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Ant1 mutant mice bridge the mitochondrial and serotonergic dysfunctions in bipolar disorder

Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.


Fluorescent in situ hybridization (FISH)
FISH was conducted as previously described 2 . DIG labeled antisense RNA probe was generated by using DIG RNA labeling kit (Sigma-Aldrich) with the primers listed in the section of primers.
Fast red (Thermo Fisher scientific) was used for the detection of Slc25a4 mRNA. Images were captured by NDP.

Mouse behavioral screening with IntelliCage
The IntelliCage apparatuses (NewBehavior AG, Zurich, Switzerland) were used for behavioral screening as described previously 3,4 . Since the transponders on two control mice did not accurately work to collect count of visits to the drinking corners and nose pokes in the caves even though they precisely lead to open the doors by nosepoking due to unknown defect, the data obtained from these two mice were excluded.
-Reward based place preference learning and reversal learning In advance of this trial, the mice were trained to learn that the time of water supply was restricted to three-hour drinking session from 9:00 p.m. (one hour after lights off) per day for three days. In place preference learning trial, in addition to the time restriction, the corner where each mouse was able to drink water was also restricted to one among all four corners in an IntelliCage for seven days. In the next reversal learning, the setting of the restricted corner was switched to the opposite corner and the ability to flexible response of the mice were measured for five days. The learning of the place was evaluated by the correct visit rates which were quotients of correct corner visits/all corner visits during drinking session.
-Impulsivity and attention test In advance of this trial, the mice were trained to learn that yellow light signal indicated above the doors was the sign of the period when the sensor to open the door was active over a week. In the first three days, lighting was randomly started at 1, 2 or 4 sec after corner entry and was held for four sec. In the next five days, the timing of the lighting was fixed at two sec after a mouse visit, if a mouse, however, conducted nose poke prior to the light-ON (premature nose poke), lighting would be canceled as fail of the trial (punishment). And the next five days, the timing of the lighting was fixed at four seconds after corner entry but the limited hold was randomly selected from 0.5, 1 and 2. In this test, the timing of the lighting was fixed at four second and the limited hold was randomly selected from 0.3, 0.5, 1 and 2. Also during the test, the lighting would be canceled by premature nose poke. The drinking session was set from 9:00 p.m. to 8:00 a.m. during this training and test. The actions of the mice had been recorded for two days and the attention to short lighting was evaluated by the success rates which were quotients of correct nose pokes/total visits.
-Delay discounting task In this task, the two bottles containing 0.5% sodium saccharin (Merck Millipore) water or purified water were juxtaposed in each corner. In advance of this trial, the mice were allowed to freely access all bottles by nosepoking to remember the side of 0.5% sodium saccharin water for four days. The last day of this habituation period was assigned to the day in which delay until the activation of the sensor to open the windows in saccharin side was 0. After the habituation period, the delay until the activation of the sensor to open the windows in saccharin side was elongated as the order of 0.1 and 1 to 8 seconds with increments of 1 second day by day. Discounting of saccharin reward by delay was evaluated by the saccharin nose poke rates which were quotients of nose poke to saccharin side in total nose poke and licking duration of each bottle.

Saccharin preference test
After behavioral analysis by using IntelliCage, these mice were applied to conventional saccharin preference test. Mice were deprived of water from 18:00 on the day before the test. On the day of the test, two bottles containing 0.5 % sodium saccharin (Merck Millipore) water or purified water were placed on the home cage for 30 minutes. Heterozygous cKO mice (Slc25a4 fl/+ ; Nes-Cre + ) (n = 7), homozygous cKO mice (Slc25a4 fl/fl ; Nes-Cre + ) (n = 10), and control mice (Slc25a4 fl/+ or Slc25a4 fl/fl ; Nes-Cre -) (n = 8) were used.
Partially deleted mitochondrial DNA (ΔmtDNA) was measured by quantitative PCR methods using

Isolation of mitochondria
Brain mitochondria were isolated using a discontinuous Percoll gradient developed by Sims 7 with minor modifications 8 . Briefly, a male mouse of each genotype was decapitated and the brain was transferred to an ice-cold isolation buffer (320 mM sucrose, 1 mM EGTA, and 10 mM MOPS; pH 7.4). The forebrain tissue was dissected and homogenized in 12% Percoll with a Dounce homogenizer. The homogenate was layered on a discontinuous gradient of 26% and 40% Percoll (GE Healthcare) in the isolation buffer and centrifuged in at 30,700 g for 5 min at 4 °C, and washed two times in the isolation buffer at 16,700 g for 5 min and 7,300 g for 10 min, respectively. Fatty acid-free bovine serum albumin (Sigma-Aldrich) was added at the last washing step (final 0.1%).
The mitochondrial pellet was suspended in a buffer (210 mM sucrose, 20 mM KCl, 3 mM glycylglycine, and 1 mM KH2PO4 (pH 7.2)) treated with Chelex 100 resin (Bio-Rad, Richmond, CA). Mitochondrial protein concentration was determined by the Bradford protein assay in the microplate protocol (Nacalai tesque, Kyoto, Japan) and adjusted to 150 µg/ml in a Ca 2+ -free buffer.

Quantification of monoamine in tissue by HPLC
The mice were euthanized by cervical dislocation and the brains were removed and placed on an ice-cold brain slicer matrix with 1 mm coronal section slice intervals (EM Japan, Tokyo, Japan).
Tissues of the nucleus accumbens were dissected by using tweezers and microscissors (World

Electrophysiological analysis
Brain slices for experiments were prepared from 8-12-week-old, male mice as described previously 9 . The mice were deeply anesthetized with halothane inhalation (~2% in air, v/v).
Following decapitation, the brains were rapidly removed and placed in ice-cold sucrose-based cutting solution (~4°C) that contained the following compounds; 252 mM sucrose, 21 mM NaHCO3,

Primers used for cloning, site directed mutagenesis and real-time PCR
Primer sequences used for these experiments are as follows.