and high (10 μM) doses of NMDA, respectively, prevented and potentiated the actions of Aβ. Blockade of NR2A or synaptic NMDAR eliminated the protective effect of 1 μM NMDA, while the effects of 10 μM NMDA were only abolished by ifenprodil. Caspase-8, acting upstream of caspase-3, was found to mediate the synaptotoxic actions of Aβ in an ifenprodil-reversible fashion. Thus, Aβ leads to a loss of synaptic proteins by suppression of NR2A function and activation of NR2B function and subsequent induction of caspase-8 and caspase-3 activities. The identified novel mechanism through which Aβ initiates synaptic dysfunction suggests that selective enhancement of NR2A activity and/or reduction of NR2B activity can halt the manifestation of a key early-stage event in AD.

Adverse early life events can induce long-lasting changes in physiology and behavior. We found that early-life stress (ELS) in mice caused enduring hypersecretion of corticosterone and alterations in passive stress coping and memory. This phenotype was accompanied by a persistent increase in arginine vasopressin (AVP) expression in neurons of the hypothalamic paraventricular nucleus and was reversed by an AVP receptor antagonist. Altered Avp expression was associated with sustained DNA hypomethylation of an important regulatory region that resisted age-related drifts in methylation and centered on those CpG residues that serve as DNA-binding sites for the methyl CpG-binding protein 2 (MeCP2). We found that neuronal activity controlled the ability of MeCP2 to regulate activity-dependent transcription of the Avp gene and induced epigenetic marking. Thus, ELS can dynamically control DNA methylation in postmitotic neurons to generate stable changes in Avp expression that trigger neuroendocrine and behavioral alterations that are frequent features in depression.

Altered corticosteroid milieu induces changes in hippocampal volume, neuronal structure, neurochemistry and cognitive function in humans and rodents. This in vivo magnetic resonance spectroscopy (1H MRS) and imaging (MRI) study investigated whether long-term alterations of the corticosteroid milieu cause: (i) metabolic and/or (ii) structural changes of the rat hippocampus. Therefore, hypocortisolism was induced by adrenalectomy (ADX), normocortisolism by ADX with low-dose corticosterone replacement, and hypercortisolism by ADX and high-dose dexamethasone treatment (for 11 weeks, respectively). All groups including a control group (n = 23) were studied by in vivo 1H MRS and MR volumetry. Effects of treatment on normalized hippocampal metabolites and volumes were tested for significance using one-factorial multivariate analysis of variance (MANOVA). Hypercortisolemic rats revealed significantly elevated glutamate. Hypocortisolemic rats showed significantly decreased myo-inositol ratio levels, and were associated with significantly reduced normalized hippocampal volumes. Our findings suggest chronic hypercortisolism to be associated with glutamate-mediated excitotoxicity in the absence of volumetric abnormalities. In contrast, hypocortisolism appears to be associated with neurodegenerative processes, altered astrocytic metabolism but preserved neuronal density.

Amyloid precursor protein (APP) mis-processing and aberrant tau hyperphosphorylation are causally related to the pathogenesis and neurodegenerative processes that characterize Alzheimer’s disease (AD). Abnormal APP metabolism leads to the generation of neurotoxic amyloid beta (Aβ), whereas tau hyperphosphorylation culminates in cytoskeletal disturbances, neuronal dysfunction and death. Many AD patients hypersecrete glucocorticoids (GC) while neuronal structure, function and survival are adversely influenced by elevated GC levels. We report here that a rat neuronal cell line (PC12) engineered to express the human ortholog of the tau protein (PC12-htau) becomes more vulnerable to the toxic effects of either Aβ or GC treatment. Importantly, APP metabolism in GC-treated PC12-htau cells is selectively shifted towards increased production of the pro-amyloidogenic peptide C99. Further, GC treatment results in hyperphosphorylation of human tau at AD-relevant sites, through the cyclin-dependent kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein kinases. Pulse-chase experiments revealed that GC treatment increased the stability of tau protein rather than its de novo synthesis. GC treatment also induced accumulation of transiently expressed EGFP-tau in the neuronal perikarya. Together with previous evidence showing that Aβ can activate cyclin-dependent kinase 5 and GSK3, these results uncover a potential mechanism through which GC may contribute to AD neuropathology.

