Brain Synaptosomal Fractions Research Articles

CB1R activates the epilepsy-associated protein Go to regulate neurotransmitter release and synaptic plasticity in the cerebellum

GNAO1 encodes the alpha subunit of the heterotrimeric Go protein. Despite being the most abundant G protein at synapses, the role of Go in the brain remains unclear, primarily because of the high mortality associated with developmental and epileptic encephalopathy (DEE) 17 in Gnao1 mutated animals. Here, we conducted proteomic analyses with a brain synaptosomal fraction to investigate the Go-interactome and then generated a non-DEE model using Gli1CreERT2 mice to selectively knockout (KO) the presynaptic Gαo within cerebellum. Our findings revealed that Gαo interacts with multiple proteins involved in neurotransmitter release, as well as cannabinoid receptor type 1 (CB1R), a key Gi/o-coupled receptor in presynaptic terminals. In Gnao1 KO mice, synapse formation was reduced in the cerebellum with a concomitant reduction in depolarization-induced suppression of excitation, a manifestation of CB1R-mediated synaptic plasticity found in the cerebellum. These mice displayed motor deficits in rotarod, grip strength, gait, and beam balance tests. Our results suggest that Go plays a critical role in regulating neurotransmitter releases at the presynaptic terminals and its absence in the entire brain may contribute to DEE pathogenesis. This study also provides valuable insights into the signaling pathways in the brain from a Go-dependent perspective.

High-Fat Diet Induces Neuroinflammation and Mitochondrial Impairment in Mice Cerebral Cortex and Synaptic Fraction.

Brain mitochondrial dysfunction is involved in the development of neurological and neurodegenerative diseases. Mitochondria specifically located at synapses play a key role in providing energy to support synaptic functions and plasticity, thus their defects may lead to synaptic failure, which is a common hallmark of neurodegenerative diseases. High-Fat Diet (HFD) consumption increases brain oxidative stress and impairs brain mitochondrial functions, although the underlying mechanisms are not completely understood. The aim of our study is to analyze neuroinflammation and mitochondrial dysfunctions in brain cortex and synaptosomal fraction isolated from a mouse model of diet-induced obesity. Male C57Bl/6 mice were divided into two groups fed a standard diet or HFD for 18 weeks. At the end of the treatment, inflammation (detected by ELISA), antioxidant state (measured by enzymatic activity), mitochondrial functions and efficiency (detected by oxidative capacity and Seahorse analysis), and brain-derived neurotrophic factor (BDNF) pathway (analyzed by western blot) were determined in brain cortex and synaptosomal fraction. In HFD animals, we observed an increase in inflammatory parameters and oxidative stress and a decrease in mitochondrial oxidative capacity both in the brain cortex and synaptosomal fraction. These alterations parallel with modulation of BDNF, a brain key signaling molecule that is linking synaptic plasticity and energy metabolism. Neuroinflammation HFD-dependent negatively affects BDNF pathway and mitochondrial activity in the brain cortex. The effect is even more pronounced in the synaptic region, where the impaired energy supply may have a negative impact on neuronal plasticity.

Ischemic Neuroprotectant PKCε Restores Mitochondrial Glutamate Oxaloacetate Transaminase in the Neuronal NADH Shuttle after Ischemic Injury.

The preservation of mitochondrial function is a major protective strategy for cerebral ischemic injuries. Previously, our laboratory demonstrated that protein kinase C epsilon (PKCε) promotes the synthesis of mitochondrial nicotinamide adenine dinucleotide (NAD+). NAD+ along with its reducing equivalent, NADH, is an essential co-factor needed for energy production from glycolysis and oxidative phosphorylation. Yet, NAD+/NADH are impermeable to the inner mitochondrial membrane and their import into the mitochondria requires the activity of specific shuttles. The most important neuronal NAD+/NADH shuttle is the malate-aspartate shuttle (MAS). The MAS has been implicated in synaptic function and is potentially dysregulated during cerebral ischemia. The aim of this study was to determine if metabolic changes induced by PKCε preconditioning involved regulation of the MAS. Using primary neuronal cultures, we observed that the activation of PKCε enhanced mitochondrial respiration and glycolysis in vitro. Conversely, inhibition of the MAS resulted in decreased oxidative phosphorylation and glycolytic capacity. We further demonstrated that activation of PKCε increased the phosphorylation of key components of the MAS in rat brain synaptosomal fractions. Additionally, PKCε increased the enzyme activity of glutamic oxaloacetic transaminase 2 (GOT2), an effect that was dependent on the import of PKCε into the mitochondria and phosphorylation of GOT2. Furthermore, PKCε activation was able to rescue decreased GOT2 activity induced by ischemia. These findings reveal novel protective targets and mechanisms against ischemic injury, which involves PKCε-mediated phosphorylation and activation of GOT2 in the MAS.

Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b.

The olfm1a and olfm1b genes in zebrafish encode conserved secreted glycoproteins. These genes are preferentially expressed in the brain and retina starting from 16h post-fertilization until adulthood. Functions of the Olfm1 gene is still unclear. Here, we produced and analyzed a null zebrafish mutant of both olfm1a and olfm1b genes (olfm1 null). olfm1 null fish were born at a normal Mendelian ratio and showed normal body shape and fertility as well as no visible defects from larval stages to adult. Olfm1 proteins were preferentially localized in the synaptosomes of the adult brain. Olfm1 co-immunoprecipitated with GluR2 and soluble NSF attachment protein receptor complexes indicating participation of Olfm1 in both pre- and post-synaptic events. Phosphorylation of GluR2 was not changed while palmitoylation of GluR2 was decreased in the brain synaptosomal membrane fraction of olfm1 null compared with wt fish. The levels of GluR2, SNAP25, flotillin1, and VAMP2 were markedly reduced in the synaptic microdomain of olfm1 null brain compared with wt. The internalization of GluR2 in retinal cells and the localization of VAMP2 in brain synaptosome were modified by olfm1 null mutation. This indicates that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain, partly through the alteration of post-translational GluR2 modifications such as palmitoylation. Olfm1 may be considered a novel regulator of the composition and function of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor complex.

Studies on sensitivity of zebrafish as a model organism for Parkinson's disease: Comparison with rat model.

Objective:To determine the utility of zebra fish as an animal model for Parkinson's disease (PD) in comparison with rat model.Materials and Methods:MTT assay was performed on rat and zebrafish brain synaptosomal fractions using rotenone as a neurotoxic agent. Quercetin and resveratrol were used as standards to compare anti-apoptotic activity in both organisms. Catalepsy was induced in zebrafish by exposing them to haloperidol (9 μM) solution. Drug-treated groups were exposed to bromocriptine and pramipexole, 30 min prior to haloperidol exposure at the dose of 2, 5, and 10 μg/mL. Swimming speed, time spent in the bottom of the tank, and complete cataleptic time were evaluated to assess behavioral changes. In rats, catalepsy was induced using haloperidol (1.25 mg/kg i.p.). Drug-treated groups received bromocriptine (2.5 mg/kg.) and pramipexole (1 mg/kg) orally. Bar test, block test, and locomotor activity were carried out to assess behavioral changes.Results:Resveratrol and quercetin showed comparable inhibition of apoptosis in rats and zebrafish. In anti-cataleptic study, bromocriptine and pramipexole-treated groups showed significant difference (P < 0.05) in behavioral parameters as compared to haloperidol control group in both the experimental organisms. Results obtained from fish model were in correlation with rat model.Conclusion:Findings of the present study revealed that zebrafish model is highly sensitive and can be used for basic screening of drugs against PD.

