Brain Protein Levels Research Articles

X11 and X11‐like proteins regulate the level of extrasynaptic glutamate receptors

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Analysis of RNA Expression Profiles Identifies Dysregulated Vesicle Trafficking Pathways in Creutzfeldt-Jakob Disease.

Functional genomics applied to the study of RNA expression profiles identified several abnormal molecular processes in experimental prion disease. However, only a few similar studies have been carried out to date in a naturally occurring human prion disease. To better characterize the transcriptional cascades associated with sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, we investigated the global gene expression profile in samples from the frontal cortex of 10 patients with sCJD and 10 non-neurological controls by microarray analysis. The comparison identified 333 highly differentially expressed genes (hDEGs) in sCJD. Functional enrichment Gene Ontology analysis revealed that hDEGs were mainly associated with synaptic transmission, including GABA (q value = 0.049) and glutamate (q value = 0.005) signaling, and the immune/inflammatory response. Furthermore, the analysis of cellular components performed on hDEGs showed a compromised regulation of vesicle-mediated transport with mainly up-regulated genes related to the endosome (q value = 0.01), lysosome (q value = 0.04), and extracellular exosome (q value < 0.01). A targeted analysis of the retromer core component VPS35 (vacuolar protein sorting-associated protein 35) showed a down-regulation of gene expression (p value= 0.006) and reduced brain protein levels (p value= 0.002). Taken together, these results confirm and expand previous microarray expression profile data in sCJD. Most significantly, they also demonstrate the involvement of the endosomal-lysosomal system. Since the latter is a common pathogenic pathway linking together diseases, such as Alzheimer's and Parkinson's, it might be the focus of future studies aimed to identify new therapeutic targets in neurodegenerative diseases.

Laser capture microdissection-targeted mass spectrometry: a method for multiplexed protein quantification within individual layers of the cerebral cortex.

The mammalian neocortex is organized into layers distinguished by the size, packing density, and connectivity of their constituent neurons. Many neuropsychiatric illnesses are complex trait disorders with etiologic factors converging on neuronal protein networks. Cortical pathology of neuropsychiatric diseases, such as schizophrenia, is often restricted to, or more pronounced in, certain cortical layers, suggesting that genetic vulnerabilities manifest with laminar specificity. Thus, the ability to investigate cortical layer-specific protein levels in human postmortem brain is highly desirable. Here, we developed and validated a laser capture microdissection-mass spectrometry (LCM-MS) approach to quantify over 200 proteins in cortical layers 3 and 5 of two cohorts of human subjects as well as a monkey model of postmortem interval. LCM-MS was readily implementable and reliably identified protein patterns that differed between cortical layers 3 and 5. Our findings suggest that LCM-MS facilitates the precise quantification of proteins within individual cortical layers from human postmortem brain tissue, providing a powerful tool in the study of neuropsychiatric disease.

Zinc Chloride Exposure Inhibits Brain Acetylcholine Levels, Produces Neurotoxic Signatures, and Diminishes Memory and Motor Activities in Adult Zebrafish.

In this study, we evaluated the acute (24, 48, 72, and 96 h) and chronic (21 days) adverse effects induced by low doses (0.1, 0.5, 1, and 1.5 mg/L) of zinc chloride (ZnCl2) exposure in adult zebrafish by using behavioral endpoints like three-dimensional (3D) locomotion, passive avoidance, aggression, circadian rhythm, and predator avoidance tests. Also, brain tissues were dissected and subjected to analysis of multiple parameters related to oxidative stress, antioxidant responses, superoxide dismutase (SOD), neurotoxicity, and neurotransmitters. The results showed that ZnCl2-exposed fishes displayed decreased locomotor behavior and impaired short-term memory, which caused an Alzheimer’s Disease (AD)-like syndrome. In addition, low concentrations of ZnCl2 induced amyloid beta (amyloid β) and phosphorylated Tau (p-Tau) protein levels in brains. In addition, significant induction in oxidative stress indices (reactive oxygen species (ROS) and malondialdehyde (MDA)), reduction in antioxidant defense system (glutathione (GSH), GSH peroxidase (GSH-Px) and SOD) and changes in neurotransmitters were observed at low concentrations of ZnCl2. Neurotoxic effects of ZnCl2 were observed with significant inhibition of acetylcholine (ACh) activity when the exposure dose was higher than 1 ppm. Furthermore, we found that zinc, metallothionein (MT), and cortisol levels in brain were elevated compared to the control group. A significantly negative correlation was observed between memory and acetylcholinesterase (AChE) activity. In summary, these findings revealed that exposure to ZnCl2 affected the behavior profile of zebrafish, and induced neurotoxicity which may be associated with damaged brain areas related to memory. Moreover, our ZnCl2-induced zebrafish model may have potential for AD-associated research in the future.

