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Lactate's role in the brain is understood as a contributor to brain energy metabolism, but it may also regulate the cerebral microcirculation. The purpose of this systematic review was to evaluate evidence of lactate as a physiological effector within the normal cerebral microcirculation in reports ranging from in vitro experiments to in vivo studies in animals and humans. Following pre‐registration of a review protocol, we systematically searched the PubMed, EMBASE, and Cochrane databases for literature covering themes of 'lactate', 'the brain', and 'microcirculation'. Abstracts were screened, and data extracted independently by two individuals. We excluded studies evaluating lactate in disease models. Twenty‐eight papers were identified, 18 of which were in vivo animal experiments (65%), four on human studies (14%), and six on in vitro or ex vivo experiments (21%). Approximately half of the papers identified lactate as an augmenter of the hyperemic response to functional activation by a visual stimulus or as an instigator of hyperemia in a dose‐dependent manner, without external stimulation. The mechanisms are likely to be coupled to NAD+/NADH redox state influencing the production of nitric oxide. Unfortunately, only 38% of these studies demonstrated any control for bias, which makes reliable generalizations of the conclusions insecure. This systematic review identifies that lactate may act as a dose‐dependent regulator of cerebral microcirculation by augmenting the hyperemic response to functional activation below 5 mmol/kg, and by initiating a hyperemic response above 5 mmol/kg. Open Science Badges: This article has received a badge for *Pre‐registration* because it made the data publicly available. The data can be accessed at www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer%5flactate%5fprotocol.aspx. 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/. This systematic review evaluated the role of lactate in the cerebral microcirculation across in vitro, in vivo, and clinical studies. We identified that when administered exogenously, at 5 mmol/kg or higher, lactate is an initiator of cerebral hyperemia and acts as an augmenter of functional hyperemia at lower doses. These findings shine light on novel aspects of lactate biochemistry in the brain. This manuscript was awarded with the Pre‐registration Badge. The pre‐registration protocol can be accessed at http://www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer%5flactate%5fprotocol.aspx. For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 672-680. Abstract Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls. [ABSTRACT FROM AUTHOR]

A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. Here, we report a procedure to record for the first time the release of endogenous amines –dopamine, serotonin and octopamine‐ in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, we carried out recordings in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, we detect an increase in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and shed new light on the contribution of these amines to olfactory processing in this animal model. [ABSTRACT FROM AUTHOR]

In adult rats, omega‐6 linoleic acid (LA, 18:2n‐6) serves as a precursor to oxidized LA metabolites (OXLAMs) known to regulate multiple signaling processes in the brain. However, little is known regarding the levels or role(s) of LA and its metabolites during brain development. To address this gap, fatty acids within various brain lipid pools, and their oxidized metabolites (oxylipins) were quantified in brains from 1‐day‐old male and female pups using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. Primary neuron‐glia co‐cultures derived from postnatal day 0–1 male and female rat neocortex were exposed to vehicle (0.1% ethanol), LA, the OXLAM 13‐hydroxyoctadecadienoic acid (13‐HODE), or prostaglandin E2 at 10–1000 nM for 48 h to test their effects on neuronal morphology. In both male and female pups, LA accounted for 1–3% of fatty acids detected in brain phospholipids and cholesteryl esters. It was not detected in triacylglycerols, and free fatty acids. Unesterified OXLAMs constituted 47–53% of measured unesterified oxylipins in males and females (vs. ~5–7% reported in adult rat brain). Of these, 13‐HODE was the most abundant, accounting for 30–33% of measured OXLAMs. Brain fatty acid and OXLAM concentrations did not differ between sexes. LA and 13‐HODE significantly increased axonal outgrowth. Separate analyses of cultures derived from male versus female pups revealed that LA at 1, 50, and 1000 nM, significantly increased axonal outgrowth in female but not male cortical neurons, whereas 13‐HODE at 100 nM significantly increased axonal outgrowth in male but not female cortical neurons. prostaglandin E2 did not alter neuronal outgrowth in either sex. This study demonstrates that OXLAMs constitute the majority of unesterified oxylipins in the developing rat brain despite low relative abundance of their LA precursor, and highlights a novel role of LA and 13‐HODE in differentially influencing neuronal morphogenesis in the developing male and female brain. [ABSTRACT FROM AUTHOR]

High‐mobility group box‐1 (HMGB‐1) acts as a pro‐inflammatory cytokine contributing to the occurrence of many central inflammatory and infectious disorders. Brain mast cells (MCs) are the first responders to peripheral inflammatory stimulation because of their rapid response to external stimuli coupled with their release of preformed and newly synthesized reactive chemicals. Little is known about the involvement of brain MCs in the pro‐inflammatory effects of HMGB‐1 on the central nervous system (CNS). Thus, we investigated the activation process of MCs by HMGB‐1 and explored whether this process is involved in the pro‐inflammatory effects of HMGB‐1 on the CNS. In this study, we used P815 cells to study the activating role of HMGB‐1 on MCs and to explore its potential mechanism in vitro. In an in vivo study, adult male Sprague‐Dawley rats received i.c.v. injection of sterile saline or cromoglycate (stabilizer of MCs) 30 min prior to i.p. injection of HMGB‐1. Increased levels of tumor necrosis factor and IL‐1β were observed in the P815 cells, as well as in the rats' brains, after HMGB‐1 treatment. Pretreatment with the receptor of advanced glycation endproducts (RAGE)‐siRNA inhibited the HMGB‐1‐induced inflammatory process in the P815 cells. Activation of the RAGE/nuclear factor‐κB (NF‐κB) pathway was observed in both the P815 cells and rats' brains. In addition, HMGB‐1 induced the accumulation of brain MCs in the hippocampal CA1 region, and the blood–brain barrier was disrupted. Pretreatment with cromoglycate, a stabilizer of MCs, mitigated these HMGB‐1‐induced pro‐inflammatory processes in rats. These findings indicate that brain MCs are involved in the pro‐inflammatory effect of HMGB‐1 on the CNS, probably via activating the RAGE/NF‐κB pathway. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidoses are lysosomal storage disorders characterised by accumulation of abnormal pathological glycosaminoglycans, cellular dysfunction and widespread inflammation, resulting in progressive cognitive and motor decline. Lysosomes are important mediators of immune cell function, and therefore accumulation of glycosaminoglycans (GAGs) and other abnormal substrates could affect immune function and directly impact on disease pathogenesis. This review summarises current knowledge with regard to inflammation in mucopolysaccharidosis, with an emphasis on the brain and outlines a potential role for GAGs in induction of inflammation. We propose a model by which the accumulation of GAGs and other factors may impact on innate immune signalling with particular focus on the Toll‐like receptor 4 pathway. Innate immunity appears to have a dominating role in mucopolysaccharidosis; however, furthering understanding of innate immune signalling would have significant impact on highlighting novel anti‐inflammatory therapeutics for use in mucopolysaccharide diseases. This article is part of the Special Issue "Lysosomal Storage Disorders". Mucopolysaccharidoses are lysosomal storage disorders characterised by accumulation of abnormal glycosaminoglycans, cellular dysfunction and widespread inflammation. Lysosomes are critical mediators of immunity, and therefore lysosomal substrate accumulation could affect immune function and directly impact on disease pathogenesis. In this review, we summarise the current knowledge with regard to inflammation in mucopolysaccharidosis with an emphasis on the brain. We propose a model by which the accumulation of GAGs may impact on innate immune signalling with particular focus on the TLR4 pathway. Furthering our understanding of inflammatory signalling in lysosomal storage disease would have significant impact on highlighting novel anti‐inflammatory therapeutics. This article is part of the Special Issue "Lysosomal Storage Disorders". [ABSTRACT FROM AUTHOR]