Influence of Maternal Alcoholism on the Brain Benzodiazepine Receptor Development in Human Embryo and Fetus during Ontogeny

Graphical Abstract: Influence of maternal alcoholism on the brain benzodiazepine receptor development in human’s embryo and fetus showed decreases their affinity and increases density as compensatory adaptation of the fetal nervous system to the effects of alcohol. Abstract: Signaling mechanisms that are required for proper neuronal development and how these processes are impaired by ethanol resulting in harmful consequences to brain development are very important for our understanding. The aim of the present work was to study the development of synaptic Benzodiazepine Receptors (BzDR) (functionally associated with the brain GABAergic system) in the brains of embryos and fetuses aged 8-15 weeks obtained from alcoholic female patients. Material and method: Abortive material (samples of brain tissue) was obtained from 33 women with grade II alcoholism (ICD-10 F10.201 and F10.202) and 30 control women were studied. The properties of BzDR were studied by radioreceptor binding with the selective ligand [3H]-flunitrazepam using a crude embryo brain synaptosomal fraction. Results: In contrast to controls, brain cells developing in conditions of prenatal alcoholization showed formation of synaptic benzodiazepine receptors and alterations in their properties: decreases in their affinity and increases in their density. A decrease in the ability of receptors to bind agonist ligands impairs the ligand: receptor protein ratio, leading to decreased binding of the major neurotransmitter GABA and impairment to synaptic transmission. Conclusion: These are interpreted as compensatory reactions promoting adaptation of the fetal nervous system to the effects of alcohol and functional deficiency of the GABAergic system.

Sensitivity to μ‐opioid receptor‐mediated anti‐nociception is determined by cross‐regulation between μ‐ and δ‐opioid receptors at supraspinal level

The perception of pain and its inhibition varies considerably between individuals, and this variability is still unexplained. The aim of the present study is to determine whether functional interactions between opioid receptors are involved in the inter-individual variability in the sensitivity to μ-opioid receptor agonists. Anti-nociceptive tests, radioligand binding, stimulation of [(35) S]GTP-γ-S binding, inhibition of cAMP production and co-immunoprecipitation experiments were performed in two strains of rat (Sprague-Dawley bred at our university - SDU - and Wistar) that differ in their sensitivity to opioids. The increased anti-nociceptive potency of µ-opioid receptor agonists in SDU rats was reversed by the δ-opioid receptor antagonist, naltrindole. Inhibition of the binding of [(3) H] naltrindole by µ-opioid receptor agonists was different in brain membranes from SDU and Wistar rats. Differences were also evident in the effect of δ-opioid receptor ligands on the binding of [(35) S]GTP-γ-S stimulated by µ-opioid receptors agonists. No strain-related differences were detected in spinal cord membranes. The potency of morphine to inhibit cAMP production in brain membranes varied between the strains, in the presence of deltorphin II and naltrindole. Co-immunoprecipitation experiments demonstrated that δ-opioid receptors were associated with μ-opioid receptors to a higher extent in brain synaptosomal fractions from SDU than in those from Wistar rats. There was increased supraspinal cross-talk between μ and δ-opioid receptors in SDU, as compared with Wistar rats. This was related to an enhanced sensitivity to anti-nociception induced by µ-opioid receptor agonists.

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2β (VIA)-deficient mice

Ca(2+)-independent phospholipase A(2)β (iPLA(2)β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M(1,3,5) receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA(2)β(-/-), iPLA(2)β(+/-), and iPLA(2)β(+/+) mice, and [1-(14)C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J(in), representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA(2)β(-/-) or iPLA(2)β(+/-) compared with iPLA(2)β(+/+) mice showed widespread and significant baseline reductions in k* and J(in) for DHA. Arecoline increased both parameters in brain regions of iPLA(2)β(+/+) mice but quantitatively less so in iPLA(2)β(-/-) and iPLA(2)β(+/-) mice. Consistent with iPLA(2)β's reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA(2)β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M(1,3,5) receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca(2+)-independent phospholipase A(2) (iPLA(2)), Ca(2+)-independent plasmalogen PLA(2) or secretory PLA(2 (sPLA2)), but not by Ca(2+)-dependent cytosolic PLA(2) (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca(2+) into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca(2+)-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca(2+) and of cPLA(2). DHA signaling may be mediated by iPLA(2), plasmalogen PLA(2), or other enzymes insensitive to low concentrations of Ca(2+). Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 +/- 4% of the GABAergic synapses and 25 +/- 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.

