Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function.

Schizophrenia is a severe psychiatric disorder with a world-wide prevalence of 1%. The pathophysiology of the illness is not understood, but is thought to have a strong genetic component with some environmental influences on aetiology. To gain further insight into disease mechanism, we used microarray technology to determine the expression of over 30 000 mRNA transcripts in post-mortem tissue from a brain region associated with the pathophysiology of the disease (Brodmann area 10: anterior prefrontal cortex) in 28 schizophrenic and 23 control patients. We then compared our study (Charing Cross Hospital prospective collection) with that of an independent prefrontal cortex dataset from the Harvard Brain Bank. We report the first direct comparison between two independent studies. A total of 51 gene expression changes have been identified that are common between the schizophrenia cohorts, and 49 show the same direction of disease-associated regulation. In particular, changes were observed in gene sets associated with synaptic vesicle recycling, transmitter release and cytoskeletal dynamics. This strongly suggests multiple, small but synergistic changes in gene expression that affect nerve terminal function.

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling.

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.

Posttranslational processing of concanavalin A precursors in jackbean cotyledons.

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation.

P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate.

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.

Differential expression of IEG mRNA in rat brain following acute treatment with clozapine or haloperidol: a semi-quantitative RT-PCR study.

Antipsychotic drugs have been shown to modulate immediate early gene (IEG) expression in rat brain regions that are associated with schizophrenia, which may be directly linked to their immediate therapeutic benefit. In this study, we analysed the expression profile of a series of IEGs (c-fos, c-jun, fra-1, Krox-20, Krox-24, arc, sgk-1, BDNF and NARP) in six rat brain regions (prefrontal cortex, hippocampus, striatum, nucleus accumbens, thalamus and cerebellum). Rats (n=5) were administered either clozapine (20 mg/kg i.p.), haloperidol (1 mg/kg i.p.) or the appropriate vehicle with pre-treatment times of 1, 6 and 24 h. IEG expression was analysed in these regions by Taqman RT-PCR. The spatial and temporal profile of IEG induction following antipsychotic drug treatment correlates with regions associated with the efficacy and side effect profile of each drug. In particular, sgk-1 expression levels after antipsychotic drug treatment may have predictive value when investigating the profile of a novel antipsychotic drug.

Amino acid neurotransmission: spotlight on synaptic vesicles.

The vesicle hypothesis describing quantal release of neurotransmitter at the cholinergic neuromuscular junction was introduced in 1956. Since then, the concept of vesicular storage and release of acetylcholine has become firmly established and extended to include other synapses and neurotransmitters. However, for the amino acids, which are the major class of neurotransmitters in the mammalian CNS, there was no direct experimental evidence of the participation of synaptic vesicles in neurotransmission. This area of research has now moved out of the shadows and this article discusses recent findings which indicate that amino acid neurotransmitters are accumulated and stored by synaptic vesicles in presynaptic nerve endings.

The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

TR3 death receptor expression in the normal and ischaemic brain.

Members of the death receptor family may play a prominent role in developmental and pathological neuronal cell death. We report the expression of the TR3 and TR7 death receptors in the adult human and rat central nervous system. Whereas expression of TR3 appears to be high in the human cerebellum, with lower levels in other brain regions, robust expression is observed in many regions of the rat brain. We also analyzed modulation of death receptor expression in an in vivo rat model of acute stroke. In contrast to tumor necrosis factor receptor 1, Fas and p75(NGFR), which all show up-regulation specifically in lesioned cortex of the permanent middle cerebral artery occlusion model of stroke. TR3 shows a rapid global increase in both lesioned and unlesioned brain. In comparison, the recently described death receptor TR7 shows no change in this model. These data indicate that the death receptors show clear differences in patterns of expression in response to ischemic injury. ¿ 2000 IBRO. Published by Elsevier Science Ltd.

Neuron-specific localisation of the TR3 death receptor in Alzheimer's disease.

Death receptors are associated with the homeostatic and pathologic induction of cell death. TR3 is a recently characterised member of the death receptor family that is expressed in the adult brain. In order to establish the role of TR3 in acute CNS disease and chronic neurodegeneration, we analysed brain regions from Alzheimer's disease (AD), stroke and neurotrauma patients, using a novel anti-peptide antibody generated to an exposed epitope in the extracellular domain of the receptor. We show a statistically significant increase in TR3 protein levels in AD brain samples but not in stroke, neurotrauma or control samples. The increase observed for TR3 was specific to neurons in regions associated with AD pathology. This is the first report describing the neuron-specific regulation of a death receptor in chronic disease and may indicate that a TR3 receptor-mediated signalling pathway is involved in AD-associated neuronal loss.

