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Migration and organization of the nucleus are essential for the proliferation and differentiation of cells, including neurons. However, the relationship between the positioning of the nucleus and cellular morphogenesis remains poorly understood. Inherited recessive cerebellar ataxia has been attributed to mutations in SYNE1, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Regardless, Syne1-mutant mice present with normal cerebellar development. The Sad1-Unc-84 homology (SUN)-domain proteins are located at the inner nuclear membrane and recruit Syne proteins through the KASH domain to the outer nuclear membrane. Here, we report an unrecognized contribution of Sun1 and Sun2 to the postnatal development of murine cerebellum. Mice depleted of Sun1 showed a marked reduction in the cerebellar volume, and this phenotype is exacerbated with additional loss of a Sun2 allele. Consistent with these histological changes, Sun1−/− and Sun1−/−Sun2+/− mice exhibited defective motor coordination. Results of immunohistochemical analyses suggested that Sun1 is highly expressed in Purkinje cells and recruits Syne2 to the periphery of the nucleus. Approximately 33% of Purkinje cells in Sun1−/− mice and 66% of Purkinje cells in Sun1−/−Sun2+/− mice were absent from the surface of the internal granule layer (IGL), whereas the proliferation and migration of granule neurons were unaffected. Furthermore, the Sun1−/−Sun2+/− Purkinje cells exhibited retarded primary dendrite specification, reduced dendritic complexity and aberrant patterning of synapses. Our findings reveal a cell-type-specific role for Sun1 and Sun2 in nucleokinesis during cerebellar development, and we propose the use of Sun-deficient mice as a model for studying cerebellar ataxia that is associated with mutation of human SYNE genes or loss of Purkinje cells.

Hepatocyte transplantation (Tx) holds promise for curing genetic liver diseases. However, a limited number of donor hepatocytes can be transplanted into the host liver. Recipient preconditioning and donor cell engineering are under investigation to improve cell engraftment. In theory, genetically engineered cells secreting therapeutic proteins with superior function could compensate for poor engraftment efficiency. We have generated a bioengineered human coagulation factor IX (FIX) with augmented specific activity (named FIX-Triple). The aim of this study was to evaluate therapeutic efficacy of cell therapy using hemophilia B (HB) as a disease model by transplanting FIX-Triple-secreting hepatocytes. The donor hepatocytes were isolated from FIX-Triple knock-in (KI) or FIX-WT (wild-type) KI mice and transplanted intrasplenically into FIX knock-out (KO) mice. FIX-Triple KI recipients exhibited fourfold higher plasma FIX clotting activity than FIX-WT KI recipients. By repeated Txs, the clotting activity of FIX-Triple KI recipients even increased to more than 10% of normal mouse plasma. The engraftment and FIX production efficiencies of transplanted cells were equivalent between the FIX-WT KI and FIX-Triple KI donors. A hemostatic function assay showed that FIX-Triple KI recipients with repeated Txs had more enhanced clot kinetics and a greater maximum rate of thrombus generation than those with a single Tx. Moreover, FIX inhibitors in these recipients rarely developed. In conclusion, hepatocyte Tx with genetically engineered donor cells is an effective therapeutic strategy for HB.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Besides its well-recognized role in hemostasis and thrombosis, thromboxane A2 synthase (TXAS) is proposed to be involved in thrombopoiesis and lymphocyte differentiation. To evaluate its various physiologic roles, we generated TXAS-deleted mice by gene targeting. TXAS–/– mice had normal bone marrow megakaryocytes, normal blood platelet counts, and normal CD4 and CD8 lymphocyte counts in thymus and spleen. Platelets from TXAS–/– mice failed to aggregate or generate thromboxane B2 in response to arachidonic acid (AA) but produced increased prostaglandin-E2 (PGE2), PGD2, and PGF2α. AA infusion caused a progressive drop of mean arterial pressure (MAP), cardiac arrest, and death in wild-type (WT) mice but did not induce shock in TXAS–/– mice or in WT and TXAS–/– mice treated with antagonist to the thromboxane-prostanoid (TP) receptor. The TXAS–/– mice were able to maintain normal MAP upon AA insult when TP was present but were unable to do so when TP was blocked by an antagonist, suggesting a role of endoperoxide accumulation in influencing MAP. We conclude that TXAS is not essential for thrombopoiesis and lymphocyte differentiation. Its deficiency causes a mild hemostatic defect and protects mice against arachidonate-induced shock and death. The TXAS-deleted mice will be valuable for investigating the roles of arachidonate metabolic shunt in various pathophysiologic processes.