The natural xanthone α-mangostin reduces oxidative damage in rat brain tissue

The antiperoxidative properties of α-mangostin, a xanthone isolated from mangosteen fruit, were tested for the first time in nerve tissue exposed to different toxic insults. Two reliable biological preparations (rat brain homogenates and synaptosomal P2 fractions) were exposed to the toxic actions of a free radical generator (ferrous sulfate), an excitotoxic agent (quinolinate), and a mitochondrial toxin (3-nitropropionate). α-Mangostin decreased the lipoperoxidative action of FeSO4 in both preparations in a concentration-dependent manner, and completely abolished the peroxidative effects of quinolinate, 3-nitropropionate and FeSO4 + quinolinate at all concentrations tested. Interestingly, when tested alone in brain homogenates, α-mangostin significantly decreased the lipoperoxidation even below basal levels. α-Mangostin also prevented the decreased reductant capacity of mitochondria in synaptosomal fractions. Our results suggest that α-mangostin exerts a robust antiperoxidative effect in brain tissue preparations probably through its properties as a free radical scavenger. In light of these findings, this antioxidant should be tested in other neurotoxic models involving oxidative stress.

Synthesis of enantiomerically pure HIP- A and HIP- B and investigation of their activity as inhibitors of excitatory amino acid transporters

The present report deals with the synthesis of the two couples of enantiomers (+)-(3a S,4 R,6a S)-/(−)-(3a R,4 S,6a R)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4- d]isoxazole-4-carboxylic acid [(+)- HIP- A and (−)- HIP- A] and (+)-(3a S,6 S,6a S)-/(−)-(3a R,6 R,6a R)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4- d]isoxazole-6-carboxylic acid [(+)- HIP- B and (−)- HIP- B], and the investigation of their inhibitory activity at EAATs. When tested in a [ 3H] d-aspartate uptake assay on native EAATs present in rat brain synaptosomal fractions, (−)- HIP- A and (+)- HIP- B turned out to be considerably more potent than their enantiomers; in particular, (−)- HIP- A showed an eudismic ratio (ER) higher than 100. Molecular modeling investigations gave clues with regard to the key amino acid residues involved in the binding with our inhibitors and provided explanations for the observed stereoselectivity.

Theanine, an ingredient of green tea, inhibits [3H]glutamine transport in neurons and astroglia in rat brain

We have previously shown that theanine (=gamma-glutamylethylamide), an ingredient of green tea, has a protective effect against ischemic neuronal death in the hippocampal CA1 region of the gerbil brain without affecting ligand binding to ionotropic receptor subtypes of the neurotransmitter glutamate structurally related to theanine. The neurotransmitter pool of glutamate is thought to be fueled by the entry of the other structural analog glutamine (Gln) and subsequent cleavage by glutaminase. Although theanine did not inhibit [3H]glutamate accumulation, [3H]theanine was actively accumulated in a temperature-dependent and saturable manner in rat brain synaptosomal fractions. The accumulation of [3H]theanine was markedly inhibited by Gln in a concentration-dependent manner, whereas [3H]Gln accumulation was inhibited by theanine vice versa. Both [3H]theanine and [3H]Gln accumulations were decreased after the replacement of sodium chloride with choline chloride, along with similarly high distribution profiles in telencephalic structures. A similar equilibrium was observed within 30 min at 30 degrees C for the accumulations of both [3H]theanine and [3H]Gln in cultured rat neocortical astroglia as well as neurons, whereas theanine inhibited [3H]Gln accumulation in a concentration-dependent manner at 0.1-10 mM. Furthermore, sustained exposure to 10 mM theanine led to a significant decrease in the level of extracellular glutamate released from cultured neurons. These results suggest that the green tea ingredient theanine would be an inhibitor of different transporters capable of transporting Gln across plasma membranes toward the modulation of the glutamate/Gln cycle required for the neurotransmitter pool of glutamate in neurons.

Involvement of Na +, K +-ATPase and Endogenous Digitalis-Like Compounds in Depressive Disorders

Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.