RETRACTED: Curcumin alleviates lipopolysaccharide-induced neuroinflammation in fetal mouse brain

Curcumin exerts multiple functions including antioxidant and anti-inflammation, and has been shown protective potential on neurological disorders. Maternal or intrauterine infection/inflammation is one of the major factors underlying perinatal brain damage. This study aimed to determine whether maternal administration of curcumin has attenuation on neuroinflammation in fetal brain caused by lipopolysaccharide (LPS) administration. LPS was used to establish mouse fetal brain injury model, and we investigated the effects of curcumin (40 mg/kg) on the fetal mouse brain by evaluating the morphological change of the neuronal cells and the expression of different pro-inflammatory cytokines and chemokines at protein and mRNA levels in the fetal brains, the maternal serum and amniotic fluid. Our results demonstrated that maternal administration of curcumin has attenuation on neuroinflammation in the fetal brain induced by LPS. Pretreatment of curcumin in the LPS-induced mice effectively reestablished the neuronal cell morphology, attenuated the expression of soluble intercellular adhesion molecule-1, sE-Selectin, macrophage chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1 in the maternal serum, decreased the expression of cyclooxygenase-2, interleukin-1 beta and chemokine (C-C motif) ligand 2 in the brain, and suppressed interleukin-6 (IL-6) mRNA transcription in the amniotic fluid. In addition, curcumin suppressed the LPS-induced microglia activation. Our study in animal models indicates that maternal administration of curcumin alleviates neuroinflammation in the fetal brain caused by LPS. Long-term consumption of curcumin might improve the neurological outcomes of premature neonates delivered from dams suffering from infection/ inflammation.

Suppressing Interferon-γ Stimulates Microglial Responses and Repair of Microbleeds in the Diabetic Brain.

Microcirculatory damage is a common complication for those with vascular risk factors, such as diabetes. To resolve vascular insults, the brain's immune cells (microglia) must rapidly envelop the site of injury. Currently, it is unknown whether Type 1 diabetes, a condition associated with chronic immune system dysfunction, alters microglial responses to damage and what mechanisms are responsible. Using in vivo two-photon microscopy in adult male mice, we show that microglial envelopment of laser-induced cerebral microbleeds is diminished in a hyperglycemic mouse model of Type 1 diabetes, which could not be fully rescued with chronic insulin treatment. Microglia were important for vessel repair because reduced microglial accumulation in diabetic mice or near-complete depletion in healthy controls was associated with greater secondary leakage of the damaged vessel. Broadly suppressing inflammation with dexamethasone in diabetic mice but not healthy controls, significantly enhanced microglial responses to microbleeds and attenuated secondary vessel leakage. These enhancements were associated with changes in IFN-γ signaling because dexamethasone suppressed abnormally high levels of IFN-γ protein levels in brain and blood serum of diabetic mice. Further, blocking IFN-γ in diabetic mice with neutralizing antibodies restored normal microglial chemotaxic responses and purinoceptor P2ry12 gene expression, as well as mitigated secondary leakage. These results suggest that abnormal IFN-γ signaling disrupts microglial function in the diabetic brain, and that immunotherapies targeting IFN-γ can stimulate microglial repair of damaged vessels.SIGNIFICANCE STATEMENT Although Type 1 diabetes is an established risk factor for vascular complications, such as microbleeds, and is known to hinder wound healing in the body, no study has examined how diabetes impacts the brain's innate immune reparative response (involving cells called microglia) to vascular injury. Here we show that microglial responses to brain microbleeds were diminished in diabetic animals, which also exacerbated secondary leakage from damaged vessels. These impairments were related to abnormally high levels of the proinflammatory cytokine IFN-γ because reducing IFN-γ with immunosuppressant drugs or blocking antibodies helped restore normal microglial responses and repair of damaged vessels. These data highlight the use of IFN-γ modulating therapeutics to enhance vascular repair in at-risk populations.

