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ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

During mammalian brain development, neural stem cells transform from neuroepithelial cells to radial glial cells and finally remain as astrocyte-like cells in the postnatal and adult brain. Neuroepithelial cells divide symmetrically and expand the neural stem cell pool; after the onset of neurogenesis, radial glial cells sequentially produce deep layer neurons and then superficial layer neurons by asymmetric, self-renewing divisions during cortical development. Thereafter, gliogenesis supersedes neurogenesis, while a subset of neural stem cells retain their stemness and lurk in the postnatal and adult brain. Thus, neural stem cells undergo alterations in morphology and the capacity to proliferate or give rise to various types of neural cells in a temporally regulated manner. To shed light on the temporal alterations of embryonic neural stem cells, we sorted the green fluorescent protein-positive cells from the dorsolateral telencephalon (neocortical region) of pHes1-d2EGFP transgenic mouse embryos at different developmental stages and performed gene expression profiling. Among dozens of transcription factors differentially expressed by cells in the ventricular zone during the course of development, several of them exhibited the activity to inhibit neuronal differentiation when overexpressed. Furthermore, knockdown of Tcf3 or Klf15 led to accelerated neuronal differentiation of neural stem cells in the developing cortex, and neurospheres originated from Klf15 knockdown cells mostly lacked neurogenic activities and only retained gliogenic activities. These results suggest that Tcf3 and Klf15 play critical roles in the maintenance of neural stem cells at early and late embryonic stages, respectively.

As the therapeutically effective blood level of mexiletine (MX), a recently marketed antiarrhythmic agent, is narrow at 0.5-2.0μg/ ml, the side-effect occurrence rate increases when the blood level of the drug exceeds 2.0μg/ ml. Therefore, close monitoring of the blood level of the drug is required after administration of the drug.We developed a new method for this measurement using high-performance liquid chromatography (HPLC), which is more convenient than the previous method by gas chromatography. The general procedure is as follows: An internal standard (4-methylmexiletine) is added to the serum sample, followed by the addition of a sodium hydroxide solution, and the whole is mixed well, then injected into an extrelut (manufactured by Merck Co.). The mixture is extracted with diethyl ether, and the extract is evaporated to dryness, and dissolved in an elution solvent, then subjected to the HPLC analysis.The values obtained by the peak-height method showed a lineality at 0.2-5.0μg/ ml. The recovery rate from the serum, to which MX was added at 2.5μg/ ml, was 80.4-90.0%. Coefficient validity was 3.1%. Good chromatographic data was also obtained in measurement of serum levels in patients administered MX.