Opiate drugs, such as codeine, morphine, and heroin, are powerful analgesics, but also are used as drugs of abuse because of their psychogenic properties. Many studies have shown that opiates impact on cellular proliferation in the adult and developing brain, although anatomical pathologies are lacking in in utero exposed infants and opioid knockout mice. Recent research has defined a context-dependent role for the opioid system in neurogenesis in the adult hippocampus with exercise. Opioids have been shown to interact with proliferating cells of the postnatal subventricular zone of the lateral ventricles. The subventricular zone is also a region of adult neurogenesis, a fact that was not well established at the time this earlier research was conducted. Although a relationship between opioids and fetal neurogenesis has yet to be firmly established, many studies have implicated the opioid system in this process. One common factor that links neurogenesis in adult, postnatal, and fetal structures is the involvement of neuronal progenitor cells of the astrocytic lineage. It is therefore of interest that opioids have been consistently shown to impact upon astrocytic proliferation. It is the intention of this paper to review the literature that has established a role for the opioid system in neurogenesis in vivo in fetal, postnatal, and adult animals and to examine the links of opioids to modulation of astrocytic proliferation. [ABSTRACT FROM AUTHOR]

Aggregation of α-synuclein, an abundant and conserved pre-synaptic brain protein, is implicated as a critical factor in several neurodegenerative diseases. These diseases, known as synucleinopathies, include Parkinson’s disease, dementia with Lewy bodies (LBs), diffuse LB disease, the LB variant of Alzheimer’s disease, multiple system atrophy, and neurodegeneration with brain iron accumulation type I. Although the precise nature of in vivoα-synuclein function remains elusive, considerable knowledge has been accumulated about its structural properties and conformational behavior. α-Synuclein is a typical natively unfolded protein. It is characterized by the lack of rigid, well-defined, 3-D structure and possesses remarkable conformational plasticity. The structure of this protein depends dramatically on its environment and it accommodates a number of unrelated conformations. This paper provides an overview of the biochemistry, biophysics, and neuropathology of α-synuclein aggregation. [ABSTRACT FROM AUTHOR]

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine ( O-Glc NAc) regulates protein homeostasis. The O-Glc NAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-Glc NAc transferase- and O-Glc NAcase-knockout alleles have helped define the role O-Glc NAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-Glc NAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-Glc NAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-Glc NAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an ' O-Glc NAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-Glc NAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-Glc NAc in the physiology of the brain and its susceptibility to neurodegenerative injury. [ABSTRACT FROM AUTHOR]

Presents abstracts of papers featured in the "Journal of Neurochemistry" that deal with proliferation, survival and differentiation processes in cells. "Influence of prenatal irradiation by 131-I on the heparin-binding activity of newborn rat brain proteins"; Cannabinoid inhibition of neuronal differentiation".

Selective serotonin reuptake inhibitors ( SSRIs) are the most frequently prescribed antidepressants. However, a major concern is their delayed onset of action, which is hypothesized to be associated with the time required for serotonin (5- HT) autoreceptors to desensitize, which should be reflected by actual neurochemical changes. Numerous in vivo microdialysis studies have been published that report on 5- HT levels in different brain sites following SSRI administration. Here, we performed a meta-analysis on dynamic changes of 5- HT neurotransmission during the course of chronic SSRI treatment. We conducted a meta-analysis on research articles of 5- HT neurotransmission measured by in vivo microdialysis in rat brain after subchronic and chronic SSRI administrations. In total, data from 42 microdialysis studies (798 rats) were analyzed. Within the first week of SSRI treatment, extracellular 5- HT concentrations drop in frontal cortex. Over the next 2 weeks of treatment, a linear increase in extracellular 5- HT levels up to 350% of prior treatment baseline is evident ( n = 269). However, in hippocampus, prefrontal cortex, nucleus accumbens, and ventral tegmental area we found increased 5- HT levels within the first 3 days of SSRI administration. The time course of 5- HT dynamics in frontal cortex is in line with the hypothesis that 5- HT autoreceptors desensitize over 2-3 weeks of SSRI treatment and thereby enhanced extracellular 5- HT levels ensue. Yet, in other regions we did not find evidence supporting the traditional autoreceptor-mediated feedback loops hypothesis and thus other neurobiological adaptation mechanisms may also play a role in the delayed onset of SSRI action. [ABSTRACT FROM AUTHOR]

Organophosphorus ( OP) insecticides are pest-control agents heavily used worldwide. Unfortunately, they are also well known for the toxic effects that they can trigger in humans. Clinical manifestations of an acute exposure of humans to OP insecticides include a well-defined cholinergic crisis that develops as a result of the irreversible inhibition of acetylcholinesterase ( AChE), the enzyme that hydrolyzes the neurotransmitter acetylcholine ( ACh). Prolonged exposures to levels of OP insecticides that are insufficient to trigger signs of acute intoxication, which are hereafter referred to as subacute exposures, have also been associated with neurological deficits. In particular, epidemiological studies have reported statistically significant correlations between prenatal subacute exposures to OP insecticides, including chlorpyrifos, and neurological deficits that range from cognitive impairments to tremors in childhood. The primary objectives of this article are: (i) to address the short- and long-term neurological issues that have been associated with acute and subacute exposures of humans to OP insecticides, especially early in life (ii) to discuss the translational relevance of animal models of developmental exposure to OP insecticides, and (iii) to review mechanisms that are likely to contribute to the developmental neurotoxicity of OP insecticides. Most of the discussion will be focused on chlorpyrifos, the top-selling OP insecticide in the United States and throughout the world. These points are critical for the identification and development of safe and effective interventions to counter and/or prevent the neurotoxic effects of these chemicals in the developing brain. This is an article for the . [ABSTRACT FROM AUTHOR]