Homocysteine-lowering gene therapy rescues signaling pathways in brain of mice with intermediate hyperhomocysteinemia

Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse cognitive dysfunction. Considering the role of the serine/threonine kinase DYRK1A, not only in developmental defects with life-long structural and functional consequences, but also in multiple neurodegenerative diseases, its protein expression and kinase activity has been analyzed in brain of heterozygous CBS deficient mice and found to be increased. We previously demonstrated that specific liver treatment with an adenovirus expressing Dyrk1A normalizes hepatic DYRK1A level and decreases hyperhomocysteinemia in mice with moderate to intermediate hyperhomocysteinemia. We here use a hepatocyte-specific recombinant adeno-associated viral (AAV) serotype 8-mediated DYRK1A gene therapy (AAV2/8-DYRK1A) to analyze the effect of hepatic Dyrk1A gene transfer on some altered molecular mechanisms in brain of mice with intermediate hyperhomocysteinemia. Our selective hepatic treatment alleviates altered DYRK1A protein level and signaling pathways in brain of mice, the MAPK/ERK and PI3K/Akt pathways initiated by receptor tyrosine kinase, the BDNF dependent TrkB pathway, and NFkB pathway. These results demonstrate the positive effect of AAV2/8-DYRK1A gene transfer on neuropathological and inflammatory processes in brain of mice with intermediate hyperhomocysteinemia.

Activation of ornithine decarboxylase protects against methylmercury toxicity by increasing putrescine

We previously reported significantly increased level of putrescine, a polyamine, in the brains of mice administered methylmercury. Moreover, addition of putrescine to culture medium reduced methylmercury toxicity in C17.2 mouse neural stem cells. In this study, the role of ornithine decarboxylase (ODC), an enzyme involved in putrescine synthesis, in response to methylmercury toxicity was investigated. Methylmercury increased ODC activity in mouse cerebrum and cerebellum, but this increase was hardly observed in the kidney and liver, where methylmercury accumulated at a high concentration. In the cerebrum and cerebellum, increased putrescine was observed with methylmercury administration. Methylmercury increased ODC activity in C17.2 cells, but this was almost completely abolished in the presence of an ODC inhibitor. Methylmercury also increased the level of ODC protein in mouse brain and C17.2 cells. In addition, C17.2 cells pretreated with ODC inhibitor showed higher methylmercury sensitivity than control cells. These results suggest that the increased ODC activity by methylmercury is involved in the increase in putrescine level, and ODC plays an important role in the reduction of methylmercury toxicity. This is the first study to provide evidence that increased ODC activity may be a protective response against methylmercury-induced neurotoxicity.

Identifying lysophosphatidic acid receptor subtype 1 (LPA1) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2

Microglia regulate immune responses in the brain, and their activation is key to the pathogenesis of diverse neurological diseases. Receptor-mediated lysophosphatidic acid (LPA) signaling has been known to regulate microglial biology, but it is still unclear which receptor subtypes guide the biology, particularly, microglial activation. Here, we investigated the pathogenic aspects of LPA receptor subtype 1 (LPA1) in microglial activation using a systemic lipopolysaccharide (LPS) administration-induced septic mouse model in vivo and LPS-stimulated rat primary microglia in vitro. LPA1 knockdown in the brain with its specific shRNA lentivirus attenuated the sepsis-induced microglia activation, morphological transformation, and proliferation. LPA1 knockdown also resulted in the downregulation of TNF-α, at both mRNA and protein levels in septic brains, but not IL-1β or IL-6. In rat primary microglia, genetic or pharmacological blockade of LPA1 attenuated gene upregulation and secretion of TNF-α in LPS-stimulated cells. In particular, the latter was associated with the suppressed TNF-α converting enzyme (TACE) activity. We reaffirmed these biological aspects using a BV2 microglial cell line in which LPA1 expression was negligible. LPA1 overexpression in BV2 cells led to significant increments in TNF-α production upon stimulation with LPS, whereas inhibiting LPA1 reversed the production. We further identified ERK1/2, but not p38 MAPK or Akt, as the underlying effector pathway after LPA1 activation in both septic brains and stimulated microglia. The current findings of the novel role of LPA1 in microglial activation along with its mechanistic aspects could be applied to understanding the pathogenesis of diverse neurological diseases that involve microglial activation.