Localized recruitment and activation of RhoA underlies dendritic spine morphology in a glutamate receptor–dependent manner

Actin is the major cytoskeletal source of dendritic spines, which are highly specialized protuberances on the neuronal surface where excitatory synaptic transmission occurs (Harris, K.M., and S.B. Kater. 1994. Annu. Rev. Neurosci. 17:341–371; Yuste, R., and D.W. Tank. 1996. Neuron. 16:701–716). Stimulation of excitatory synapses induces changes in spine shape via localized rearrangements of the actin cytoskeleton (Matus, A. 2000. Science. 290:754–758; Nagerl, U.V., N. Eberhorn, S.B. Cambridge, and T. Bonhoeffer. 2004. Neuron. 44:759–767). However, what remains elusive are the precise molecular mechanisms by which different neurotransmitter receptors forward information to the underlying actin cytoskeleton. We show that in cultured hippocampal neurons as well as in whole brain synaptosomal fractions, RhoA associates with glutamate receptors (GluRs) at the spine plasma membrane. Activation of ionotropic GluRs leads to the detachment of RhoA from these receptors and its recruitment to metabotropic GluRs. Concomitantly, this triggers a local reduction of RhoA activity, which, in turn, inactivates downstream kinase RhoA-specific kinase, resulting in restricted actin instability and dendritic spine collapse. These data provide a direct mechanistic link between neurotransmitter receptor activity and the changes in spine shape that are thought to play a crucial role in synaptic strength.

Effects of chronic ethanol administration on the activities and relative synthetic rates of myelin and synaptosomal plasma membrane-associated sialidase in the rat brain

In an attempt to understand the possible mechanism of chronic ethanol-induced generation of asialoconjugates in the brain and consequent behavioral abnormalities, we have studied the effects of chronic ethanol feeding to rats on the plasma membrane sialidase status in the various subcellular fractions of the brain. We determined sialidase activity using 3H-monosialoganglioside ( 3H-GM 3), 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid (4-MU-NeuAC) substrates and Amplex™ Red (Sialidase) kit. We determined the plasma membrane sialidase protein by Western blot using the anti-plasma membrane sialidase. We also determined its relative synthetic rate (RSR) by the 60 min incorporation of intracranially infused [ 35S]-methionine (50 μCi/100 g) into immunoprecipitable plasma membrane sialidase. Chronic ethanol administration stimulated the sialidase activity in the total brain homogenate as well as the myelin and synaptosomal membrane fractions, respectively, in all the three experimental models. Chronic ethanol also increased the concentration of the rat brain plasma membrane sialidase protein relative to that of glyceraldehyde-3-phosphate dehydrogenase by 2.4-, 1.62- and 1.51-fold in the total brain homogenate, myelin and synaptosomal membrane fractions, respectively. These increases in plasma membrane sialidase activity and its protein content were due to concomitant increases in their relative synthetic rates by 115% ( p < 0.01) and 72% ( p < 0.01) in the myelin and synaptosomal membrane fractions, respectively. Thus, our studies clearly show that chronic ethanol induced deglycosylation of brain gangliosides is in part, due to specific up-regulation of plasma membrane sialidase in the myelin and synaptosomal membrane fractions of the brain. This increase in plasma membrane sialidase may be responsible for chronic-ethanol-induced physiological and neurological impairment in the brain, presumably due to deglycosylation of gangliosides that are essential for crucial cellular and metabolic activities.

A novel alanine-insensitive d-serine transporter in rat brain synaptosomal membranes

d-Serine is an endogenous modulator of brain N-methyl- d-aspartate receptors. This study investigates serine transport in brain synaptosomal fractions. Saturable, temperature-dependent uptake of both d- and l-[ 3H]serine was observed. Alanine was only partially effective in blocking transport, arguing against involvement of system ASC. Inhibitors of the systems A, L and GLY were also ineffective. Saturation studies suggested a submillimolar K m for both d- and l-serine. These data suggest the presence of a novel serine transport system in rodent synaptosomes.

Neuregulin‐1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin‐1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin‐1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N‐terminal fragment containing the bioactive EGF‐like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140‐kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X‐100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low‐density fractions enriched in PSD‐95, a known lipid raft protein. In transfected cells expressing the neuregulin I‐β1a or the III‐β1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X‐100, and neuregulin proproteins and C‐terminal fragments were detected in lipid raft fractions. In contrast, the III‐β1a N‐terminal fragment was detected only in the detergent‐soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.