Accumulation of ceramide is implicated in mediating the cellular responses to stress and aberrant sphingolipid metabolism is frequently associated with metabolic and neurodegenerative diseases. It is often assumed that (i) peripheral disturbances in sphingolipid concentrations are reflective of processes occurring in the brain, or (ii) circulating sphingolipids directly influence cerebral sphingolipid abundance. In order to address these assumptions, this study explores, in a physiological system, the metabolic pathways regulating sphingolipid metabolism in the brain and plasma of mice. Male C57Bl/6 were maintained on a low fat (control diet) or saturated fat enriched ( SFA) diet with, or without the provision of sphingolipid modulating agents. Following 6 months of feeding, the abundance of seven sphingolipid classes was assessed by LC- ESI- MS/ MS in the hippocampus ( HPF), cerebral cortex ( CTX), and plasma. Long-term consumption of the SFA diet increased ceramide and dihydroceramide in the plasma. Inhibiting de novo synthesis ameliorated this effect, while inhibition of acidic sphingomyelinase, or the sphingosine-1-phosphate receptor agonist did not. SFA feeding did not influence sphingolipid levels in either the HPF or CTX. De novo synthesis inhibition reduced ceramide in the CTX, while treatment with a sphingosine-1-phosphate receptor agonist reduced ceramides in the HPF. Analysis of the individual ceramide species revealed the effects were chain-length dependent. Both positive and negative correlations were observed between plasma and HPF/ CTX ceramide species. The findings in this study show that HPF and CTX sphingolipid concentration are influenced by distinct pathways, independent of peripheral sphingolipid concentration. [ABSTRACT FROM AUTHOR]

The endocannabinoid system shifts energy balance toward storage and fat accumulation, especially in the context of diet-induced obesity. Relatively little is known about factors outside the central nervous system that may mediate the effect of high-fat diet ( HFD) on brain endocannabinoid levels. One candidate is the liver fatty acid binding protein ( FABP1), a cytosolic protein highly prevalent in liver, but not detected in brain, which facilitates hepatic clearance of fatty acids. The impact of Fabp1 gene ablation ( LKO) on the effect of high-fat diet ( HFD) on brain and plasma endocannabinoid levels was examined and data expressed for each parameter as the ratio of high-fat diet/control diet. In male wild-type mice, HFD markedly increased brain N-acylethanolamides, but not 2-monoacylglycerols. LKO blocked these effects of HFD in male mice. In female wild-type mice, HFD slightly decreased or did not alter these endocannabinoids as compared with male wild type. LKO did not block the HFD effects in female mice. The HFD-induced increase in brain arachidonic acid-derived arachidonoylethanolamide in males correlated with increased brain-free and total arachidonic acid. The ability of LKO to block the HFD-induced increase in brain arachidonoylethanolamide correlated with reduced ability of HFD to increase brain-free and total arachidonic acid in males. In females, brain-free and total arachidonic acid levels were much less affected by either HFD or LKO in the context of HFD. These data showed that LKO markedly diminished the impact of HFD on brain endocannabinoid levels, especially in male mice. [ABSTRACT FROM AUTHOR]

Liver fatty acid-binding protein ( FABP1, L- FABP) has high affinity for and enhances uptake of arachidonic acid ( ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids ( EC) such as arachidonoylethanolamide ( AEA) and 2-arachidonoylglycerol (2- AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins ( FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2- AG was examined in wild-type ( WT) and FABP1 null ( LKO) male mice. LKO increased brain levels of AA-containing EC ( AEA, 2- AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non- ARA that contain potentiating endocannabinoids ( EC*) such as oleoyl ethanolamide ( OEA), PEA, 2- OG, and 2- PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non- ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis ( NAPEPLD, DAGLα) or cytosolic EC chaperone proteins ( FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors ( CB1, TRVP1). These data show for the first time that the non- CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids ( AEA, 2- AG) as well as their non- ARA potentiating endocannabinoids. [ABSTRACT FROM AUTHOR]

Hyperexcitability of the hippocampus is a commonly observed phenomenon in the years preceding a diagnosis of Alzheimer's disease ( AD). Our previous work suggests a dysregulation in glutamate neurotransmission may mediate this hyperexcitability, and glutamate dysregulation correlates with cognitive deficits in the rTg(TauP301L)4510 mouse model of AD. To determine whether improving glutamate regulation would attenuate cognitive deficits and AD-related pathology, TauP301L mice were treated with riluzole (~ 12.5 mg/kg/day p.o.), an FDA-approved drug for amyotrophic lateral sclerosis that lowers extracellular glutamate levels. Riluzole-treated TauP301L mice exhibited improved performance in the water radial arm maze and the Morris water maze, associated with a decrease in glutamate release and an increase in glutamate uptake in the dentate gyrus, cornu ammonis 3 ( CA3), and cornu ammonis 1 ( CA1) regions of the hippocampus. Riluzole also attenuated the TauP301L-mediated increase in hippocampal vesicular glutamate transporter 1, which packages glutamate into vesicles and influences glutamate release; and the TauP301L-mediated decrease in hippocampal glutamate transporter 1, the major transporter responsible for removing glutamate from the extracellular space. The TauP301L-mediated reduction in PSD-95 expression, a marker of excitatory synapses in the hippocampus, was also rescued by riluzole. Riluzole treatment reduced total levels of tau, as well as the pathological phosphorylation and conformational changes in tau associated with the P301L mutation. These findings open new opportunities for the development of clinically applicable therapeutic approaches to regulate glutamate in vulnerable circuits for those at risk for the development of AD. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid ( LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1- LPA6. LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [35S] GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. Ma LPA1-null mice (a variant of LPA1-null) lack [35S] GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI- IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. [ABSTRACT FROM AUTHOR]

The article presents abstracts of research papers on demyelination. They include "Acute depletion of glutathione leads to carbonylation of rat brain proteins," "Mechanism of protein s-nitrosylation in rat spinal cord," "Immediate early gene expression patterns map neuronal responses to cuprizone-induced demyelination," and "Lipoic acid inhibits expression of ICAM-1 and VCAM-1 by CNS endothelial cells and T cell migration into the spinal cord in EAE."