The balance in actions mediated by mineralocorticoid (MR) and glucocorticoid (GR) receptors in certain regions of the brain, predominantly in the limbic system, appears critical for neuronal activity, stress responsiveness, and behavioral programming and adaptation. Alterations in the MR/GR balance appear to make nervous tissue vulnerable to damage; such damage can have adverse effects on the regulation of the stress response and may increase the risk for psychopathology. Besides the hippocampal formation, other subpopulations of neurons in extra-hippocampal brain areas have been also shown recently to be sensitive to changes in the corticosteroid milieu. From a critical analysis of the available data, the picture that emerges is that the balance (or imbalance) between MR/GR activation influences not only cell birth and death, but also other forms of neuroplasticity. MR occupation appears to promote pro-survival actions, while exclusive GR activation favors neurodegeneration. Interestingly, the sustained co-activation of both receptors, for example in chronic stress conditions, usually results in less drastic effects, restricted to dendritic atrophy and impaired synaptic plasticity. As our knowledge of the plastic changes underpinning the wide spectrum of behavior effects triggered by corticosteroids/stress growths, researchers should be able to better define new targets for therapeutic intervention in stress-related disorders.

Chronic stress is a powerful modulator of emotional behaviour. Previous studies have shown that distinct neuronal pathways modulate different emotional behaviours: while the amygdala plays a key role in fear-conditioned-to-cue stimuli, the bed nucleus of stria terminalis (BNST) is implicated in anxiety behaviour and responses to contextual stimuli. In addition, the BNST is directly involved in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis. In the present study, we assessed anxiety (measured in the elevated-plus maze and acoustic startle apparatus) and fear-conditioned responses to light stimuli in rats that had been exposed to either chronic unpredictable stress or corticosterone for 28 days; thereafter, stereological estimates of the BNST and amygdaloid complex were performed, followed by three-dimensional morphometric dendritic analysis. Results show that chronic stress induces hyperanxiety without influencing fear conditioning or locomotion and exploratory activity. Stress-induced hyperanxiety was correlated with increased volumes of the BNST but not of the amygdala. Dendritic remodelling was found to make a significant contribution to the stress-induced increase in BNST volume, primarily due to changes in the anteromedial area of the BNST, an area strongly implicated in emotional behaviour and in the neuroendocrine control of the stress response. Importantly, all of the effects of stress were recapitulated by exogenous corticosterone. In conclusion, this study shows that chronic stress impacts on BNST structure and function; its findings pertain to the modulation of emotional behaviour and the maladaptive response to stress.

Stress, acting through glucocorticoids (GC), has profound effects on brain physiology and pathology and is causally implicated in depressive illness. Here, we consider the information derived from genetic models generated to probe the role of the hypothalamo-pituitary-adrenal axis in depression. This essay also briefly reviews the status of knowledge regarding GC actions on neuronal birth, survival and death from the perspective of the importance of these phenomena in depression.

Understanding the mechanisms that regulate postnatal neurogenesis is becoming increasingly relevant since its modulation has been implicated in the pathogenesis of certain neuropsychiatric disorders. Lithium is a mood stabilizer known to increase hippocampal neurogenesis. Lithium also results in increased levels of the angiogenic factor vascular endothelial growth factor (VEGF). Since VEGF was recently shown to have neurogenic properties, we were interested to examine whether lithium administration might also be accompanied by alterations in VEGF expression in the hippocampus of normal and stressed rats; the latter treatment was introduced to reproduce some of the psychopathological signs for which lithium is used therapeutically. The expression of VEGF in the hippocampus in stressed animals was lower than that in controls, but the effect of stress was significantly attenuated in animals concomitantly receiving lithium. Double staining for VEGF and specific markers for immature neurons, mature neurons and astroglia revealed that immature neurons were most sensitive to the VEGF-inhibiting effects of stress. Confirming the involvement of a known regulatory pathway in these actions of lithium, we demonstrated that lithium co-administration prevented the stress-induced upregulation of glycogen synthase kinase-3beta (GSK-3beta) and down-regulation of beta-catenin expression; GSK-3beta is a known primary lithium target and its inhibition by this mood stabilizer leads to an upregulation of beta-catenin and subsequently, an increase of VEGF. Our results suggest that the actions of lithium, and possibly its therapeutic efficacy as a mood stabilizer also, are mediated by VEGF., Rui Silva is a pre-doctoral fellow of the Portuguese Foundation for Science and Technology (SFRH/BD/9756/2003). This work was supported by a grant (JG0495) from the Gulbenkian Foundation.

