Abstract

A fundamental molecular component of neural connectivity is the SNARE (SNAP receptor) protein complex, which consists of three proteins, syntaxin, SNAP-25 and VAMP. Under appropriate conditions, the SNARE complex can be formed in vitro. To investigate the hypothesis that dysregulation of SNARE proteins or their interactions could be abnormal in severe mental disorders, the three SNARE proteins and the complex were studied in post-mortem anterior frontal cortex homogenates. An ELISA was used to quantify SNARE protein immunoreactivities in cortical homogenates from four groups: patients with schizophrenia who died of causes other than suicide (n = 6), patients with schizophrenia and suicide (n = 7), patients with depression and suicide (n = 11), and controls (n = 11). Differences between groups in patterns of SNARE protein immuno-reactivities were demonstrated [Wilks' Lambda F(9,68) = 3.57, P = 0.001]. Protein-by-protein analyses indicated a significant reduction in SNAP-25 immunoreactivity in the schizophrenia non-suicide group [28% decrease relative to controls, F(3,31) = 6.45, P = 0.002, Student–Newman–Keuls test, P < 0.01]. The intercorrelations between SNARE protein and synaptophysin immunoreactivities were high in controls, but lower in the other groups, further indicating disturbances in relationships between these proteins. The extent of SNARE complex formation in vitro was studied using immuno-blotting. Significant differences related to group membership were observed for the SNARE complexes identified by SNAP-25 [Wilks' Lambda F(3,31) = 4.76, P = 0.008] and by syntaxin immunostaining [Wilks' Lambda F(3,31) = 9.16, P = 0.0002]. In both groups with suicide as a cause of death, relatively more SNAP-25 and syntaxin was present in the heterotrimeric SNARE complex than in other molecular forms. These abnormalities in the SNARE complex could represent a molecular substrate for abnormalities of neural connectivity in severe mental disorders.

Introduction

Severe mental illnesses such as schizophrenia and affective disorders appear to be the consequence of abnormalities of neural connections, rather than neuronal loss (Andreasen, 1997; Lewis, 1997; Selemon and Goldman-Rakic, 1999). The effects of abnormalities of neural connections can be visualized with functional neuroimaging, and may have correlates in disturbances of cognition. These types of studies particularly implicate dysfunction of the prefrontal cortex in schizophrenia, depression and suicidal behaviour (Andreasen, 1997; Goldman-Rakic and Selemon, 1997; Mann, 1998). The molecular substrates for disturbed neural connectivity in severe mental illness remain uncertain, with evidence for involvement of multiple neuro-transmitter systems.

A candidate molecular substrate for one type of abnormal neural connectivity is a group of synapse-enriched proteins called SNAREs (= SNAP receptors; SNAP = soluble NSF attachment protein; NSF = N-ethylmaleimide-sensitive factor), which serve as a fundamental molecular mechanism governing neuro-transmission (Söllner et al., 1993a,b). Two groups of SNARE proteins, the Q-SNAREs (characterized by a glutamine residue in the central complex-forming region) and R-SNAREs (characterized by an arginine residue) participate in the closely regulated process of synaptic vesicle exocytosis and recycling (Fasshauer et al., 1998). Molecular components of the SNARE mechanism include three main proteins: the R-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), and the Q-SNAREs synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin (Söllner et al., 1993a,b). Other proteins, including synaptophysin and the complexins, are endogenous modifiers of SNARE protein interactions (Edelmann et al., 1995; McMahon et al., 1995).

Although the SNARE mechanism is thought to be implicated in all forms of chemical neurotransmission, some evidence suggests differential distribution of isoforms of the proteins. SNAP-25 appears to be present in most, but not all synapses (Oyler et al., 1989; Geddes et al., 1990; Duc and Catsicas, 1995). Syntaxin was found to be particularly enriched in asymmetric synapses in one animal study (Sesack and Snyder, 1995). The mRNA coding for different isoforms of VAMP also has specific patterns of cellular distribution (Trimble et al., 1990). Although the correspondence between SNARE protein isoforms and neurotransmitters is unclear, mice hemizygous for a deletion of the gene coding for SNAP-25 display regionally specific abnormalities of dopaminergic, serotonergic and glutamatergic transmission (Raber et al., 1997). SNARE proteins appear to be present in both small synaptic vesicles as well as large, densecore vesicles (Langley and Grant, 1997; Winkler, 1997; Winkler and Fischer-Colbrie, 1998). Targeted cleavage of SNARE proteins by botulinum or tetanus neurotoxins results in broad failure of synaptic neurotransmission (Hayashi et al., 1994), and can have therapeutic value in the treatment of dystonias.

Although each of the three SNARE proteins are vital to neuro-transmission, considerable experimental evidence indicates that different stimuli can have different effects on individual SNARE and other presynaptic proteins. As examples in animal models, transient forebrain ischaemia was associated with increased SNAP-25 immunoreactivity in hippocampal mossy fibres, but no change in syntaxin (Martí et al., 1998). Brain-derived neurotrophic factor (BDNF) appeared to exert effects that were selective for VAMP and synaptophysin, without affecting SNAP-25 or syntaxin (Pozzo-Miller et al., 1999). Kainate administration to rats resulted in increased hippocampal SNAP-25 immuno-reactivity (Martí et al., 1999), while syntaxin immunoreactivity was unchanged, and syntaxin-1A mRNA was reported to be decreased (Fujino et al., 1997). In contrast, stimuli associated with induction of long-term potentiation (LTP) resulted in increased hippocampal syntaxin-1B mRNA without change in SNAP-25 (Kamphuis et al., 1995; Hicks et al., 1997). Spatial learning was without effect on hippocampal syntaxin-1B (Richter-Levin et al., 1998), further supporting the selective nature of the relationship between certain specific stimuli and regulation of SNARE mechanism proteins.

Potential roles for the SNARE mechanism proteins in disease are largely unexplored. In dementia, cognition is impaired in proportion to reduction of neocortical and hippocampal synaptophysin, which likely serves as a marker of diffuse synaptic terminal loss (Terry et al., 1991; Lassmann et al., 1992; Wakabayashi et al., 1994; Sze et al., 1997; Heffernan et al., 1998). Corresponding reductions of individual SNARE proteins in frontal and temporal cortex are described in the end stages of dementia (Gabriel et al., 1997; Shimohama et al., 1997; Mukaetova-Ladinska et al., 2000). In one study, reduction in syntaxin immunoreactivity in the molecular layer of the dentate gyrus provided a better association with dementia and antemortem cognition than did reductions in synaptophysin (Wakabayashi et al., 1994). In severe mental disorders, studies of individual SNARE proteins as well as modifier proteins such as synaptophysin and the complexins indicate abnormalities in prefrontal cortex and hippocampus in schizophrenia (Davidsson et al., 1999; Honer et al., 2000) and in hippocampus and cingulate cortex in depression or bipolar disorder (Jørgensen and Riederer, 1985; Eastwood et al., 2000a; Eastwood and Harrison, 2001; Webster et al., 2001). Since the SNARE proteins interact and are differentially regulated, we tested the hypothesis that abnormalities of the SNARE mechanism might be present in severe mental disorder by studying the three core proteins, and their interactions in frontal cortex.

