1.6.1 Heterogeneity of the synaptosomal preparation
A quantitative electron microscopic study of synaptosomes prepared by the sucrose-density gradient procedure, reported that 49.1 % of structures visible in the EM were synaptosomes (Dodd et al., 1981). Free mitochondria contributed 2.3 % of particles, while myelin fragments amounted to 3 %. The majority of the remaining 46.6 % were unidentified structures, most likely of glial origin (Cotman et al., 1971; Henn et al., 1976). Henn et al. have estimated that about 40 % of gliosomes present in the homogenate are copurified in the synaptosome preparations. Thus the synaptosome fraction is enriched in synaptic particles of different neurotransmitter phenotypes but also contains a large number of non-synaptic particles of neuronal and glial origin. Despite this heterogeneity synaptosomes represent a fraction that is enriched in synaptic particles and therefore allows a much more synapse-specific analysis than the analysis of unfractionated tissue homogenates. For this reason the analysis of the protein content of synaptosomes and synaptosome-derived fractions such as SVs, synaptic plasma membranes and postsynaptic density fractions has lead to an explosion of knowledge about the biochemical makeup of synapses. (for reviews see (Abul-Husn and Devi, 2006; Bai and Witzmann, 2007; Bayés and Grant, 2009; Tribl et al., 2006;
Tribl et al., 2008).
1.6.2 Synaptosome proteomics
The amount of data made available through proteomic analysis of synaptosomes and derived fractions is overwhelming and only few examples can be mentioned here. Schrimpf et al. (2005) utilized the ICAT-approach to further reduce the complexity of the synaptosome proteome. They identified 1131 proteins, 631 of which were identified reproducibly. The identified proteins cover a large spectrum of pre- and postsynaptic proteins, but also include the known glial contaminations. This approach did not identify VGLUTs. On the other hand macroglial contaminations were evident through the identification of GLT-1, MBP, PLP (Schrimpf et al., 2005). Another proteomic study combined 2-DE-MALDI-TOF and a LC-MS/MS shotgun approach to catalogue the synaptosomal proteome. Over 900 proteins were identified in this study including many known presynaptic and postsynaptic proteins. A striking feature of this study is that it failed to identify any of the vesicular neurotransmitter transporters. In addition to the identification of proteins, this study also identified several
posttranslational modifications in synaptosomal proteins, such as glycosylation and acetylation (Witzmann et al., 2005).
Proteomics of synaptosomes has also been applied to study protein expression changes in pathology. A study analyzed changes in synaptsomal protein expression of spinal cord ganglia after spinal nerve injury and proteomics identified 27 proteins to be differentially expressed (Singh et al., 2009). Also a study on the relationship between the administration of antipsychotic drugs used in the treatment of schizophrenia and changes in synaptic protein expression and yielded a total of 17 proteins whose expression levels are differentially regulated as assessed by silver staining after 2-D-gel electrophoresis of synaptosomal proteins (Ji et al., 2009).
The few examples given above illustrate that proteomics of synaptosomes can provide a wealth of information about the protein composition of synaptic fractions and can also be applied to study changes in protein expression in the context of neurological diseases. Even though synaptosomal preparations are enriched in synaptic particles, contaminations by non-synaptic and glial particles also contribute to the proteins identified in proteomic studies of synaptosomes. Furthermore, the synaptosomal preparation contains a mixture of synaptosomes derived from all the different neurotransmitter systems described earlier. Therefore, analysis of synaptosomal proteins reflects the average protein composition of synaptosomal particles from all these neurotransmitter systems. The synaptosomal preparation contains thousands of different proteins, however mass spectrometry based protein identification can not yet fully represent this high complexity and tends to preferentially identify the more abundant proteins within a mixture of proteins (Patterson and Aebersold, 2003). This is exemplified by the fact that the afore mentioned proteomics approaches did not identify the VGLUT proteins and indicates that a further subfractionation of the synaptosomal preparation may be necessary to allow more comprehensive description of it’s individual components.
