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Regional and developmental brain expression patterns of SNAP25 splice variants.

Prescott GR, Chamberlain LH - BMC Neurosci (2011)

Bottom Line: Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b.The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus.The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK.

ABSTRACT

Background: SNAP25 is an essential SNARE protein for regulated exocytosis in neuronal cells. Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b. These splice variants differ by only 9 amino acids, and studies of their expression to date have been limited to analysis of the corresponding mRNAs. Although these studies have been highly informative, it is possible that factors such as differential turnover of the SNAP25 proteins could complicate interpretations based entirely on mRNA expression profiles.

Results: We report the generation and characterization of antibodies that distinguish between SNAP25a and SNAP25b isoforms, and their use to investigate the expression profile of these proteins in rat and human brain. In rat brain, SNAP25b protein expression increased dramatically during post-natal development, whereas the increase in SNAP25a expression was more modest and variable. The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus. The SNAP25 isoforms also displayed distinct regional expression patterns, with SNAP25a very weakly expressed in both rat and human cerebellum. Quantitative analysis revealed that SNAP25b was the dominant isoform in all adult human brain regions examined.

Conclusions: SNAP25a and SNAP25b display distinct developmental and regional expression profiles in rat and human brain. These differences might reflect distinct functions of these highly conserved isoforms in membrane fusion pathways in the brain. The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

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Semi-quantitative comparison of SNAP25 isoform expression levels in human brain. A) Lysates prepared from post-mortem brain samples (pooled from 6 individual patients) were resolved by SDS-PAGE alongside various dilutions of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23. Gels were transferred to nitrocellulose and immunoblotted using antibodies against SNAP25a (3067), SNAP25b (3068) or SNAP23. The position of EGFP-tagged and endogenous proteins are highlighted, and molecular weight markers are shown on the left side of all blots. B) The relative levels of SNAP25a, SNAP25b and SNAP23 in brain were calculated by comparison of band intensities to those of the respective EGFP-tagged proteins. Highest expression levels were for SNAP25b in cortex and this was given an arbitrary value of 1, with all other values expressed as a fraction of this.
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Figure 4: Semi-quantitative comparison of SNAP25 isoform expression levels in human brain. A) Lysates prepared from post-mortem brain samples (pooled from 6 individual patients) were resolved by SDS-PAGE alongside various dilutions of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23. Gels were transferred to nitrocellulose and immunoblotted using antibodies against SNAP25a (3067), SNAP25b (3068) or SNAP23. The position of EGFP-tagged and endogenous proteins are highlighted, and molecular weight markers are shown on the left side of all blots. B) The relative levels of SNAP25a, SNAP25b and SNAP23 in brain were calculated by comparison of band intensities to those of the respective EGFP-tagged proteins. Highest expression levels were for SNAP25b in cortex and this was given an arbitrary value of 1, with all other values expressed as a fraction of this.

Mentions: Having characterized regional expression patterns of SNAP25a and SNAP25b in rat brain, we next examined expression in post-mortem samples of human brain. For this, lysates of cortex, hippocampus, cerebellum and thalamus were prepared from 6 post-mortem brains. Equal amounts of the individual lysates from each brain region were then pooled and resolved on SDS-PAGE gels alongside a dilution series of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23 (Figure 4A). Densitometry was then used to estimate the relative expression levels of SNAP25a, SNAP25b and SNAP23 in the different brain regions by comparison with the EGFP-tagged standards (Figure 4B). This semi-quantitative analysis revealed that expression of SNAP25b was highest and SNAP23 expression lowest in every brain region examined. The expression pattern of SNAP25a in human brain was grossly similar to that observed in rat brain, with this isoform highest in cortex and only weakly expressed in cerebellum.


Regional and developmental brain expression patterns of SNAP25 splice variants.

Prescott GR, Chamberlain LH - BMC Neurosci (2011)

Semi-quantitative comparison of SNAP25 isoform expression levels in human brain. A) Lysates prepared from post-mortem brain samples (pooled from 6 individual patients) were resolved by SDS-PAGE alongside various dilutions of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23. Gels were transferred to nitrocellulose and immunoblotted using antibodies against SNAP25a (3067), SNAP25b (3068) or SNAP23. The position of EGFP-tagged and endogenous proteins are highlighted, and molecular weight markers are shown on the left side of all blots. B) The relative levels of SNAP25a, SNAP25b and SNAP23 in brain were calculated by comparison of band intensities to those of the respective EGFP-tagged proteins. Highest expression levels were for SNAP25b in cortex and this was given an arbitrary value of 1, with all other values expressed as a fraction of this.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3104942&req=5

Figure 4: Semi-quantitative comparison of SNAP25 isoform expression levels in human brain. A) Lysates prepared from post-mortem brain samples (pooled from 6 individual patients) were resolved by SDS-PAGE alongside various dilutions of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23. Gels were transferred to nitrocellulose and immunoblotted using antibodies against SNAP25a (3067), SNAP25b (3068) or SNAP23. The position of EGFP-tagged and endogenous proteins are highlighted, and molecular weight markers are shown on the left side of all blots. B) The relative levels of SNAP25a, SNAP25b and SNAP23 in brain were calculated by comparison of band intensities to those of the respective EGFP-tagged proteins. Highest expression levels were for SNAP25b in cortex and this was given an arbitrary value of 1, with all other values expressed as a fraction of this.
Mentions: Having characterized regional expression patterns of SNAP25a and SNAP25b in rat brain, we next examined expression in post-mortem samples of human brain. For this, lysates of cortex, hippocampus, cerebellum and thalamus were prepared from 6 post-mortem brains. Equal amounts of the individual lysates from each brain region were then pooled and resolved on SDS-PAGE gels alongside a dilution series of HEK293T lysates from cells transfected with EGFP-SNAP25a, EGFP-SNAP25b or EGFP-SNAP23 (Figure 4A). Densitometry was then used to estimate the relative expression levels of SNAP25a, SNAP25b and SNAP23 in the different brain regions by comparison with the EGFP-tagged standards (Figure 4B). This semi-quantitative analysis revealed that expression of SNAP25b was highest and SNAP23 expression lowest in every brain region examined. The expression pattern of SNAP25a in human brain was grossly similar to that observed in rat brain, with this isoform highest in cortex and only weakly expressed in cerebellum.

Bottom Line: Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b.The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus.The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK.

ABSTRACT

Background: SNAP25 is an essential SNARE protein for regulated exocytosis in neuronal cells. Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b. These splice variants differ by only 9 amino acids, and studies of their expression to date have been limited to analysis of the corresponding mRNAs. Although these studies have been highly informative, it is possible that factors such as differential turnover of the SNAP25 proteins could complicate interpretations based entirely on mRNA expression profiles.

Results: We report the generation and characterization of antibodies that distinguish between SNAP25a and SNAP25b isoforms, and their use to investigate the expression profile of these proteins in rat and human brain. In rat brain, SNAP25b protein expression increased dramatically during post-natal development, whereas the increase in SNAP25a expression was more modest and variable. The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus. The SNAP25 isoforms also displayed distinct regional expression patterns, with SNAP25a very weakly expressed in both rat and human cerebellum. Quantitative analysis revealed that SNAP25b was the dominant isoform in all adult human brain regions examined.

Conclusions: SNAP25a and SNAP25b display distinct developmental and regional expression profiles in rat and human brain. These differences might reflect distinct functions of these highly conserved isoforms in membrane fusion pathways in the brain. The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

Show MeSH
Related in: MedlinePlus