FGF2 gene's antisense protein, NUDT6, plays a depressogenic role by promoting inflammation and suppressing neurogenesis without altering FGF2 signalling.

Fibroblast growth factor-2 (FGF2) is involved in the regulation of affective behaviour and shows antidepressant effects through the Akt and extracellular signal regulated kinase (ERK) 1/2 pathways. Nudix hydrolase 6 (NUDT6) protein is encoded from FGF2 gene's antisense strand and its role in the regulation of affective behaviour is unknown. Here, we overexpressed NUDT6 in the hippocampus and investigated its behavioural effects and the underlying molecular mechanisms affecting the behaviour. We showed that increasing hippocampal NUDT6 results in depression-like behaviour in rats without changing FGF2 levels or activating its downstream effectors, Akt and ERK1/2. Instead, NUDT6 acted by inducing inflammatory signalling, specifically by increasing S100 calcium binding protein A9 (S100A9) levels, activating nuclear factor-kappa B-p65 (NF-κB-p65), and elevating microglia numbers along with a reduction in neurogenesis. Our results suggest that NUDT6 could play a role in major depression by inducing a proinflammatory state. This is the first report of an antisense protein acting through a different mechanism of action than regulation of its sense protein. The opposite effects of NUDT6 and FGF2 on depression-like behaviour may serve as a mechanism to fine-tune affective behaviour. Our findings open up new venues for studying the differential regulation and functional interactions of sense and antisense proteins in neural function and behaviour, as well as in neuropsychiatric disorders. KEY POINTS: Hippocampal overexpression of nudix hydrolase 6 (NUDT6), the antisense protein of fibroblast growth factor-2 (FGF2), increases depression-like behaviour in rats. Hippocampal NUDT6 overexpression triggers a neuroinflammatory cascade by increasing S100 calcium binding proteinA9 (S100A9) expression and nuclear NF-κB-p65 translocation in neurons, in addition to microglial recruitment and activation. Hippocampal NUDT6 overexpression suppresses neurogenesis. NUDT6 exerts its actions without altering the levels or downstream signalling pathways of FGF2.

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Cortical spreading depression can be triggered by sensory stimulation in primed wild type mouse brain: a mechanistic insight to migraine aura generation.

BACKGROUND: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight into how migraine starts in an otherwise healthy brain, remain largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. METHODS: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low concentration of Na+/K+-ATPase blocker ouabain, allowing longer than 30-min intervals between CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake, with shRNA. Stimulation-triggered CSDs and extracellular K+ changes were monitored in vivo electrophysiologically and a K+-sensitive fluoroprobe (IPG-4), respectively. RESULTS: After priming with ouabain, photic stimulation significantly increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p < 0.001). Whisker stimulation also significantly increased the CSD incidence, albeit less effectively (14.9 vs. 2.4%, p = 0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested with KCl but triggered CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during sensory stimulation after ouabain or shRNA treatment unlike controls. In line with the higher CSD susceptibility observed, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the probability of stimulus-evoked CSDs, we applied an A1-receptor antagonist (DPCPX) to the occipital cortex, because adenosine formed during stimulation from ATP can reduce CSD susceptibility. DPCPX induced spontaneous CSDs but only small-DC shifts along with suppression of EEG spikes during photic stimulation, suggesting that the inhibition co-activated with sensory stimulation could limit CSD ignition when K+ uptake was not sufficiently suppressed as with ouabain. CONCLUSIONS: Normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has potential to trigger CSD when co-existing conditions bring extracellular K+ and glutamate concentrations over CSD-ignition threshold and stimulation-evoked inhibitory mechanisms are overcome.

cAMP/PKA-CREB-BDNF signaling pathway in hippocampus of rats subjected to chemically-induced phenylketonuria.

