- Email: [email protected]
WHAT PROTEOMICS WILL ADD TO OUR UNDERSTANDING OF THE NEUROPATHOLOGY OF SCHIZOPHRENIA
Abstracts
include an awareness of a need to use integrative approaches and an acknowledgement that there is a continued need for detailed neuropathology using newer technologies and studying multiple cortical regions and assessing different developmental time periods. Dr Kleinman in his role of discussant will integrate the conclusions of these talks and provide a summary regarding future work in this area. doi:10.1016/j.schres.2010.02.115
ABNORMAL N-GLYCOSYLATION OF PROTEINS OF THE GLUTAMATE SYNAPSE IN SCHIZOPHRENIA James Meador Woodruff University of Alabama at Birmingham, Alabama, USA Recent data suggest abnormalities of glutamate transmission in schizophrenia. While many findings of abnormal glutamate receptor expression in schizophrenia have been published, findings are subtle or even contradictory. These conflicting studies on the expression of these receptors leads to a reconsideration of the “glutamate hypothesis of schizophrenia” as not “too many” or “too few” receptors, but rather one of alterations in the cell biological processes that manage the total pool of receptors. Recent data point to abnormalities of glutamate receptor trafficking, delivery, dendritic localization, recycling, and degradation in the brain in schizophrenia. In this study, we extend our findings of abnormalities of intracellular trafficking of the AMPA subtype of glutamate receptor in schizophrenia. AMPA receptor trafficking starts in the endoplasmic reticulum (ER), in which AMPA receptor subunits are postranslationaly N-glycosylated. N-linked high mannose containing sugars added to AMPA subunits, which are trimmed and replaced by more elaborate sugars in the Golgi, after which AMPA subunits are trafficked for insertion into the plasma membrane. We assayed N-glycosylation status of AMPA subunits in prefrontal cortex in schizophrenia. N-glycosylation was assessed following digestion with endoglycosidase H (Endo H), which removes immature high mannose containing sugars, or with peptide-Nglycosidase F (PNGase F), which removes all N-linked sugars. We found that both GluR2 and GluR4 were sensitive to Endo H and PNGase F treatment, indicating that they are N-glycosylated; neither GluR1 nor GluR3 are N-glycosylated in human brain. GluR2 was found to have less N-linked high mannose and/or hybrid sugars in schizophrenia. This was confirmed by immunoprecipitation of GluR2 and probing with Concavalin A, a mannose-specific lectin. GluR2 immunoprecipitated from schizophrenia cortex was significantly less reactive to Con A comparing to the comparison group. These results indicate that GluR2 is abnormally glycosylated in schizophrenia, consistent with abnormal assembly or trafficking of the AMPA receptor. Abnormalities of glycosylation in schizophrenia may be more extensive, as we also have preliminary results suggesting abnormalities of N- glycosylation of NMDA subunits and two of the glutamate transporters. These results suggest that there are changes in glutamate receptors in schizophrenia that involve abnormalities of intracellular processes that effectively reduce receptor function even though total cellular levels of these receptors may be normal. Such findings are important because they point to the complexity of molecular and intracellular abnormalities in schizophrenia, and highlight novel sites that may be profitably targeted for drug.
