Novel mechanism of increased Ca2+ release following oxidative stress in neuronal cells involves type 2 inositol-1,4,5-trisphosphate receptors

Abstract

Dysregulation of Ca(2+) signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca(2+) signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca(2+) release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca(2+) release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca(2+) release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca(2+) dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca(2+) signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders.

Figure 1. Identification of sub-lethal tBHP concentrations

(A) Exposure of HT-22 cells to tBHP concentrations of up to 10 µM for 16–20 hrs. had no significant effect on normalized calcein-AM fluorescence. (B) Absorbance of the MTT assay was similar to control up to concentrations of 10 µM tBHP. As determined by both assays, the approximate LC₅₀ of tBHP in HT-22 cells is around 25µM. (C) Representative images of HT-22 cell culture (passage 14), taken with an inverted tissue culture microscope with phase contrast, shows that morphology of HT-22 cells in vitro is unaffected by sublethal oxidative stress with 10 µM tBHP. There is no evidence of rounded cell morphology, typically observed at higher tBHP concentrations. (D) Level of oxidative stress was measured after 6 hrs. exposure to tBHP or vehicle using carboxy-2’, 7’-dichlorodihydrofluorescein diacetate (carboxy-H₂ DCFDA). 10 µM tBHP resulted in a significant 4.2% increase in DCFDA fluorescence compared with control (n=3; P<0.05). Higher concentrations of tBHP correlated with higher DCFDA fluorescence, indicating increased levels of oxidative stress. *P<0.05; ** P<0.01; *** P<0.001.

Figure 2. tBHP-mediated oxidative stress causes increased nuclear Ca²⁺ release

(A) Representative example of IP₃-AM stimulated Ca²⁺ release in HT-22 cells, following 16–20 hrs. exposure to either vehicle or 10 µM tBHP. Fluorescence is higher in the nucleus in the tBHP condition compared with control. (B) Overall magnitude of the response, expressed as increases in the amplitude of Ca²⁺ transients and presented as peak normalized change in fluorescence (F/F₀) is statistically not significantly different following exposure to oxidative stress (n=6, ANOVA P=0.096). Data is shown as mean ± s.e.m. (C) The specificity of the agonist (IP₃-AM) for IP₃Rs is confirmed by using Ca²⁺ channel blockers. The RyR blocker dantrolene (20 µM) did not affect overall Ca²⁺ release following IP₃-AM stimulation, whereas the IP₃R antagonist Xestospongin D (10 µM) abolished the response (n=2). Data is shown as mean ± s.d. (D) Subcellular analysis and quantification revealed a significant 32% increase in Ca²⁺ release into the nucleoplasm from membranes of the nuclear envelope (n=6, P<0.0%). No difference in Ca²⁺ release was found in the cytosol (n=6, P=0.84). Data is presented as peak normalized change in fluorescence (F/F₀). (E) The slope of the rising phase of the Ca²⁺ transient (20–80% of the peak) did not change in the presence of tBHP, suggesting the normal complement of IP₃Rs. * P<0.05. Scale bar: 10 µM.

Figure 3. Selective up-regulation of IP₃R2s following oxidative stress

(A) Specific immunoreactivity for IP₃R2s is increased following overnight (16–20 hrs.) treatment with 10 µM tBHP. Scale bar 25 µm. (B) Microfluorimetric analysis reveals a significant 62% increase of specific IP₃R2 immunoreactivity following 10 µM tBHP treatment, but not 2 µM or control. We observed a small, non-significant increase of immunoreactivity in the cytosol (n=3, P<0.05). (C) Using Taqman chemistry, we observed a three-fold increase in IP₃R2 mRNA levels following 10 µM tBHP oxidative stress for overnight (n=3, P<0.01). A modest increase following 2 µM treatment did not reach statistical significance. The endogenous control, mouse β-actin, did not change in response to oxidative stress. A representative experiment is shown. (D) Representative example of immunoblotting of total protein lysate against IP₃R2s shows an increase of specific IP₃R2 immunoreactivity at the expected size of around 270 kDa. Mouse β-actin was used as endogenous control. Quantification of three separate experiments revealed a statistically significant doubling of IP₃R2-specific signal. * P<0.05; ** P<0.01.

Figure 4. Absence of IP₃R1 and IP₃R3 involvement

(A/B) Immunocytochemistry (left) and microfluorimetric analysis (right) did not reveal any changes in specific immunoreactivity for IP₃R1s (n=3, P=0.81) and IP₃R3s (n=3, P=0.92). Scale bar: 25 µM. (C/D) Similarly, immunoblotting against IP₃R1 and IP₃R3 was similar following oxidative stress, compared with control. Representative blots are shown. Mouse β-actin was used as endogenous control. Quantification of three separate experiments revealed no statistically significant difference between sham and tBHP treatments.