Cerebrospinal Fluid Biomarkers Are Associated With Glial Fibrillary Acidic Protein and αII-spectrin Breakdown Products in Brain Tissues Following Penetrating Ballistic-Like Brain Injury in Rats.

Treatments to improve outcomes following severe traumatic brain injury (TBI) are limited but may benefit from understanding subacute-chronic brain protein profiles and identifying biomarkers suitable for use in this time. Acute alterations in the well-known TBI biomarkers glial fibrillary acidic protein (GFAP), αII-spectrin, and their breakdown products (BDPs) have been well established, but little is known about the subacute-chronic post-injury profiles of these biomarkers. Thus, the current study was designed to determine the extended profile of these TBI-specific biomarkers both in brain tissue and cerebral spinal fluid (CSF). Protein abundance was evaluated in brain tissue samples taken from regions of interest and in CSF at 24 h, 3 days, 7 days, 1 month, and 3 months following severe TBI in rats. Results showed increased full length GFAP (GFAP-FL) and GFAP-BDPs starting at 24 h that remained significantly elevated in most brain regions out to 3 months post-injury. However, in CSF, neither GFAP-FL nor GFAP-BDPs were elevated as a consequence of injury. Regional-specific reduction in αII-spectrin was evident in brain tissue samples from 24 h through 3 months. In contrast, SBDP-145/150 was robustly elevated in most brain regions and in CSF from 24 h through 7 days. Correlation analyses revealed numerous significant relationships between proteins in CSF and brain tissue or neurological deficits. This work indicates that TBI results in chronic changes in brain protein levels of well-known TBI biomarkers GFAP, αII-spectrin, and their BDPs and that SBDP-145/150 may have utility as an acute-chronic biomarker.

Upregulation of PSMB8 and cathepsins in the human brains of dementia with Lewy bodies

Proteasome and lysosome are responsible for the homeostasis of proteins, lipids and carbohydrates in cells. Numerous reports indicate the proteolytic pathways have altered functions during neurodegeneration and aging. Dementia with Lewy bodies (DLB) is one of the leading forms of dementia, and the proteolytic alteration in DLB has not yet been fully investigated. This study shows that the components of proteasome and lysosome had selectively altered gene expression and enzymatic functions. Specifically, PSMB8, an inducible proteasomal β subunit, had elevated mRNA level and protein level in DLB brain compared with age-matched controls. The proteasomal caspase-like peptidase showed significant decreased activity in DLB brains and the trypsin-like/chemotrypsin-like activities did not reach statistical significance. Lysosomal cathepsin B and D had elevated mRNA levels while only cathepsin B showed elevated enzymatic activity in DLB brains. This data indicate that the alteration of proteolytic pathways is highly selective and comprehensive. Further study to elucidate the correlation between neurodegenerative development and the alteration of proteolytic pathways would be important for therapeutic development.

Characteristics of Insulin-degrading Enzyme in Alzheimer's Disease: A Meta-Analysis.