Materials and Methods

Samples

Frozen samples were obtained from the Clinical Brain Disorders Branch (NIMH, St Elizabeth's, Washington, DC; n = 14) or from tissue banks of the authors (n = 21). Control and mental illness tissues were obtained from each site. Age and post-mortem time did not differ between samples obtained at the different sites, the mean storage time for the NIMH samples was longer than the other samples [119 versus 78 months, F(1,33) = 16.4, P = 0.0003]. Hemispheres were cut into 1.5 cm coronal sections, and samples of cortical grey matter were dissected from the anterior half of the superior or middle frontal gyrus (NIMH set) or the anterior half of the middle or inferior frontal gyrus (author's set). Hemisphere sampled was not known consistently, nor were anatomical sections taken to permit cytoarchitectonic analysis. Death was sudden (trauma, myocardial infarction or suicide) in nearly all cases. Samples were available from seven patients with schizophrenia and suicide as a cause of death (sch/sui), six patients with schizophrenia (sch/nonsui), 11 patients with major depression and suicide as a cause of death (dep/sui) and 11 controls (con). A summary of demographic variables appears in Table 1; detailed description of individual cases was published previously (Honer et al., 1999). Diagnoses were made according to DSM-III-R criteria, or for some earlier schizophrenia cases, the Diagnostic Evaluation After Death (Feighner criteria) following chart review by a research psychiatrist (Zalcman and Endicott, 1983). These criteria are largely consistent for the diagnosis of schizophrenia. For 25 of the cases, toxicological testing was performed on brain or serum samples, or detailed notes describing medications at the time of death were available. Antipsychotic drugs were detected in two cases, and antidepressant drugs in three. All the cases with schizophrenia were likely to have been previously prescribed antipsychotic drugs.

Antibodies and Fusion Protein Studies

Monoclonal antibodies to investigate synapse-enriched proteins were prepared by immunizing mice with a crude synaptic preparation from human frontal cortex homogenate (Honer et al., 1993). The hybridoma cells derived were screened for production of antibodies reactive with human cortical homogenates in an enzyme-linked immunoadsorbent assay (ELISA), with formalin fixed brain tissue using immunocyto-chemistry, and with single bands on immunoblots. With this approach, antibodies presumed to be reactive with SNAP-25 (antibody SP12) and syntaxin (SP6) were identified and their targets subsequently confirmed with immunoblotting of fusion proteins (Honer et al., 1997). Immuno-blotting and immunocytochemical studies indicated antibody SP10 was likely reactive with VAMP (Honer et al., 1993). As part of the present study, fusion protein experiments were performed for further characterization.

VAMP-1 and VAMP-2 cDNAs were subcloned into the bacterial expression vector pGEX-KG (Guan and Dixon, 1991) to produce in-frame fusion proteins with the bacterial glutathione-S-transferase (GST) protein. The fragments chosen gave rise to full length VAMP-1 (amino acids 1–118) and VAMP-2 (amino acids 1–116) proteins, as well as truncated fragments of VAMP-2 (amino acids 1–96, 33–116 and 33–96). Recombinant fusion proteins were induced, the bacteria lysed and protein determinations were made prior to immunoblotting with SP10. Controls for the immunoblotting studies were proteins obtained from bacteria transfected with syntaxin-1A cDNA. As well, immunostaining of all fusion proteins was carried out using tissue culture conditioned medium from the parent non-secreting myeloma, as an additional negative control.

ELISA Studies of Frontal Cortex Homogenates

An ELISA was chosen for quantification of immunoreactivity since this assay format exhibits a good linear range, allows multiple samples to be studied easily and simultaneously, and allows whole homogenates rather than semi-purified extracts to be studied. Previous studies demonstrated a very high correlation between results obtained with the ELISA and quantitative immunoblotting (Honer et al., 1997), with a superior linear range for the ELISA. Frozen samples were rapidly thawed and homogenized in Tris-buffered saline (TBS: 10 mM Tris–HCl, 140 mM NaCl, pH 7.4) prior to determination of protein concentration, and dilution to a concentration of 60 μg/ml with distilled water. All samples were studied together in duplicate, blinded to diagnosis. Serial dilutions of each sample were dried onto ELISA plates, as described in detail (Honer et al., 1999). Tissue culture supernatants containing antibodies SP12 and SP6 were used at a dilution of 1:10, purified antibody SP10 was used at 1:500. The assay format provided a duplicate serial dilution curve of antigen immunoreactivity for each sample. For syntaxin and SNAP-25, these curves were used to determine the protein concentration required for an optical density of 0.5 for each sample. These protein concentration values were used for comparisons between groups. The ELISAs were linear over a 16-fold range of homogenate protein concentration for SNAP-25 and a 32-fold range for syntaxin. Run-to-run correlations for three runs of all samples were 0.77–0.96. For VAMP, in some cases the optical density readings were less than 0.5, so the integrated sum of optical density for each sample over an 8-fold dilution range of homogenate was used for comparisons (Vincent et al., 1996).

Studies of SNARE Protein–Protein Interactions

Previous studies of SNARE fusion proteins or native proteins extracted from animal brain indicated the capacity of these proteins in vitro to exist as monomers as well as to form heterodimers, heterotrimers, and multimers of these heterotrimers (Hayashi et al., 1994, 1995; Pevsner et al., 1994). These complexes have the property of being sodium dodecyl sulphate (SDS)-resistant, and can be detected as higher molecular weight bands with immunoblotting of non-boiled samples. Complex formation in vivo is necessary for neurotransmission. To investigate in vitro complex formation in human frontal cortex, frozen tissue samples were rapidly thawed, homogenized in TBS and incubated with 0.1% Triton X-100 for 30 min on ice, followed by centrifugation at 13 000 g for 15 min and collection of the supernatant. This approach allowed the samples for immunoblotting to be as similar as possible to samples used for the quantitative ELISA, where higher concentrations of detergent could disrupt binding to the plates. Polyacrylamide gel electrophoresis (SDS–PAGE) was performed on boiled and non-boiled samples, using sample buffer containing 64 mM Tris-base pH 6.8, 1% SDS and 2% beta-mercaptoethanol (boiled samples only), and 10% or 12% acrylamide gels. Following transfer to nitrocellulose, immunoblotting was carried out with antibodies SP10 (ascites 1:200), SP12 (tissue culture supernatant 1:50) and SP6 (tissue culture supernatant 1:20) using the ECL detection system. Secondary antibodies (Jackson Immunolabs, West Grove, PA) were used at 1:1000 (SP10) or 1:2000 (SP6 and SP12). Conditioned tissue cultured medium was used as a negative control.

