Effect of high K⁺ stimulation of rat brain synaptosomes on cleavage of fGAD65.

<p><b>A.</b> Representative immuno-blot image showing progression in cleavage of fGAD65 to form tGAD65 when rat brain synaptosomes were stimulated with increasing K⁺ concentration (lanes 2–4). A non-stimulated synaptosomal fraction (lane 1) served as a control. The samples were analyzed by immuno-blotting using monoclonal GAD65-specific antibody; GAD6. The 58 KDa cleaved product tGAD65 was found to accumulate proportional to the increase in KCl concentration. <b>B.</b> Densitometric analyses of the conversion of fGAD65 to tGAD65 in rat brain synaptosomes treated with increasing K⁺ concentration expressed as % of control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g001" target="_blank">Fig. 1B</a>, Ctrl). Data are presented as mean ± SEM, n = 4 independent experiments. <i>*P</i><0.05 and <i>^#P</i><0.05 are statistically significant differences between the levels of fGAD65 and tGAD65 of 50 mM KCl treatment group and the control respectively. <i>**P</i><0.001 and <i>^##P</i><0.001 are statistically significant differences in the levels of fGAD65 and tGAD65 between 100 mM KCl treatment group and the control respectively.</p

Time dependent differential expression of GAD65 and GAD67 mRNA upon exposure to 100 mM KCl in primary rat neuronal cell cultures.

<p>A. Representative gel image of GAD65 and GAD67 mRNA separated on a 1.5% agarose gel. Lane 1: Control; Lanes 2–5: 10 min, 1 hr, 4 hr and 8 hr exposure to 100 mM KCl respectively. GAD65 and GAD67 mRNA were differentially regulated under the same conditions of exposure to 100 mM KCl for different time points as indicated in lanes 2–5. B. Quantitative analyses of GAD65 and GAD67 mRNA expression shown as % of control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g004" target="_blank">Fig. 4B</a>, Ctrl). Values are mean of ± SEM (n = 4); <i>^##P</i><0.05 statistically significant differences between GAD67 mRNA expression of 10 min exposure group and the control. <i>*P</i><0.05 statistically significant differences between GAD65 mRNA expression of 1 hr and 4 hr exposure groups with the control respectively. <i>**P</i><0.005 is the statistically significant difference between GAD65 mRNA expression between 8 hr exposure group and the control.</p

Effect of calpain treatment of GABAergic SVs on newly synthesized GABA uptake.

<p>Uptake of newly synthesized GABA was conducted in the presence (•) or absence (▪) of calpain enzyme. [³H] Glu was used as a substrate for the synthesis of newly synthesized [³H] GABA. Values are mean of ± SEM (n = 3). The specific uptake was obtained by subtracting the non-specific uptake from the total uptake and was expressed as pmol per mg of protein.</p

Proposed model of the SV-associated fGAD65 cleavage mediated by calpain under sustained neuronal stimulation.

<p>Under sustained neuronal stimulation, SV-associated fGAD65 which is in association with VGAT via partnership with HSC70 and CSP is cleaved by hyper-active calpain to liberate the functional part of the enzyme. This resulted in the disruption of the fGAD65-VGAT functional coupling process, largely reducing the releasable pool of GABA loaded SVs. The GABA generated by SV-associated fGAD65 is shown in magenta; GABA generated by fGAD67 is shown in teal. The stars on GAD67 and GAD65 represent the active states of the enzymes. GAD67 is activated by dephosphorylation, whereas GAD65 is active in its phosphorylated state. Certain areas of the pathway which are directly affected by calpain-mediated cleavage of fGAD65 are shown in red crosses. The sequence of events leading to liberation of tGAD65 is discussed in the text.</p

Cleavage of SV-associated fGAD65 as a function of KCl concentration:

<p><b>A.</b> Representative immuno-blot demonstrating the isolation of enriched SVs, relatively free from any contaminating cytosolic fractions, other than that of Golgi and ER fractions, as indicated by immuno-blotting against synaptosophysin (SV marker), calnexin (ER marker) and 58K Protein (Golgi marker). <b>B.</b> Representative immuno-blot demonstrating cleavage of SV-associated fGAD65 directly on the SVs, on account of a dose-dependent KCl stimulation. <b>C.</b> Quantitative densitometric comparison, expressed as relative percentage with respect to SVs, of the levels of expression of fGAD65 and synaptophysin in SVs vs Cyt (cytosolic) fractions. Values are mean of ± SEM (n = 3). *<i>P</i><0.05 and <i>**P</i><0.001 are statistically significant differences in the levels of expression between fGAD65 and synaptophysin in the SV fractions when compared tocyt fractions. <b>D.</b> Densitometric analyses of the changes in the expressions of fGAD65 and tGAD65 in the SV fractions. Untreated SVs served as a control. Data points are mean of ± SEM (n = 3). <i>*P</i><0.05 and <i>^#P</i><0.05 are statistically significant differences between 50 mM KCl treatment group and the control group respectively. **P<0.005 and ##P<0.005 are statistically significant differences between 100 mM KCl group and the control group.</p

Effect of calpain-mediated cleavage of SVs on GABA synthesis.