β-amyloid (Aβ) accumulates abnormally to senile plaque which is the initiator of Alzheimer's disease (AD). As one of the Aβ-degrading enzymes, Insulin-degrading enzyme (IDE) remains controversial for its protein level and activity in Alzheimer's brain. The electronic databases PubMed, EMBASE, The Cochrane Library, OVID and Sinomed were systemically searched up to Sep. 20th, 2017. And the published case-control or cohort studies were retrieved to perform the meta-analysis. Seven studies for IDE protein level (AD cases = 293; controls = 126), three for mRNA level (AD cases = 138; controls = 81), and three for enzyme activity (AD cases = 123; controls = 75) were pooling together. The IDE protein level was significantly lower in AD cases than in controls (SMD = - 0.47, 95% CI [-0.69, -0.24], p < 0.001), but IDE mRNA and enzyme activity had no significant difference (SMD = 0.02, 95% CI [-0.40, 0.43] and SMD = 0.06, 95% CI [-0.41, 0.53] respectively). Subgroup analyses found that IDE protein level was decreased in both cortex and hippocampus of AD cases (SMD = -0.43, 95% CI [-0.71, -0.16], p = 0.002 and SMD = -0.53, 95% CI [-0.91, -0.15], p = 0.006 respectively). However, IDE mRNA was higher in cortex of AD cases (SMD = 0.71, 95% CI [0.14, 1.29], p = 0.01), not in hippocampus (SMD = -0.26, 95% CI [-0.58, 0.06]). Our results indicate that AD patients may have lower IDE protease level. Further relevant studies are still needed to verify whether IDE is one of the factors affecting Aβ abnormal accumulation and throw new insights for AD detection or therapy.

PR-957 mediates neuroprotection by inhibiting Th17 differentiation and modulating cytokine production in a mouse model of ischaemic stroke.

Acute ischaemic stroke can induce secondary brain injury by activating an inflammatory response that contributes to clinical impairment. As a specificinhibitor of the immunoproteasome subunit low molecular weightpolypeptide 7 (LMP7), PR-957 may participate in regulating pathophysiological and inflammatory responses in multiple diseases of the central nervous system (CNS). We investigated the neuroprotective properties of PR-957 in a mouse model of stroke, induced by middle cerebral artery occlusion (MCAO). After MCAO and injections of PR-957 or vehicle, we evaluated mice behaviourally using modified Neurological Severity Scores (mNSS) and sensorimotor tests, including the adhesive-removal test, a foot-fault test and an inclined plane test. Infarct volume was measured 24 and 72h after MCAO. Infiltration by different lymphocyte subpopulations was evaluated by flow cytometry and immunofluorescent staining of brain tissue from the penumbral area. Quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay were used to measure the expression of proinflammatory cytokines: interkeukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) protein levels in brain was measured by immunoblot. MCAO mice treated with PR-957 showed a significant decrease in infarct volume and had mild neurological deficits compared to vehicle-treated mice. PR-957 administration also significantly decreased IL-1β, IL-6, IL-12, IL-17A and TNF-α. PR-957 provides neuroprotection via inhibiting T lymphocyte infiltration and decreasing T helper type 17 (Th17) cell differentiation in MCAO mice, which may result from the reduced expression of pSTAT-3. The neuroprotective effect of PR-957 indicates its potential utility as anti-inflammatory therapy for ischaemic stroke.

PKA-CREB-BDNF signaling pathway mediates propofol-induced long-term learning and memory impairment in hippocampus of rats

Studies have found that propofol can induce widespread neuroapoptosis in developing brains, which leads to cause long-term learning and memory abnormalities. However, the specific cellular and molecular mechanisms underlying propofol-induced neuroapoptosis remain elusive. The aim of the present study was to explore the role of PKA-CREB-BDNF signaling pathway in propofol-induced long-term learning and memory impairment during brain development. Seven-day-old rats were randomly assigned to control, intralipid and three treatment groups (n = 5). Rats in control group received no treatment. Intralipid (10%, 10 mL/kg) for vehicle control and different dosage of propofol for three treatment groups (50, 100 and 200 mg/kg) were administered intraperitoneally. FJB staining, immunohistochemistry analysis for neuronal nuclei antigen and transmission electron microscopy were used to detect neuronal apoptosis and structure changes. MWM test examines the long-term spatial learning and memory impairment. The expression of PKA, pCREB and BDNF was quantified using western blots. Propofol induced significant increase of FJB-positive cells and decrease of PKA, pCREB and BDNF protein levels in the immature brain of P7 rats. Using the MWM test, propofol-treated rats demonstrated long-term spatial learning and memory impairment. Moreover, hippocampal NeuN-positive cell loss, long-lasting ultrastructural abnormalities of the neurons and synapses, and long-term down-regulation of PKA, pCREB and BDNF protein expression in adult hippocampus were also found. Our results indicated that neonatal propofol exposure can significantly result in long-term learning and memory impairment in adulthood. The possible mechanism involved in the propofol-induced neuroapoptosis was related to down-regulation of PKA-CREB-BDNF signaling pathway.