Besides its well-recognized role in hemostasis and thrombosis, thromboxane A2synthase (TXAS) is proposed to be involved in thrombopoiesis and lymphocyte differentiation. To evaluate its various physiologic roles, we generated TXAS-deleted mice by gene targeting. TXAS–/–mice had normal bone marrow megakaryocytes, normal blood platelet counts, and normal CD4 and CD8 lymphocyte counts in thymus and spleen. Platelets from TXAS–/–mice failed to aggregate or generate thromboxane B2in response to arachidonic acid (AA) but produced increased prostaglandin-E2(PGE2), PGD2, and PGF2α. AA infusion caused a progressive drop of mean arterial pressure (MAP), cardiac arrest, and death in wild-type (WT) mice but did not induce shock in TXAS–/–mice or in WT and TXAS–/–mice treated with antagonist to the thromboxane-prostanoid (TP) receptor. The TXAS–/–mice were able to maintain normal MAP upon AA insult when TP was present but were unable to do so when TP was blocked by an antagonist, suggesting a role of endoperoxide accumulation in influencing MAP. We conclude that TXAS is not essential for thrombopoiesis and lymphocyte differentiation. Its deficiency causes a mild hemostatic defect and protects mice against arachidonate-induced shock and death. The TXAS-deleted mice will be valuable for investigating the roles of arachidonate metabolic shunt in various pathophysiologic processes.

Abstract 5129

Preeclampsia is a pregnancy-induced disorder. If a pregnant woman suffered from preeclampsia, her worse developed-placenta might result in growth retardation of the fetus, and in the most severe cases, it would cause fetal and maternal death. Several previous studies showed that circulating thromboxane A2 in preeclampsic women was higher than that of normal pregnant women, and higher thromboxane A2 level might induce thrombosis and hypertension in these patients. Thus, thromboxane A2 was thought to be an important metabolite relevant to the development of preeclampsia.In order to clearly clarify the roles of thromboxane A2 on preeclampsia, wild type and thromboxane A2 synthase knockout pregnant mice were fed with drinking water containing high concentration of sodium chloride. The effect of thromboxane A2 on preeclampsia was observed by measuring blood pressure, urinary protein, systemic edema in pregnant mice, and placenta weight, fetal weight, fetal size and fetal sections were also measured and stained.In the groups of pregnant mice fed with high salt, maternal body weight, fetal size and fetal weight were severely decreased in wild type mice as compared to thromboxane A2 synthase knockout mice. Furthermore, abnormal cell degeneration was observed in fetus of wild type pregnant mice, but less in thromboxane A2 synthase knockout fetus.These results demonstrate that knockout pregnant mice were more resistant to high salt treatment, implying the roles of thromboxane A2 on the development of maternal and fetal defects during high salt treatment.No relevant conflicts of interest to declare.

Hemophila B is an X-linked inherited disorder caused by the lack of a functional Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. However, the high cost associated with the protein concentrates poses a financial burden to protein replacement therapy. Other treatments, such as gene therapy and tissue implant techniques, are also under study but not yet developed for clinical use. An alternative approach is to develop a FIX molecule with higher specific activity that will reduce the amount of the clotting factor consumed in treatment. For this purpose, we used alanine-scanning mutagenesis and generated 7 alanine-replaced FIX variants that were expressed in HEK-293 cells and purified. These variants have either single (n=3), double (n=3) or triple (n=1) amino acid changes and each was characterized by ELISA and aPTT assay. We observed that the FIX triple mutation variant (R86A/E277A/R338A, FIX-Triple) exhibited 10-12 times higher clotting activity than wild-type FIX (FIX-WT). The affinity of IX-Triple to human Factor VIIIa increased 10-fold (Kd = 0.19 nM vs. 2.4 nM, FIX-Triple vs. FIX-WT) in a Factor Xa-generation assay, consistent with the fact that the amino acid substitutions were in two domains important for factor VIII interaction. Protein infusion into hemophilia B (HB) mice showed that recombinant FIX-Triple was also more effective than FIX-WT in shortening the clotting time in these HB mice. To further evaluate the medium and long-term efficacy of FIX-Triple expression, we used two methods of gene delivery in (HB) mice. For medium-term expression, we performed tail vein hydrodynamic plasmid injections of either FIX-WT or FIX-Triple, expressed from a liver-specific promoter. We assayed specific activity (clotting activity/antigen, U/mg) 24h post-delivery and found that FIX-Triple had a 3.5-fold higher specific activity than FIX-WT. For long-term expression, we performed tail vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple from a liver-specific promoter at vector doses of 4x1012 (high dose) and 8x1010 (low dose) vector genomes (v.g.)/kg. These mice were sacrificed 8 weeks post vector administration and blood samples were analyzed for FIX antigen (ELISA) and clotting activity (aPTT). Corroborating our previous observations, we found that the FIX-Triple variant had 7 times higher specific activity vs. FIX-WT, for either vector dose used. More importantly, mice that received AAV8-FIX-Triple exhibited activity of 23% of normal pooled human plasma (12-38%, n=6), in contrast to mice that received FIX-WT that only reached 4% (1-8%, n = 4), following 8x1010 v.g./kg vector administration. Activity and antigen level for both transgenes was dose-dependent. Lastly, we generated knock-in mice for FIX-WT and FIX-Triple (FIX-KI-WT and FIX-KI-Triple) by exchanging the mouse sequence for the human, keeping the endogenous promoter intact. Confirming our observations with hydrodynamic injection and AAV administration, mice with FIX-KI-Triple exhibited 7 fold higher specific activity than those with FIX-KI-WT (2040 ± 253 vs 279 ± 33 U/mg). Collectively, our results indicate that FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to Factor VIIIa, as demonstrated both in vitro and in vivo. Therefore, Factor IX-Triple is a good candidate for further evaluation in protein replacement therapy as well as other, gene-based therapeutic strategies.