Following parallel studies of boiled and non-boiled samples to establish the properties of the SNARE antigens in human frontal cortex, all individual samples were studied in the non-boiled condition using SP12 and SP6 for immunoblotting of crude homogenates. SP10 results required loading much more protein, were more variable, and were not obtained for individual samples. Homogenate samples containing 5 μg protein were loaded. Densitometry and NIH Image v1.58 (Rasband, 1992) were used to measure the within-sample relative immunoreactivities of the bands of complexed and uncomplexed proteins. An aliquot of a reference sample was loaded onto each gel to allow correction for variability in immuno-staining between gels. The reference sample was also used in serial dilution to obtain a standard curve, with 8-fold linear range. Greyscale values obtained from the individual immunostained samples were used with the standard curve to obtain antigen concentration for each sample relative to the reference standard. The coefficient of variation was 4–6% for the reference samples used as internal controls for each gel.

Statistical Analysis

Previous studies of synaptophysin in the same series of samples reported here indicated the importance of separating groups by both diagnosis and suicide/non-suicide as a cause of death (Honer et al., 1999). Since the SNARE proteins are potentially interacting molecules, a multivariate analysis of variance (MANOVA) was used initially to determine if SNARE immunoreactivity determined by ELISA differed between the four groups (con, sch/nonsui, sch/sui and dep/sui). Linear regression analysis was used to investigate potential effects of age, post-mortem interval and storage time on ELISA results. These measures were also used as covariates in a MANOVA to control for the possible influence of these variables on results. Possible effects of site of origin of tissues and gyrus sampled were investigated similarly.

The immunoblot studies of protein–protein interactions resulted in four bands of immunoreactivity for SNAP-25 in the non-boiled samples, and three for syntaxin. The percentage of total immunoreactivity in each band was determined. These values were subject to a MANOVA, SNAP-25 and syntaxin data were kept separate. The same four groups were used as for the ELISA data.

Results

Characterization of Antibody SP10 and SNARE Protein–Protein Interactions

Previous immunoblotting and immunocytochemical studies suggested antibody SP10 was reactive with VAMP. This was confirmed by positive immunoblotting results for both VAMP-1 and VAMP-2 fusion proteins (Fig. 1).

At least three higher molecular weight complexes were observed in immunoblots of the non-boiled samples (Fig. 2). The immunoreactivity pattern of the highest molecular weight band was consistent with formation of the ternary SNARE complex of VAMP, syntaxin and SNAP-25. Lower molecular weight complexes likely represent formation of (multiple) dimers of VAMP with SNAP-25, and syntaxin with SNAP-25. In the non-boiled condition, the majority of SNAP-25 was observed in higher molecular weight complexes, while for VAMP and syntaxin both monomeric and complexed proteins were identified. The complexes detected are most likely to have formed in vitro. The results demonstrate that SNARE proteins in human frontal cortex have similar properties to those in other experimental systems, and that the panel of antibodies used in the present study detected monomeric as well as complexed proteins.

Quantification of SNARE Proteins with ELISA

There were no statistically significant correlations between age or post-mortem interval and immunoreactivity of any of the SNARE proteins. The only significant correlation with storage interval was with syntaxin immunoreactivity (r = 0.41, P = 0.02 uncorrected for multiple comparisons); longer storage was associated with increased syntaxin immunoreactivity. This result is opposite to what would be expected if degradation of the antigen was related to longer storage time, and may represent a chance finding among nine comparisons.

Separation of the sample into four groups (con, sch/nonsui, sch/sui and dep/sui) indicated a difference in SNARE immuno-reactivity between groups [Wilks' Lambda F(9,68) = 3.57, P = 0.001]. Addition of covariates, site of origin of tissue or gyrus sampled to the analysis did not alter the significance of the result. This finding also remained significant if three groups were used [schizophrenia, depression and control; Wilks' Lambda F(9,58) = 2.93, P = 0.01]. Inspection of the results in Figure 3 suggests the relationship of immunoreactivity of the SNARE proteins to each other differed between groups. This observation was supported by tests of correlation between the individual SNARE protein immunoreactivities (Table 2), and between the SNARE protein and synaptophysin immunoreactivities (measured previously) (Honer et al., 1999). In controls, correlations between all protein immunoreactivities were high, with the exception of syntaxin and VAMP. In the illness groups, correlations were weaker, and in some cases clearly nonsignificant. In the non-suicide schizophrenia group, SNAP-25 was poorly correlated with the other proteins, while in the depressed suicide group VAMP was poorly correlated with the others. In analyses of the total amounts of immunoreactivity of individual proteins, only SNAP-25 showed a significant difference between groups [F(3,31) = 6.45, P = 0.002]. The non-suicide schizophrenia group had 28% lower SNAP-25 immunoreactivity than controls, and was also significantly different from the other two groups (Student–Newman– Keuls test, P < 0.01, both comparisons).

Quantification of SNARE Protein–Protein Interactions

The non-denaturing gels allow assessment of the capacity of the SNARE proteins to spontaneously form complexes in vitro. Total SNAP-25 immunoreactivity can be resolved into a monomer, a heterodimer consisting of SNAP-25 and VAMP, a heterodimer consisting of SNAP-25 and syntaxin, and a heterotrimer consisting of the three SNARE proteins. The relative immunoreactivity of each of the four bands was quantified for each sample, and a MANOVA was used to analyse differences between the four groups (con, sch/nonsui, sch/sui and dep/sui). A significant overall difference was observed [Wilks' Lambda F(12,74) = 1.90, P < 0.05], with the heterotrimer representing the full SNARE complex demonstrating the most significant difference [ANOVA F(3,31) = 4.76, P = 0.008, Fig. 4]. Compared with control samples, both groups of samples from cases where suicide was the cause of death demonstrated relatively more SNAP-25 immunoreactivity in the heterotrimeric complex than in the monomeric or heterodimeric forms (Student–Newman–Keuls P < 0.05, both comparisons). In the schizophrenia/non-suicide group the ELISA of overall SNAP-25 immunoreactivity showed a decrease compared with controls. The immunoblot study indicated the relative proportions of SNAP-25 in the different molecular complexes were unchanged for this group compared with controls.

A similar analysis was applied to syntaxin immunoreactivity, which was present as a monomer, a heterodimer composed of syntaxin and SNAP-25, and the SNARE heterotrimer (no syntaxin-VAMP heterodimers were detected). The MANOVA indicated a significant difference between groups [Wilks' Lambda F(9,71) = 3.78, P = 0.0006, Fig. 4]. The results for the heterotrimeric complex identified with syntaxin immunoreactivity were similar to the SNAP-25 immunostaining [ANOVA F(3,31) = 9.16, P = 0.0002]. Compared with control samples, both groups of samples from cases where suicide was the cause of death demonstrated relatively more syntaxin immunoreactivity in the heterotrimeric complex than in other forms (Student–Newman– Keuls P < 0.05, both comparisons).

Discussion

The results support the hypothesis that abnormalities of SNARE proteins may be present in severe mental disorders. Quantitative disturbances in SNARE protein immunoreactivities were observed, as well as perturbations in the likelihood of formation of ternary SNARE complexes in vitro.