Phenylketonuria (PKU) is an inborn error disease in phenylalanine metabolism resulting from defects in the stages of converting phenylalanine to tyrosine. Although the pathophysiology of PKU is not elucidated yet, the toxic effect of phenylalanine on the brain causes severe mental retardation. In relation to learning and memory, the hippocampal PKA / CREB / BDNF pathway may play a role in learning deficits in PKU patients. This study aimed to investigate PKA/CREB/BDNF pathway in hippocampus of chemically induced PKU rats with regard to gender. Sprague-Dawley rat pups were randomized into two groups of both genders. To chemically induce PKU, animals received subcutaneous administration of phenylalanine (5.2 mmol / g) plus p-chlorophenylalanine, phenylalanine hydroxylase inhibitor (0.9 mmol / g); control animals received 0.9% NaCl. Injections started on the 6th day and continued until the 21st day after which locomotor activity, learning and memory were tested. In male PKU rats, locomotor activity was reduced. There were no differences in learning and memory performances of male and female PKU rats. In PKU rats, pCREB / CREB levels in males was unchanged while it decreased in females. Elevated PKA activity, BDNF levels and decreased pCREB/CREB ratio found in female PKU rats were not replicated in PKU males in which BDNF is decreased. Our results display that in this disease model a gender specific differential activation of cAMP/PKA-CREB-BDNF signaling pathway in hippocampus occurs investigation of which can help us to a better understanding of disease pathophysiology.

Ion Channel Dysfunction and Neuroinflammation in Migraine and Depression.

Migraine and major depression are debilitating disorders with high lifetime prevalence rates. Interestingly these disorders are highly comorbid and show significant heritability, suggesting shared pathophysiological mechanisms. Non-homeostatic function of ion channels and neuroinflammation may be common mechanisms underlying both disorders: The excitation-inhibition balance of microcircuits and their modulation by monoaminergic systems, which depend on the expression and function of membrane located K+, Na+, and Ca+2 channels, have been reported to be disturbed in both depression and migraine. Ion channels and energy supply to synapses not only change excitability of neurons but can also mediate the induction and maintenance of inflammatory signaling implicated in the pathophysiology of both disorders. In this respect, Pannexin-1 and P2X7 large-pore ion channel receptors can induce inflammasome formation that triggers release of pro-inflammatory mediators from the cell. Here, the role of ion channels involved in the regulation of excitation-inhibition balance, synaptic energy homeostasis as well as inflammatory signaling in migraine and depression will be reviewed.

Brain Peptides for the Treatment of Neuropsychiatric Disorders.

The realization of the importance of growth factors in adult CNS led to several studies investigating their roles in neuropsychiatric disorders. Based on the observations that chronic stress decreases brain-derived neurotrophic factor (BDNF) and antidepressant treatments reverse BDNF to normal levels, "neurotrophic hypothesis of depression" was proposed. Subsequent studies found that several other growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor, nerve growth factor were also decreased by chronic stress. Growth factors promote stem cell survival, angiogenesis and neurogenesis in addition to having anti-apoptotic and anti-inflammatory effects, all of which make them potential drug candidates as neuroprotective or neurorestorative agents. Indeed, certain peptides have consistently been shown to improve stroke outcome in experimental models of cerebral ischemia. Recent developments in nanotechnology appear promising in overcoming the blood-brain barrier and in delivering sufficient amounts of these large peptides to the brain after systemic administration. In addition to the translational potential resulting from application of nanotechnical approaches for delivering these large peptide growth factors, recent success obtained with small molecule and peptide antagonists of calcitonin gene-related peptide has created renewed enthusiasm to elucidate the role of neuropeptides in migraine headache, one of the most common health problems in the world. In this review, we will first focus on the role of FGF2 in mood disorders as well as in ischemic stroke. We will also introduce the nanomedicines developed to efficiently deliver FGF2 to the brain. In the last section, we will explore roles of the neuropeptides in migraine and its acute and prophylactic treatment.

Nuclear expansion and pore opening are instant signs of neuronal hypoxia and can identify poorly fixed brains.

The initial phase of neuronal death is not well characterized. Here, we show that expansion of the nuclear membrane without losing its integrity along with peripheralization of chromatin are immediate signs of neuronal injury. Importantly, these changes can be identified with commonly used nuclear stains and used as markers of poor perfusion-fixation. Although frozen sections are widely used, no markers are available to ensure that the observed changes were not confounded by perfusion-induced hypoxia/ischemia. Moreover, HMGB1 was immediately released and p53 translocated to mitochondria in hypoxic/ischemic neurons, whereas nuclear pore complex inhibitors prevented the nuclear changes, identifying novel neuroprotection targets.