Clozapine and haloperidol differentially regulate dendritic spine formation and synaptogenesis in rat hippocampal neurons.

Antipsychotic drugs are the primary therapeutic treatment for schizophrenia. In addition to their dopaminergic/serotonergic function, atypical antipsychotics differ from conventional antipsychotics in the way they affect glutamatergic receptor function. A cellular correlate of this may be the modulation of dendritic spines (DS). Here, we demonstrate that in rat dissociated hippocampal neurons 1.0 microM clozapine administration increased DS-enriched protein spinophilin by 70%, increased post-synaptic protein shank1a puncta density by 26% and increased overall primary dendrite DS density by 59%. Filopodia and mushroom DS were particularly affected by clozapine. Conversely, 0.1 microM haloperidol decreased spinophilin protein by 40%, caused a 25% decrease in shank1a puncta and reduced the numbers of filopodia. In contrast, neither haloperidol nor clozapine induced any change in the levels of the pre-synaptic protein synapsin. This indicates that clozapine and haloperidol differentially regulate DS and post-synaptic plasticity. These findings may provide a molecular and cellular correlate to the superior therapeutic profile of clozapine when compared with haloperidol.

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.

Bacteriorhodopsin drives the glutamate transporter of synaptic vesicles after co-reconstitution.

Active accumulation of neurotransmitters by synaptic vesicles is an essential component of the synaptic transmission cycle. Isolated vesicles show energy-dependent uptake of several transmitters by processes which are apparently mediated by a proton electrochemical potential across the vesicle membrane. Although this energy gradient is probably generated by a proton ATPase, the functional separation of ATP cleavage and transmitter uptake activity has only been shown clearly for monoamine transport. We report here that the light-driven proton pump, bacteriorhodopsin, can replace the endogenous proton ATPase in proteoliposomes reconstituted from vesicular detergent extracts. The system shows light-dependent uptake of glutamate with properties very similar to those observed in intact vesicles, e.g. chloride dependence or stimulation by NH4+. Our experiments show that the proton pump and the glutamate transporter are separate entities and provide a powerful tool for further characterization of the glutamate carrier.

Synaptic vesicles: key organelles involved in neurotransmission.

1. This article summarizes some of the recent advances in the understanding of structural and functional properties of isolated small synaptic vesicles (SSV) from mammalian brain. 2. SSV contain a set of integral membrane proteins which are highly specific for this organelle and which occur on all SSV of the central and peripheral nervous system irrespective of their transmitter content. In contrast, these proteins are absent from the membrane of peptide-containing large dense-core vesicles indicating that the two types of organelle have a different membrane composition. The availability of antibodies for these proteins has allowed the evaluation of the purity of vesicle preparations which is instrumental for functional studies. 3. Recent advances in the study of neurotransmitter uptake have revealed that SSV contain specific carrier systems for glutamate and GABA. They are different from the transporters of the plasma membrane, and are dependent on the energy of a proton electrochemical gradient. The uptake of glutamate has been characterized in some detail and the mechanistic and physiological implications of these findings are discussed.

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.

Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes.

The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.

A study of maturation events in jackbeans (Canavalia ensiformis).

Maturation events have been studied in developing jackbean (Canavalia ensiformis) cotyledons by using a combination of analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, overlays with 125I-concanavalin A (Con A) and the use of anti-(Con A) after Western transfer. The number of polypeptides recognized by 125I-Con A varies during maturation, until at maturity only one remains. Several molecular forms of the lectin occur during development; one, corresponding to Mr 33 000 and found only in immature seeds, interacts with 125I-Con A, suggesting that it is glycosylated.

A transgenic mouse model for human hereditary neuropathy with liability to pressure palsies.

Mutations in the gene encoding peripheral myelin protein 22 (PMP22) account for several inherited peripheral neuropathies in humans. We now show that transgenic mice expressing antisense PMP22 RNA exhibit modestly reduced levels of PMP22 together with a phenotype that is reminiscent of hereditary neuropathy with liability to pressure palsies (HNPP), a human disease caused by a 1.5-Mb deletion of a chromosome 17 region that contains the PMP22 gene. Transgenic antisense homozygotes display a striking movement disorder and a slowing of nerve conduction that worsens with age. Morphological analysis of peripheral nerves demonstrates that a subset of axons have thickened myelin sheaths and tomacula in young adults, with significant myelin degeneration detected in older animals. Together with other recent work, these data suggest that dosage of the PMP22 gene alone underlies the pathophysiology observed in HNPP and related disorders.

Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production. Potential involvement of endogenous EDG-4 receptors.

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.