Synaptosomal Protein Research Articles

Phosphorylation of Adult Type Sept5 (CDCrel-1) by Cyclin-dependent Kinase 5 Inhibits Interaction with Syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.

Verapamil abolished the enhancement of protein phosphorylation of brainstem mitochondria and synaptosomes from the hens dosed with tri- o-cresyl phosphate

To explore the changes of the endogenous phosphorylation of brainstem mitochondrial and synaptosomal proteins in adult hens dosed with tri- o-cresyl phosphate (TOCP) following the development of organophosphate-induced delayed neurotoxicity (OPIDN). Verapamil (7 mg/(kg day), i.m.) was given for 4 days. A dose of TOCP (750 mg/kg, p.o.) was administrated in second day after verapamil. Phosphorylation of the proteins from brainstem mitochondria and synaptosomes was assayed in vitro by using [γ- 32P]ATP as phosphate donor. Radiolabeled proteins were separated by SDS-PAGE and visualized by autoradiography. The results showed that TOCP administration enhanced the phosphorylation of the cell organelle proteins (mitochondria: 60, 55, 45, and 20 kDa; synaptosomes: 65, 60, and 20 kDa), while verapamil abolished the enhancement induced by TOCP. Additionally, the reaction for the phosphorylation is catalyzed by the calcium/calmodulin protein kinase. Therefore, TOCP can enhance the phosphorylation of the brainstem mitochondrial and synaptosomal proteins from the hens with OPIDN; however, protection from the enhancement of the phosphorylation should be involved in the mechanisms of the amelioration of TOCP-induced delayed neurotoxicity by verapamil.

Permanent brain ischemia induces marked increments in hsp72 expression and local protein synthesis in synapses of the ischemic hemisphere

Transient focal ischemia induced in rat brain by occlusion of the middle cerebral artery (MCAo) elicits a generalized induction of the 72 kDa heat-shock protein (hsp72) heralding functional recovery. As this effect implies activation of protein synthesis, and local systems of protein synthesis are present in brain synapses, and may be analyzed in preparations of brain synaptosomes, we evaluated hsp72 expression and protein synthesis in synaptosomal fractions of spontaneously hypertensive rats (SHRs) subjected to permanent MCAo. SHRs were randomly divided in ischemics and sham controls, anaesthesia controls and passive controls. Focal ischemia was induced under chloral hydrate anaesthesia by unilateral permanent MCAo. Protein synthesis was determined by [ 35S]methionine incorporation into synaptosomal proteins from ischemic and contralateral cortex/striatum, and from cerebellum. Hsp72 expression was measured in the same fractions by immunoblotting. Our data demonstrate that under these conditions synaptic hsp72 markedly increases in the ischemic hemisphere 1 and 2 days after MCAo, progressively declining in the following 2 days, while no significant change occurs in control rats. In addition, in the ischemic hemisphere the rate of synaptic protein synthesis increases more than two-fold between 1 and 4 days after MCAo, without showing signs of an impending decline. The present data provide the first demonstration that synaptic protein synthesis is massively involved in brain plastic events elicited by permanent focal ischemia.

O-GlcNAc modification in diabetes and Alzheimer's disease

Similar to phosphorylation, O-GlcNAcylation (or simply GlcNAcylation) is an abundant, dynamic, and inducible post-translational modification. In some cases, GlcNAcylation and phosphorylation occur at the same or adjacent sites, modulating each other. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription, among others. GlcNAcylation affects protein-protein interactions, activity, stability, and expression. Several GlcNAcylated proteins are involved in diabetes and Alzheimer's disease. Hyperglycemia increases GlcNAcylation of proteins within the insulin signaling pathway and contributes to insulin resistance. In addition, hyperinsulinemia and hyperlipidemia are also associated with increased GlcNAcylation, which affect and regulate several insulin signaling proteins, as well as proteins involved on the pathology of diabetes. With respect to Alzheimer's disease, several proteins involved in the etiology of the disease, including tau, neurofilaments, beta-amyloid precursor protein, and synaptosomal proteins are GlcNAcylated in normal brain. The impairment of brain glucose uptake/metabolism is a known metabolic defect in Alzheimer's neurons. Data support the hypothesis that hypoglycemia within the brain may reduce the normal GlcNAcylation of tau, exposing kinase acceptor sites, thus leading to hyperphosphorylation, which induces tangle formation and neuronal death. Alzheimer's disease and type II diabetes represent two metabolic disorders where dysfunctional protein GlcNAcylation/phosphorylation may be important for disease pathology.

