Optogenetically Induced Seizure and the Longitudinal Hippocampal Network Dynamics

Epileptic seizure is a paroxysmal and self-limited phenomenon characterized by abnormal hypersynchrony of a large population of neurons. However, our current understanding of seizure dynamics is still limited. Here we propose a novel in vivo model of seizure-like afterdischarges using optogenetics, and report on investigation of directional network dynamics during seizure along the septo-temporal (ST) axis of hippocampus. Repetitive pulse photostimulation was applied to the rodent hippocampus, in which channelrhodopsin-2 (ChR2) was expressed, under simultaneous recording of local field potentials (LFPs). Seizure-like afterdischarges were successfully induced after the stimulation in both W-TChR2V4 transgenic (ChR2V-TG) rats and in wild type rats transfected with adeno-associated virus (AAV) vectors carrying ChR2. Pulse frequency at 10 and 20 Hz, and a 0.05 duty ratio were optimal for afterdischarge induction. Immunohistochemical c-Fos staining after a single induced afterdischarge confirmed neuronal activation of the entire hippocampus. LFPs were recorded during seizure-like afterdischarges with a multi-contact array electrode inserted along the ST axis of hippocampus. Granger causality analysis of the LFPs showed a bidirectional but asymmetric increase in signal flow along the ST direction. State space presentation of the causality and coherence revealed three discrete states of the seizure-like afterdischarge phenomenon: 1) resting state; 2) afterdischarge initiation with moderate coherence and dominant septal-to-temporal causality; and 3) afterdischarge termination with increased coherence and dominant temporal-to-septal causality. A novel in vivo model of seizure-like afterdischarge was developed using optogenetics, which was advantageous in its reproducibility and artifact-free electrophysiological observations. Our results provide additional evidence for the potential role of hippocampal septo-temporal interactions in seizure dynamics in vivo. Bidirectional networks work hierarchically along the ST hippocampus in the genesis and termination of epileptic seizures.     

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.     

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.     

Generation of hybridomas producing human monoclonal antibodies against herpes simplex virus after in vitro stimulation

Hybridomas producing human monoclonal antibodies against herpes simplex virus were generated by in vitro antigen stimulation before cell fusion. The cell fusion with tonsillar lymphocytes which were stimulated with antigen and/or pokeweed mitogen generated many hybridomas producing human IgG against the virus. A combination of antigen and pokeweed mitogen synergistically enhanced the generation of virus-specific hybridomas. Furthermore, the higher the antibody response of the tonsil, the more virus-specific hybridomas were generated by the cell fusion. These results suggest that cell fusion with in vitro stimulated lymphocytes can be applied to a variety of clinically relevant viruses.     

A new esteroproteinase (proteinase F) from the submandibular glands of female mice

One of the esteroproteinases present in the submandibular glands of female mice was purified and characterized. The enzyme, designated proteinase F in this report, had a pI value of 4.6 and a molecular weight of 27 600, being comprised of two subunits of 10 000 and 18 000 daltons. The amino acid composition of proteinase F resembled that of the epidermal growth factor-binding protein, but antiserum against proteinase F only reacted weakly against the binding protein. Proteinase F had an optimum pH at around 9.0 and was strongly inhibited by Cu²⁺ and Hg²⁺ (42 and 76% inhibition, respectively, at a concentration of 4·10^?6 M). It was also inhibited by aprotinin, phenylmethylsulfonylfluoride, iodoacetamide, leupeptin, antipain, and benzamidine but neither by trypsin inhibitors from pancreas, soybean or ovomucoid, nor by TLCK, TPCK, and ?-amino-n-caproic acid. Although its actual physiological function has yet to be determined, these properties indicate that proteinase F is a new enzyme, being distinguished from known proteinases, kallikrein, plasmin, trypsin, chymotrypsin, tonin, angiotensin-converting enzyme, proteinase A (β-nerve growth factor endopeptidase), proteinase D (epidermal growth factor-binding protein), P-esterase, renin A, and renin C. Proteinase F was present in the submandibular glands of female mice more abundantly than in those of males, but it increased in males following castration. Thus, proteinase F appears to be affected by male hormones in vivo.