Synaptosomal Structures Research Articles

Antipurinergic therapy corrects the autism-like features in the Fragile X (Fmr1 knockout) mouse model.

BackgroundThis study was designed to test a new approach to drug treatment of autism spectrum disorders (ASDs) in the Fragile X (Fmr1) knockout mouse model.MethodsWe used behavioral analysis, mass spectrometry, metabolomics, electron microscopy, and western analysis to test the hypothesis that the disturbances in social behavior, novelty preference, metabolism, and synapse structure are treatable with antipurinergic therapy (APT).ResultsWeekly treatment with the purinergic antagonist suramin (20 mg/kg intraperitoneally), started at 9 weeks of age, restored normal social behavior, and improved metabolism, and brain synaptosomal structure. Abnormalities in synaptosomal glutamate, endocannabinoid, purinergic, and IP3 receptor expression, complement C1q, TDP43, and amyloid β precursor protein (APP) were corrected. Comprehensive metabolomic analysis identified 20 biochemical pathways associated with symptom improvements. Seventeen pathways were shared with human ASD, and 11 were shared with the maternal immune activation (MIA) model of ASD. These metabolic pathways were previously identified as functionally related mediators of the evolutionarily conserved cell danger response (CDR).ConclusionsThe data show that antipurinergic therapy improves the multisystem, ASD-like features of both the environmental MIA, and the genetic Fragile X models. These abnormalities appeared to be traceable to mitochondria and regulated by purinergic signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/2040-2392-6-1) contains supplementary material, which is available to authorized users.

The Molecular Synaptosomal Deficit in Down Syndrome Starts Early in Life

Information about brain abnormalities in early prenatal life leading to or representing mental retardation in Down syndrome (DS) is limited. Synaptic dysgenesis was described and is considered the morphological correlate of mental retardation. The availability of appropriate fetal brain samples along with the advent of proteomic technologies unambiguously identifying proteins concomitantly made us study synaptosomal structures at the molecular level in the fetal cortex of controls and DS fetuses. Cortical tissue of seven controls (mean 18.8 weeks of gestation) and eight DS fetuses (mean 19.8 weeks of gestation) was homogenized, proteins were extracted and run on two-dimensional gel electrophoresis followed by mass spectrometrical analysis using MALDI-TOF (matrix-assisted laser desorption ionization – time of flight). Levels of synaptic proteins N-ethylmaleimide-sensitive fusion protein (controls: mean 0.030 ± 0.015; DS: mean 0.011 ± 0.008 O.D.), synaptotagmin I (controls: mean 0.038 ± 0.021; DS: mean 0.010 ± 0.002 O.D.) and septin 3 (controls: mean 0.061 ± 0.002; DS: mean 0.019 ± 0.012 O.D.) were significantly decreased in brains of fetuses with DS. It is concluded that an altered expression of synaptosomal proteins at the time of the early second trimester of gestation may form the structural basis for the brain deficit in DS early in life.

Increased RNA levels of the 25 kDa synaptosomal associated protein in brain samples of adult patients with Down Syndrome

The synaptosomal associated protein of 25kDa (SNAP-25) is widely distributed in the brain and reduced in neurodegenerative diseases. In a previous paper we have shown reduced amounts of SNAP-25 protein in adult Down Syndrome (DS) brain. Neuronal cell death and downregulation at the transcriptional level may be responsible for the decrease. Therefore SNAP-25 mRNA levels were determined in frontal cortex and cerebellum of adult DS by a competitive reverse transcription-polymerase chain reaction. We found significantly increased mRNA levels in DS either related to 10 ng total RNA ( P<0.05 level in cerebellum: DS 2622±1081 attogr mean±SEM and controls 154± 37 attogr. mean± SEM) or normalized versus the house keeping gene beta-actin ( P<0.05 level in frontal cortex: DS 1324± 504 attogr. mean± SEM and control 131± 32 attogr. mean± SEM; P<0.01 in cerebellum: DS 632± 189 attogr. mean± SEM and control 21± 2 attogr. mean± SEM). The main finding of this study shows elevated mRNA levels of SNAP-25 in adult DS brain whereas histological and protein-chemical evidence for decreased synaptosomal structures including SNAP-25 in a comparable cohort has been reported. We suggest compensatory mechanisms for the upregulation at the transcriptional level. We propose that SNAP-25 as many other brain proteins are regulated by protein stability rather than at the mRNA level.

