Overexpression of Mitochondrial Calcium Uniporter Causes Neuronal Death

Figure 4

MCU overexpression accelerates neuronal cell death after excitotoxic insults. Mouse primary cortical neurons were infected with synapsin-driven EGFP (synGFP) or MCU-EGFP (synMCU-GFP) adenoviral particles. After 48 hours, cells were loaded with TMRM (20 nM, 30 minutes). (a) TMRM fluorescence intensity in unstimulated resting conditions. (b) Representative images of TMRM staining. The scale bars represent 10 μm. (c) Representative traces of relative TMRM fluorescence intensity. Glutamate (100 μM) and CCCP (10 μM) were added when indicated. (d) Half-life of TMRM fluorescence decay after glutamate treatment. (e, f) Mouse primary cortical neurons were infected with synapsin-driven pEGFP (synGFP) or MCU-GFP (synMCU-GFP) adenoviral particles. After 48 hours, cells were loaded with Fura-FF-AM (5 μM, 30 minutes). (e) Representative traces of Fura-FF fluorescence ratio (excitation at 340 and 380 nm). Glutamate (100 μM) was added when indicated. (f) Average values recorded 19 minutes after glutamate treatment. For each condition, at least 30 cells from 3 different preparations were analyzed. (g, h) Mouse primary cortical neurons were infected with synapsin-driven EGFP (synGFP) or MCU-EGFP (synMCU-GFP) adenoviral particles. After 48, 72, and 96 hours, cells were treated with glutamate (100 μM, 1 hour) and then fixed and stained by TUNEL assay. (g) Representative images of GFP (green)- and TUNEL (red)-positive neurons. The scale bars represent 10 μm. (h) Quantification of TUNEL-positive neurons for the indicated conditions. At least 60 random fields from 3 different preparations were analyzed. (i) Resting cytosolic Ca²⁺ level of mouse primary cortical neurons cotransfected for 24 hours with GCaMP6f probe and either empty vector mCherry as control (Ctrl) or MCU-mCherry (MCU), in absence or in presence of Ca_v1 (nimodipine 5 μM) or Ca_v2 inhibitors (GVIA 2 μM and M7C 1 μM for Ca_v2.2 and Ca_v2.1, respectively). Each measurement was performed in at least 100 neurons from 3 different preparations. compared to control. Detailed statistics are described in Table 1.

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