Chieko Aoyama

A Rostrocaudal Muscular Dystrophy Caused by a Defect in Choline Kinase Beta, the First Enzyme in Phosphatidylcholine Biosynthesis *

Muscular dystrophies include a diverse group of genetically heterogeneous disorders that together affect 1 in 2000 births worldwide. The diseases are characterized by progressive muscle weakness and wasting that lead to severe disability and often premature death. Rostrocaudal muscular dystrophy (rmd) is a new recessive mouse mutation that causes a rapidly progressive muscular dystrophy and a neonatal forelimb bone deformity. The rmd mutation is a 1.6-kb intragenic deletion within the choline kinase beta (Chkb) gene, resulting in a complete loss of CHKB protein and enzymatic activity. CHKB is one of two mammalian choline kinase (CHK) enzymes (α and β) that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid phosphatidylcholine. While mutant rmd mice show a dramatic decrease of CHK activity in all tissues, the dystrophy is only evident in skeletal muscle tissues in an unusual rostral-to-caudal gradient. Minor membrane disruption similar to dysferlinopathies suggest that membrane fusion defects may underlie this dystrophy, because severe membrane disruptions are not evident as determined by creatine kinase levels, Evans Blue infiltration, and unaltered levels of proteins in the dystrophin-glycoprotein complex. The rmd mutant mouse offers the first demonstration of a defect in a phospholipid biosynthetic enzyme causing muscular dystrophy, representing a unique model for understanding mechanisms of muscle degeneration.

A Congenital Muscular Dystrophy with Mitochondrial Structural Abnormalities Caused by Defective De Novo Phosphatidylcholine Biosynthesis

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

Early Embryonic Lethality Caused by Disruption of the Gene for Choline Kinase α, the First Enzyme in Phosphatidylcholine Biosynthesis

Choline kinase α (CK-α) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-α with an embryonic stem cell line containing an insertional mutation in the gene for CK-α (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-α is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka+/-) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by ∼30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka+/- mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka+/- mice. Triacylglycerol and cholesterol ester were elevated ∼2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka+/- and wild-type (Chka+/+) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.

Muscular dystrophy with large mitochondria associated with mutations in the CHKB gene in three British patients: Extending the clinical and pathological phenotype

Three patients with CHKB deficient muscular dystrophy are described which broadens the previously described phenotype. Blood smear in one patient showed Jordans anomaly (vacuolated leukocytes). Gastrointestinal features occurred in two patients and there appeared to be acute deterioration with infection/general anaesthesia. Brain imaging showed no structural changes but brain magnetic resonance proton spectroscopy (MRS) demonstrated significant reduction in choline:N-acetyl aspartate and choline:creatine ratios in keeping with a general decrease in the amount of choline and phosphocholine-based substrate. Muscle pathology showed either myopathic or dystrophic features, uneven oxidative enzyme staining, COX deficient fibres and peripherally located large mitochondria. CHKB activity was reduced in all three patients and complex 1 activity was significantly reduced in one patient.

Choline kinase alpha expression during RA-induced neuronal differentiation: Role of C/EBPβ

Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria.

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40–50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1

Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 μM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.

Identification of nuclear localization and nuclear export signals in Ets2, and the transcriptional regulation of Ets2 and CTP:phosphocholine cytidylyltransferase α in tetradecanoyl-13-acetate or macrophage-colony stimulating factor stimulated RAW264 cells

PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase alpha (CTalpha), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTalpha transcription by binding at the Ets binding site (-49/-47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTalpha transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404-410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1-392 present) was localized to the cytoplasm and site-specific mutation in the region 364-372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTalpha-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTalpha was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTalpha transcription by TPA and macrophage-colony stimulating factor.

The heterotrimeric G protein subunits Gαq and Gβ1 have lysophospholipase D activity

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gαq and Gβ1 respectively. When FLAG-tagged Gαq or Gβ1 was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg2+ dependency and substrate specificity of Gαq were similar to those of lysoPLD purified from the rat brain. Mutation of Gαq at amino acids Lys52, Thr186 or Asp205, residues that are predicted to interact with nucleotide phosphates or catalytic Mg2+, dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gαq overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated Km and Vmax values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gαq and Gβ1 as an enzyme with lysoPLD activity. Tag-purified Gα11 also exhibited a high lysoPLD activity, but Gαi and Gαs did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gαq and Gα11 siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.