The proliferative capacity of individual naive CD4(+) T cells is amplified by prolonged T cell antigen receptor triggering.

Strong antigenic encounter by T cells rapidly induces immunological synapse formation and surface T cell receptor (TCR) downregulation. Although surface TCR expression can remain low for several days, T cells can still sustain antigenic signaling. It has been unclear whether prolonged antigenic signaling occurs in the absence of surface TCR replenishment, being maintained by a few “nondownregulatable” surface TCRs that might reside in a synaptosomal structure. Alternatively, the low surface TCR level induced by antigen might represent a dynamic state of expression involving continual surface TCR replenishment, reengagement by antigen, and ongoing downregulation. To resolve this issue, we studied in vivo–generated, dual-specificity primary naive CD4+ T cells. On these cells, antigenic stimulus exclusively downregulated antigen-specific, but not antigen-nonspecific, TCRs. In addition to providing a means to track TCR engagement, this also allowed us to use the antigen nonspecific TCR to track TCR expression in isolation from TCR engagement by antigen. Surface TCR replenishment began within the first day of stimulation, and occurred synchronously with continuous antigen-specific TCR engagement and downregulation. Furthermore, by enhancing CD25 expression, extended signaling through surface-replenishing TCRs significantly amplified the number of daughter cells generated by naive CD4+ T cells that had already committed to proliferate. This effect required TCR engagement and could not be substituted for by interleukin 2. These data demonstrate that TCR triggering and consumption can occur over an extended period of time, with a significant impact on the effector responses evoked from naive CD4+ T cells.

Fetal life in Down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure.

Information on fetal brain in Down Syndrome (DS) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exists. Histological methods are also hampered by inherent problems of morphometry of neuronal structures. It was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with Down Syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. This technique identifies proteins unambiguously and concomitantly on the same gel. Fetal cortex samples were taken at autopsy with low post-mortem time, homogenized and neuron specific enolase (NSE) determined as a marker for neuronal density, the synaptosomal associated proteins alpha SNAP [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein], beta SNAP, SNAP 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (DRB) as marker for dendritic spines. NSE, chapsyn 110 and beta SNAP were comparable in the control fetus panel and in Down Syndrome fetuses. Drebrin was significantly and remarkably reduced and not even detectable in several Down Syndrome brain samples. Quantification of SNAP 25 revealed significantly reduced values in DS cortex and alpha SNAP was only present in half of the DS individuals. We conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha SNAP and SNAP 25 were significantly reduced indicating impaired synaptogenesis. Early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of Down Syndrome from infancy.

Embryonic genes expressed in Alzheimer's disease brains

Alzheimer's disease (AD)-specific or characteristic gene expression was explored by the identification of cDNA clones by means of differential screening for embryonic brain cDNA library with 32P-labeled cDNA probes prepared from mRNA of AD and normal human brains. To isolate neuronal genes in degenerating neurons, we used rat embryonic cDNA library at stage day 15 when glial cells developed poorly in the brain. Seventeen embryonic genes were identified as embryonic α-tubulin, embryonic β-tubulin, RNP, protein l-sioaspartyl methyltransferase (PIMT), ferritin heavy chain, type IV collagen, actin-binding protein cofilin, profilin and nine novel sequences designated as A1–9. We characterized these genes by Northern blot analysis. RNase protection assay and immunohistochemical studies, showing that PIMT and a novel gene designated as A5 showed the transcriptional up-regulation in AD brains. In addition, the immunohistochemical studies showed PIMT, type IV collagen, and cofilin were associated with neurofibrillary tangles in degenerating neurons, brain vessels in affected regions, and synaptosomal structures in AD brains, respectively. The catalogue presented here also showed the involvement of cytoskeletal proteins, cytoskeleton-associated proteins, and an iron-storage protein, suggesting the presence of regenerating activity and the abnormal metabolisms in affected neurons of AD brains.

Calcium‐Dependent and ‐Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 x 10(-7) M), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 x 10(-7) M to 10(-5) M, the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10(-6) M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations (> or = 10(-5) M), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.

