Article Figures & Data
Figures
- Figure 1.
cPLA2 phosphorylation by Cdk5. A, Cdk5 kinase assay with cPLA2 wt and its mutants along with TFP5 treatment in vitro by its pure proteins. B–E, Western blot images of phosphorylated cPLA2 (i.e., p-cPLA2 268 and p-cPLA2 505 expressions after Cdk5 kinase assay with cPLA2 wt and its mutants as substrate). Total cPLA2 was considered as standard. F, The Western blot image shows the expression of cPLA2 and its mutants post-transfection in HEK293T cells. G, Bar diagram showing Cdk5 kinase activity with cPLA2 wt and mutant protein as substrates of Cdk5 along with Cdk5 inhibitor TFP5. H, cPLA2 kinase activity. The bar graph shows cPLA2 activity of cPLA2 wt along with its three mutants S505A, T268A, and DM, also a comparative analysis of its mutants with the cPLA2 wt activity. Data are presented as the mean ± Standard error of mean (SEM), and analysis was performed using one-way ANOVA and Bonferroni’s test; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, not significant (ns).
- Figure 2.
Comparison of wild-type and mutant structural forms using MD simulations. A, Representative snapshots of MD simulations derived structure at the initial (0 ns) and last (1 μs) of wild and mutant systems. The protein structures are colored according to cPLA2 domain architecture, where C2 domain is highlighted in blue, linker in magenta, and catalytic domain (KD) in orange. B, Angular distribution between domains in wild and mutant systems during the last 150 ns of the trajectory. C, Secondary structure evolution of the catalytic domain residues (144–49) according to DSSP analysis in wild and mutant trajectories. D, Intramolecular network near the mutant sites for wild and double mutant systems at 1 μs. The size of the nodes represents the degree. The edges are represented as types of interaction, where hydrogen bonds are highlighted in blue, and contacts in gray. E, Snapshots highlighting these sites in ball and stick representation and surface hydrophobicity shown in blue and gold.
- Figure 3.
The expression of cPLA2 wt and its mutants in the membrane and cytosolic protein fraction. A, The expression pattern of cPLA2 and its mutants in a membrane and cytosolic fraction of HEK293T protein lysate cotransfected with cPLA2 and its mutants along with Cdk5/p35 and Cdk5/p25. The reaction standard used for membrane fraction is calnexin, and for cytosolic fraction is β-tubulin. B, Cytosolic and membrane fraction fold changes over control. C, Membrane translocation of phosS268 cPLA2 in the presence of active Cdk5 kinase. D, Membrane translocation of phospho-T505 cPLA2 in the presence of active Cdk5 Kinase. E, Densitometry of relative expressions of p-cPLA2-268 in cytosolic and membrane fractions. F, Densitometry of p-cPLA2-505 in cytosolic and membrane fractions. Calnexin is used as membrane fraction control, and β-tubulin as cytosolic frantic control. Data are presented as the mean ± SEM, and analysis was performed using a one-way ANOVA followed by Bonferroni’s post hoc test; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 4.
Colocalization of Cdk5 and cPLA2. A, Colocalization of Cdk5 and cPLA2. Immunocytochemistry analysis of Cdk5 and cPLA2 showing their expressions and colocalization. B, Western blot data of Cdk5 and cPLA2 pull-down IPs developed with cPLA2 and Cdk5 antibodies, respectively.
- Figure 5.
