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. 2024 Sep:75:103254.
doi: 10.1016/j.redox.2024.103254. Epub 2024 Jun 26.

NADPH oxidase 2 activity disrupts Calmodulin/CaMKIIα complex via redox modifications of CaMKIIα-contained Cys30 and Cys289: Implications in Parkinson's disease

Filippo Pullara  1 , Madison C Forsmann  2 , Ignacio J General  3 , Joseph C Ayoob  4 , Emily Furbee  4 , Sandra L Castro  2 , Xiaoping Hu  2 , J Timothy Greenamyre  2 , Roberto Di Maio  5
Affiliations

NADPH oxidase 2 activity disrupts Calmodulin/CaMKIIα complex via redox modifications of CaMKIIα-contained Cys30 and Cys289: Implications in Parkinson's disease

Filippo Pullara et al. Redox Biol. 2024 Sep.

Abstract

Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) signaling in the brain plays a critical role in regulating neuronal Ca2+ homeostasis. Its dysfunctional activity is associated with various neurological and neurodegenerative disorders, including Parkinson's disease (PD). Using computational modeling analysis, we predicted that, two essential cysteine residues contained in CaMKIIα, Cys30 and Cys289, may undergo redox modifications impacting the proper functioning of the CaMKIIα docking site for Ca2+/CaM, thus impeding the formation of the CaMKIIα:Ca2+/CaM complex, essential for a proper modulation of CaMKIIα kinase activity. Our subsequent in vitro investigations confirmed the computational predictions, specifically implicating Cys30 and Cys289 residues in impairing CaMKIIα:Ca2+/CaM interaction. We observed CaMKIIα:Ca2+/CaM complex disruption in dopamine (DA) nigrostriatal neurons of post-mortem Parkinson's disease (PD) patients' specimens, addressing the high relevance of this event in the disease. CaMKIIα:Ca2+/CaM complex disruption was also observed in both in vitro and in vivo rotenone models of PD, where this phenomenon was associated with CaMKIIα kinase hyperactivity. Moreover, we observed that, NADPH oxidase 2 (NOX2), a major enzymatic generator of superoxide anion (O2●-) and hydrogen peroxide (H2O2) in the brain with implications in PD pathogenesis, is responsible for CaMKIIα:Ca2+/CaM complex disruption associated to a stable Ca2+CAM-independent CaMKIIα kinase activity and intracellular Ca2+ accumulation. The present study highlights the importance of oxidative stress, in disturbing the delicate balance of CaMKIIα signaling in calcium dysregulation, offering novel insights into PD pathogenesis.

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Conflict of interest statement