doi:10.1016/j.schres.2010.02.116
141
WHAT PROTEOMICS WILL ADD TO OUR UNDERSTANDING OF THE NEUROPATHOLOGY OF SCHIZOPHRENIA David Cotter RCSI, Dublin, Ireland Proteomic studies of the post-mortem brain offer direct insights into the pathogenesis of schizophrenia at the level of protein expression. Until recently, the methods have been hampered by technological difficulties, not least the lack of a high though-put protein confirmation method. Fortunately, mass spectrometry methods have advanced greatly and these will have an impact on the field in the short term. We have reviewed the proteomic studies of post mortem brain studies of schizophrenia and will summarises the major protein pathways implicated in the disease pathology. The findings generally point towards prominent cellular assembly and organisation abnormalities, diminished cytoskeletal integrity, synaptic pruning and plasticity, and metabolic dysfunction. In our own data we have observed changes in several pathways which have the potential for providing novel insights into schizophrenia. For example, we have observed differential protein expression of a number of proteins involved in clathrin–mediated endocytosis and NMDA receptor recycling. We have also observed changes ironhomeostasis proteins, which are responsible for oligodendroglial function, in schizophrenia. Together these finding have the potential to provide novel insights into our understanding of the proposed NMDA hypofunction and, myelin changes in schizophrenia. Considering the complexity of the proteome it should be appreciated that we have only begun to explore the proteomic 'iceberg' that has relevance to schizophrenia. Future studies will be able to focus on previously inaccessible proteins by enriching for various membrane, synaptic, and cytoskeletal sub-proteomes. This will be an improvement on previous studies which have been based largely on relatively crude whole tissue preparations. Large scale validation of the various protein pathways will also need to be done. The advent of the mass spectrometry multiple reaction monitoring method (MRM) for absolute quantification may be the answer in this regard. However, integrating the findings from various 'omic' fields should provide answers that will not be apparent from the study of one field alone. Thus, there is an argument for future work in schizophrenia to apply more integrative approaches that incorporate the findings relating to envirome, genome, transcriptome in addition to the proteome. doi:10.1016/j.schres.2010.02.117
THE FUTURE OF POST-MORTEM RESEARCH -1 Maree Webster SMRI, Washington, USA Much emphasis has been placed on investigating the genetic susceptibility of the major mental disorders. However, the recent identification of a brain-specific isoform as a risk gene for schizophrenia and the identification of polymorphisms with clear tissue-specific effects on both gene expression and splicing indicate that the genetics of gene expression will need to be studied in cells that represent, and are most relevant to, the disease state. Thus, it is becoming apparent that in order to identify and understand the susceptibility genes and the most relevant associated biological pathways, post-mortem brain tissue will need to be utilized. To facilitate this effort future studies may need to integrate genomic and genome-wide expression data as well as neuropathological, proteomic, and neurochemical data from the same set of brains in
include an awareness of a need to use integrative approaches and an acknowledgement that there is a continued need for detailed neuropathology using newer technologies and studying multiple cortical regions and assessing different developmental time periods. Dr Kleinman in his role of discussant will integrate the conclusions of these talks and provide a summary regarding future work in this area. doi:10.1016/j.schres.2010.02.115
ABNORMAL N-GLYCOSYLATION OF PROTEINS OF THE GLUTAMATE SYNAPSE IN SCHIZOPHRENIA James Meador Woodruff University of Alabama at Birmingham, Alabama, USA Recent data suggest abnormalities of glutamate transmission in schizophrenia. While many findings of abnormal glutamate receptor expression in schizophrenia have been published, findings are subtle or even contradictory. These conflicting studies on the expression of these receptors leads to a reconsideration of the “glutamate hypothesis of schizophrenia” as not “too many” or “too few” receptors, but rather one of alterations in the cell biological processes that manage the total pool of receptors. Recent data point to abnormalities of glutamate receptor trafficking, delivery, dendritic localization, recycling, and degradation in the brain in schizophrenia. In this study, we extend our findings of abnormalities of intracellular trafficking of the AMPA subtype of glutamate receptor in schizophrenia. AMPA receptor trafficking starts in the endoplasmic reticulum (ER), in which AMPA receptor subunits are postranslationaly N-glycosylated. N-linked high mannose containing sugars added to AMPA subunits, which are trimmed and replaced by more elaborate sugars in the Golgi, after which AMPA subunits are trafficked for insertion into the plasma membrane. We assayed N-glycosylation status of AMPA subunits in prefrontal cortex in schizophrenia. N-glycosylation was assessed following digestion with endoglycosidase H (Endo H), which removes immature high mannose containing sugars, or with peptide-Nglycosidase F (PNGase F), which removes all N-linked sugars. We found that both GluR2 and GluR4 were sensitive to Endo H and PNGase F treatment, indicating that they are N-glycosylated; neither GluR1 nor GluR3 are N-glycosylated in human brain. GluR2 was found to have less N-linked high mannose and/or hybrid sugars in schizophrenia. This was confirmed by immunoprecipitation of GluR2 and probing with Concavalin A, a mannose-specific lectin. GluR2 immunoprecipitated from schizophrenia cortex was significantly less reactive to Con A comparing to the comparison group. These results indicate that GluR2 is abnormally glycosylated in schizophrenia, consistent with abnormal assembly or trafficking of the AMPA receptor. Abnormalities of glycosylation in schizophrenia may be more extensive, as we also have preliminary results suggesting abnormalities of N- glycosylation of NMDA subunits and two of the glutamate transporters. These results suggest that there are changes in glutamate receptors in schizophrenia that involve abnormalities of intracellular processes that effectively reduce receptor function even though total cellular levels of these receptors may be normal. Such findings are important because they point to the complexity of molecular and intracellular abnormalities in schizophrenia, and highlight novel sites that may be profitably targeted for drug.