Molecular Species of Choline and Ethanolamine Phospholipids in Rat Cerebellum During Development

The molecular species composition of rat cerebellar phospholipid subclasses has been studied by HPLC after phospholipase C treatment and dinitrophenyl derivatization. During rat cerebellum development (3-90 days postpartum), cholinephosphoglycerides and ethanolamine phosphoglycerides represented approximately 80% of all phospholipids, with their relative amount changing after 1 month. Among ethanolamine phosphoglycerides, the molar ratio of diacylglycerophosphoethanolamine (diacylGPE) to alkenylacylGPE decreased from approximately 1.4 at 3 days to approximately 0.5 after 10 days. The phospholipids investigated contained up to 12 different molecular species. The rate of accumulation of the various molecular species of diacylglycerophosphocholine (diacylGPC), diacylGPE, and alkenylacylGPE during cerebellar development allowed a classification into three main groups. The overall increase of the molecular species of the first group (6-diacylGPC, 5-diacylGPE, and 4-alkenylacylGPE) was approximately 18-fold between 3 and 90 days, with a faster rate of accumulation between 3 and 30 days. Those of the second group (3-diacylGPC, 5-diacylGPE, and 5-akenylacylGPE) increased by approximately 45-fold during the same developmental period, at a slow rate before day 15 and a faster one thereafter. The molecular species of the third group (3-alkenylacylGPE) increased by greater than 250-fold between 3 and 90 days, at a very slow rate before day 21 and more quickly thereafter. The different rates of accumulation of the components of the three groups during cerebellar development suggest a preferential location of the first group in membranes of neuronal perikaryons, glial cells, and synaptosomal structures. Those of the second group appear to be located in both synaptosomal membranes and myelin sheets, and those of the third group can be considered as myelin markers.

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4°C and 37°C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4°C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.

Superoxide radical-mediated alteration of synaptosome membrane structure and high-affinity gamma-[14C]aminobutyric acid uptake.

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.

High-performance liquid chromatographic analysis of ‘specifically bound’ label after [ 125I]angiotensin II binding to rat brain membranes

The combined hypothalamus-thalamus-septum and anteroventral third ventricular region (HTSA) of the rat was examined for [ 125I]angiotensin II ([ 125I]AII) binding using two protocols: one that preserved synaptosomal structure and a second that did not. Although maximum binding (B max) and dissociation constants ( K d ) were similar in both preparations, high-performance liquid chromatographic analyses revealed that [ 125I]AII made up the majority of specifically bound label in the synaptosome preserved preparation while [ 125I]tyrosine ([ 125I]Tyr) represented most of the specifically bound label in the disrupted preparation. These results indicate that [ 125I]Tyr accumulation occurred subsequent to binding and degradation of [ 125I]AII and are consistent with the notion that rapid internalization of the receptor-[ 125I]AII complex occurs in those preparations where the synaptosomal structure remains intact.

Optimal fixation of chick synaptosomes using different osmotic pressures.

Chick synaptosomes were found to remain intact following fixation in the presence of 0.2 M sucrose or 0.3 M sucrose. Increasing the sucrose concentration up to 0.4 M significantly decreased the number of intact synaptosomes; however, typical intrasynaptosomal organelles such as synaptic vesicles and mitochondria were visible in all preparations. This work suggests possibly important species differences in synaptosomal structure between chick and rat synaptosomes.

Metabolism of gamma-hydroxy-[1-14C] butyrate by rat brain: relationship to the Krebs cycle and metabolic compartmentation of amino acids.

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ‐hydroxy‐[1‐14C]butyric acid (GHB) or [1‐14C] succinate. The loss of isotope (as 14CO2) was similar for both substances. The [1‐14C]GHB metabolites lost 75% of the label and the [1‐14C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1‐14C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a‐amino group. These results also indicate that the label from [1‐14C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1‐14C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l‐14C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.

Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain.

The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.

Regulatory action of lithium in manic-depressive illness.

<h2>Abstract</h2> It is proposed, on the basis of recent observations, that lithium prevents recurrent attacks of mania and depression by regulating cell-membrane A.T.P.ases. The stabilising effect is thought to be achieved by a combination of factors. Periodic fluctuations in the distribution of sodium and potassium across cell membranes during episodes of depression and mania result in secondary changes in the distribution of lithium, allowing it to exhibit certain unique therapeutic characteristics: immediate and readily reversible potassium-like stimulation of the sodium pump as an external cation; competition with sodium for the pump as an internal cation; alteration in the cell/ plasma magnesium ratio; and stimulation of the magnesium-activated A.T.P.ase on prolonged administration. The proposed regulatory mechanism has been tested using the red blood-cell as a model, and is supported by experiments on synaptosomal structures.