Stress modulates cortical excitability via α-2 adrenergic and glucocorticoid receptors: As assessed by spreading depression.

An increase in cortical excitability may be one of the factors mediating stress-induced vulnerability to neuropsychiatric disorders. Since stress increases extracellular glutamate and predisposes to migraine with aura attacks, we aimed to study the effect of stress on cortical spreading depression (CSD), the biological substrate of migraine aura and a measure of cortical excitability. CSD was induced by increasing concentrations of KCl applied over the dura with 5-minute intervals and recorded from parieto-occipital cortex to assess the CSD-induction threshold in acutely-stressed, chronically-stressed and naive mice. To study the mechanisms of acute stress-induced decrease in CSD threshold, we systemically administered clonidine, yohimbine, propranolol, CRH1 receptor antagonist NBI27914, mifepristone and spironolactone at doses shown to be effective on stress as well as a central noradrenergic neurotoxin (DSP-4) before acute stress. CSD threshold was significantly lowered by acute and chronic stress as well as central noradrenergic denervation. Clonidine and mifepristone further decreased the CSD threshold below the acute stress-induced levels, whereas yohimbine reversed the acute stress-induced decrease in CSD threshold compared to the saline-injected and stressed control groups. Propranolol, NBI27914 and spironolactone did not modify the effect of acute stress on CSD threshold. Stress increases cortical excitability as illustrated by a decrease in CSD-induction threshold. This action of acute stress is mediated by α2-adrenergic and glucocorticoid receptors. The decrease in CSD threshold may account for the stress-induced susceptibility to migraine. CSD may be used as a tool to study the link between stress-related disorders and cortical excitability.

Inadequate brain glycogen or sleep increases spreading depression susceptibility.

OBJECTIVE: Glycogen in astrocyte processes contributes to maintenance of low extracellular glutamate and K+ concentrations around excitatory synapses. Sleep deprivation (SD), a common migraine trigger, induces transcriptional changes in astrocytes, reducing glycogen breakdown. We hypothesize that when glycogen utilization cannot match synaptic energy demand, extracellular K+ can rise to levels that activate neuronal pannexin-1 channels and downstream inflammatory pathway, which might be one of the mechanisms initiating migraine headaches. METHODS: We suppressed glycogen breakdown by inhibiting glycogen phosphorylation with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and by SD. RESULTS: DAB caused neuronal pannexin-1 large pore opening and activation of the downstream inflammatory pathway as shown by procaspase-1 cleavage and HMGB1 release from neurons. Six-hour SD induced pannexin-1 mRNA. DAB and SD also lowered the cortical spreading depression (CSD) induction threshold, which was reversed by glucose or lactate supplement, suggesting that glycogen-derived energy substrates are needed to prevent CSD generation. Supporting this, knocking down the neuronal lactate transporter MCT2 with an antisense oligonucleotide or inhibiting glucose transport from vessels to astrocytes with intracerebroventricularly delivered phloretin reduced the CSD threshold. In vivo recordings with a K+ -sensitive/selective fluoroprobe, Asante Potassium Green-4, revealed that DAB treatment or SD caused a significant rise in extracellular K+ during whisker stimulation, illustrating the critical role of glycogen in extracellular K+ clearance. INTERPRETATION: Synaptic metabolic stress caused by insufficient glycogen-derived energy substrate supply can activate neuronal pannexin-1 channels as well as lower the CSD threshold. Therefore, conditions that limit energy supply to synapses (eg, SD) may predispose to migraine attacks, as suggested by genetic studies associating glucose or lactate transporter deficiency with migraine. Ann Neurol 2018;83:61-73.

[Cognitive Dysfunctions in Posttraumatic Stress Disorder]. / Travma Sonrasi Stres Bozuklugunda Bilissel Bozukluklar.