doi:10.1016/j.schres.2010.02.116
141
WHAT PROTEOMICS WILL ADD TO OUR UNDERSTANDING OF THE NEUROPATHOLOGY OF SCHIZOPHRENIA David Cotter RCSI, Dublin, Ireland Proteomic studies of the post-mortem brain offer direct insights into the pathogenesis of schizophrenia at the level of protein expression. Until recently, the methods have been hampered by technological difficulties, not least the lack of a high though-put protein confirmation method. Fortunately, mass spectrometry methods have advanced greatly and these will have an impact on the field in the short term. We have reviewed the proteomic studies of post mortem brain studies of schizophrenia and will summarises the major protein pathways implicated in the disease pathology. The findings generally point towards prominent cellular assembly and organisation abnormalities, diminished cytoskeletal integrity, synaptic pruning and plasticity, and metabolic dysfunction. In our own data we have observed changes in several pathways which have the potential for providing novel insights into schizophrenia. For example, we have observed differential protein expression of a number of proteins involved in clathrin–mediated endocytosis and NMDA receptor recycling. We have also observed changes ironhomeostasis proteins, which are responsible for oligodendroglial function, in schizophrenia. Together these finding have the potential to provide novel insights into our understanding of the proposed NMDA hypofunction and, myelin changes in schizophrenia. Considering the complexity of the proteome it should be appreciated that we have only begun to explore the proteomic 'iceberg' that has relevance to schizophrenia. Future studies will be able to focus on previously inaccessible proteins by enriching for various membrane, synaptic, and cytoskeletal sub-proteomes. This will be an improvement on previous studies which have been based largely on relatively crude whole tissue preparations. Large scale validation of the various protein pathways will also need to be done. The advent of the mass spectrometry multiple reaction monitoring method (MRM) for absolute quantification may be the answer in this regard. However, integrating the findings from various 'omic' fields should provide answers that will not be apparent from the study of one field alone. Thus, there is an argument for future work in schizophrenia to apply more integrative approaches that incorporate the findings relating to envirome, genome, transcriptome in addition to the proteome. doi:10.1016/j.schres.2010.02.117
THE FUTURE OF POST-MORTEM RESEARCH -1 Maree Webster SMRI, Washington, USA Much emphasis has been placed on investigating the genetic susceptibility of the major mental disorders. However, the recent identification of a brain-specific isoform as a risk gene for schizophrenia and the identification of polymorphisms with clear tissue-specific effects on both gene expression and splicing indicate that the genetics of gene expression will need to be studied in cells that represent, and are most relevant to, the disease state. Thus, it is becoming apparent that in order to identify and understand the susceptibility genes and the most relevant associated biological pathways, post-mortem brain tissue will need to be utilized. To facilitate this effort future studies may need to integrate genomic and genome-wide expression data as well as neuropathological, proteomic, and neurochemical data from the same set of brains in