This study evaluated the developmental gene and protein expression of proton-coupled oligopeptide transporters ( POTs: peptide transporter, PepT1 and PepT2; peptide-histidine transporter, PhT1 and PhT2) in different regions of rodent brain, and the age-dependent uptake of a POT substrate, glycylsarcosine (GlySar), in brain slices. Slices were obtained from cerebral cortex, cerebellum and hippocampus of wildtype and PepT2 null mice, and from rats at different ages. Gene and protein expression were determined by real-time PCR and immunoblot analyses. Brain slice uptakes of radiolabeled glycylsarcosine were determined in the absence and presence of excess unlabeled glycylsarcosine or l-histidine, the latter being an inhibitor of PhT1/2 but not PepT1/2. As PepT2 and PhT1 transcripts were abundantly expressed in all three regions of mouse brain, little to no expression was observed for PepT1 and PhT2. PhT1 protein was present in brain regions of adult but not neonatal mice and expression levels increased with age in rats. Glycylsarcosine uptake, inhibition and transporter dominance did not show regional brain or species differences. However, there were clear age-related differences in functional activity, with PepT2 dominating in neonatal mice and rats, and PhT1 dominating in adult rodents. These developmental changes may markedly impact the neural activity of both endogenous and exogenous (drug) peptides/mimetics. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 576–586. Alzheimer’s disease (AD) is characterized by the presence of extracellular deposits referred to beta-amyloid (Aβ) complexes or senile plaques. Aβ peptide is firstly produced as monomers, readily aggregating to form multimeric complexes, of which the smallest aggregates are known to be the most neurotoxic. In AD patients, abundant reactive microglia migrate to and surround the Aβ plaques. Though it is well known that microglia are activated by Aβ, little is known about the peptide conformation and the signaling cascades responsible for this activation. In this study, we have stimulated murine microglia with different Aβ(1-42) forms, inducing an inflammatory state, which was peptide conformation-dependent. The lightest oligomeric forms induced a more violent inflammatory response, whereas the heaviest oligomers and the fibrillar conformation were less potent inducers. BocMLF, a formylpeptide chemotactic receptor 2 antagonist, decreased the oligomeric Aβ-induced inflammatory response. The Aβ-induced signal transduction was found to depend on phosphorylation mechanisms mediated by MAPKs and on activator protein 1/nuclear factor kappa-light-chain-enhancer of activated B cells pathways activation. These results suggest that the reactive microgliosis intensity during AD might depend on the disease progression and consequently on the Aβ conformation production. The recognition of Aβ by the formylpeptide chemotactic receptor 2 seems to be a starting point of the signaling cascade inducing an inflammatory state. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 150–159. A previous study from our laboratory demonstrated the presence within the rat striatum of dopaminergic terminals in different dynamical states, determined at least in part by the extent to which terminals are subject to autoinhibition. The present study is designed to test the hypothesis that heterogeneity in the basal tonic extracellular dopamine concentration contributes to the variable extent of autoinhibition. We probed basal extracellular dopamine concentrations using a previously demonstrated strategy that utilizes intrastriatal microinfusion of kynurenate, a substance that according to voltammetric measurements decreases extracellular dopamine from its basal concentration. In the striatum, however, we find that the response to kynurenate infusion is itself heterogeneous, allowing a broad classification of sites within the striatum as kynurenate-insensitive and kynurenate-sensitive, respectively. These newly identified kynurenate-insensitive and sensitive sites yield substantially and significantly different evoked dopamine release as measured by voltammetry during electrical stimulation of the medial forebrain bundle. Our findings confirm the hypothesis that heterogeneity in the local basal concentration of dopamine is responsible for the variable extent of autoinhibition within the striatum and support the conclusion that the steady state and dynamical components of extracellular dopamine in this brain region are coupled. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 113, 1426–1435. The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current ( Ig), which was half maximal ( KM) at 74 μM (EAAT3) or at 4 μM (EAAT4) glutamate. Co-expression of constitutively active γR70QAMPK or of wild type AMPK did not affect KM but significantly decreased the maximal Ig in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant αK45RAMPK [α1(K45R)β1γ1] did not appreciably affect Ig. According to confocal microscopy and chemiluminescence co-expression of γR70QAMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na+-coupled glutamate transport. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 10.1111/j.1471-4159.2009.06544.x The brain forms a vascular barrier system comprised of the blood–brain barrier (BBB) and the blood–CSF barriers. Together they prevent the passage of a number of drugs from the bloodstream into the brain parenchyma, because their molecules are either hydrophilic, too large or both. In many disorders affecting the CNS, these barriers are physically intact, which limits the entry of large molecules with potentially important therapeutic implications. The BBB is the most relevant barrier against drug delivery to the brain as the area of the BBB is about 1000 times larger than that of the blood–CSF barrier. Moreover, the transport through the choroid plexus is directed to the ventricular system, only allowing the transported molecules to access cells near the ventricular and subarachnoid surfaces. This review outlines possible routes for targeted entry of macromolecules like polypeptides, siRNA and cDNA. In the vascular compartment, targeting molecules should interact specifically with proteins expressed exclusively by these barrier cells, and therefore prevent uptake elsewhere in the body. Preferably, the targeting molecule should be conjugated to a drug carrier that allows uptake of a defined cargo. However, evidence for transport of such targetable drug-carrier complexes through the barriers, in particular the BBB, is contentious, and is discussed with emphasis on the different attempts that have evinced transport through the BBB not only from blood-to-endothelium, but also from endothelium-to-brain. [ABSTRACT FROM AUTHOR]

In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how certain ligands and drugs are transported across the blood-brain barrier and choroid plexus out of brain and CSF. In the CNS, there are several separate efflux transporters with very broad specificity that are responsible for much of the efflux transport. This review focuses on three such transporters: organic acid transporter-3, peptide transporter-2 and P-glycoprotein for which there is substantial new information including ‘knockout’ models in mice and, in one case, dogs. Moreover, the structural biology and transport mechanism of P-glycoprotein at 3.8 angstroms is described. The overall objective is to show how this new knowledge provides a more thorough understanding (e.g., of molecular mechanisms) of efflux transport and in several cases leads to clinically relevant information that allows better treatment of certain CNS disorders (e.g., meningitis and brain cancer). [ABSTRACT FROM AUTHOR]

