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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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Related in: MedlinePlus

Developmental expression patterns of SNAP25a and SNAP25b in rat brain. A) Left panel, Equal amounts of whole brain lysates prepared from rats at different developmental stages (supplied by Zyagen Inc.) were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, panSNAP25, SNAP23 and actin antibodies. Right panel shows the mean (+/-SEM) fold increase in expression levels of SNAP23, SNAP25a and SNAP25b between a late embryonic stage (E18-E20) and P30-32 (n = 3). The fold increase in SNAP25b expression was significantly greater than that of either SNAP25a (P < 0.05) or SNAP23 (p < 0.02), analysed using a one-way ANOVA. B) Equal amounts of brain lysates from gender-determined E20 litter mates were resolved on SDS-PAGE gels beside an aliquot of P32 brain lysate and analyzed as in panel A. C) Lysates from the hippocampus, cerebellum and cortex were prepared from pooled samples collected from 3 animals at P2 or P20. Samples were analyzed as in Panel A. Position of molecular weight standards are shown on the left of all panels.
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Figure 2: Developmental expression patterns of SNAP25a and SNAP25b in rat brain. A) Left panel, Equal amounts of whole brain lysates prepared from rats at different developmental stages (supplied by Zyagen Inc.) were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, panSNAP25, SNAP23 and actin antibodies. Right panel shows the mean (+/-SEM) fold increase in expression levels of SNAP23, SNAP25a and SNAP25b between a late embryonic stage (E18-E20) and P30-32 (n = 3). The fold increase in SNAP25b expression was significantly greater than that of either SNAP25a (P < 0.05) or SNAP23 (p < 0.02), analysed using a one-way ANOVA. B) Equal amounts of brain lysates from gender-determined E20 litter mates were resolved on SDS-PAGE gels beside an aliquot of P32 brain lysate and analyzed as in panel A. C) Lysates from the hippocampus, cerebellum and cortex were prepared from pooled samples collected from 3 animals at P2 or P20. Samples were analyzed as in Panel A. Position of molecular weight standards are shown on the left of all panels.

Mentions: SNAP25 displays a marked increase in expression from around post-natal week two in mice, and analysis of mRNA levels demonstrated a specific increase in the SNAP25b transcript at this stage [15]. However, expression of SNAP25a/b proteins will be affected by both transcriptional and post-transcriptional (e.g. protein stability) effects. Therefore, we examined how SNAP25a/b protein levels change during post-natal development. Brain lysates prepared from Sprague Dawley rats at different ages were probed with the 3067 and 3068 antibodies. SNAP25b exhibited a dramatic up-regulation in expression beginning at around post-natal week two (Figure 2A); similar increases in SNAP25b expression were observed in every set of samples that we tested. SNAP25a also exhibited a developmental increase but this was more modest and variable than SNAP25b (Figure 2A). Expression levels of SNAP23 were relatively constant throughout development (Figure 2A). For quantification purposes, we compared mean values of the fold increase in brain expression levels of the SNAP25/23 isoforms between a late embryonic stage (E18-E20) and ~ 1 month after birth (P30-P32). The graph in Figure 2A highlights the robust increase in SNAP25b levels during this period compared with SNAP25a and SNAP23. No gender-specific differences in SNAP25a/b expression levels were apparent at E20 (Figure 2B).


Regional and developmental brain expression patterns of SNAP25 splice variants.

Prescott GR, Chamberlain LH - BMC Neurosci (2011)

Developmental expression patterns of SNAP25a and SNAP25b in rat brain. A) Left panel, Equal amounts of whole brain lysates prepared from rats at different developmental stages (supplied by Zyagen Inc.) were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, panSNAP25, SNAP23 and actin antibodies. Right panel shows the mean (+/-SEM) fold increase in expression levels of SNAP23, SNAP25a and SNAP25b between a late embryonic stage (E18-E20) and P30-32 (n = 3). The fold increase in SNAP25b expression was significantly greater than that of either SNAP25a (P < 0.05) or SNAP23 (p < 0.02), analysed using a one-way ANOVA. B) Equal amounts of brain lysates from gender-determined E20 litter mates were resolved on SDS-PAGE gels beside an aliquot of P32 brain lysate and analyzed as in panel A. C) Lysates from the hippocampus, cerebellum and cortex were prepared from pooled samples collected from 3 animals at P2 or P20. Samples were analyzed as in Panel A. Position of molecular weight standards are shown on the left of all panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Developmental expression patterns of SNAP25a and SNAP25b in rat brain. A) Left panel, Equal amounts of whole brain lysates prepared from rats at different developmental stages (supplied by Zyagen Inc.) were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, panSNAP25, SNAP23 and actin antibodies. Right panel shows the mean (+/-SEM) fold increase in expression levels of SNAP23, SNAP25a and SNAP25b between a late embryonic stage (E18-E20) and P30-32 (n = 3). The fold increase in SNAP25b expression was significantly greater than that of either SNAP25a (P < 0.05) or SNAP23 (p < 0.02), analysed using a one-way ANOVA. B) Equal amounts of brain lysates from gender-determined E20 litter mates were resolved on SDS-PAGE gels beside an aliquot of P32 brain lysate and analyzed as in panel A. C) Lysates from the hippocampus, cerebellum and cortex were prepared from pooled samples collected from 3 animals at P2 or P20. Samples were analyzed as in Panel A. Position of molecular weight standards are shown on the left of all panels.
Mentions: SNAP25 displays a marked increase in expression from around post-natal week two in mice, and analysis of mRNA levels demonstrated a specific increase in the SNAP25b transcript at this stage [15]. However, expression of SNAP25a/b proteins will be affected by both transcriptional and post-transcriptional (e.g. protein stability) effects. Therefore, we examined how SNAP25a/b protein levels change during post-natal development. Brain lysates prepared from Sprague Dawley rats at different ages were probed with the 3067 and 3068 antibodies. SNAP25b exhibited a dramatic up-regulation in expression beginning at around post-natal week two (Figure 2A); similar increases in SNAP25b expression were observed in every set of samples that we tested. SNAP25a also exhibited a developmental increase but this was more modest and variable than SNAP25b (Figure 2A). Expression levels of SNAP23 were relatively constant throughout development (Figure 2A). For quantification purposes, we compared mean values of the fold increase in brain expression levels of the SNAP25/23 isoforms between a late embryonic stage (E18-E20) and ~ 1 month after birth (P30-P32). The graph in Figure 2A highlights the robust increase in SNAP25b levels during this period compared with SNAP25a and SNAP23. No gender-specific differences in SNAP25a/b expression levels were apparent at E20 (Figure 2B).

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