Raw data, a2Ca3-dKO phenotype and dystonic spell of a3-HT

We found brain haemorrhage phenotype of Atp1a2 and Atp1a3 doule knockout (α2α3-dKO) foetuses. Here we present two kinds of α2α3-dKO line, α2-Nα3-dKO andα2-Cα3-dKO, both show brain haemorrhage upon birth. They were similar to that of homozygous knockout of the gene encoding ascorbic acid (ASC orNvitamin C) transporter, SVCT2 (SVCT2-KO).  We made SVCT2-knockout mouse line by CRIPR/CAS9 method. We present SVCT2-KO, showing brain hemorrage as α2α3-dKO. The α2α3-dKO and SVCT2-KO brain showed significantly decreased level of ASC compared with the wild-type (WT) and single knockout (here, raw data). We found that the ASC content in the basal ganglia and cerebellum was significantly lower in the adult Atp1a3 heterozygous knockout mouse (α3-HT) than in the WT (here, raw data). We did not observe increased oxidative stress in them (here raw data). Interestingly, we observed a significant decrease in the ASC level in the basal ganglia and cerebellum of α3-HTin the peripartum period, during which mice are under physiological stress(here, raw data). Here, we show dystonic spell of α3-HT in peripartum. These observations indicate that the α2 and α3 subunits independently contribute to the ASC level in the foetal brain and that the α3 subunit contributes to ASC transport in the adult basal ganglia and cerebellum. We propose that decreases in ASC levels may affect neural network development and are linked to the pathophysiology of ATP1A2- and ATP1A3-related neurologic disorders.

Measurement of malondialdehyde (MDA) and protein sulfhydryl (SH) levels

Frozen brain regions (the cortex including hippocampus, basal ganglia, and cerebellum) prepared as described above were used. Analyses of MDA and SH levels were performed as previously described [35-38]with some modifications. Briefly, each sample was homogenized in a small volume of 1 ml of 5% trichloroacetic acid. The volume was made up to 1 ml, and the samples were centrifuged at 13,000 rpm at room temperature for 5 min. Then, 250 μl of each sample was mixed with the same volume of 0.02 M thiobarbituric acid for 35 min at 95 °C, and the reaction was terminated by incubation at 4 °C for 10 min. MDA levels in 200 μl samples of the final product were quantified in duplicate by measuring fluorescence in a 96-well plate (Synergy H4 plate reader; excitation at 515, emission at 553). The final concentration was calculated relative to the wet weight with reference to a standard curve generated from a stock solution of 50 pmol/μl MDA that was treated in an identical manner to the samples. For SH level determination, the samples were homogenized in 10 volumes of sodium phosphate buffer (pH 7.4) with 140 mM KCl and a protease inhibitor pellet. Each sample was then centrifuged at 750 x g at 4 °C for 10 min. The supernatant was diluted 1:1 in homogenization buffer. Protein content was calculated using a standard BCA assay, and 10 μl of sample was reacted with 6 μl of 10 mM DTNB in 0.2 M potassium phosphate solution (pH 8) with an additional volume of 200 μl of 1x PBS (pH 7.4) containing 1 mM EDTA. The samples were incubated at room temperature in the dark for 30 min, and the absorption was then measured at 412 nm.

Measurement of malondialdehyde (MDA) and protein sulfhydryl (SH) levels

Frozen brain regions (the cortex including hippocampus, basal ganglia, and cerebellum) prepared as described above were used. Analyses of MDA and SH levels were performed as previously described [35-38]with some modifications. Briefly, each sample was homogenized in a small volume of 1 ml of 5% trichloroacetic acid. The volume was made up to 1 ml, and the samples were centrifuged at 13,000 rpm at room temperature for 5 min. Then, 250 μl of each sample was mixed with the same volume of 0.02 M thiobarbituric acid for 35 min at 95 °C, and the reaction was terminated by incubation at 4 °C for 10 min. MDA levels in 200 μl samples of the final product were quantified in duplicate by measuring fluorescence in a 96-well plate (Synergy H4 plate reader; excitation at 515, emission at 553). The final concentration was calculated relative to the wet weight with reference to a standard curve generated from a stock solution of 50 pmol/μl MDA that was treated in an identical manner to the samples. For SH level determination, the samples were homogenized in 10 volumes of sodium phosphate buffer (pH 7.4) with 140 mM KCl and a protease inhibitor pellet. Each sample was then centrifuged at 750 x g at 4 °C for 10 min. The supernatant was diluted 1:1 in homogenization buffer. Protein content was calculated using a standard BCA assay, and 10 μl of sample was reacted with 6 μl of 10 mM DTNB in 0.2 M potassium phosphate solution (pH 8) with an additional volume of 200 μl of 1x PBS (pH 7.4) containing 1 mM EDTA. The samples were incubated at room temperature in the dark for 30 min, and the absorption was then measured at 412 nm.

Measurement of ASC content

The mice were sacrificed by quick decapitation under deep anaesthesia using inhaled isoflurane. The whole brains or dissected regions of the brain (the cortex including the hippocampus, basal ganglia, and cerebellum) were immediately removed from foetuses/neonates or 2- to 4-month-old adult mice (female and male), respectively, quickly frozen in liquid nitrogen, and stored at -80 °C until needed. Frozen samples were suspended in 500 µl of a solution of 0.1 M phosphate buffer (pH 2.5) plus 10 mg/L EDTA-2Na and 20 µg 3,4-dihydroxybenzylamine hydrobromide (DHBA, internal standard), and then 400 µl of methanol was added. The samples were homogenized for 1 min using an ultrasound sonicator and left on ice for 30 min. After centrifugation (20,000 x g, 15 min, 4 °C), the supernatants were diluted with 0.1 M phosphate buffer (pH 3.5) plus 1 mM EDTA-2Na. A 20 µl of filtered diluent (passed through a 0.22-µm filter) was applied to a separation column (150×3.0 mm, Eicompak SC-5ODS). High-performance liquid chromatography (HPLC) with electrochemical (EC) detection was performed at +0.8 V vs. Ag/AgCl with a flow rate of 450 µl/min (HITEC-500, EICOM, Kyoto, Japan). Quantitation of ASC content in each sample was achieved by comparing peak heights with those obtained after injections of known quantities of ASC and DHBA (standard, in 20 μl, 40 pg and 200 pg, respectively). The data were analysed using the EPC-300 system at a 10/sec sample speed (EICOM). The values were normalized to the weights of the specimen (whole brain or parts of the brain).