1.6.3 Proteomic analyses of synaptic vesicles
One way to further reduce the complexity of the synaptosomal preparation is to break down the synaptosomes into smaller components such as synaptic plasma membranes or SVs. Several investigators have employed these further fractionations in their proteomic studies.
An early, gel based proteomic study on cortical SVs identified 36 vesicle proteins among which were GAPDH, annexin III, α-internexin, VDAC1, and Rab14, ZnT3 in cortical SVs. (Coughenour et al., 2004). Later immunoisolation of SV2 containing SVs and synaptic plasma membrane fractions (membranes with docked vesicles – Active zones) and analysis by 2D-16-BAC/SDS PAGE identified 72 SV proteins using MALDI-TOF-MS. (Morciano et al.,
2005). These studies identified several previously known SV proteins and SV associated proteins. Subsequently, a carefully conducted study by Shigeo Takamori in the laboratory of Reinhard Jahn aimed at providing a quantitative description of the lipid and protein composition of average SVs. Following 16-BAC/SDS-PAGE and 1D-SDS PAGE, a total of 410 proteins was identified. These authors also monitored the distribution of 85 proteins among different subcellular fractions by Western blotting, in order to investigate the specificity of these proteins to the SV fraction. Among the SV proteins identified in proteomics were the vesicular neurotransmitter transporters, such as VGLUT1, VGLUT2 and VGAT and the less abundant transporters VAChT and VMAT2 could be identified by Western blotting. The presence of all the different vesicular neurotransmitter transporters illustrates nicely that the SVs isolated here are originating from a variety of different synaptic particles.
The authors noted that they observed an unexpected diversity of SNARE and Rab proteins in their isolated SVs. Considering that the diversity of synapses from which synaptic vesicles were isolated this might have reflected the diversity of SVs between different neurotransmitter types. Also the diversity of synaptic, non-synaptic or even non-neuronal particles within the synaptosomal preparation could result in the presence of non-synaptic vesicles in the SV fraction.
These issues were addressed in a later study of the same laboratory. In this study, a quantitative proteomic comparison of immunoisolated VGLUT1 and VIAAT containing SVs based on iTRAQ identified and quantified over 450 proteins (Grønborg et al., 2010). Only 50 proteins were found to be differentially expressed between the two vesicle populations of which only very few were proper SV proteins. These included, ZnT3, SV2A, SV31, which preferentially associated with VGLUT1, and SV2C that was preferentially associated with VIAAT. SV2B was equally distributed between the two vesicle populations. In addition, this study identified a novel VGLUT1-SV protein, called MAL-2. The fact that only few differences could be resolved between SVs of different neurotransmitter systems indicates that the protein correlates of functional specializations of these synapses are not determined by SVs alone, but are probably reflected in the molecular composition of the whole synapse, including cytosolic, vesicular and plasma membrane proteins of the pre-and postsynapse.
1.6.4 A need for further fractionation of the synaptosomal preparation
The description above illustrates two key points: (i) The mass-spectrometry based identification of proteins is a powerful tool, which can generate a wealth of data on the protein composition of subcellular fractions in health and disease (ii) Unless quantitative comparative analyses are made it is difficult to differentiate between protein identifications that originate from contaminations and proteins that are specifically enriched in the organelle under investigation.
As a result of the second point, in the context neurological disease models one can not be certain about the subcellular origin of a phenotype specific change observed by investigations of proteomic differences in the synaptosomal preparation. It would therefore be of great value if it were possible to further purify and sub fractionate the particles contained in the conventional synaptosome preparations. The need for such protocols has already been recognized in the “pre-proteomic era”, and in the past several groups have introduced further purification steps, in an attempt to isolate specific subpopulations of synaptosomes at higher purity (reviewed in part by (Whittaker, 1993)).