Analysis of brain microtubule protein from patients with Alzheimer’s disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function. [ABSTRACT FROM AUTHOR]

Recombinant baculoviral vectors efficiently transduce several types of cells in the brain. To characterize host responses to virus challenge, thus verifying the suitability of using baculovirus for the development of gene therapy strategies in the central nervous system, we used cDNA microarray technology to examine in vitro and in vivo global cellular gene expression profiles in the rat brain, cultured human astrocytes and human neuronal cells after viral transduction. We demonstrated that the transduction induced host antiviral responses as a major reaction in all three types of samples profiled. The related genes were mainly those associated with innate immunity, including several of the genes involved in Toll-like receptor signaling pathway and cytokine-cytokine receptor interaction. These findings should be useful in understanding the molecular basis for neural cell response to baculoviral transduction and in guiding rational therapeutic applications of baculoviral vectors in the central nervous systems. [ABSTRACT FROM AUTHOR]

The learning process sets in motion a prolonged, reproducible, and complicated pattern of brain activation, which provides information about biochemical reactions in activated brain. Study of this pattern during one-trial aversive bead discrimination in day-old chick is facilitated by precise timing of sequential metabolic events occurring between a 10-s learning period, in which the chicks learn to associate a red bead with aversive taste, and memory consolidation, indicated by unwillingness to peck at untainted red beads while freely pecking at corresponding blue beads. Inhibition of learning by metabolic inhibitors and restoration of memory by specific substrates at specific times allow determination of specific metabolic events and their neuronal or astrocytic localization. Downstream metabolism of glycogen and of glucose to pyruvate/lactate is segregated into separate pools. Glucose metabolism via pyruvate dehydrogenation provides energy in both neurons and astrocytes and may include gap junction-mediated lactate transport into astrocytes. A key role is played by glycogenolysis, stimulated by β2-adrenergic and/or 5-HT2-receptor stimulation along with α2-adrenergic stimulation of glycogen synthesis. The importance of glycogen reflects that it selectively supports de novo synthesis of transmitter glutamate by combined pyruvate dehydrogenation and carboxylation in astrocytes. [ABSTRACT FROM AUTHOR]

Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N-terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research. [ABSTRACT FROM AUTHOR]

GABAA receptor function is involved in regulating proliferation, migration, and differentiation of rodent neural progenitor cells (NPCs). However, little is known about the molecular composition and functional relevance of GABAA receptors in human neural progenitors. Here, we investigated human fetal midbrain-derived NPCs in respect to their GABAA receptor function and subunit expression using electrophysiology, calcium imaging, and quantitative real-time PCR. Whole-cell recordings of ligand- and voltage-gated ion channels demonstrate the ability of NPCs to generate action potentials and to express functional GABAA receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular characterizations indicate a predominance of GABAA receptor heteromers containing subunits α2, β1, and/or β3, and γ. Intracellular Ca2+ measurements and the expression profile of the Na+–K+–Cl− co-transporter 1 and the K+–Cl− co-transporter 2 in differentiated NPCs suggest that GABA evokes depolarizations mediated by GABAA receptors. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential GABAA receptor properties during neuronal maturation in vitro. [ABSTRACT FROM AUTHOR]

The mechanisms underlying developmental myelination have therapeutic potential following CNS injury and degeneration. We report that transplanted central glial (CG)-4 cells had a diminished myelinating capacity in myelin-deficient ( md) rats when cells express a mutated form of Tau (Tau [688]), which binds Fyn but not the microtubules. In the brain of the md rats, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth and myelination; in the spinal cord the extent of myelination rostral and caudal to the injection site was decreased. In contrast, control Tau [605]-transfected CG-4 cells formed long cellular processes and substantial areas of myelin both in the brain and spinal cord. In culture, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth, and Fyn localized largely in the cell body, not the processes. Thus, Tau in oligodendrocytes plays a key role in myelination, and a functional Tau-Fyn interaction might have therapeutic potential during demyelination and myelin repair following CNS injury and degeneration. [ABSTRACT FROM AUTHOR]

This study utilised the pond snail, Lymnaea to examine the contribution that alterations in serotonergic signalling make to age-related changes in feeding. Age-related decreases in 5-HIAA levels in feeding ganglia were positively correlated with a decrease in the number of sucrose-evoked bites and negatively correlated with an increase in inter-bite interval, implicating alterations in serotonergic signalling in the aged phenotype. Analysis of the serotonergic cerebral giant cell (CGC) input to the protraction motor neurone (B1) demonstrated that fluoxetine (10–100 nM) increased the amplitude/duration of the evoked EPSP in both young and middle aged but not in old neurones, suggesting an age-related attenuation of the serotonin transporter. 5-HT evoked a concentration-dependent increase in the amplitude/duration of B1 EPSP, which was greater in old neurones compared to both young and middle aged. Conversely, the 5-HT-evoked depolarisation and conditional bursting of the swallow motor neurone (B4) were attenuated in old neurones, functions critical for a full feeding rhythm. The CGCs’ ability to excite B1 was blocked by cinanserin but not by methysergide. Conversely, the CGC to B4 connection was completely blocked by methysergide and only partially by cinanserin suggesting that age-related changes may be receptor-specific. In summary, synapse-specific attenuation of the CGC-B4 connection and enhancement of the CGC-B1 connection would slow the swallow phase and maintain protraction, consistent with behavioural observations. [ABSTRACT FROM AUTHOR]

The consequences of maternal linolenic acid (LNA, 18:3n-3) dietary deficiency on key dopamine (DA)-associated regulatory proteins in mesolimbic and mesocortical structures of the postnatal rat brain have been investigated. A marked (4.5-fold) decrease of the DA-synthesizing enzyme tyrosine hydroxylase accompanied by a down-regulation (approx 7.5-fold) of the vesicular monoamine transporter (VMAT-2) and a depletion of VMAT-associated vesicles in the hippocampus were observed in deficient offspring compared with adequately fed controls. The DA transporter (DAT) was not affected by the LNA deficiency indicative of a DAT/VMAT-2 ratio increase that may enhance the risk of damage of the dopaminergic (DAergic) terminal. A robust increase in DA receptor (DAR1 and DAR2) levels was noticed in the cortex and striatum structures possibly to compensate for the low levels of DA in synaptic clefts. Microglia activation characterized by enhanced levels of ED1 antibody and nuclear internalization of p65 NFκB was noticed following LNA deficiency. Diminished levels of 22:6n-3 docosahexaenoic acid ( Schiefermeier and Yavin 2002 ), the most ubiquitous metabolite generated by LNA is proposed to reduce the anti-oxidant arsenal in the developing brain and cause microglia activation and enhanced oxidative stress to increase the risk of certain DA-associated neurological disorders. [ABSTRACT FROM AUTHOR]