SNARE Protein–Protein Interactions

The results indicate the present panel of monoclonal antibodies can detect SNARE proteins in a range of forms in human frontal cortex. The properties of protein–protein interactions and formation of SDS-resistant complexes previously demonstrated in animal brain, or with studies of fusion proteins (Söllner et al., 1993a,b; Hayashi et al., 1994, 1995; Pevsner et al., 1994), were also detected in human tissue.

The high molecular weight complex comprised of SNAP-25, syntaxin and VAMP is likely a multiple heterotrimer, consistent with observations of others (Hayashi et al., 1994, 1995; Poirier et al., 1998). Such large macromolecular complexes appear particularly prone to form when low detergent concentrations are used with native proteins having intact membrane anchors (Hohl et al., 1998). The pattern of immunoreactivity of the lower molecular weight complexes appeared to indicate interactions of VAMP with SNAP-25, and of syntaxin with SNAP-25. These findings are consistent with in vitro studies which report a high-affinity interaction between SNAP-25 and syntaxin, a moderate affinity interaction of SNAP-25 and VAMP, and much reduced affinity interaction between VAMP and syntaxin (Calakos and Scheller, 1994; Hayashi et al., 1994; Pevsner et al., 1994). The present panel of antibodies detected monomers as well as complexes of SNARE proteins. In the ELISA, proteins could exist in multiple forms, and some anti-SNARE antibodies appear to have their binding sites masked by complex formation (Garcia et al., 1995; Pellegrini et al., 1995). This was not the case for SP6, SP10 and SP12, indicating the ELISA results with these antibodies would not be biased by differences between samples in the extent of in vitro complex formation.

Quantification of SNARE Mechanism Proteins

Studies of individual SNARE proteins in severe mental disorders indicate quantitative abnormalities in SNAP-25 in hippocampus and frontal cortex (Jørgensen and Riederer, 1985; Thompson et al., 1998; Young et al., 1998; Karson et al., 1999), and syntaxin in cingulate cortex (Gabriel et al., 1997; Honer et al., 1997). Previous studies of VAMP are not reported. The present approach of simultaneous analysis suggests that differences in patterns of protein expression may be related to behaviours associated with successful suicide. Suicide was associated with relatively increased VAMP and SNAP-25, and relatively higher likelihood of formation of the heterotrimeric SNARE complex in vitro. Of note, the availability of SNAP-25 appears to be an important factor influencing the interaction in vitro of VAMP with the SNARE complex (Hayashi et al., 1995). Elevations in either VAMP or SNAP-25 could be responsible for the relative increase in likelihood of formation of the ternary SNARE complex. Alternatively, alteration in one or more of a host of proteins which interact with and modulate SNARE complex formation and disassembly could be present (Mochida, 2000). A recent microarray study is of interest in this regard (Mirnics et al., 2000). Reduced mRNA coding for the protein NSF was described in BA 9 in schizophrenia. NSF protein acts to help disassemble the ternary SNARE complex, and perturbation in NSF could influence the in vitro stability of the SNARE complex. An additional factor which may influence SNARE complex formation is altered dopaminergic neurotransmission. Administration of dopamine to rat striatal slices resulted in increased SNARE complex formation, which was partially blocked by haloperidol (Fisher and Braun, 2000). Differences in neuro-transmission between cases that died by suicide compared with other causes could contribute to the altered SNARE protein interactions.

The effects of relative increases in SNARE proteins are not well studied. Treatment of mouse hippocampal slice preparations with BDNF increased VAMP and synaptophysin, and was associated with increased electrophysiological responses to high frequency stimulation (Pozzo-Miller et al., 1999). Overexpression of SNAP-25 in cultured hippocampal neurons resulted in impaired synaptic transmission (Owe-Larsson et al., 1999). Disturbances in neurotransmission related to overexpression of SNARE proteins in frontal cortex could be associated with abnormal behaviour. The ventral prefrontal cortex contributes to the executive function of inhibition, and impairment in this function could contribute to aspects of suicidal behaviour including disinhibition, impulsivity and aggression (Mann, 1998).

Differences in SNARE protein immunoreactivities and interactions between schizophrenia with suicide as a cause of death and non-suicides were observed. Such observations are not without precedent. Considering neurotransmitter studies, increased AMPA binding in caudate was described for patients with psychotic illness and suicide compared to psychotic illness alone (Freed et al., 1993). In schizophrenia, significantly decreased affinity of paroxetine binding to the serotonin transporter in hippocampus was observed only in cases with suicide (Dean et al., 1995). In contrast, in prefrontal cortex, serotonin 5HT2 receptor density in psychotic patients with suicide was not different from controls, but in non-suicide psychotic patients was decreased relative to controls (Laruelle et al., 1993). These studies and the present results concerning presynaptic proteins suggest that there may be shared features of the neurobiology of suicide that are independent of psychiatric diagnosis.

Schizophrenia in the absence of suicide was associated with relatively reduced SNAP-25 immunoreactivity. The consequences of reduced SNARE proteins are somewhat better studied than overexpression. For SNAP-25, two genetic model systems are described. In Caenorhabditis elegans, a reduction of function mutation in SNAP-25 resulted in resistance to the acetylcholinesterase inhibitor aldicarb, indicating impaired cholinergic neurotransmission (van Swinderen et al., 1999). In mice, the coloboma (Cm/+) mutant has a deletion of the chromosomal segment containing the Snap gene which encodes SNAP-25 (Hess et al., 1992).The homozygous mutation is lethal in utero; heterozygous animals have a 40–50% reduction in SNAP-25 and a range of behavioural and neurochemical abnormalities (Hess et al., 1992, 1996; Heyser et al., 1995). In cortical synaptosomes, glutamate content was observed to be reduced and release mechanisms were impaired (Raber et al., 1997). Hippocampal LTP was also reported to be attenuated (Steffensen et al., 1996). These findings suggest reduced SNAP-25 could contribute to abnormalities of neurotransmission in anterior frontal cortex in schizophrenia.

Relationship to Previous Studies

The majority of previous studies of synaptic proteins in frontal cortex in schizophrenia have focused on synaptophysin. Several studies indicated reduced synaptophysin immunoreactivity, using ELISA or immunoblotting studies of homogenates (Perrone-Bizzozero et al., 1996; Honer et al., 1999; Karson et al., 1999), however other studies have reported no change (Gabriel et al., 1997; Davidsson et al., 1999; Eastwood et al., 2000b). An immunocytochemical study of fixed tissue reported decreased synaptophysin (Glantz and Lewis, 1997). The latter technique has the advantage of more precise cytoarchitectonic delineation of subregions of cortex, and the possibility of analysing laminar differences. In schizophrenia, synaptophysin immunoreactivity appeared to be reduced proportionately in all layers (Glantz and Lewis, 1997). Synaptophysin immunoreactivity was also studied in the samples used in the present investigation, and a significant reduction in the non-suicide schizophrenia homogenates was reported (Honer et al., 1999). Correlations between synaptophysin and the SNARE proteins in control samples in the present study were high, as were correlations between the SNARE proteins except for VAMP and syntaxin. There was no significant correlation between SNAP-25 and synaptophysin or syntaxin in the individuals with schizophrenia who died of causes other than suicide. Similarly, a disturbed relationship between VAMP and synaptophysin as well as the other SNARE protein immunoreactivities was noted in the group of depressed patients who died of suicide. These disturbances in patterns of correlation provide further support for the concept that abnormal relationships between SNARE and other presynaptic proteins may contribute to expression of illness. Higher resolution analysis of relationships between SNARE and other synaptic proteins in tissue samples using confocal microscopy would allow further testing of this hypothesis, and the possible relationship to neural circuit models of cortical dysfunction in schizophrenia (Lewis and Anderson, 1995).