Posttraumatic stress disorder (PTSD) is a condition that occurs after a traumatic event, and its diagnostic criteria include attention and memory deficits as well as symptoms of anxiety. We aimed to review the literature related to attention, memory, and executive functions in PTSD. Although studies on the subject are limited (in that there is no uniformity in terms of trauma type, selection of the control groups or types of neuropsychological tests used), most reported similar deficits in PTSD subjects in terms of memory and executive functions including attention. Since the presence of psychiatric comorbidities may disrupt neuropsychological functions, results of studies that have not controlled comorbidity may be questionable. However, studies that excluded the comorbid conditions reported similar deficits in cognitive functions in PTSD patients. The relationship between recovery from PTSD symptoms and change in cognitive functions has been examined in only a few studies, and most have reported an improvement for both memory and executive functions in remitted patients. The improvement in executive functions, however, has been limited by the difficulty of task. Cognitive deficits have been among the major causes of disabilities in PTSD patients. Therefore, the amount of improvement in cognitive dysfunctions by current treatments of PTSD deserves more attention.

Dysregulated fibroblast growth factor (FGF) signaling in neurological and psychiatric disorders.

The role of the fibroblast growth factor (FGF) system in brain-related disorders has received considerable attention in recent years. To understand the role of this system in neurological and psychiatric disorders, it is important to identify the specific members of the FGF family that are implicated, their location and the various mechanisms they can be modulated. Each disorder appears to impact specific molecular players in unique anatomical locations, and all of these could conceivably become targets for treatment. In the last several years, the issue of how to target this system directly has become an area of increasing interest. To date, the most promising therapeutics are small molecule inhibitors and antibodies that modulate FGF receptor (FGFR) function. Beyond attempting to modify the primary players affected by a given brain disorder, it may prove useful to target molecules, such as membrane-bound or extracellular proteins that interact with FGF ligands or FGFRs to modulate signaling.

Posttraumatic growth after earthquake trauma is predicted by executive functions: a pilot study.

Although positive personal change after adverse events (posttraumatic growth [PTG]) is repeatedly shown to occur after a range of traumatic or distressing events, there is still a debate on the validity of the concept. Using the objective measurement of cognitive functions, we attempted to show that PTG is a scientifically valid construct in a group of earthquake survivors. This is the first study to associate PTG with cognitive functioning. We found that growth was predicted by executive functions and not by memory or processing speed, showing that the correlation between cognitive functions and growth is a specific one. In addition, a specific form of PTG, namely personal growth, was related to cognitive functions, whereas relational growth was not. Our findings provide support for the validity of the PTG concept.

Effects of acute and chronic electroconvulsive shocks on glycogen synthase kinase 3ß level and phosphorylation in mice.

OBJECTIVES: Glycogen synthase kinase 3ß (GSK-3ß) is recently proposed as a novel target in the treatment of mood disorders. Recent evidence has suggested that acute and chronic administration of antidepressants led to inhibition of GSK-3ß via phosphorylation of Serine9 residue. Acute electroconvulsive shock (ECS) has been reported to increase GSK-3ß phosphorylation transiently. In this study, the changes in the level of GSK-3ß and its phoshorylated form (phospho-Ser9-GSK-3ß) following chronic ECS (cECS) were investigated in mice. METHODS: Mice were given daily ECS via bilateral corneal electrodes for 10 consecutive days in the chronic group and a single ECS in the acute group. Electrodes were applied without stimulation in corresponding sham groups. Immunoblotting for GSK-3ß and phospho-Ser9-GSK-3ß was performed with the frontal cortex and hippocampus samples, extracted 10 minutes after single ECS, and 24 hours after the last ECS in the chronic group. The optical densities of the bands obtained were compared between the active treatment and sham groups for each condition separately. RESULTS: The level of phospho-Ser9-GSK-3ß was not different following chronic ECS, but significantly higher following acute ECS, compared with the corresponding sham group, in the hippocampus and frontal cortex. The level of GSK-3ß was similar to sham following both acute and chronic ECS, in both regions. CONCLUSIONS: The transient increase in GSK-3ß phosphorylation observed following acute ECS in the mice hippocampus and frontal cortex was not found to persist 24 hours following chronic ECS. The mechanism of action of ECS does not seem to involve persistent change in the level and phosphorylation of GSK-3ß.

