Deletion of TRPC6 Attenuates NMDA Receptor-Mediated Ca2+ Entry and Ca2+-Induced Neurotoxicity Following Cerebral Ischemia and Oxygen-Glucose Deprivation

Abstract

Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na⁺ and Ca²⁺ and are widely expressed in the brain. In this study, the role of TRPC6 was investigated following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild-type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca²⁺ levels ([Ca²⁺] _i ), we found that OGD induced a significant higher increase in glutamate-evoked Ca²⁺ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca²⁺] _i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca²⁺] _i in TRPC6 infected cells, indicating that such a Ca²⁺ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca²⁺ influx was blunted by blockade of Na⁺ entry in TRPC6 infected cells. Finally, OGD-enhanced Ca²⁺ influx was reduced, but not completely blocked, in the presence of voltage-dependent Na⁺ channel blocker tetrodotoxin (TTX) and dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na⁺ entry which activated NMDAR, thus leading to a damaging Ca²⁺ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage.

Keywords: Ca2+; NMDA; TRPC6; ischemia; neurotoxicity; oxygen-glucose deprivation (OGD).

Figure 1

Ischemia/Reperfusion (I/R) and OGD increased TRPC6 expression. (A,B) Time course analysis of TRPC6 mRNA (A) and protein (B) expressions in the ipsilateral and contralateral cortices of mice subjected to I/R. Quantitative analyses of the changes in TRPC6 mRNA and protein levels at the indicated time points after I/R were determined by densitometry. Fold increases are relative to Sham (S). ^*P < 0.01 compared to corresponding values in the contralateral cortices (n = 4/group). (C) TRPC 6 protein in WT cortical neurons subjected to OGD. Immunoblotting was performed with 50 μg of total proteins and probed with antibodies to TRPC6. β-actin was used as a loading control. Three independent experiments at each time point (n = 3). ^*P < 0.01, compared with control groups (0 h).

Figure 2

TRPC6 deletion attenuated I/R-induced brain infarct size and protected neurons from OGD-induced cell death. (A) Images of the brain sections from WT and TRPC6^−/− mice 24 h after a 60 min-MCAO that were stained with TTC. Bar graph shows the infarct volume. Infarct volume was calculated by integration of infarct areas that were not stained by TTC for all slices of each brain. ^*P < 0.01 (n = 6 /group). Regional cerebral blood flow (rCBF) in TRPC6^−/− and WT mice at the indicated time points before (PRE), during 60 min MCAO and 45 min reperfusion (n = 3–4/group). rCBF was measured over the MCA territory by laser Doppler flowmetry. Baseline rCBF measured before MCA occlusion was defined to be 100% flow. (B) Propidium iodide (PI) staining of nuclei and cell bodies (red) and immunocytochemical labeling of MAP2 (green) of cell body and dendrites in primary neurons from WT and TRPC6^−/− mice following OGD pretreatment. Scale bar, 10 μm. The small, bright condensed nuclei (<2 μm in diameter) without stomata and dendrites indicate dead neurons as indicated by arrows. Bar graph shows the percentage of cell death in WT and TRPC6^−/− primary neurons with or without OGD pretreatment ^*P < 0.01 (n = 3 independent experiments/group). In each experiment, three wells and >100 neurons/well were counted.

Figure 3

TRPC6 deletion protected primary cortical neurons from glutamate- or NMDA-induced neuronal death.(A) Representative images using propidium iodide (PI) staining in WT and TRPC6^−/− primary neurons with glutamate (100 μM) or NMDA (50 μM) pretreatments, respectively. The small, bright condensed nuclei (<2 μm in diameter) (arrow) show the dying neurons. Scale bar, 10 μm. (B,C) Cell death after glutamate and NMDA pretreatment in WT and TRPC6^−/− neurons. ^*P < 0.01 (n = 3 independent experiments/group). In each experiment, three wells and >100 neurons/well were counted.