Limitations and Potentially Confounding Factors

The availability of tissue for the investigation resulted in limitations in analyses related to behaviour and diagnosis. Samples from patients with depression who died of causes other than suicide were not available, limiting possible comparisons. As well, although every attempt was made to control for differences between groups that could be related to differences in age, this remains a possible factor that could affect results. Age at death in control subjects was not correlated with SNARE immunoreactivity. In other studies, while significant effects of ageing (range 22–87) were reported on synaptophysin mRNA expression, no significant relationship with synaptophysin protein immunoreactivity was seen in the same samples (Eastwood et al., 2000b). In completely different series than reported here, we noted no relationship between age and SNARE protein immunoreactivity over a range of 53–99 years in control subjects (Minger et al., 2001). Recently, aged rats with intact spatial learning were reported to show no differences in hippocampal synaptophysin immunoreactivity from young rats, while learning-impaired aged rats were different from both other groups (Smith et al., 2000).

Neurotransmission can be altered by psychotropic medications. Differences in presynaptic proteins between the samples of patient and control brains could be due to drug treatment history. In the present study, the treatment histories of the suicides with schizophrenia and those with depression were likely to have differed, yet the SNARE proteins were altered in parallel. Similarly, while individuals with schizophrenia who died of suicide, as well as those who died of other causes were all likely to have been exposed to antipsychotics, the pattern of SNARE protein differences from controls differed. As well, other studies have failed to observe effects of antipsychotic medications on SNAP-25 or syntaxin immunoreactivity in human brain (Gabriel et al., 1997; Honer et al., 1997), or in SNAP-25 immunoreactivity in rats administered perphenazine (Fog et al., 1976). One study of mRNA expression in rats following haloperidol treatment reported a decrease in syntaxin message, but only in the nucleus accumbens and no changes in SNAP-25, VAMP or syntaxin mRNA in cortex were observed (Nakahara et al., 1998). The effects of antipsychotic medications appear to be more pronounced on postsynaptic proteins such as neuro-receptors rather than presynaptic molecules (Harrison, 1999).

The present findings suggest abnormalities of SNARE mechanism proteins are present in schizophrenia and other severe mental disorders. Heterogeneity in the pattern of disturbance of SNARE proteins was observed, which may contribute to the clinical heterogeneity of these illnesses. Further studies of SNARE proteins in severe mental disorders, and of the possibility of developing agents that could modify SNARE protein expression or interactions may be of interest.

Notes

W.G.H. was supported by a Scientist Award from the Canadian Institutes of Health Research. Grant support was provided by CIHR (MT14037), NARSAD and the National Institutes of Mental Health (MT46745, MH40210 and MH60877). Drs Joel Kleinman and Manuel Casanova provided tissue samples.

Table 1

Demographic variables for the sample, mean (SD)

 Control Schizophrenia (non-suicide) Schizophrenia (suicide) Depression (suicide) 
The mean age of patients with schizophrenia who died of natural causes differed from both groups who died of suicide (Student–Newman–Keuls, P < 0.05), but not controls. Mean storage time for the schizophrenia suicide group was longer than controls or the depressed suicide group (Student–Newman–Keuls, P < 0.05). 
Males/females  8/3  3/4  3/3  5/6 
Age (years) 46.3 (17.0) 58.0 (16.9)  36.2 (9.0) 34.7 (6.9) 
Post-mortem time (h) 17.8 (4.8) 12.2 (5.6)  15.7 (6.5) 11.0 (5.3) 
Storage time (months) 97.0 (26.8) 93.3 (11.4) 129.5 (64.1) 73.4 (26.8) 
 Control Schizophrenia (non-suicide) Schizophrenia (suicide) Depression (suicide) 
The mean age of patients with schizophrenia who died of natural causes differed from both groups who died of suicide (Student–Newman–Keuls, P < 0.05), but not controls. Mean storage time for the schizophrenia suicide group was longer than controls or the depressed suicide group (Student–Newman–Keuls, P < 0.05). 
Males/females  8/3  3/4  3/3  5/6 
Age (years) 46.3 (17.0) 58.0 (16.9)  36.2 (9.0) 34.7 (6.9) 
Post-mortem time (h) 17.8 (4.8) 12.2 (5.6)  15.7 (6.5) 11.0 (5.3) 
Storage time (months) 97.0 (26.8) 93.3 (11.4) 129.5 (64.1) 73.4 (26.8) 
Table 2

Correlation matrix of presynaptic protein immunoreactivities, with approximate P-values calculated from Fisher's r to z transformation

  Synaptophysin Syntaxin SNAP-25 
Values shown are r and (P). Nonparametric analysis with Spearman's correlation yielded similar results. 
Control Syntaxin  0.591 (0.05)   
 SNAP-25  0.885 (0.0001) 0.643 (0.03)  
 VAMP  0.854 (0.0003) 0.424 (0.20) 0.820 (0.001) 
sch/nonsui Syntaxin  0.807 (0.05)   
 SNAP-25 –0.102 (0.86) 0.095 (0.87)  
 VAMP  0.604 (0.23) 0.600 (0.23) 0.457 (0.39) 
sch/sui Syntaxin  0.885 (0.02)   
 SNAP-25  0.744 (0.10) 0.879 (0.02)  
 VAMP  0.870 (0.02) 0.641 (0.19) 0.653 (0.18) 
Depression Syntaxin  0.839 (0.0006)   
 SNAP-25  0.722 (0.01) 0.748 (0.006)  
 VAMP  0.087 (0.81) 0.165 (0.64) 0.264 (0.44) 
  Synaptophysin Syntaxin SNAP-25 
Values shown are r and (P). Nonparametric analysis with Spearman's correlation yielded similar results. 
Control Syntaxin  0.591 (0.05)   
 SNAP-25  0.885 (0.0001) 0.643 (0.03)  
 VAMP  0.854 (0.0003) 0.424 (0.20) 0.820 (0.001) 
sch/nonsui Syntaxin  0.807 (0.05)   
 SNAP-25 –0.102 (0.86) 0.095 (0.87)  
 VAMP  0.604 (0.23) 0.600 (0.23) 0.457 (0.39) 
sch/sui Syntaxin  0.885 (0.02)   
 SNAP-25  0.744 (0.10) 0.879 (0.02)  
 VAMP  0.870 (0.02) 0.641 (0.19) 0.653 (0.18) 
Depression Syntaxin  0.839 (0.0006)   
 SNAP-25  0.722 (0.01) 0.748 (0.006)  
 VAMP  0.087 (0.81) 0.165 (0.64) 0.264 (0.44) 
Figure 1.