Initial studies on neural stem cell biology were performed mainly with embryonic stem cells, but exciting discoveries and advances in knowledge about tissue-specific stem cells have emerged in the last few years. This review focuses on stem and/or progenitor cells in the brain that drive adult neurogenesis in mammals. Neuronal precursor cells are found in two regions of the adult brain: the subventricular zone and the hippocampus. Adult neurogenesis in the subventricular zone has implications for behavior and olfactory function and, in the hippocampus, is involved in mood, learning and memory. Several neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's disease and Parkinson's disease) are increasing in frequency as the population is aging. Understanding the hormonal aspects of how adult neurogenesis is regulated could lead to advances in understanding, managing and eventually, treating neurodegenerative disorders. In this review, we summarize what is currently known about the influence of hormones on adult neurogenesis. Many hormones that act through nuclear receptors are implicated in regulating neural progenitor cell biology. Given that nuclear receptors are well defined, drugable targets, further research on their mechanisms of action in adult neurogenesis are likely to engender new replacement, repair and therapeutic approaches.

Endocrine dysfunction is a common effect of traumatic brain injury (TBI). In addition to affecting the regulation of important body functions, the disruption of endocrine physiology can significantly impair mental functions, such as attention, memory, executive function, and mood. This mini-review focuses on alterations in mental functioning that are associated with neuroendocrine disturbances in adults who suffered TBI. It summarizes the contribution of hormones to the regulation of mental functions, the consequences of TBI on mental health and neuroendocrine homeostasis, and the effects of hormone substitution on mental dysfunction caused by TBI. The available empirical evidence suggests that comprehensive assessment of mental functions should be standard in TBI subjects presenting with hormone deficiency and that hormone replacement therapy should be accompanied by pre- and post-assessments.

Tau-mediated neurodegeneration is a central event in Alzheimer's disease (AD) and other tauopathies. Consistent with suggestions that lifetime stress may be a clinically-relevant precipitant of AD pathology, we previously showed that stress triggers tau hyperphosphorylation and accumulation; however, little is known about the etiopathogenic interaction of chronic stress with other AD risk factors, such as sex and aging. This study focused on how these various factors converge on the cellular mechanisms underlying tau aggregation in the hippocampus of chronically stressed male and female (middle-aged and old) mice expressing the most commonly found disease-associated Tau mutation in humans, P301L-Tau. We report that environmental stress triggers memory impairments in female, but not male, P301L-Tau transgenic mice. Furthermore, stress elevates levels of caspase-3-truncated tau and insoluble tau aggregates exclusively in the female hippocampus while it also alters the expression of the molecular chaperones Hsp90, Hsp70, and Hsp105, thus favoring accumulation of tau aggregates. Our findings provide new insights into the molecular mechanisms through which clinically-relevant precipitating factors contribute to the pathophysiology of AD. Our data point to the exquisite sensitivity of the female hippocampus to stress-triggered tau pathology., This study was supported by the Japanese Soci- ety for the Promotion of Science (IS), the Portuguese Foundation for Science & Technology (IS, AJR, NS), the Max Planck Society (OFXA), and the European Union FP7 Project SwitchBox (OFXA and NS). AT is supported by Research Funding for Longevity Sci- ences (23–39) from National Center for Geriatrics and Gerontology, the Strategic Research Program for Brain Science (“Integrated Research on Neuropsychiatric Disorders”) and a Grant-in-Aid for Scientific Research on Innovative Areas (“Brain Environment”) from the Ministry of Education, Science, Sports and Culture of Japan.

Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore., European Union Directorate General for Research & Innovation through the CRESCENDO Consortium (O.F.X.A. and D.S) and the NINA Initial Training Program (D.S. and O.F.X.A)

Amyloid-β (Aβ) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Aβ oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Aβ in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Aβ1-40reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Aβ11-40-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Aβ treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Aβ triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

Prova tipográfica (In Press), An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7a-(acetylthio)-3-oxo-17a-pregn- 4-ene,21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7a,17a)-17-Hydroxy-3-oxo-7- propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR.

