Pin1 Is Regulated by CaMKII Activation in Glutamate-Induced Retinal Neuronal Regulated Necrosis

Abstract

In our previous study, we reported that peptidyl-prolyl isomerase 1 (Pin1)-modulated regulated necrosis (RN) occurred in cultured retinal neurons after glutamate injury. In the current study, we investigated the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in Pin1-modulated RN in cultured rat retinal neurons, and in an animal in vivo model. We first demonstrated that glutamate might lead to calcium overloading mainly through ionotropic glutamate receptors activation. Furthermore, CaMKII activation induced by overloaded calcium leads to Pin1 activation and subsequent RN. Inactivation of CaMKII by KN-93 (KN, i.e., a specific CaMKII inhibitor) application can decrease the glutamate-induced retinal neuronal RN. Finally, by using an animal in vivo model, we also demonstrated the important role of CaMKII in glutamate-induced RN in rat retina. In addition, flash electroretinogram results provided evidence that the impaired visual function induced by glutamate can recover after CaMKII inhibition. In conclusion, CaMKII is an up-regulator of Pin1 and responsible for the RN induced by glutamate. This study provides further understanding of the regulatory pathway of RN and is a complementary mechanism for Pin1 activation mediated necrosis. This finding will provide a potential target to protect neurons from necrosis in neurodegenerative diseases, such as glaucoma, diabetic retinopathy, and even central nervous system diseases.

Keywords: CaMKII; Pin1; calcium; glutamate; regulated necrosis.

FIGURE 1

Calcium concentrations in retinal neuron cultures treated with 50 μM glutamate injury for different time points. (A) Calcium concentrations indicated by Fluo-4 signals. (B) Quantitative analysis of Fluo-4 fluorescence signal intensity. (C) The fold change of calcium concentrations was determined by cell calcium assay kit. Data were analyzed using one-way ANOVA. ^*p < 0.05 vs. CTL group. These data are representative of results from five independent experiments. Scale bar = 20 μm in all panels.

FIGURE 2

Calcium concentrations in retinal neuron cultures after different GluRs inhibition. (A) Calcium concentrations indicated by Fluo-4 signals. (B) Quantitative analysis of Fluo-4 signals intensity. (C) The fold change of calcium concentrations was determined by cell calcium assay commercial kit. MK, CN, and MC, which respectively block NMDAR, AMPA/KA receptor and mGluRs, were added to the cultures at a final concentration of 10, 10, and 50 μM, 30 min before 15 min glutamate insult. Experiments were repeated three times. Data were analyzed using one-way ANOVA. ^*p < 0.05 vs. CTL group, ^# p < 0.05 vs. Glu group. Scale bar = 20 μm in all panels.

FIGURE 3

The mRNA level of various subtypes of ionotropic glutamate receptors after antagonist and glutamate treatment. (A,B) Fold change of various subtypes of glutamate receptors mRNA level detected by real-time PCR; MK, CN, and MC were used at a concentration of 10, 10, and 50 μ$\mathrm{M}$, 30 min before 15 min glutamate insult. To ensure consistency of the results, experiments were repeated four times. Data were analyzed using one-way ANOVA. ^*p < 0.05, ^∗∗p < 0.01 vs. respective CTL group, ^#p < 0.05 vs. respective Glu group.

FIGURE 4

Expression of CaMKII, p-CaMKII and Pin1 in retinal neurons following glutamate treatment. (A) Western blot of CaMKII, p-CaMKII and Pin1 expression. (B) The statistical analysis of p-CaMKII/CaMKII expression. (C) The statistical analysis of Pin1 expression. MC was used at a concentration of 50 μ$\mathrm{M}$, 30 min before glutamate insult. N = 3 cultures. Data were analyzed using one-way ANOVA. ^*p < 0.05 vs. CTL group.

FIGURE 5

The regulatory role of CaMKII in Pin1 activity. (A) Three dimensional structure of proteins by homologous modeling (Swiss Model). (B) Protein surface electrostatic potential energy are calculated by APBS (Figures are produced by using PyMOL). (C) The initial binding mode is constructed by manually binding areas with opposite electrostatic potential of two proteins and then the protein complex is energy minimized using molecular dynamics software NAMD v2.12. (D) IP assay of CaMKII and Pin1. Pin1 was pull-down with CaMKII antibody suggested by a 18 KDa band as observed. (E) Western blot of CaMKII, p-CaMKII, and Pin1 expression after Glu, MC, BA, and KN treatment. (F) The statistical analysis of p-CaMKII/CaMKII expression. (G) The statistical analysis of Pin1 expression. MC, BA, and KN were used 30 min before glutamate treatment at a concentration of 50, 10, and 10 μM, respectively. N = 3 cultures. Data were analyzed using one-way ANOVA. ^*p < 0.05 vs. CTL group, ^#p < 0.05 vs. Glu group.

FIGURE 6

Immunofluorescence staining of p-CaMKII and Pin1 after glutamate and drugs treatment. (A) Immunofluorescence staining of p-CaMKII (red) and Map2 (green). (B) Immunofluorescence staining of Pin1 (red) and Map2 (green). N = 3 cultures. MC, BA, and KN were used 30 min before 12 h glutamate treatment at a concentration of 50, 10, and 10 μM, respectively. Scale bar = 10 μm in all panels.

FIGURE 7

Glutamate-induced necrosis decreased by KN treatment. (A) Retinal necrotic neurons were stained with PI (red). Nuclei were counterstained with DAPI (blue). (B) Statistical analysis of PI-positive retinal neurons. (C) The percentage of necrotic neurons after glutamate treatment and pretreated with 50 μM MC, 10 μM BA, and 10 μM KN was determined by LDH release assay. N = 3 cultures. Data were analyzed using one-way ANOVA. ^*p < 0.05 vs. CTL group, ^#p < 0.05 vs. Glu group. Scale bar = 20 μm in all panels.

FIGURE 8

Necrosis and visual function in retina in vivo. (A,C,E,G,I,K) Nuclei were stained with DAPI (blue). (B,D,F,H,J,L) Retinal necrotic neurons were stained with PI (red). Nuclei and PI stained cells in GCL as indicated are shown in the merged frame areas (A′,C′,E′,G′,I′,K′). (M,N,O,P,Q,R) Representative fERG results of groups treated with glutamate and pretreated with different drugs. (S) Statistical analysis of PI stained cells in GCL and INL in retina. (T) The percentage of necrosis cells were determined by LDH release assay. (U) Statistical analysis of the b wave amplitudes. ^*p < 0.05 vs. CTL group, ^#p < 0.05 vs. Glu group. Each group was composed of four animals. Data were analyzed using one-way ANOVA. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar = 20 μm in all panels.

FIGURE 9

Possible molecular pathway underlying the effect of glutamate in RN of retinal neurons. The role of iGluRs-CaMKII-Pin1-CAST/calpain pathway induced by excessive glutamate in RN of retinal neurons.