Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals

The recent identification of the mitochondrial Ca2+ uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca2+ uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca2+ levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca2+, resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca2+ overload when cytoplasmic Ca2+ levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca2+ and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca2+ uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.

Neurons were transfected with the indicated siRNA, plus an expression vector for cyt-GCaMP and treated ± NMDA as indicated, following the same protocol as Fig. 4i. 24 h after NMDA treatment, cytoplasmic Ca2+ levels were calculated from the cyt-GCaMP signal followed by calculation of F max and F min as per the methods (10-24 cells analysed per condition).

Subcellular fractionation.
Fractionation of mitochondrial and cytoplasmic fractions was performed using the Cell Fractionation kit (Abcam, ab109719) according to the manufacturer's instructions with slight modifications. Briefly, mouse hippocampal neurons (0.5 x 10 6 ) were washed with PBS and harvested into 350 μl ice-cold PBS containing 1x protease inhibitor cocktail (Complete, Roche).
The cells were washed with 1 ml Buffer A and resuspended in 350 μl Buffer A. After Detergent I treatment for 20 min at RT, cells were centrifuged to collect cytosolic proteins. The pellet was re-suspended in Buffer A and treated with Detergent II for 10 min at RT. The suspension was centrifuged and supernatant was collected as mitochondria fraction.

Transfection and nucleofection
Neurons were transfected in trophic transfection medium 55 with plasmids (2µg/ml total) and/or siRNA (100nM) using Lipofectamine 2000. The sequence targeted by Mcu-directed siRNA is cgaccuagagaaauacaau; control siRNA used was siGENOME Non-Targeting siRNA 2 (Dharmacon). Experiments were performed 48h post-transfection. Nucleofection was performed using the Amaxa rat Neuron Nucleofector Kit (Lonza). Briefly, for every sample, mouse cortical neuron pellets containing 4-5 x 10 6 cells were mixed with 100l nucleofector solution and either 3g plasmid DNA or 300mol siRNA. Nucleofection was carried out using a Nucleofector II system. Following nucleofection, 50l pre-equilibrated Opti-MEM was added to each sample and the cells gently plated down in 24-well dish. Nucleofection efficiency was determined by eGFP expression 48 h after transfection and was routinely 50%-60%.

Plasmids and virus generation
The vector containing the mouse CaMKIIα promoter used to construct and package recombinant adeno-associated virus (rAAV) has been described previously 56  The non-targeting shRNA (scrambled) is the following: gtgccaagacgggtagtca. All rAAV vectors were generated by standard molecular biology techniques and verified by sequencing. Viral particles were produced and purified as described previously 56 . Neurons were infected with 10 11 rAAV particles/μL at DIV 4. Infection efficiencies were determined at DIV 10 by analyzing the fluorescence of eGFP or mCherry; they ranged from 80 to 90% of the viable neurons. rAAV expression vectors for Npas4, CaMBP4 and mCherry have been described previously 35, 56 .

Studying transfected neurons after excitotoxic insult:
Excitotoxicity experiments performed on Lipofectamine-transfected neurons were performed as  City, CA, USA). Total RNA was extracted using RNeasy kit (Qiagen GmbH, Germany) with additional on-column DNase I digestion during RNA purification. cDNA was generated from 1 μg of total RNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

Quantitative reverse transcriptase PCR
QRT-PCR was carried out using TaqMan