Keiji Masuda

Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice

Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1−/− mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1−/− mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1−/− murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.

DNA polymerases η and θ function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase η (POLH) exhibited an ∼80% reduction of the mutations at A/T, whereas absence of polymerase θ (POLQ) resulted in ∼20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq–/–Polh–/– mice, however, did not show a further decrease of A/T mutations as compared with Polh–/– mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene Hypermutation

REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. To explore the role of its catalytic activity in Ig gene hypermutation in mammalian cells, we have generated mice expressing a catalytically inactive REV1 (REV1AA). REV1AA mice developed normally and were fertile on a pure C57BL/6 genetic background. B and T cell development and maturation were not affected, and REV1AA B cells underwent normal activation and class switch recombination. Analysis of Ig gene hypermutation in REV1AA mice revealed a great decrease of C to G and G to C transversions, consistent with the disruption of its deoxycytidyl transferase activity. Intriguingly, REV1AA mice also exhibited a significant reduction of C to T and G to A transitions. Moreover, each type of nucleotide substitutions at A:T base pairs was uniformly reduced in REV1AA mice, a phenotype similar to that observed in mice haploinsufficient for Polh. These results reveal an unexpected role for REV1 in the generation of C:G transitions and A:T mutations and suggest that REV1 is involved in multiple mutagenic pathways through functional interaction with other polymerases during the hypermutation process.

Regulation of bcl-xL expression and Fas susceptibility in mouse B cells by CD40 ligation, surface IgM crosslinking and IL-4.

CD40 is one of the key molecules involved in the survival, growth and differentiation of B lymphocytes. In contrast, Fas (Apo-1, CD95) mediates apoptosis of a variety of cell types, including lymphocytes. Recent studies have found that Fas expression on mouse B cells could be strongly induced by CD40 ligation, a helper T cell-derived signal. Here, evidence is provided that CD40 ligation induced two distinct signals: one leading to the upregulation of Fas and the other leading to the enhanced Fas susceptibility. B lymphoma cell lines, CH31 and WEHI279, expressed Fas on cell surfaces, but were resistant to anti-Fas antibody (Ab) induced apoptosis. Treatment with CD40 ligand (CD40L), however, greatly enhanced Fas susceptibility of these cells. Similarly, normal splenic B cells became highly susceptible to Fas-mediated apoptosis following prolonged signaling through CD40. While CD40 ligation enhanced Fas-mediated apoptosis, stimulation with anti-IgM and IL-4 partially protected CD40L-activated B cells from Fas-mediated apoptosis. It was found that bcl-xL gene expression in normal splenic B cells was induced drastically by treatment with anti-IgM and IL-4, but not CD40L. By contrast, the expression of bcl-2 or bax was not significantly affected by these treatments. Moreover, in three of the four B lymphoma cell lines tested, Fas susceptibility correlated with the status of bcl-xL expression. The data suggest that an increase in bcl-xL expression may protect B cells from Fas-mediated apoptosis.

Role of DNA polymerase θ in tolerance of endogenous and exogenous DNA damage in mouse B cells

DNA polymerase θ (Polθ) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Polθ in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Polθ polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, γ‐irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Polθ is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Polθ in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross‐links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.

Expression levels of adiponectin receptors and periodontitis

BACKGROUND AND OBJECTIVE We recently showed that adiponectin, an adipocyte-derived cytokine, may function as a negative regulator of the Toll-like receptor signaling pathway and of osteoclast formation in periodontal disease. In this study, we investigated whether the expression levels of adiponectin receptors (AdipoR1 and AdipoR2) are related to the presence of periodontitis. MATERIAL AND METHODS We initially examined, using RT-PCR, the expression of the AdipoR1 and AdipoR2 genes at the mRNA level in several oral tissues of C57BL mice. Next, we investigated (using real-time PCR assays) whether inflammatory cytokines, such as tumor necrosis factor-alpha, could affect the expression levels of these genes in human gingival fibroblasts. Lastly, we compared the expression levels of these receptor proteins in gingival tissues between two healthy subjects and five patients with severe periodontal disease using western blotting analysis. RESULTS The AdipoR1 and AdipoR2 receptors were ubiquitously expressed in the oral tissues of mice. We observed that treatment with tumor necrosis factor-alpha could significantly reduce the expression levels of both AdipoR1 and AdipoR2 genes in human gingival fibroblasts. Moreover, we found that the expression of both receptors was lower in periodontal tissues from patients with severe periodontitis than in patients with healthy periodontal tissues. CONCLUSION These observations suggest that adiponectin may not function efficiently in sites of periodontal disease because of a decrease in the number of its receptors, and this probable dysfunction may play a role in worsening periodontitis in patients.

Liposteroid against refractory pulmonary haemorrhage in idiopathic pulmonary haemosiderosis

We describe two Japanese children with idiopathic pulmonary haemosiderosis (IPH), whose refractory haemorrhages were treated with an intravenous lipid emulsion containing dexamethasone (liposteroid). A 22-month-old boy and a 14-month-old girl have been observed with similar symptoms; periodic bouts of anaemia, reticulocytosis, diffuse infiltrates on chest X-ray and the finding of siderophages in sputum or gastric lavage fluid. The MRI of the lung was useful for the diagnosis. Methylprednisolone pulse therapy was successful in treating acute massive bleeding. Subsequent oral prednisolone could not prevent chronic recurrent haemorrhages. However, the intermittent administration of liposteroid (0.05 mg/kg/dose IV) led to a cessation of bleeding; the haemoglobin concentration rose to normal levels. This observation emphasizes the usefulness of liposteroid in the management of refractory IPH.

DNA polymerase η is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation

DNA polymerase η (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh+/− mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh+/+ and Polh−/− mice. These observations suggest that Polh is haplo‐insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh+/− mice as compared with WT mice. Polh−/− mice produced decreased serum titers of high‐affinity antibodies against a T‐dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high‐affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh−/− mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.

Mitochondria Regulate the Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth

Stem cells from human exfoliated deciduous teeth (SHED) are isolated from the dental pulp tissue of primary teeth and can differentiate into neuronal cells. Although SHED are a desirable type of stem cells for transplantation therapy and for the study of neurological diseases, a large part of the neuronal differentiation machinery of SHED remains unclear. Recent studies have suggested that mitochondrial activity is involved in the differentiation of stem cells. In the present work, we investigated the neuronal differentiation machinery of SHED by focusing on mitochondrial activity. During neuronal differentiation of SHED, we observed increased mitochondrial membrane potential, increased mitochondrial DNA, and elongated mitochondria. Furthermore, to examine the demand for mitochondrial activity in neuronal differentiation, we then differentiated SHED into neuronal cells in the presence of rotenone, an inhibitor of mitochondrial respiratory chain complex I, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupler, and found that neuronal differentiation was inhibited by treatment with rotenone and CCCP. These results indicated that increased mitochondrial activity was crucial for the neuronal differentiation of SHED.Key words: mitochondria, differentiation, stem cells, dental pulp, exfoliated deciduous teeth.