Cdk5 expression, generation of p25, its kinase activity, and release of arachidonic acid and prostaglandin E2 synthesis by astrocytes exposed to MPP+ and TFP5 treatment. A, The image shows a blot with the expression of Cdk5 in control versus MPP+ (10 μm) along with MPP+ (10 μm) versus MPP+ (10 μm) plus TFP5 and SCP treatment in the protein lysate of astrocytes. B, Bar diagram showing densitometric analysis of Cdk5 expression over β-actin used as a standard for control, MPP+ (10 μm), MPP+ (10 μm) + TFP5 and MPP+ (10 μm) + SCP, respectively. C, The bar diagram represents Cdk5 activity in the astrocyte protein lysate with control versus MPP+ (10 μm) and MPP+ (10 μm) versus MPP+ (10 μm) + TFP5. D, The expression pattern of p35 and p25 in control, MPP+ (10 μm), MPP+ (10 μm) + TFP5, and MPP+ (10 μm) + SCP, respectively. β-Actin was used as a standard. E, The blot representing the expression pattern of cPLA2 in control, MPP+ (10 μm), MPP+ (10 μm) + TFP5, and MPP+ (10 μm) + SCP, respectively. F, The densitometric graph showing cPLA2 expression over β-actin of the above blot. G, The bar graph showing fold changes in cPLA2 activity in astrocyte lysate of control versus MPP+ (10 μm) and MPP+ (10 μm) versus MPP+ (10 μm) + TFP5. H, The scatter plot showing 3H-arachidonic acid release from control astrocytes along with MPP+ (10 μm), MPP+ (10 μm) + TFP5, and MPP+ (10 μm) + SCP. I, The bar graph showing prostaglandin E2 synthesis in control versus MPP+ (10 μm) and MPP+ (10 μm) versus MPP+ (10 μm) + TFP5 along with MPP+ (10 μm) + SCP as the standard for inhibitor treatment. Data are presented as the mean ± SEM, and analysis was performed using one-way ANOVA and Bonferroni’s test; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, not significant (ns).
- Figure 6.
cPLA2 expression, its activity, and prostaglandin E2 synthesis in neuroglia cells with MPP+ and TFP5 treatment. A, The blot showing cPLA2 expression pattern of control, MPP+ (10 μm), MPP+ (10 μm) + TFP5 and MPP+ (10 μm) + SCP in protein lysate of neuronal–glial culture along with its densitometric analysis. B, The bar graph for the fold of change in cPLA2 activity of control versus MPP+ (10 μm) and MPP+ (10 μm) versus MPP+ (10 μm) + TFP5 in neuronal–glial culture. C, The bar graph shows prostaglandin E2 amount in control versus MPP+ (10 μm) and MPP+ (10 μm) versus MPP+ (10 μm) + TFP5 in neuronal–glial culture. The MPP+ (10 μm) + SCP is used as a standard reaction for inhibitor treatment; n = 3. Data are presented as the mean ± SEM, and analysis was done using one-way ANOVA and Bonferroni’s test. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 7.
Generation of p25, Cdk5 activity, cPLA2 activity, and PGE2 levels along with the expression pattern of cPLA2 and phospho-cPLA2 in control versus PD mice model. A, The blot shows the expression pattern of Cdk5, p35, and p25 in control versus PD mouse brain protein lysate (n = 3). B, The densitometric analysis of Cdk5 expression and p25/p35 ratio over its standard, β-tubulin, in control versus PD mice (n = 6). C, The bar graph shows Cdk5 activity that correlates with the luminescence (in relative light units) measure of control versus PD mouse brain protein lysate (n = 6). D, The bar graph shows cPLA2 activity in the brain protein lysate of control versus PD mice model (n = 6). E, The bar graph shows the PGE2 level (in picograms per milliliter) of control versus PD mice model. F, Western blot analysis of cPLA2-total, cPLA2-505, and cPLA2-268 expression in control versus PD mice. G, The bar graph shows fold changes of cPLA2-total, cPLA2-505, and cPLA2-268 expression over β-actin in control versus PD mice. Data are presented as the mean ± SEM, and analysis was performed using unpaired t test with Holm–Sidak method. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 8.
Astrocyte activation along with total and phospho-cPLA2 (S505 and T268) enhanced expression in the PD mouse brain. A, Brain sections (10 μm) of control and PD mice were stained with GFAP, cPLA2 total, phospho-cPLA2 at T-268, and phospho-cPLA2 at S-505 in control versus PD mouse brain sections of the substantia nigra region. B, GFAP immunodensity/mm2 in control versus PD (n = 3 mice/group). Scale bar, 100 μm. C, cPLA2 and phospho-cPLA2 immunodensity/mm2 in control versus PD (n = 3 mice/group). All data are presented as the mean ± SEM, and analysis was performed using an unpaired t test and the Holm–Sidak method; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 9.
A, Workflow of the conjoint differential expression analysis of four PD transcriptomics datasets. B, Heatmap showing upregulated genes in the arachidonic acid and prostaglandin synthesis and inflammatory pathways. C, Heatmap showing downregulated genes in the AA and prostaglandin synthesis and inflammatory pathways. D, The model proposed to explain the mechanism of Cdk5/p25-mediated cPLA2 activation that leads to neuroinflammation.
- Figure 10.
A–D, Principal component analysis without (A, B) and with (C, D) normalized expression profiles.
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