Declaration of competing interest The Authors declare no conflicts of interest.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Dodecameric model of CaMKIIα and dynamic molecular dynamics prediction of C30 and C289 distance.(A) Top and side views of the full dodecamer model of CaMKIIα. In this conformer there are four CaMKII subunits in the open conformation and the remaining eight in the closed conformation. (B) A detailed view of two adjacent CaMKII subunits. The C30 and C289 are represented by two larger yellow spheres. (C) Snapshots from one of the “all atoms” molecular dynamics simulations highlighting the open and closed conformations. The double yellow arrows indicate the distance between C30 and C289. The graph shows the time evolution of the distance between those two residues. Note that the minimum Cys-Cys distance reached during this simulation is less than 6 Å.
Fig. 2
Fig. 2
Close-up view of two subunits of CaMKIIα. The computational analysis predicts a localization of Cys30 and Cys289 compatible with the formation of a disulfide bridge (S–S). As showed, T305 and T306 (binding region of CaM), are exposed when the C30–C289 bridge is not formed (left panel), allowing CaMKII-CaM interaction. When the bridge is formed (right panel), the accessible area to the binding site becomes greatly reduced, possibly preventing CaM binding.
Fig. 3
Fig. 3
Redox changes of Cys30 and Cys289 affect CaMKIIα:CaM interaction.(A) Representative image of PLA for CaMKIIα:CaM interaction (green) in Drosophila S2R + cells expressing human CaMWT associated with the expression of CaMKIIαWT, CaMKIIαC30A, CaMKIIαC289A or CaMKIIαC30A−C289A (scale bar: 15μm).(B) Quantification of the CaMKIIα:CaM PLA-related fluorescence intensity in Drosophila S2R + cell lines. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed for each independent experiment (200–250 cells/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments). (C) Redox WB of lysates from genetically modified Drosophila S2R + cells. (D) Quantification of the bands intensity reported as normalized ratio CaMKIIα/β-actin (upper graph) or as normalized CaMKIIα85KDa/CaMKIIα65KDa (lower graph). Symbols represent the normalized bands intensities ratios (with vehicle treatment being set at 1) analyzed for each independent experiment Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments). In plots B and D, *denotes p < 0.0001 significance compared vehicle.
Fig. 4
Fig. 4
Oxidative stress-related CaMKIIα:CaM interaction.(A) Image of PLA for CaMKIIα:CaM interaction (green) in DA neurons (Red: TH) in primary VMB cultures. (Scale bar: 35 μm). (B) Quantification of the CaMKIIα:CaM PLA-related fluorescence intensity in primary VMB cultures. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed for each independent experiment (100–150 neurons/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments). (C) CaMKIIα Redox WB assay performed with a non-selective reduction of thiol residues (TCEP) or with a S–NO selective reduction step performed with ascorbic acid. (D) Quantification of the bands intensity reported as normalized ratio CaMKIIα/β-actin (upper graph) or as normalized CaMKIIα85KDa/CaMKIIα65KDa (lower graph). Symbols represent the normalized bands' intensities ratios (with vehicle treatment being set at 1) analyzed for each independent experiment. Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments) In plots B and D, *denotes p < 0.0001 significance compared to vehicle.
Fig. 5
Fig. 5
NOX2 activity affects CaMKIIα:CaM interaction and modulates CaM-independent CaMKIIa kinase activity in rotenone-treated ventral midbrain neurons.(A) Representative image of PLA for CaMKIIα:CaM interaction (green) in DA neurons (Red: TH) in primary VMB cultures. (Scale bar: 35 μm). (B) Image representing levels of pThr286CaMKIIα (blue) in DA neurons (Red: TH) in primary VMB cultures (Scale bar: 35 μm). (C) Quantification of the CaMKIIα:CaM PLA-related fluorescence intensity in primary VMB cultures (light blue violins) associated with quantification of fluorescence relative to phosphor Thr286 (yellow violins). Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed for each independent experiment (100–150 neurons/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments). C, * denotes p < 0.0001 significance of PLA compared vehicle. # denotes p < 0.0001 significance of normalized intensity for pThr286CaMKIIα compared to vehicle.
Fig. 6
Fig. 6
NOX2 activity-induced CaM-independent CaMKIIα kinase activity affects Ca2+homeostasis in rotenone-treated ventral midbrain neurons.(A) Representative images acquired by live imaging assay of Fluo-8 AM related fluorescent signal (green) in primary VMB cultures. (Scale bar: 50 μm). (B) Quantification of the time-dependent accumulation of intracellular Ca2+ in response to rotenone measured as fluorescence intensity relative to Fluo-8 AM in primary VMB cultures. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed in 3 independent experiments (100–150 neurons/treatment group per independent experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 3 independent experiments). In plot C, * denotes p < 0.0001 significance compared to vehicle.
Fig. 7
Fig. 7
NOX2-mediates CaMKIIα/CaM disruption the rotenone model of PD in rat. (A) Representative image of PLA for CaMKIIα:CaM interaction (red) in rat nigrostriatal DA neurons (Blue: TH) (Scale bar: 50 μm). (B) Quantification of the CaMKIIα:CaM PLA-related fluorescence intensity in rat SNpc. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed for each independent experiment (35–50 nigrostriatal neurons per hemisphere). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 6 animals/group). (C) Image representing levels of phospho-Thr286 (red) in nigrostriatal DA neurons in rats (blue: TH). (Scale bar: 50 μm) (D) Quantification of CaMKIIα(pThr286) in rat SNpc. Symbols represent the normalized means of the intensities (with vehicle treatment being set at 100 %) analyzed for each independent experiment (35–50 nigrostriatal neurons per hemisphere). Statistical analysis was performed as one-way ANOVA with Bonferroni's correction (n = 6 animals/group). In plots B and D, *denotes p < 0.0001 significance compared to vehicle.
Fig. 8
Fig. 8
Relevance of CaMKIIα/CaM interaction in idiopathic PD.(A) PLA assay for CaMKIIα and CaM in human brain. (Scale bar: 30 μm). (B) Quantification of the CaMKIIα:CaM PLA-related fluorescence intensity in Human SNpc. Symbols represent the normalized means of the intensities (with controls being set at 100 %) analyzed for each independent experiment (10–20 nigrostriatal neurons/sample). Statistical testing by 2-tailed unpaired t-test with Welch's correction. *Denotes p < 0.0001 significance compared controls.
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