Sex‐selective effects of prior binge‐like drinking followed by intermittent stress and ethanol drinking on alterations in several brain protein levels and neurosteroids

Alcohol misuse and abuse are serious problems in our society and stress may increase the risk for escalation of drinking from recreational to problematic. We are continuing to explore the effect of prior binge drinking on subsequent drinking behaviors and the response to intermittent predator odor stress exposure, which is a model of traumatic stress. Adult male and female C57BL/6 mice had either 7 binge ethanol sessions (binge) or drank water (control). Following a 30‐day abstinence period, all mice were provided free access to 10% ethanol (10E) and water via lickometers for 4 weeks, with 30 min exposure to predator odor during the middle 2 weeks (2X/week). At 24 hrs after the final drinking session, mice were euthanized, and brains were harvested and frozen for Western Blot analyses vs separate naïve age‐matched mice. Dissected prefrontal cortex (PFC) and hippocampus (HIPP) were assayed for levels of the GABAA receptor a2 and a4 subunits, P450scc (an enzyme involved in steroidogenesis), and synaptophysin and ARC (activity‐regulated cytoskeletal protein), two proteins involved in synaptic plasticity. Western blot analyses found divergent responses across proteins and by sex. For the GABAA receptor a2 and a4 subunits, treatments (binge and control) decreased levels by 20–25% in male PFC but not HIPP versus naïve mice. Binge and control females displayed a similar decrease in PFC levels of the a4 subunit, whereas PFC levels of the a2 GABAA receptor subunit were increased by 25% compared to naïve mice. Additional sex differences were observed for the effect of the binge and control treatment groups versus naïve mice on P450scc levels: a 20% decrease in relative protein levels in male PFC versus a 15–30% increase in levels for both the female PFC and HIPP. Treatments also increased levels of synaptophysin in male and female HIPP by 20–30%, compared to naïve mice, with only the female PFC having a 30% increase in ARC levels. Alterations in relative protein levels were similar for the control and binge animals versus naïve mice, suggesting that the repeated predator odor stress and ethanol drinking had the greatest influence on assayed neuroaptations in the PFC and HIPP. Simultaneous analysis of ten plasma neurosteroid levels by gas chromatography‐mass spectrometry showed that treatment significantly increased levels of several GABAA receptor active neurosteroids in these male and female mice versus naïve animals. Taken together, these data reinforce the importance of assessing sex differences, as there were divergent neurochemical responses between males and females. These data also implicate the influence of neurosteroids on responses to the impact of prior binge‐like drinking on subsequent ethanol drinking and effects of stress.Support or Funding InformationSupported by BX001070 and BX002966 (DF) from the Department of Veteran AffairsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated monoamine oxidase A activity and protein levels in rodent brain during acute withdrawal after chronic intermittent ethanol vapor exposure

BackgroundA key component of alcohol dependence (AD), a severe form of alcohol use disorder, is the negative emotional state during withdrawal. Monoamine oxidase A (MAO-A) is an important enzyme that metabolizes monoamines and creates oxidative stress. Elevations in MAO-A level, especially in the prefrontal and anterior cingulate cortex (PFC and ACC), are associated with low mood states, including the dysphoria of early alcohol withdrawal in humans. The aim of the present study was to determine whether chronic alcohol vapor exposure causes an upregulation of MAO-A activity or level in the PFC and ACC of rodents during acute withdrawal. MethodsSprague-Dawley rats were exposed to ethanol vapor or control condition for 17 h per day for 8 weeks. MAO-A activity and protein levels were measured immediately after exposure, acute withdrawal (24 h), protracted withdrawal (4 day), and protracted abstinence (3 weeks) (n = 16/group; 8 alcohol exposed, 8 control). ResultsChronic ethanol vapor exposure significantly elevated MAO-A activity and protein levels in the PFC and ACC at 24-h withdrawal (multivariate analysis of variance (MANOVA), activity: F2,13 = 3.82, p = .05, protein: F2,13 = 5.13, p = .02). There were no significant changes in MAO-A level or activity at other timepoints. ConclusionsThe results of this study suggest a causal relationship between acute alcohol withdrawal and elevated MAO-A levels and activity, clarifying the observation of greater MAO-A binding in human alcohol withdrawal. This has important implications for developing methods of targeting MAO-A and/or sequelae of its dysregulation in alcohol dependence.