Hemophila B is an X-linked inherited disorder caused by the lack of a functional Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. However, the high cost associated with the protein concentrates poses a financial burden to protein replacement therapy. Other treatments, such as gene therapy and tissue implant techniques, are also under study but not yet developed for clinical use. An alternative approach is to develop a FIX molecule with higher specific activity that will reduce the amount of the clotting factor consumed in treatment. For this purpose, we used alanine-scanning mutagenesis and generated 7 alanine-replaced FIX variants that were expressed in HEK-293 cells and purified. These variants have either single (n=3), double (n=3) or triple (n=1) amino acid changes and each was characterized by ELISA and aPTT assay. We observed that the FIX triple mutation variant (R86A/E277A/R338A, FIX-Triple) exhibited 10-12 times higher clotting activity than wild-type FIX (FIX-WT). The affinity of IX-Triple to human Factor VIIIa increased 10-fold (Kd = 0.19 nM vs. 2.4 nM, FIX-Triple vs. FIX-WT) in a Factor Xa-generation assay, consistent with the fact that the amino acid substitutions were in two domains important for factor VIII interaction. Protein infusion into hemophilia B (HB) mice showed that recombinant FIX-Triple was also more effective than FIX-WT in shortening the clotting time in these HB mice. To further evaluate the medium and long-term efficacy of FIX-Triple expression, we used two methods of gene delivery in (HB) mice. For medium-term expression, we performed tail vein hydrodynamic plasmid injections of either FIX-WT or FIX-Triple, expressed from a liver-specific promoter. We assayed specific activity (clotting activity/antigen, U/mg) 24h post-delivery and found that FIX-Triple had a 3.5-fold higher specific activity than FIX-WT. For long-term expression, we performed tail vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple from a liver-specific promoter at vector doses of 4x1012(high dose) and 8x1010(low dose) vector genomes (v.g.)/kg. These mice were sacrificed 8 weeks post vector administration and blood samples were analyzed for FIX antigen (ELISA) and clotting activity (aPTT). Corroborating our previous observations, we found that the FIX-Triple variant had 7 times higher specific activity vs. FIX-WT, for either vector dose used. More importantly, mice that received AAV8-FIX-Triple exhibited activity of 23% of normal pooled human plasma (12-38%, n=6), in contrast to mice that received FIX-WT that only reached 4% (1-8%, n = 4), following 8x1010v.g./kg vector administration. Activity and antigen level for both transgenes was dose-dependent. Lastly, we generated knock-in mice for FIX-WT and FIX-Triple (FIX-KI-WT and FIX-KI-Triple) by exchanging the mouse sequence for the human, keeping the endogenous promoter intact. Confirming our observations with hydrodynamic injection and AAV administration, mice with FIX-KI-Triple exhibited 7 fold higher specific activity than those with FIX-KI-WT (2040 ± 253 vs 279 ± 33 U/mg). Collectively, our results indicate that FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to Factor VIIIa, as demonstrated both in vitro and in vivo. Therefore, Factor IX-Triple is a good candidate for further evaluation in protein replacement therapy as well as other, gene-based therapeutic strategies.