P25α is a protein normally expressed in oligodendrocytes and subcellular relocalization of p25α occurs in multiple system atrophy, Parkinson’s disease and Lewy body dementia along with ectopic expression in neurons. Moreover, it accumulates in Lewy body inclusions with aggregated α-synuclein and is a potent stimulator of α-synuclein aggregation. P25α is a phosphoprotein and post-translational modifications (PTMs) may play a role in its disease-related abnormalities. To investigate the spectrum of PTMs on p25α we cloned porcine p25α and isolated the protein from porcine brain. Using several complementary tandem mass spectrometry techniques for peptide mass analysis and amino acid sequencing, a comprehensive analysis of the PTMs on porcine p25α was performed. It was found that porcine p25α is heavily modified with a variety of modifications: phosphorylation, di- and trimethylation, citrullination and a HexNAc group. The modifications are localized within p25α’s unfolded terminal domains and suggest that their functional states are regulated. This comprehensive mapping of p25α’s PTMs will form the basis for future functional studies and investigations of p25α’s potential role as a biomarker. [ABSTRACT FROM AUTHOR]

Brain defective cholesterol homeostasis has been associated with neurologic diseases, such as Alzheimer’s and Huntington’s disease. The elimination of cholesterol from the brain involves its conversion into 24(S)-hydroxycholesterol by CYP46A1, and the efflux of this oxysterol across the blood–brain barrier. Herein, we identified the regulatory elements and factors involved the human CYP46A1 expression. Functional 5′deletion analysis mapped a region spanning from nucleotides -236/-64 that is indispensable for basal expression of this TATA-less gene. Treatment of SH-SY5Y cells with mithramycin A resulted in a significant reduction of promoter activity, suggesting a role of Sp family of transcription factors in CYP46A1 regulation. Combination of Sp1, Sp3, and Sp4 over-expression studies in Drosophila SL-2 cells, and systematic promoter mutagenesis identified Sp3 and Sp4 binding to four GC-boxes as required and sufficient for high levels of promoter activity. Moreover, Sp3 and Sp4 were demonstrated to be the major components of the protein-DNA complexes observed in primary rat cortical extracts. Our results suggest that the cell-type specific expression of Sp transcription factors – substitution of Sp1 by Sp4 in neurons – is responsible for the basal expression of the CYP46A1 gene. This study delineates for the first time the mechanisms underlying the human CYP46A1 transcription and thereby elucidates potential pathways underlying cholesterol homeostasis in the brain. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effect of chronic repeated restraint (RR) on prolactin-releasing peptide (PrRP) expression. In the brainstem, where PrRP colocalize with norepinephrine in neurons of the A1 and A2 catecholaminergic cell groups, the expression of tyrosine hydroxylase (TH) has also been examined. In the brainstem, but not in the hypothalamus, the basal PrRP expression in female rats was higher than that in the males that was abolished by ovariectomy. RR evoked an elevation of PrRP expression in all areas investigated, with smaller reaction in the brainstems of females. There was no gender-related difference in the RR-evoked TH expression. Elevation of PrRP was relatively higher than elevation of TH, causing a shift in PrRP/TH ratio in the brainstem after RR. Estrogen α receptors were found in the PrRP neurons of the A1 and A2 cell groups, but not in the hypothalamus. Bilateral lesions of the hypothalamic paraventricular nucleus did not prevent RR-evoked changes. Elevated PrRP production parallel with increased PrRP/TH ratio in A1/A2 neurons indicate that: (i) there is a clear difference in the regulation of TH and PrRP expression after RR, and (ii) among other factors this may also contribute to the changed sensitivity of the hypothalamo-pituitary–adrenal axis during chronic stress. [ABSTRACT FROM AUTHOR]

Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine-mediated-cellular responses. In the present study, we investigated the modulation of rat A2A receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A2A receptor binding and functioning. On the contrary, in striatum, a significant decrease in A2A ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A2A receptor-G protein coupling. On the contrary, A2A receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up-regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A2A ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non-ischemic hemisphere and in sham-operated rats A2A ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time-dependent protective effects of specific A2A active compounds. [ABSTRACT FROM AUTHOR]

A novel radioligand, 6-chloro-3-((2-( S)-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The Kd and T1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[18F]fluoro-3-(2( S)-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α4β2* nAChRs distribution. Comparison between [18F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [18F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [18F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α4β2* versus α3β4* nAChRs. These results suggest that [18F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (ApoE), encoded by the apolipoprotein E gene (APOE), plays an important role in the pathogenesis of Alzheimer’s disease (AD). The APOE ε4 variant is strongly associated with AD. APOE promoter polymorphisms have also been reported to associate with higher AD risk. Mouse models of APOE expression have long been used to study the pathogenesis of AD. Elucidating the role of the APOE gene in AD requires understanding of how its regulation differs between mouse and human APOE genes, and how the differences influence AD risk. We compared the structure and function of both the human APOE gene promoter (hAPOEP) and mouse APOE gene promoter (mAPOEP) regions. Homology is less than 40% at 180 bp or more upstream of the two species’ transcription start site (TSS, +1). Functional analysis revealed both similarities and important differences between the two sequences, significantly affected by human versus rodent cell line origin. We likewise probed nuclear extracts from several cell lines of different origins (astrocytic, glial, and neuronal) and mouse brain with specific hAPOEP and mAPOEP fragments. Each fragment shared DNA–protein interactions with the other but, notably, also bound distinct factors, demonstrated by gel shift and southwestern analyses. We determined possible identities for these distinct factors. These results suggest that regulation of mouse and human APOE genes may be sufficiently unique to justify the use of both the human APOE promoter sequence in transgenic rodent models and non-rodent AD models for studying factors involved in AD pathogenesis. [ABSTRACT FROM AUTHOR]