Effect of glucagon-like peptide-1 analogue; Exendin-4, on cognitive functions in type 2 diabetes mellitus; possible modulation of brain derived neurotrophic factor and brain Visfatin

BackgroundBrain derived neurotrophic factor (BDNF) is one of the most essential neurotrophic factors in the brain. BDNF is involved in learning, memory and locomotion suggesting it as a target in type 2 diabetes mellitus (T2DM) associated cognitive changes. Visfatin; an adipokine discovered to be expressed in the brain; was found to have multiple effects including its participation in keeping energy supply to the cell and is consequentially involved in cell survival. Its role in cognitive functions in T2DM was not studied before. Recent studies point to the possible neuro-protective mechanisms of glucagon-like peptide 1 analogue: Exendin-4 (Ex-4) in many cognitive disorders, but whether BDNF or Visfatin are involved or not in its neuro-protective mechanisms; is still unknown. Aimsto study the changes in cognitive functions in T2DM, either not treated or treated with Glucagon-like peptide 1 (GLP-1) analogue: Ex-4, and to identify the possible underlying mechanisms of these changes and whether BDNF and brain Visfatin are involved. MethodsA total of 36 adult male wistar albino rats were divided into 4 groups; Control, Exendin-4 control, Diabetic and Exendin-4 treated groups. At the end of the study, Y-maze and open field tests were done the day before scarification to assess spatial working memory and locomotion, respectively. Fasting glucose and insulin, lipid profile and tumor necrosis factor- alpha (TNF-α) were measured in the serum. Homeostasis model assessment insulin resistance was calculated. In the brain tissue, malondialdehyde (MDA) level, gene expression and protein levels of BDNF and Visfatin, area of degenerated neurons, area of glial cells and area % of synaptophysin immunoexpression were assessed. ResultsCompared with the control, the untreated diabetic rats showed insulin resistance, dyslipidemia and elevation of serum TNF-α. The brain tissue showed down-regulation of BDNF gene expression and reduction of its protein level, up-regulation of Visfatin gene expression and elevation of its protein level, increase in MDA, area of degenerated neurons and area of glial cells and reduction in area % of synaptophysin immunoexpression. These changes were paralleled with significant deterioration in spatial working memory and locomotion. Treatment of diabetic rats with Ex-4 reversed all these changes. ConclusionT2DM has a negative impact on cognitive functions through different pathological and subcellular mechanisms. The current study provides evidence for involvement of BDNF and brain Visfatin in T2DM- associated cognitive dysfunction. BDNF and brain Visfatin were also found to contribute to the neuro-protective effect of Ex-4 via modulation of inflammation, oxidative stress, neuro-degeneration and synaptic function.

Erythromyeloid-Derived TREM2: A Major Determinant of Alzheimer's Disease Pathology in Down Syndrome.