<p>Residual GAD activity of calpain-treated and untreated SVs. Lane 1: Control SVs; Lane 2: SVs treated with 0.15 U of calpain enzyme. Data are mean of ± SEM (n = 3). <i>*P</i><0.005 is the statistically significant difference in the GAD activity between the calpain-treated SV group and the control.</p

Time dependent cleavage of fGAD65 and corresponding cell viability upon exposure to 100 mM K⁺ stimulation in primary rat neuronal cultures.

<p>E17 primary rat neuronal cell cultures at 11 DIV were exposed to 100 mM KCl for different time intervals as shown. <b>A.</b> Representative immuno-blot analysis of fGAD65 cleavage post 100 mM KCl treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g003" target="_blank">Fig. 3A</a>, lanes 2–5). Lane 1 represents the untreated control. <b>B.</b> Quantitative analysis of fGAD65 cleavage in primary rat neuronal cells expressed as % of control is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g003" target="_blank">Fig. 3B</a>. Values are mean of ± SEM (n = 3); <i>*P</i><0.001 is the statistically significant differences between the levels of expression of fGAD65 in 1 hr, 4 hr and 8 hr treatment groups and the control group respectively; <i>^##P</i><0.005 and <i>^#P</i><0.05 are statistically significant differences between the levels of expression of tGAD65 in the 1 hr, 4 hr and 8 hr treatment groups with the control group respectively. <b>C.</b> Cell viability of rat primary neuronal cells in culture under similar conditions as in Expt. 3A, which promoted fGAD65 cleavage. Values are mean of ± SEM (n = 4); <i>*P</i><0.05 is the statistically significant difference between 10 min exposure and the control group. <i>*P</i><0.001 is the statistically significant differences between 1 hr, 4 hr and 8 hr treatment with the control group respectively.</p

Sequence of primers used in the RT-PCR reaction.

<p>Abbreviations used: G3PDH: Glyceraldehyde-3-phosphate dehydrogenase.</p

Identification of truncated forms of GAD and corresponding elevation of glutamate levels in a MCAO model.

<p><b>A.</b> Representative immuno-blot demonstrating the presence of truncated forms of GAD65 and GAD67 and corresponding elevation of calpastatin. N, Normal; I, Ischemic. <b>B.</b> Densitometric analysis of the immuno-blot images. Data points are mean of ± SEM (n = 3). **<i>P</i><0.005 and *<i>P</i><0.05 are statistically significant differences when compared to their respective controls. <b>C.</b> Corresponding changes in the levels of glutamate before and during ischemia. Values are mean of ± SEM (n = 4). *<i>P</i><0.005 is the statistically significant differences between during occlusion and before occlusion.</p

Effect of high K⁺ stimulation of rat brain synaptosomes on total GAD65 activity.

<p><b>A.</b> Total GAD65 activity in the rat brain synaptosomes was measured under conditions promoting cleavage of fGAD65. Synaptosomes were stimulated with increasing concentrations of K⁺ as indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>; 10 mM KCl group, 50 mM KCl group and 100 mM KCl group). Unstimulated synaptosomes served as the control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>; Ctrl group). Total GAD65 activity comprising the activity of both fGAD65 and tGAD65 after stimulation was measured using a combinational approach of immunoprecipitation and radiometric GAD activity assays. First, soon after stimulation, total GAD65 was pulled down by immunoprecipitation using monoclonal GAD65 antibody; GAD6. Next, after normalizing the synaptosomal lysates between groups, the pulled down complex was analyzed using the classical radiometric method for measuring GAD activity. The values are mean ± SEM (n = 4); <i>*P</i><0.05 and <i>**P</i><0.005 are statistically significant differences between 50 mM KCl and 100 mM KCl treatment groups with the control group respectively. <b>B.</b> Effect of calpain inhibitor on total GAD65 activity was evaluated by pre-incubating the synaptosomes with 1 µM of calpain inhibitor peptide as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2B</a>. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2A</a>, the synaptosomes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033002#pone-0033002-g002" target="_blank">Fig. 2B</a>, were subjected to 50 mM KCl treatment with or without the calpain inhibitor, followed by synaptosomal lysis, normalization, immunoprecipitation and then the total GAD65 activity was measured by radiometric GAD activity assays. Data are represented as ± mean SEM (n = 3); <i>*P</i><0.05 are the statistically significant differences between K⁺ (50 mM) and the control groups. <i>^#P</i><0.05 are the statistically significant differences between K⁺ (50 mM) and the K⁺+Calp Inhi (50 mM KCl and 1 µM calpain inhibitor) groups.</p