Figure 4

TRPC6 deletion decreased OGD-induced Ca²⁺ overload. (A) Ratiometric fluorescence images of Fura-2 loaded WT and TRPC6^−/− primary neurons in response to glutamate (100 μM) application with and without OGD pretreatments. Images were taken every 10 s during the recording. Intracellular [Ca²⁺]i intensity is indicated by the ratio of 340/380 nm and encoded by pseudocolor as shown in the color bar. (B) Traces show the time course of [Ca²⁺]i which was analyzed using the Nikon live cell imaging software. Glutamate was added as indicated by an arrow before 60 s. Ten neurons were sampled and analyzed in each experiment. F/F0 is expressed as the Fura-2 fluorescence ratio 340/380 nm (F) relative to the initial fluorescence ratio (F0) at each time point. At time 0, F/F0 is 1. (C) Bar graph shows the mean peaks of F/F0 traces of the 4 groups. ^*P < 0.01 (n = 3 independent experiments/group).

Figure 5

Overexpression of TRPC6 increased glutamate-evoked Ca²⁺ influx in WT cortical neurons: effects of AMPA receptor antagonist, NMDA receptor antagonist, and Na⁺-free medium. (A) AdCMV-GFP and AdCMV-TRPC6-GFP were infected WT neurons with GFP fluorescence which are indicated as GFP in the right column. Ratiometric fluorescence images of Fura-2 AM loaded AdCMV-GFP (control) and AdCMV-TRPC6-GFP (TRPC6 overexpression) neurons after glutamate (100 μM). Images were taken every 10 s during recording. [Ca²⁺]_i was analyzed using Fura-2 as ratio of 340/380 nm and encoded by pseudocolor as in Figure 4. (B) AdCMV-TRPC6-GFP neurons exhibited a significantly higher glutamate-evoked [Ca²⁺]_iincrease compared to AdCMV-GFP control neurons in the presence of the AMPA antagonist (10 μM CNQX). The bar graph shows the peak of F/F0 in AdCMV-GFP and AdCMV-TRPC6-GFP neurons after glutamate. Three neurons were collected per experiment. ^*P < 0.01 (n = 3 independent experiments/group). (C) TRPC6-mediated Ca²⁺ influx was blocked by an irreversible NMDAR channel blocker MK-801 (20 μM). The bar graph shows the peaks of F/F0 in AdCMV-GFP (control) and AdCMV-TRPC6-GFP (TRPC6 overexpression) infected neurons treated with or without MK801 in response to glutamate (100 μM) application. Three neurons per experiment were analyzed. n = 3 independent experiments/group. ^*P < 0.01. (D) Glutamate-evoked increase in [Ca²⁺]_i was significantly reduced in control (AdCMV-GFP) and AdCMV-TRPC6-GFP neurons after Na⁺ was removed and replaced by NMDG in the bath solution (Na⁺-free bath). The bar graph shows the peaks of F/F0 in AdCMV-GFP or AdCMV-TRPC6-GFP primary neurons treated with or without NMDG replacement in response to glutamate. Three neurons per experiment were used. n = 3 independent experiments/group. ^*P < 0.01, ^#P < 0.05.

Figure 6

OGD-induced [Ca²⁺]i increase may be attributed to voltage-sensitive Na⁺ channels and AMPA receptor channels. (A) Ca²⁺ influx in OGD-pretreated WT primary neurons in response to glutamate (100 μM) application in the bath containing TTX (0.1 μM) and CNQX (10 μM) to block voltage-sensitive channels and AMPA receptors. (B) Traces show the time course of the intracellular [Ca²⁺]i. (C) The bar graph shows the peaks of F/F0 traces. Ten neurons per experiment were analyzed. n = 3 independent experiments/group. ^*P < 0.01.

Figure 7

A schematic drawing to show the role of TRPC6 in I/R- and ODG-induced Ca2+ influx via regulation of NMDARs. TRPC6 expression was increased in neurons following I/R in mice brain and OGD in primary cortical neurons. TRPC6 up-regulation following I/R and OGD induced cortical neuronal death may in part through Na⁺ entry, leading to over-activation of NMDA receptors, and causing an overload of Ca2⁺. Deletion of TRPC6 protects against cerebral ischemia-induced brain damage in vivo and OGD- and excitatory neurotoxin-induced cell death in vitro. This protection is mediated partially through TRPC6-mediated NMDA pathways.