Young parkin null (pk−/−) mice have subtle abnormalities of behaviour, dopamine (DA) neurotransmission and free radical production, but no massive loss of DA neurons. We investigated whether these findings are maintained while ageing. Pk−/− mice have reduced life span and age-related reduced exploratory behaviour, abnormal walking and posture, and behaviours similar to those of early Parkinson’s disease (PD), reduced number of nigrostriatal DA neurons and proapoptotic shifts in the survival/death proteins in midbrain and striatum. Contrary to young pk−/− animals 24-month-old pk−/− mice do not have compensatory elevation of GSH in striatum, glutathione reductase (GR) and glutathione peroxidase (GPx) activities are increased and catalase unchanged. Aged pk−/− mice accumulate high levels of tau and fail to up-regulate CHIP and HSP70. Our results suggest that aged pk−/− mice lack of the compensatory mechanisms that maintain a relatively normal DA function in early adulthood. This study could help to explain the effects of ageing in patients with genetic risks for Parkinson’s disease. [ABSTRACT FROM AUTHOR]

Excessive N-methyl-d-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms. Lithium and carbamazepine, effective against bipolar mania, are reported in rats to reduce brain transcription of an arachidonic acid selective calcium-dependent cytosolic phospholipase A2 (cPLA2), as well as expression of one of its transcription factors, activator protein (AP)-2. In this study, we determined if chronic administration of NMDA (25 mg/kg i.p.) to rats would increase brain cPLA2 and AP-2 expression, as these antimanic drugs are known to down-regulate excessive NMDA signaling. Administration of a daily subconvulsive dose of NMDA to rats for 21 days decreased frontal cortex NMDA receptor (NR)-1 and NR-3A subunits and increased cPLA2 activity, phosphorylation, protein, and mRNA levels. The activity and protein levels of secretory phospholipase A2 or calcium-independent phospholipase A2 were not changed significantly. Chronic NMDA also increased the DNA-binding activity of AP-2 and the protein levels of its α and β subunits. These changes were absent following acute (3 h earlier) NMDA administration. The changes, opposite to those found following chronic lithium or carbamazepine, are consistent with up-regulated arachidonic acid release due to excessive NR signaling and may be a contributing factor to bipolar mania. [ABSTRACT FROM AUTHOR]

An increase in the concentration of glycerol in the ischemic brain is assumed to reflect degradation of phospholipids of plasma membranes. However, glycerol could, theoretically, be formed from glucose, which after glycolytic conversion to dihydroxyacetone phosphate, could be converted to glycerol-3-phosphate and hence to glycerol. We show here that 13C-labeled glycerol accumulate in incubation media of cultured cerebellar granule cells and astrocytes incubated with [13C]glucose, 3 mmol/L, demonstrating the formation of glycerol from glucose. Co-incubation of cerebellar granule cells with kainate, 50 µmol/L, led to increased glucose metabolism and increased accumulation of [13C]glycerol. Accumulation of [13C]glycerol and its precursor, [13C]glycerol-3-phosphate, was evident in brain, but not in serum, of kainate-treated rats that received [U-13C]glucose, 5 µmol/g bodyweight, intravenously and survived for 5 min. Global ischemia induced by decapitation also caused accumulation of [13C]glycerol and [13C]glycerol-3-phosphate. These results show that glycerol can be formed from glucose in brain; they also demonstrate the existence of a cerebral glycerol-3-phosphatase activity. Ischemia-induced increases in brain glycerol may, in part, reflect an altered metabolism of glucose, in which glycerol formation, like lactate formation, acts as a redox sink. [ABSTRACT FROM AUTHOR]

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-13C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured 13C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-13C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-13C]glucose, [2-13C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 ± 4% of the rate of glutamine synthesis, or 0.02 μmol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial–venous differences and radiotracer methods. [ABSTRACT FROM AUTHOR]

EAAC1/EAAT3 is a transporter of glutamate (Glu) present at the post-synaptic neuronal element, in opposition to the two other main transporters, GLAST/EAAT1 and GLT1/EAAT2, expressed at the excitatory amino acid (EAA) synapse by surrounding astrocytes. Although, in the adult, EAAC1/EAAT3 exhibits a rather low expression level and is considered to make a minor contribution to Glu removal from the synapse, its early expression during brain development, before the astrocytes are functional, suggests that such a neuronal transporter is involved in the developmental effects of EAA and, possibly, in the biosynthesis and trophic role of GABA, which is excitatory in nature in different brain regions during the earlier stages of brain development. This neuronal Glu transporter is considered to have a dual action as it is apparently involved in the neuronal uptake of cysteine, which acts as a key substrate for the synthesis of glutathione, a major anti-oxidant, because the neurones do not express the Xc– transport system in the mature brain. Interestingly, EAAC1/EAAT3 activity/expression was shown to be highly regulated by neuronal activity as well as by intracellular signalling pathways involving primarily α protein kinase C (αPKC) and phosphatidylinositol-3-kinase (PI3K). Such regulatory processes could act either at the post-traductional level or at the transcriptional level. It is worth noting that EAAC1/EAAT3 exhibits specificity, compared with other EAA transporters, because it is present mainly in the intracellular compartment and only for about 20% at the plasma membrane. Variations in neuronal Glu uptake were shown to be associated with rapid changes in the trafficking of the transporter protein altering the membranar location of the transporter. More recent data show that astrocyte-secreted factors such as cholesterol could also influence rapid changes in the location of EAAC1/EAAT3 between the plasma membrane and the cytoplasmic compartment. Such a highly regulated process of EAAC1/EAAT3 activity/expression may have implications in the physiopathology of major diseases affecting EAA brain signalling, which is further supported by data obtained in animal models of hypoxia–anoxia, for example. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) and other neurotrophins induce a unique prolonged activation of mitogen-activated protein kinase (MAPK) compared with growth factors. Characterization and kinetic and spatial modeling of the signaling pathways underlying this prolonged MAPK activation by BDNF will be important in understanding the physiological role of BDNF in many complex systems in the nervous system. In addition to Shc, fibroblast growth factor receptor substrate 2 (FRS2) is required for the BDNF-induced activation of MAPK. BDNF induces phosphorylation of FRS2. However, BDNF does not induce phosphorylation of FRS2 in cells expressing a deletion mutant of TrkB (TrkBΔPTB) missing the juxtamembrane NPXY motif. This motif is the binding site for SHC . NPXY is the consensus sequence for phosphotyrosine binding (PTB) domains, and notably, FRS2 and SHC contain PTB domains. This NPXY motif, which contains tyrosine 484 of TrkB, is therefore the binding site for both FRS2 and SHC. Moreover, the proline containing region (VIENP) of the NPXY motif is also required for FRS2 and SHC phosphorylation, which indicates this region is an important component of FRS2 and SHC recognition by TrkB. Previously, we had found that the phosphorylation of FRS2 induces association of FRS2 and growth factor receptor binding protein 2 (Grb2). Now, we have intriguing data that indicates BDNF induces association of the SH2 domain containing protein tyrosine phosphatase, Shp2, with FRS2. Moreover, the PTB association motif of TrkB containing tyrosine 484 is required for the BDNF-induced association of Shp2 with FRS2 and the phosphorylation of Shp2. These results imply that FRS2 and Shp2 are in a BDNF signaling pathway. Shp2 is required for complete MAPK activation by BDNF, as expression of a dominant negative Shp2 in cells attenuates BDNF-induced activation of MAPK. Moreover, expression of a dominant negative Shp2 attenuates Ras activation showing that the protein tyrosine phosphatase is required for complete activation of MAPKs by BDNF. In conclusion, Shp2 regulates BDNF signaling through the MAPK pathway by regulating either Ras directly or alternatively, by signaling components upstream of Ras . Characterization of MAPK signaling controlled by BDNF is likely to be required to understand the complex physiological role of BDNF in neuronal systems ranging from the regulation of neuronal growth and survival to the regulation of synapses. [ABSTRACT FROM AUTHOR]