Synaptosomal protein synthesis is selectively modulated by learning

Synaptosomes from rat brain have long been used to investigate the properties of synaptic protein synthesis. Comparable analyses have now been made in adult male rats trained for a two-way active avoidance task to examine the hypothesis of its direct participation in brain plastic events. Using Ficoll-purified synaptosomes from neocortex, hippocampus and cerebellum, our data indicate that the capacity of synaptosomal protein synthesis and the specific activity of newly synthesized proteins were not different in trained rats in comparison with home-caged control rats. On the other hand, the synthesis of two proteins of 66.5 kDa and 87.6 kDa separated by SDS-PAGE and analyzed by quantitative densitometry was selectively enhanced in trained rats. In addition, the synthesis of the 66.5 kDa protein, but not of the 87.6 kDa protein, correlated with avoidances and escapes and inversely correlated with freezings in the neocortex, while in the cerebellum it correlated with avoidances and escapes. The data demonstrate the participation of synaptic protein synthesis in plastic events of behaving rats, and the selective, region-specific modulation of the synthesis of a synaptic 66.5 kDa protein by the newly acquired avoidance response and by the reprogramming of innate neural circuits subserving escape and freezing responses.

Asymmetric accumulation of hippocampal 7S SNARE complexes occurs regardless of kindling paradigm

Modifications of neurotransmission may contribute to the synchronization of neuronal networks that are a hallmark of epileptic seizures. In this study we examine the synaptosomal proteins involved in neurotransmitter release to determine if alterations in their interactions correlate with the chronic epileptic state. Using quantitative western blotting, we measured the levels of 7S SNARE complexes and SNARE effectors in the effected hippocampi from animals that were electrically kindled through stimulation from one of three different foci. All three kindling paradigms, amygdalar, entorhinal, and septal, were associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus, measured 1 month after completion of kindling. Of the eight SNARE effectors examined (alpha-SNAP, NSF, SV2A/B, Munc18a/nSec1, Munc13-1, Complexins 1 and 2, and synaptotagmin I), there was a statistically significant bihemispheric increase of hippocampal SV2 and decrease of NSF upon kindling; neither by itself would be expected to account for the asymmetry of SNARE complex distribution. These data suggest that an ipsilateral hippocampal accumulation of SNARE complexes is a permanent alteration of kindling-induced epilepsy, regardless of stimulation pathway. The significance of these findings toward a molecular understanding of epilepsy will be discussed.

Effects of Peroxisome Proliferator-Activated Receptor Gamma Agonists on Brain Glucose and Glutamate Transporters after Stress in Rats

Repeated stress causes an energy-compromised status in the brain, with a decrease in glucose utilization by the brain cells, which might account for excitotoxicity processes seen in this condition. In fact, brain glucose metabolism mechanisms are impaired in some neurodegenerative disorders, including stress-related neuropsychopathologies. More recently, it has been demonstrated that some synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increase glucose utilization in rat cortical slices and astrocytes, as well as inhibit brain oxidative damage after repeated stress, which add support for considering these drugs as potential neuroprotective agents. To assess if stress causes glucose utilization impairment in the brain and to study the mechanisms by which this effect is achieved, young-adult male Wistar rats (control and immobilized for 6 h during 7 or 14 consecutive days, S7, S14) were i.p. injected with the natural ligand 15-deoxy-Delta-12,14-prostaglandin J2 (PGJ2, 120 microg/kg) or the high-affinity ligand rosiglitazone (RG, 3 mg/kg) at the onset of stress. Repeated immobilization during 1 or 2 weeks produces a decrease in brain cortical synaptosomal glucose uptake, and this effect was prevented by treatment with both natural and synthetic PPARgamma ligands by restoring protein expression of the neuronal glucose transporter, GLUT-3 in membrane fractions. On the other hand, treatment with PPARgamma ligands prevents stress-induced ATP loss in rat brain. Finally, repeated immobilization stress also produces a decrease in brain cortical synaptosomal glutamate uptake, and this effect was prevented by treatment with PPARgamma ligands by restoring synaptosomal protein expression of the glial glutamate transporter, EAAT2. In summary, our results demonstrate that 15d-PGJ2 and the thiazolidinedione rosiglitazone increase neuronal glucose metabolism, restore brain ATP levels and prevent the impairment in glutamate uptake mechanisms induced by exposure to stress, suggesting that this class of drugs may be therapeutically useful in conditions in which brain glucose levels or availability are limited after exposure to stress.