Antibody SP10 recognizes VAMP, but not syntaxin fusion protein. Bacteria expressing fusion proteins of portions of VAMP or syntaxin proteins with glutathione-S-transferase were lysed and 1 μg of protein from each was electrophoresed, blotted and probed with SP10. The portion of the VAMP or syntaxin protein present in the fusion construct is indicated above. Proteolytic degradation products are frequently detected with full-length VAMP fusion proteins.

Figure 1.

Antibody SP10 recognizes VAMP, but not syntaxin fusion protein. Bacteria expressing fusion proteins of portions of VAMP or syntaxin proteins with glutathione-S-transferase were lysed and 1 μg of protein from each was electrophoresed, blotted and probed with SP10. The portion of the VAMP or syntaxin protein present in the fusion construct is indicated above. Proteolytic degradation products are frequently detected with full-length VAMP fusion proteins.

Figure 2.

Composite immunoblot of frontal cortex homogenate prepared under reducing conditions with boiling (+), or under non-reducing conditions without boiling (–), then probed with antibodies reactive with VAMP, syntaxin (synt) or SNAP-25. Multiple complexes were visualized under the non-boiling conditions. The highest molecular weight complex was immunoreactive with all three antibodies. Other complexes consistent with VAMP/SNAP-25 and syntaxin/SNAP-25 dimers were observed. Arrows adjacent to the non-boiled α-SNAP-25 lane indicate the position, from top down, of SNAP-25/syntaxin/VAMP heterotrimers, SNAP-25/syntaxin heterodimers and SNAP-25/VAMP heterodimers.

Figure 2.

Composite immunoblot of frontal cortex homogenate prepared under reducing conditions with boiling (+), or under non-reducing conditions without boiling (–), then probed with antibodies reactive with VAMP, syntaxin (synt) or SNAP-25. Multiple complexes were visualized under the non-boiling conditions. The highest molecular weight complex was immunoreactive with all three antibodies. Other complexes consistent with VAMP/SNAP-25 and syntaxin/SNAP-25 dimers were observed. Arrows adjacent to the non-boiled α-SNAP-25 lane indicate the position, from top down, of SNAP-25/syntaxin/VAMP heterotrimers, SNAP-25/syntaxin heterodimers and SNAP-25/VAMP heterodimers.

Figure 3.

ELISA results. The mean and standard error of immunoreactivity of syntaxin (synt), SNAP-25 (S-25) and VAMP is plotted relative to control. This presentation of the results emphasizes relative patterns of SNARE protein immunoreactivities. The schizophrenia/non-suicide group and the two suicide groups appeared to differ from controls. Controls (con), schizophrenia where the cause of death was not suicide (sch/nonsui), schizophrenia with suicide (sch/sui) and depressed suicides (dep/sui).

Figure 3.

ELISA results. The mean and standard error of immunoreactivity of syntaxin (synt), SNAP-25 (S-25) and VAMP is plotted relative to control. This presentation of the results emphasizes relative patterns of SNARE protein immunoreactivities. The schizophrenia/non-suicide group and the two suicide groups appeared to differ from controls. Controls (con), schizophrenia where the cause of death was not suicide (sch/nonsui), schizophrenia with suicide (sch/sui) and depressed suicides (dep/sui).

Figure 4.

Non-denaturing gel results for quantification of heterotrimeric SNARE complex formation relative to total immunoreactivity. SNARE complexes were detected by antibodies reactive with SNAP-25 or with syntaxin on immunoblots. Increased complex formation was detected in the suicide cases with both antibodies. Percentiles are indicated by boxes (25, 50, 75) and bars (10, 90) with points outside these ranges also shown. *P < 0.05 compared with control.

Figure 4.

Non-denaturing gel results for quantification of heterotrimeric SNARE complex formation relative to total immunoreactivity. SNARE complexes were detected by antibodies reactive with SNAP-25 or with syntaxin on immunoblots. Increased complex formation was detected in the suicide cases with both antibodies. Percentiles are indicated by boxes (25, 50, 75) and bars (10, 90) with points outside these ranges also shown. *P < 0.05 compared with control.

References

Andreasen NC (
1997
) Linking mind and brain in the study of mental illnesses: a project for a scientific psychopathology.
Science
 
275
:
1586
–1593.
Calakos N, Scheller RH (
1994
) Protein–protein interactions contributing to the specificity of intracellular vesicular trafficking.
Science
 
263
:
1146
–1149.
Davidsson P, Gottfries J, Bogdanovic N, Ekman R, Karlsson I, Gottfries C-G, Blennow K (
1999
) The synaptic-vesicle-specific proteins rab3a and synaptophysin are reduced in thalamus and related cortical brain regions in schizophrenic brains.
Schizophr Res
 
40
:
23
–29.
Dean B, Opeskin K, Pavey G, Naylor L, Hill C, Keks N, Copolov DL (
1995
) [3H]Paroxetine binding is altered in the hippocampus but not the frontal cortex or caudate nucleus from subjects with schizophrenia.
J Neurochem
 
64
:
1197
–1202.
Duc C, Catsicas S (
1995
) Ultrastructural localization of SNAP-25 within the rat spinal cord and peripheral nervous system.
J Comp Neurol
 
356
:
152
–163.
Eastwood SL, Burnet PWJ, Harrison PJ (
2000
) Expression of complexin I and II mRNAs and their regulation by antipsychotic drugs in the rat forebrain.
Synapse
 
36
:
167
–177.
Eastwood SL, Cairns NJ, Harrison PJ (
2000
) Synaptophysin gene expression in schizophrenia.
Brit J Psychiatry
 
176
:
236
–242.
Eastwood SL, Harrison PJ (
2001
) Synaptic pathology in the anterior cingulate cortex in schizophrenia and mood disorders.
Brain Res Bull
 
55
:
569
–578.
Edelmann L, Hanson PI, Chapman ER, Jahn R (
1995
) Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.
EMBO J
 
14
:
224
–231.
Fasshauer D, Sutton RB, Brunger AT, Jahn R (
1998
) Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q-and R-SNAREs.
Proc Natl Acad Sci USA
 
95
:
15781
–15786.
Fisher H, Braun JEA (
2000
) Modulation of the SNARE core complex by dopamine.
Can J Physiol Pharmacol
 
78
:
856
–859.
Fog R, Pakkenberg H, Juul P, Bock E, Jørgensen OS, Andersen J (
1976
) High-dose treatment of rats with perphenazine.
Psychopharmacology
 
