Increased excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from bipolar disorder patients

Abstract

Reports of cognitive decline, symptom worsening and brain atrophy in bipolar disorder (BD) suggest that the disease progresses over time. The worsening neuropathology may involve excitotoxicity and neuroinflammation. We determined protein and mRNA levels of excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from 10 BD patients and 10 age-matched controls. The brain tissue was matched for age, postmortem interval and pH. The results indicated statistically significant lower protein and mRNA levels of the N-methyl-D-aspartate receptors, NR-1 and NR-3A, but significantly higher protein and mRNA levels of interleukin (IL)-1beta, the IL-1 receptor (IL-1R), myeloid differentiation factor 88, nuclear factor-kappa B subunits, and astroglial and microglial markers (glial fibrillary acidic protein, inducible nitric oxide synthase, c-fos and CD11b) in postmortem frontal cortex from BD compared with control subjects. There was no significant difference in mRNA levels of tumor necrosis factor alpha or neuronal nitric oxide synthase in the same region. These data show the presence of excitotoxicity and neuroinflammation in BD frontal cortex, with particular activation of the IL-R cascade. The changes may account for reported evidence of disease progression in BD and be a target for future therapy.

Figures

Figure 1

Representation of IL-1R cascade activation by IL-1β. Activation of the type I IL-1R by IL-1 leads to recruitment of IRAK to the receptor complex via its association with the IL-1receptor accessory protein and an adaptor protein MyD88. Upon association, IRAK becomes highly phosphorylated and subsequently dissociates from the receptor complex to interact with tumor necrosis factor receptor-associated factor 6 (TRAF6), which in turn is involved in nuclear I kappa kinase (NIK) and NF-κB activation. AP-2 transcription factor also is activated by IL-1β.

Figure 2

Mean NR-1 (A), NR-2A (B), NR-2B (C) and NR-3A (D) protein (with representative immunoblots) levels as percent of control levels in frontal cortex from control (n = 10) and BD (n = 10) subjects. Data are optical densities relative to that of β-actin. Mean mRNA as percent of control of NR-1 (E), NR-2A (F), NR-2B (G) and NR-3A (H) in frontal cortex from control (n = 10) and BD (n =10) subjects, measured using RT-PCR. Data are normalized to the endogenous control (β-globulin) and expressed relative to the control (calibrator), using the ΔΔCT method. Mean ± SEM, *p < 0.05, **p < 0.01.

Figure 3

Mean IL-1β (A), IL-1R (B) and MyD88 (C) protein (with representative immunoblots) as percent of control in frontal cortex, from control (n = 10) and BD (n = 10) subjects. Data are optical densities relative to that of β-actin. IL-1β (D), IL-1R (E) and MyD88 (F) mRNA levels in the frontal cortex from controls (n = 10) and BD patients (n = 10), measured using RT-PCR. Data are normalized to the endogenous control (β-globulin) and expressed relative to the control (calibrator), using the ΔΔCT method. Mean ± SEM, *p < 0.05, ** p < 0.01, ***p < 0.001.

Figure 4

Mean NF-κB p50 (A) and NF-κB p65 (B) protein levels (with representative immunoblots) in frontal cortex from control (n = 10) and BD (n = 10) subjects. Bar graphs are ratios of optical densities of NF-κB p50 or NF-κB p65 to that of β-actin, expressed as percent of control. NF-κB p50 (C) and NF-κB p65 (D) mRNA in postmortem frontal cortex from the control (n = 10) and BD (n = 10) subjects, measured using RT-PCR. Data are levels of NF-κB p50 and NF-κB p65 in BD normalized to the endogenous control (β-globulin) and relative to the control (calibrator), using the ΔΔCT method. Mean ± SEM, * p<0.05, **p < 0.01, ***p < 0.001.

Figure 5

Mean GFAP (A), iNOS (B) and nNOS (C) protein (with representative immunoblots) in control (n = 10) and BD (n = 10) frontal cortex. Data are optical densities of GFAP, iNOS and nNOS proteins to β-actin, expressed as percent of control. mRNA levels of GFAP (D), iNOS (E) and iNOS (F) in postmortem control (n = 10) and BD (n =10) frontal cortex, measured using RT-PCR. Data are levels of GFAP, iNOS and nNOS in the BD patients normalized to the endogenous control (β-globulin) and relative to control level (calibrator), using the ΔΔCT method. Mean ± SEM, *p < 0.05, ** p < 0.01.

Figure 6

Mean c-fos (A), and CD11B (C) protein (with representative immunoblots) in control (n = 10) and BD (n = 10) frontal cortex. Data are optical densities of c-fos and CD11B proteins to β-actin, expressed as percent of control. mRNA levels of c-Fos (B), CD11B (D) and TNFα (E) in postmortem control (n = 10) and BD (n =10) frontal cortex, measured using RT-PCR. Data are levels of cfos, CD11B and TNFα in the BD patients normalized to the endogenous control (β-globulin) and relative to control level (calibrator), using the ΔΔCT method. Mean ± SEM, *p < 0.05, ** p < 0.01. Lower right (F) Representative histology of control and BD. Microglial activation was characterized by HLA-DR antibody and visualized by DAB. Astrocytes were detected by GFAP antibody and stained on an IHC Omni-Ultra MAP HRP. Sections were stained with Harris hematoxylin. Scale bar: 25 μm▶ Arrows indicate hypertrophic astrocytes and activated microglia in BD tissue.