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months). [ABSTRACT FROM AUTHOR]

This study has used receptor autoradiography to characterize imidazoline binding sites (I-BS) in monoamine oxidase (MAO) A knockout and wild-type mice. A comparison between MAO-A and MAO-B, binding of the endogenous β-carboline [3H]harmane, and I-BS, has been made using sections from brain and kidney. The loss of binding to MAO-A in the knockout animals was confirmed using the selective radioligand [3H]Ro41–1049, with labelling reduced to background levels. The binding of [3H]Ro19–6327 to MAO-B was unaffected, indicating no change in this isoform in response to the loss of MAO-A. A reduction in binding to the I2-BS, as labelled by both [3H]idazoxan and [3H]2-BFI (2-(2-benzofuranyl)-2-imidazoline), was seen in the MAO-A knockout animals in both brain and kidney sections, whereas binding to the I1-BS in kidney sections remained unchanged. The loss of I2 binding was found to be regionally dependent and was positively correlated with the relative expression of MAO-A in specific regions in the wild-type animals. Using the MAO-A knockout mice it was also possible to demonstrate a non-MAO-A population of binding sites labelled by the putative I-BS endogenous ligand, harmane. 2 [ABSTRACT FROM AUTHOR]

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A2 (PLA2) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–14C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–14C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2–/–) and wild-type (COX-2+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2–/– compared with COX-2+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2–/– compared with COX-2+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA2-released AA to prostanoids. [ABSTRACT FROM AUTHOR]

This study was conducted to determine whether provision of preformed dietary docosapentaenoic acid (DPAn-6) can replace docosahexaenoic acid (DHA) for brain function as assessed by spatial task performance. A newly modified artificial rearing method was employed to generate n-3 fatty acid-deficient rats. Newborn pups were separated from their mothers at 2 days of age and given artificial rat milk containing linoleic acid (LA), or LA supplemented with 1% DHA (DHA), 1% DPAn-6 (DPA) or 1% DHA plus 0.4% DPAn-6 (DHA/DPA). The animals were then weaned onto similar pelleted diets. At adulthood, behavioural tasks were administered and then the brains were collected for fatty acid analysis. The LA and DPA groups showed a lower (63–65%) brain DHA than the dam-reared, DHA and DHA/DPA groups and this loss was largely compensated for by an increase in brain DPAn-6. The brain fatty acid composition in the DPA group was the same as that in the LA group at adulthood. In the Morris water maze, the LA and DPA groups exhibited a longer escape latency than the dam-reared and DHA groups and had a defect in spatial retention. In conclusion, DPAn-6 could not replace DHA for brain function, indicating a highly specific structural requirement for DHA. [ABSTRACT FROM AUTHOR]

Activation of neural pathways originating in the cerebellar fastigial nucleus (FN) protects the brain from the deleterious effects of cerebral ischemia and excitotoxicity, a phenomenon termed central neurogenic neuroprotection. The neuroprotection is, in part, mediated by suppression of apoptosis. We sought to determine whether FN stimulation exerts its anti-apoptotic effect through mitochondrial mechanisms. Mitochondria were isolated from the cerebral cortex of rats in which the FN was stimulated for 1 h (100 μA; 1 s on/1 s off), 72 h earlier. Stimulation of the dentate nucleus (DN), a brain region that does not confer neuroprotection, served as control. Mitochondria isolated from FN-stimulated rats exhibited a marked increase in their ability to sequester Ca2+ and an increased resistance to Ca2+-induced membrane depolarization and depression in respiration. FN stimulation also leads to reduction in the release in cytochrome c, induced either by Ca2+ or the mitochondrial toxin mastoparan. Furthermore, in brain slices, FN stimulation reduced the staurosporine-induced insertion of the pro-apoptotic protein Bax into the mitochondria, a critical step in the mitochondrial mechanisms of apoptosis. Collectively, these results provide evidence that FN stimulation protects the mitochondria from dysfunction induced by Ca2+ loading, and inhibits mitochondrial pathways initiating apoptosis. These mitochondrial mechanisms are likely to play a role in the neuroprotection exerted by FN stimulation. [ABSTRACT FROM AUTHOR]

Ten years after the isoforms of mammalian phospholipase D (PLD), PLD1 and 2, were cloned, their roles in the brain remain speculative but several lines of evidence now implicate these enzymes in basic cell functions such as vesicular trafficking as well as in brain development. Many mitogenic factors, including neurotransmitters and growth factors, activate PLD in neurons and astrocytes. Activation of PLD downstream of protein kinase C seems to be a required step for astroglial proliferation. The characteristic disruption of the PLD signaling pathway by ethanol probably contributes to the delay of brain growth in fetal alcohol syndrome. The post-natal increase of PLD activities concurs with synapto- and myelinogenesis in the brain and PLD is apparently involved in neurite formation. In the adult and aging brain, PLD activity has antiapoptotic properties suppressing ceramide formation. Increased PLD activities in acute and chronic neurodegeneration as well as in inflammatory processes are evidently due to astrogliosis and may be associated with protective responses of tissue repair and remodeling. ARF-regulated PLD participates in receptor endocytosis as well as in exocytosis of neurotransmitters where PLD seems to favor vesicle fusion by modifications of the shape and charge of lipid membranes. Finally, PLD activities contribute free choline for the synthesis of acetylcholine in the brain. Novel tools such as RNA interference should help to further elucidate the roles of PLD isoforms in brain physiology and pathology. [ABSTRACT FROM AUTHOR]