Background:Down syndrome (DS; trisomy 21) individuals have a spectrum of hematopoietic and neuronal dysfunctions and by the time they reach the age of 40 years, almost all develop Alzheimer’s disease (AD) neuropathology which includes senile plaques and neurofibrillary tangles. Inflammation and innate immunity are key players in AD and DS. Triggering receptor expressed in myeloid cells-2 (TREM2) variants have been identified as risk factors for AD and other neurodegenerative diseases.Objective:To investigate the effects of TREM2 and the AD-associated R47H mutation on brain pathology and hematopoietic state in AD and DS.Methods:We analyzed peripheral blood, bone marrow, and brain tissue from DS, AD, and age-matched control subjects by immunohistochemistry and western blotting. TREM2-related phagocytosis was investigated using a human myeloid cell line.Results:TREM2 protein levels in brain and sera declined with age and disease progression in DS. We observed soluble TREM2 in brain parenchyma that may be carried by a subset of microglia, macrophages, or exosomes. Two DS cases had the AD-associated TREM2-R47H mutation, which manifested a morphologically extreme phenotype of megakaryocytes and erythrocytes in addition to impaired trafficking of TREM2 to the erythroid membrane. TREM2 was shown to be involved in phagocytosis of red blood cells. TREM2 was seen in early and late endosomes. Silencing TREM2 using siRNA in THP1 cells resulted in significant cell death.Conclusion:We provide evidence that peripheral TREM2 originating from erythromyeloid cells significantly determines AD neuropathology in DS subjects. Understanding the molecular signaling pathways mediated by TREM2 may reveal novel therapeutic targets.

High-concentration sevoflurane exposure in mid-gestation induces apoptosis of neural stem cells in rat offspring.

Sevoflurane is the most commonly used volatile anesthetic during pregnancy. The viability of neural stem cells directly affects the development of the brain. However, it is unknown whether the use of sevoflurane during the second trimester affects the survival of fetal neural stem cells. Therefore, in this study, we investigated whether exposure to sevoflurane in mid-gestation induces apoptosis of neural stem cells and behavioral abnormalities. On gestational day 14, pregnant rats were anesthetized with 2% or 3.5% sevoflurane for 2 hours. The offspring were weaned at 28 days and subjected to the Morris water maze test. The brains were harvested to examine neural stem cell apoptosis by immunofluorescence and to measure Nestin and SOX-2 levels by western blot assay at 6, 24 and 48 hours after anesthesia as well as on postnatal day (P) 0, 14 and 28. Vascular endothelial growth factor (VEGF) and phosphoinositide 3-kinase (PI3K)/AKT pathway protein levels in fetal brain at 6 hours after anesthesia were assessed by western blot assay. Exposure to high-concentration (3.5%) sevoflurane during mid-gestation increased escape latency and path length to the platform, and it reduced the average duration spent in the target quadrant and platform crossing times. At 6, 24 and 48 hours after anesthesia and at P0, P14 and P28, the percentage of Nestin/terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells was increased, but Nestin and SOX-2 protein levels were decreased in the hippocampus of the offspring. At 6 hours after anesthesia, VEGF, PI3K and phospho-AKT (p-AKT) levels were decreased in the fetal brain. These changes were not observed in animals given low-concentration (2%) sevoflurane exposure. Together, our findings indicate that exposure to a high concentration of sevoflurane (3.5%) in mid-gestation decreases VEGF, PI3K and p-AKT protein levels and induces neural stem cell apoptosis, thereby causing learning and memory dysfunction in the offspring.

English

The study evaluated the biochemical effects of methanolic seed extract of Aframomum melegueta (MEAM) on biochemical parameters of the brain of rats treated with monosodium glutamate (MSG) with a view to considering the possibility of using the plant as a remedy for the management of neurotoxic disorders. MEAM was prepared according to standard methods, phytochemically screened and followed by evaluation of its antioxidant and anti-inflammatory potentials as well as its biological activities. Phytochemical screening of the methanolic extract (ME) revealed the presence of alkaloids, cardiac glycosides, flavonoids, saponins, steroids, tannins and terpenoids. Administration of MSG (2 g/kg bwt) caused alterations in the levels of glutathione, nitric oxide, Vitamin C and E, as well as in the reduction of activities of enzymatic antioxidants. There were increases in the activities of brain marker enzymes, protein and peroxidation levels. Histopathological observations of brain sections revealed that MEAM protected the brain from glutamate-induced neurotoxicity. Conclusively, extract of A. melegueta elicited appreciable and potent neuroprotective potentials against neuro-degeneration caused by MSG oral administration, in a concentration dependent manner. Key words: Neurotoxicity, Aframomum melegueta, methanolic extract, monosodium glutamate, antioxidant enzymes, neurotransmitters, brain biomarkers.