Genetische Befunde bei der Aufmerksamkeitsdefizit- und Hyperaktivitätsstörung (ADHS)

Attention-Deficit and Hyperactivity Disorder (ADHD) is a common child and adolescent psychiatric disorder with a prevalence rate of 3-7%. Formal genetic studies provided an estimated heritability of 0.6-0.8 and an approximately five-fold elevated risk for ADHD in first-degree relatives. Currently, four genome scans have led to the identification of chromosomal regions potentially relevant in ADHD; especially the evidence for linkage to chromosome 5p13 is convincing. Meta-analyses of a large number of candidate gene studies suggest association with gene variants of the dopaminergic receptors DRD4 and DRD5, the serotonergic receptor HTR1B, and the synaptosomal receptor protein (SNAP-25). Hyperactivity has been investigated particularly in animal models, focusing on knockout- and quantitative trait loci (QTL) designs, with promising results for the dopaminergic system. It is likely that several gene polymorphisms with moderate to small effect sizes contribute to the phenotype ADHD; different combinations of such predisposing variants presumably underlie ADHD in different individuals. Therefore, large samples for molecular genetic studies are mandatory to detect these polymorphisms. Accordingly, several of today's findings have to be regarded as preliminary. The understanding of ADHD's neurobiology may be advanced by new technologies, such as SNP-based genome scans performed with gene chips comprising 10,000-1,000,000 SNPs, as well as using more sophisticated animal model designs.

Structure-Toxicity Analysis of Type-2 Alkenes: In Vitro Neurotoxicity

Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The alpha,beta-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1 microM-10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other alpha,beta-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (>or= 25 mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2-conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups.

Oral administrations of methamidophos and TOCP have different effects on in vitro protein phosphorylation levels from subcellular fractions of hen brain

The effects of methamidophos and tri- o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ- 32P]ATP as phosphate donor. Tri- o-cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.

SNAP-25 in hippocampal CA3 region is required for long-term memory formation

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593–1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation.

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.

Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells

SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca(2+)-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic beta-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca(2+)-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant (tau) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be approximately 300 nm and every beta-cell contained approximately 400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca(2+)-current (-40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca(2+)-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in beta-cells.

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin alpha-1 chain, beta-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-alpha, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, alpha-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE.

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca 2+-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca 2+-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca 2+ signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.

Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2–10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3 mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 μM), increased KCl and 0.3 mM 4AP-evoked Ca 2+-dependent release of glutamate, but had little effect upon exocytosis evoked by 1 mM 4AP. PP1 did not affect the release of FM 2–10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.

Secretory role for human uterodomes (pinopods): secretion of LIF

The differentiation of human endometrial epithelium is a dynamic event, which occurs throughout the menstrual cycle in preparation for pregnancy. The appearance of uterodomes (pinopods) in this regard was first introduced in rodents with an established pinocytotic function, whereas little evidence was available in humans in this context. This study was undertaken to identify the potential physiological roles of uterodomes in the implantation process. To address this, endometrial biopsies from early, mid- and late luteal phases of the menstrual cycle of 23 fertile female patients with regular menses were used. Scanning and transmission electron microscopies (SEM and TEM) as well as immunofluorescence and immunogold TEM were performed to study the morphological changes and the expression pattern of leukaemia inhibitory factor (LIF) at uterodomes. Our results illustrated a high level of LIF expression in the human uterodomes, which was colocalized with the well-known biochemical markers of exocytosis, including syntaxin-1, 25-kDa synaptosomal protein (SNAP-25) and vesicle-associated membrane protein-2 (VAMP-2). Our morphological and immunocytochemical findings illustrated a secretory function for human uterodomes for the first time. In conclusion, this novel function for uterodomes provides an important clue in detection of their physiological function(s) during the process of the plasma membrane transformation.

Identification of synaptic plasma membrane proteins co‐precipitated with fibrillar β‐amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.

The Application of Proteomics in Neurology

Rapidly progressing proteomics techniques have been widely adopted in most areas of biology and medicine. In neurology and neuroscience, many applications of proteomics have involved neurotoxicology and neurometabolism, as well as in the determination of specific proteomic aspects of individual brain areas and body fluids in neurodegeneration. Investigation of brain protein groups in neurodegeneration, such as enzymes, cytoskeleton proteins, chaperones, synaptosomal proteins and antioxidant proteins, is in progress as phenotype related proteomics. The concomitant detection of several hundred proteins on a gel provides sufficiently comprehensive data to determine a pathophysiological protein network and its peripheral representatives. The rapid spread of proteomics technology, which principally consists of twodimensional gel electrophoresis (2-DE) with in-gel protein digestion of protein spots and identification by massspectrometry, has provided an explosive amount of results. An additional advantage is that hitherto unknown proteins have been identified as brain proteins. The current proteomics methods, however, have shortcomings and disadvantages. We would emphasize the failure to separate hydrophobic proteins as a major problem. So far, we have been unable to analyze the vast majority of these proteins in gels on 2-DE. There are several other analytical problems which also need to be overcome, and once solved, will allow for a more comprehensive analysis of the individual disease process. Here, we have reviewed the recent progress in proteomics research on neurodegeneration, with reference to its technological utility and problems in clinical application. Keywords: genome, proteome, proteomics, neurodegeneration, alzheimers disease, plasma, csf, two-dimensional gel electrophoresis

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.