Spreading depression triggers headache by activating neuronal Panx1 channels.

The initial phase in the development of a migraine is still poorly understood. Here, we describe a previously unknown signaling pathway between stressed neurons and trigeminal afferents during cortical spreading depression (CSD), the putative cause of migraine aura and headache. CSD caused neuronal Pannexin1 (Panx1) megachannel opening and caspase-1 activation followed by high-mobility group box 1 (HMGB1) release from neurons and nuclear factor κB activation in astrocytes. Suppression of this cascade abolished CSD-induced trigeminovascular activation, dural mast cell degranulation, and headache. CSD-induced neuronal megachannel opening may promote sustained activation of trigeminal afferents via parenchymal inflammatory cascades reaching glia limitans. This pathway may function to alarm an organism with headache when neurons are stressed.

Psychiatric disorders and epigenetics.

The term epigenetic refers to long-lasting changes in gene expression that are beyond the DNA base sequence. Understanding the dynamic role of epigenetic mechanisms in the regulation of gene expression in adulthood has led researchers to investigate epigenetic mechanisms in psychiatric disorders. The aim of this review was to describe epigenetic mechanisms and to discuss the role of epigenetic modifications in stress, depression, schizophrenia, and substance dependence. For this purpose PubMed was searched using the keywords epigenetic, stress, depression, schizophrenia, and substance dependence; studies published between 2000 and 2011 were reviewed. Different maternal behavioral patterns and early life stress have been reported to yield heritable changes in gene expression via epigenetic mechanisms, which are reversible. Studies that investigated the role of epigenetic modifications in stress and depression focused on the proteins involved in the regulation of the hypothalamo-pituitary adrenal (HPA) axis, whereas epigenetic studies of schizophrenia primarily focused on changes in the GABAergic system. Studies on substance dependence, on the other hand, showed that substance use might change the expression of many genes by causing short- or long-lasting epigenetic modifications. These findings have led to the development of new therapeutic strategies that target epigenetic mechanisms. Among these strategies, histone deacetylase inhibitors are especially promising. More studies are needed to improve our understanding of the role of epigenetic modifications in the development of psychiatric disorders, and to aid in the development of new treatment strategies that focus on epigenetic mechanisms.

Short-hairpin RNA silencing of endogenous fibroblast growth factor 2 in rat hippocampus increases anxiety behavior.

BACKGROUND: The fibroblast growth factor system has been implicated in the pathophysiology of mood disorders in humans and in affective behavior in animal models. However, the studies have been either correlative or involved exogenous administration of fibroblast growth factor 2 (FGF2). None of them have directly linked endogenous FGF2 to changes in emotional responses. Therefore, we began a series of studies to knockdown FGF2 by RNA interference to examine the role of brain FGF2 in emotional responsiveness. METHODS: We assessed the efficacy of short-hairpin RNA (shRNA) sequences targeted to FGF2 in COS7 cells transfected with a plasmid vector containing the full-length FGF2 sequence. We then sought to assess the effects of knocking down FGF2 gene expression in vivo on behavior. We microinjected a lentiviral vector containing either a shRNA targeting FGF2 or a nonsilencing sequence bilaterally into the dentate gyrus of the rat. RESULTS: In a reporter assay system, three different shRNA sequences resulted in significant FGF2 knockdown in vitro. Five weeks following a single microinjection of one of those sequences in vivo, we observed a significant decrease in FGF2 gene expression by messenger RNA in situ hybridization in the hippocampus. The FGF2 knockdown increased the time spent in the closed arms of the elevated-plus maze, a test of anxiety behavior. CONCLUSIONS: The FGF2 knockdown in the hippocampus resulted in an anxiogenic effect. Together with our findings of an inverse correlation between anxiety and FGF2 expression levels, these results implicate FGF2 in the genesis and expression of anxiety disorders.