50
:
305
–307.
Freed WJ, Dillon-Carter O, Kleinman JE (
1993
) Properties of [3H]AMPA binding in postmortem human brain from psychotic subjects and controls: increases in caudate nucleus associated with suicide.
Exp Neurol
 
121
:
48
–56.
Fujino I, Fujiwara T, Akagawa K (
1997
) Transient decrease of HPC-1/ syntaxin-1A mRNA in the rat hippocampus by kainic acid.
Neurosci Res
 
28
:
243
–247.
Gabriel SM, Haroutunian V, Powchik P, Honer WG, Davidson M, Davies P, Davis KL (
1997
) Increased concentrations of presynaptic proteins in the cingulate cortex of schizophrenics.
Arch Gen Psychiatry
 
54
:
559
–566.
Garcia EP, McPherson PS, Chilcote TJ, Takei K, De Camilli P (
1995
) rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin.
J Cell Biol
 
129
:
105
–120.
Geddes JW, Hess EJ, Hart RA, Kesslak JP, Cotman CW, Wilson MC (
1990
) Lesions of hippocampal circuitry define synaptosomal-associated protein-25 (SNAP-25) as a novel presynaptic marker.
Neuroscience
 
38
:
515
–525.
Glantz LA, Lewis DA (
1997
) Reduction of synaptophysin immuno-reactivity in the prefrontal cortex of subjects with schizophrenia: regional and diagnostic specificity.
Arch Gen Psychiatry
 
54
:
943
–952.
Goldman-Rakic PS, Selemon LD (
1997
) Functional and anatomical aspects of prefrontal pathology in schizophrenia.
Schizophr Bull
 
23
:
437
–458.
Guan KL, Dixon JE (
1991
) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal Biochem
 
192
:
262
–267.
Harrison PJ (
1999
) The neuropathological effects of antipsychotic drugs.
Schizophr Res
 
40
:
87
–99.
Hayashi T, Yamasaki S, Nauenburg S, Binz T, Niemann H (
1995
) Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.
EMBO J
 
14
:
2317
–2325.
Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H (
1994
) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.
EMBO J
 
13
:
5051
–5061.
Heffernan JM, Eastwood SL, Nagy Z, Sanders MW, McDonald B, Harrison PJ (
1998
) Temporal cortex synaptophysin mRNA is reduced in Alzheimer's disease and is negatively correlated with the severity of dementia.
Exp Neurol
 
150
:
235
–239.
Hess EJ, Jinnah HA, Kozak CA, Wilson MC (
1992
) Spontaneous locomotor hyperactivity in a mouse mutant with a deletion including the Snap gene on chromosome 2.
J Neurosci
 
12
:
2865
–2874.
Hess EJ, Collins KA, Wilson MC (
1996
) Mouse model of hyperkinesis implicates SNAP-25 in behavioral regulation.
J Neurosci
 
16
:
3104
–3111.
Heyser CJ, Wilson MC, Gold LH (
1995
) Coloboma hyperactive mutant exhibits delayed neurobehavioral developmental milestones.
Dev Brain Res
 
89
:
264
–269.
Hicks A, Davis S, Rodger J, Helme-Guizon A, Laroche S, Mallet J (
1997
) Synapsin I and syntaxin 1B: key elements in the control of neurotransmitter release are regulated by neuronal activation and long-term potentiation in vivo.
Neuroscience
 
79
:
329
–340.
Hohl TM, Parlati F, Wimmer C, Rothman JE, Söllner TH, Engelhardt H (
1998
) Arrangements of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes.
Mol Cell
 
2
:
539
–548.
Honer WG, Hu L, Davies P (
1993
) Human synaptic proteins with a heterogeneous distribution in cerebellum and visual cortex.
Brain Res
 
609
:
9
–20.
Honer WG, Falkai P, Young C, Wang T, Xie J, Bonner J, Hu L, Boulianne GL, Lu Z, Trimble WS (
1997
) Cingulate cortex synaptic terminal proteins and neural cell adhesion molecule in schizophrenia.
Neuroscience
 
78
:
99
–110.
Honer WG, Falkai P, Chen C, Arango V, Mann JJ, Dwork AJ (
1999
) Synaptic and plasticity associated proteins in anterior frontal cortex in severe mental illness.
Neuroscience
 
91
:
1247
–1255.
Honer WG, Young C, Falkai P (2000) Synaptic pathology. In: The neuropathology of schizophrenia (Harrison PJ, Roberts GW, eds), pp. 105–136. Oxford: Oxford University Press.
Jørgensen OS, Riederer P (
1985
) Increased synaptic markers in hippo-campus of depressed patients.
J Neural Transm
 
64
:
55
–66.
Kamphuis W, Smirnova T, Hicks A, Hendriksen H, Mallet J, Lopes da Silva FH (
1995
) The expression of syntaxin 1B/GR33 mRNA is enhanced in the hippocampal kindling model of epileptogenesis.
J Neurochem
 
65
:
1974
–1980.
Karson CN, Mrak RE, Schluterman KO, Stumer WQ, Sheng JG, Griffin WST (
1999
) Alterations in synaptic proteins and their encoding mRNAs in prefrontal cortex in schizophrenia: a possible neuro-chemical basis for ‘hypofrontality’.
Mol Psychiatry
 
4
:
39
–45.
Langley K, Grant NJ (
1997
) Are exocytosis mechanisms neurotransmitter specific?
Neurochem Int
 
31
:
739
–757.
Laruelle M, Abi-Dargham A, Casanova MF, Toti R, Weinberger DR, Kleinman JE (
1993
) Selective abnormalities of prefrontal serotonergic receptors in schizophrenia.
Arch Gen Psychiatry
 
50
:
810
–818.
Lassmann H, Weiler R, Fischer P, Bancher C, Jellinger K, Floor E, Danielczyk W, Seitelberger F, Winkler H (
1992
) Synaptic pathology in Alzheimer's disease: immunological data for markers of synaptic and large dense-core vesicles.
Neuroscience
 
46
:
1
–8.
Lewis DA (
1997
) Development of the prefrontal cortex during adolescence: insights into vulnerable neural circuits in schizophrenia.
Neuropsychopharmacology
 
16
:
385
–398.
Lewis DA, Anderson SA (
1995
) The functional architecture of the prefrontal cortex and schizophrenia.
Psychol Med
 
25
:
887
–894.
Mann JJ (
1998
) The neurobiology of suicide.
Nature Med
 
4
:
25
–30.
Martí E, Blasi J, Gomez de Aranda I, Ribera R, Blanco R, Ferrer I (
1999
) Selective early induction of synaptosomal-associated protein (molecular weight 25,000) following systemic administration of kainate at convulsant doses in the rat.
Neuroscience
 
90
:
1421
–1432.
Martí E, Ferrer I, Ballabriga J, Blasi J (
1998
) Increase in SNAP-25 immunoreactivity in the mossy fibers following transient forebrain ischemia in the gerbil.
Acta Neuropathol
 
95
:
254
–260.
McMahon HT, Missler M, Li C, Südhof TC (
1995
) Complexins: cytosolic proteins that regulate SNAP receptor function.
Cell
 