Mineralocorticoid (MR) and glucocorticoid (GR) receptors are two closely-related members of the steroid nuclear receptor family of transcription factors that bind common ligands in the brain (corticosterone and cortisol) and supposedly have identical hormone response elements. This raises the important question of how they can elicit differential biological actions in neurons in which they are often colocalized. One plausible explanation is that they differentially recruit proteins (coregulators or other receptor-interacting factors) through cell-specific interactions with regions that diverge between MR and GR to modulate target gene transcription in a receptor-specific manner. We therefore performed a yeast-two-hybrid screening of a human brain cDNA library with an AF1-containing region of the human MR as bait. This screening revealed several potential MR-interacting partners; among them were several clones bearing homology to DAXX, FLASH, and FAF-1, all previously implicated in apoptosis. Coexpression of candidate clones in a mouse hippocampal cell line confirmed these interactions in a mammalian neural cell environment as well. In transient transactivation assays, DAXX and FLASH influenced MR- and GR-driven transcription of the MMTV-Luc reporter similarly; in contrast, although FAF-1 did not transactivate GR, it did selectively stimulate MR-mediated transcription. Thus, the present findings, that 1) DAXX, FLASH, and FAF-1 modulate the transcriptional activities of MR and GR and that 2) FAF-1 selectively coactivates only MR, provide possible clues for how these closely related receptors might differentially influence neuronal function.

The enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) is involved in the generation of neuroactive steroids through ring-A-reduction of hormonal precursors. We examined the developmental regulation of, gender differences in, and effects of hormonal manipulations on the expression of 3 alpha-HSD in the rat hippocampus. High levels of 3 alpha-HSD mRNA were found on postnatal day 7, coinciding with the stress hyporesponsive period in the rat. Gender differences in 3 alpha-HSD expression were documented during puberty, but not in adulthood. Adrenalectomy and gonadectomy, and supplementation with individual steroid hormones influenced 3 alpha-HSD expression in a gender-specific mode. We also demonstrate that the manifestation of behavioral and endocrine consequences of early life stress depends on the individual's gender and gonadal status. Males are liable to aftereffects of neonatal maternal deprivation, regardless of their adult gonadal status. In females, however, anxiogenic aftereffects of neonatal stress become apparent only after gonadectomy. These data suggest that (i) transient increase of neurosteroid biosynthesis may contribute to stress hyporesponsiveness during early infancy; (ii) gonadal steroids regulate 3 alpha-HSD expression in the hippocampus in a sex-specific mode; (iii) physiological sex steroid secretions in females may mask behavioral consequences of adverse early life events, and (iv) concomitant treatment with the neurosteroid THP counteracts behavioral and endocrine dysregulation induced by neonatal stress in both genders.

The regional distribution, developmental profiles, and gonadectomy- and estrogen-induced changes in the density of transcripts encoding the steroid receptor coactivator-1 (SRC-1) were examined in the female rat brain by semiquantitative in situ hybridization. The results demonstrate striking differences in the abundance of SRC-1 mRNA in discrete brain regions throughout ontogeny. Whereas transcript densities gradually decreased with age in the cerebral cortex, they peaked prominently during the peripubertal period in the hypothalamic medial preoptic area (MPOA) and ventromedial nucleus (VMN). Gonadectomy and estrogen substitution influenced SRC-1 mRNA levels in sexually mature animals in a region-specific fashion. Ovariectomy resulted in a down-regulation of SRC-1 mRNA levels in the VMN, a brain region richly endowed with estrogen receptors and playing a major role in neuroendocrine control of reproductive functions. In contrast, SRC-1 transcript levels were significantly up-regulated after estradiol treatment. Interestingly, SRC-1 expression in the cortex was refractory to alterations of the estrogen milieu. The obtained SRC-1 mRNA expression profiles during development clearly demonstrate brain region specificity and regulation by estrogen, thus it is proposed that SRC-1 amplifies estrogen receptor-dependent transcription in a temporally and spatially coordinated manner and therefore contributes to the functional specialization of brain areas involved in the regulation of reproduction.

The undesired side-effects of haloperidol treatment include a number of extrapyramidal side-effects which have been proposed to result from drug-induced damage to the basal ganglia. The drug also causes irregular movements and locomotor patterns in experimental animals. Here we show that haloperidol treatment in rats is associated with increases in the expression of p53 and the ratio of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2/Bcl-x(L)) proteins in the hippocampus and caudate putamen (CPu). In addition, haloperidol induces the DNA binding activity of the redox-sensitive nuclear factor-kappa B (NF-kappaB) and concomitantly upregulates the levels of the phosphorylated form of IkappaBalpha protein in vivo. Similar responses are observed when a mouse hippocampal cell line (HT-22) is treated with haloperidol and/or vitamin E. Interestingly, all of these biochemical effects of haloperidol are significantly attenuated when animals or cultured cells are pretreated with alpha-tocopherol (vitamin E). Consistent with this, vitamin E is demonstrated to substantially reduce the haloperidol-induced impairment of locomotor activity in rats. Collectively, the data indicate the usefulness of vitamin E as an adjunct to haloperidol treatment and provide initial clues about the underlying molecular mechanisms involved in these effects.