83
:
111
–119.
Minger SL, Honer WG, Esiri MM, McDonald B, Keene J, Nicoll JAR, Carter J, Hope T, Francis PT (
2001
) Synaptic pathology in prefrontal cortex is present only with severe dementia in Alzheimer's disease.
J Neuropath Exp Neurol
 
60
:
929
–936.
Mirnics K, Middleton FA, Marquez A, Lewis DA, Levitt P (
2000
) Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex.
Neuron
 
28
:
53
–67.
Mochida S (
2000
) Protein–protein interactions in neurotransmitter release.
Neurosci Res
 
36
:
175
–182.
Mukaetova-Ladinska EB, Garcia-Siera F, Hurt J, Gertz H-J, Xuereb JH, Hills R, Brayne C, Huppert FA, Paykel ES, McGee M, Jakes R, Honer WG, Harrington CR, Wischik CM (
2000
) Staging of cytoskeletal and α-amyloid changes in human isocortex reveals biphasic synaptic protein response during progression of Alzheimer's disease.
Am J Pathol
 
157
:
623
–636.
Nakahara T, Nakamura K, Tsutsumi T, Hashimoto K, Hondo H, Hisatomi S, Motomura K, Uchimura H (
1998
) Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain.
Mol Brain Res
 
61
:
238
–242.
Owe-Larsson B, Berglund MM, Kristensson K, Garoff H, Larhammar D, Brodin L, Löw P (
1999
) Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector.
Eur J Neurosci
 
11
:
1981
–1987.
Oyler GA, Higgins GA, Hart RA, Battenberg E, Billingsley M, Bloom FE, Wilson MC (
1989
) The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.
J Cell Biol
 
109
:
3039
–3052.
Pellegrini LL, O'Connor V, Lottspeich F, Betz H (
1995
) Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.
EMBO J
 
14
:
4705
–4713.
Perrone-Bizzozero NI, Sower AC, Bird ED, Benowitz LI, Ivins KJ, Neve RL (
1996
) Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia.
Proc Natl Acad Sci USA
 
93
:
14182
–14187.
Pevsner J, Hsu S-C, Braun JEA, Calakos N, Ting AE, Bennett MK, Scheller RH (
1994
) Specificity and regulation of a synaptic vesicle docking complex.
Neuron
 
13
:
353
–361.
Poirier MA, Hao JC, Malkus PN, Chan C, Moore MF, King DS, Bennett MK (
1998
) Protease resistance of syntaxinSNAP-25VAMP complexes: implications for assembly and structure.
J Biol Chem
 
273
:
11370
–11377.
Pozzo-Miller LD, Gottschalk W, Zhang L, McDermott K, Du J, Gopalakrishnan R, Oho C, Sheng Z-H, Lu B (
1999
) Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice.
J Neurosci
 
19
:
4972
–4983.
Raber J, Mehta PP, Kreifeldt M, Parsons LH, Weiss F, Bloom FE, Wilson MC (
1997
) Coloboma hyperactive mutant mice exhibit regional and transmitter-specific deficits in neurotransmission.
J Neurochem
 
68
:
176
–188.
Rasband W (1992) NIH image. Bethesda, MD: NIMH.
Richter-Levin G, Thomas KL, Hunt SP, Bliss TVP (
1998
) Dissociation between genes activated in long-term potentiation and in spatial learning in the rat.
Neurosci Lett
 
251
:
41
–44.
Selemon LD, Goldman-Rakic PS (
1999
) The reduced neuropil hypothesis: a circuit based model of schizophrenia.
Biol Psychiatry
 
45
:
17
–25.
Sesack SR, Snyder CL (
1995
) Cellular and subcellular localization of syntaxin-like immunoreactivity in the rat striatum and cortex.
Neuroscience
 
67
:
993
–1007.
Shimohama S, Kamiya, Taniguchi T, Akagawa K, Kimura J (
1997
) Differential involvement of synaptic vesicle and presynaptic plasma membrane proteins in Alzheimer's disease.
Biochem Biophys Res Commun
 
236
:
239
–242.
Smith TD, Adams MM, Gallagher M, Morrison JH, Rapp PR (
2000
) Circuit-specific alterations in hippocampal synaptophysin immuno-reactivity predict spatial learning impairment in aged rats.
J Neurosci
 
20
:
6587
–6593.
Söllner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (
1993
) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation and fusion.
Cell
 
75
:
409
–418.
Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE (
1993
) SNAP receptors implicated in vesicle targeting and fusion.
Nature
 
362
:
318
–324.
Steffensen SC, Wilson MC, Hendriksen SJ (
1996
) Coloboma contiguous gene deletion encompassing Snap alters hippocampal plasticity.
Synapse
 
22
:
281
–289.
Sze C-I, Troncoso JC, Kawas C, Mouton P, Price DL, Martin LJ (
1997
) Loss of the presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive decline in Alzheimer disease.
J Neuropathol Exp Neurol
 
56
:
933
–944.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (
1991
) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment.
Ann Neurol
 
30
:
572
–580.
Thompson PM, Sower AC, Perrone-Bizzozero NI (
1998
) Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia.
Biol Psychiatry
 
43
:
239
–243.
Trimble W, Gray T, Elferink L, Wilson M, Scheller R (
1990
) Distinct patterns of expression of two VAMP genes within the rat brain.
J Neurosci
 
10
:
1380
–1387.
van Swinderen B, Saifee O, Shebester L, Roberson R, Nonet ML, Crowder CM (
1999
) A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans.
Proc Natl Acad Sci USA
 
96
:
2479
–2484.
Vincent I, Rosado M, Davies P (
1996
) Mitotic mechanisms in Alzheimer's disease?
J Cell Biol
 
132
:
413
–425.
Wakabayashi K, Honer WG, Masliah E (
1994
) Synapse alterations in the hippocampal–entorhinal formation in Alzheimer's disease with and without Lewy body disease.
Brain Res
 
667
:
24
–32.
Webster MJ, Weickert CS, Herman MM, Hyde TM, Kleinman JE (
2001
) Synaptophysin and GAP-43 mRNA levels in the hippocampus of subjects with schizophrenia.
Schizophr Res
 
49
:
61
–70.
Winkler H (
1997
) Membrane composition of adrenergic large and small dense core vesicles and of synaptic vesicles: consequences for their biogenesis.
Neurochem Res
 
22
:
921
–932.
Winkler H, Fischer-Colbrie R (
1998
) Regulation of the biosynthesis of large dense-core vesicles in chromaffin cells and neurons.
Cell Mol Neurobiol
 
18
:
193
–209.
Young CE, Arima K, Xie J, Hu L, Beach TG, Falkai P, Honer WG (
1998
) SNAP-25 deficit and hippocampal connectivity in schizophrenia.
Cereb Cortex
 
8
:
261
–268.
Zalcman S, Endicott J (1983) Diagnostic evaluation after death. Bethesda, MD: Neurosciences Research Branch, National Institute of Mental Health.