Reexamination of N-terminal domains of Syntaxin-1 in vesicle fusion from central murine synapses

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open-conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.

Reexamination of N-terminal domains of Syntaxin-1 in vesicle fusion from central murine synapses

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight binary complex through two major binding modes: one through STX1’s N-peptide, the other through STX1’s closed conformation driven by its Habc-domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1’s N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1’s N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1’s open conformation governs the vesicle fusogenicity. Strikingly, we also show that neurotransmitter release still proceeds when both the interaction modes between STX1 and Munc18-1 are presumably intervened together, necessitating a refinement of the conceptualization of STX1–Munc18-1 interaction.

EXPRESSION OF E8^E2 IS REQUIRED FOR WART FORMATION BY MOUSE PAPILLOMAVIRUS 1 IN VIVO

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells. IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

An HIV-Tat inducible mouse model system of childhood HIV-associated nephropathy

BackgroundModern antiretroviral therapies (ART) have decreased the prevalence of HIV-associated nephropathy (HIVAN). Nonetheless, we continue to see children and adolescents with HIVAN all over the world. Furthermore, once HIVAN is established in children, it is difficult to revert its long-term progression, and we need better animal models of childhood HIVAN to test new treatments.ObjectivesTo define whether the HIV-1 trans-activator (Tat) gene precipitates HIVAN in young mice, and to develop an inducible mouse model of childhood HIVAN.Design/MethodsAn HIV-Tat gene cloned from a child with HIVAN was used to generate recombinant adenoviral vectors (rAd-Tat). rAd-Tat and LacZ control vectors (2 × 109) were expressed in the kidney of newborn wild type and HIV-transgenic (Tg26) FVB/N mice without significant proteinuria (n = 5 - 8 per group). Mice were sacrificed 7 and 35 days later to assess their renal outcome, the expression of HIV-genes and growth factors, and markers of cell growth and differentiation by RT-qPCR, immunohistochemistry, and/or Western blots.ResultsHIV-Tat induced the expression of HIV-1 genes (env) and heparin binding growth factors in the kidney of HIV-Tg26 mice, and precipitated HIVAN in the first month of life. No significant renal changes were detected in wild type mice infected with rAd-Tat vectors, suggesting that HIV-Tat alone does not induce renal disease.ConclusionThis new mouse model of childhood HIVAN highlights the critical role that HIV-Tat plays in the pathogenesis of HIVAN, and could be used to study the pathogenesis and treatment of HIVAN in children and adolescents.Summary statementWe developed a new inducible mouse model system of childhood HIV-associated nephropathy, and demonstrated that HIV-Tat plays a critical role in this renal disease acting in synergy with other HIV-1 genes and heparin binding cytokines.

DDIS-29. BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT, ST-401, KILLS GLIOBLASTOMA THROUGH A NOVEL MECHANISM

Glioblastomas are particularly sensitive to mitotic disruption when compared with nonmalignant cells and thus microtubule-targeting agents (MTA) represent promising therapeutics to treat patients with glioblastomas; but few such compounds pass the blood brain barrier. We developed a series of modified carbazoles, evaluated their anti-cancer activity in glioblastoma cells in culture and identified ST-401 as the most potent compound (IC50, 10 – 102 nM, depending on the cell line). Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity does not correlate with any of the compounds tested thus far through this platform but showed weak correlations for taxol (p=0.46) and vinblastine (p=0.34). Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function. Biochemical analysis indicates that ST-401 binds to the colchicine site of tubulin and inhibits tubulin assembly. Real-time imaging of MT dynamics in cells in culture shows that ST-401 reduces MT assembly rates but in a reversible fashion. ST-401 potently blocks mitotic progression and triggers cell death in multiple glioblastoma lines in culture, including patient-derived glioblastomas of the proneural, mesenchymal and classical subtypes. We established the maximum tolerated dose (MTD) of ST-401 in mice (20 mg/kg/bdip) and found that its acute i.p. injection results in micromolar amounts of ST-401 in mouse brain. Using this treatment regimen, we found that ST-401 reduces tumor growth and doubles overall survival in a human tumor xenograft mouse model system. ST-401 also increases by 2-fold overall survival in the genetic RCAS-PDGF glioblastomas mouse model treated with standard care (radiation and Temodar® treatments). Histological analysis of RCAS-PDGF glioblastoma tissue shows that ST-401 triggers mitotic arrest of glioblastoma cells. ST-401 represents a promising lead compound for the treatments for patients diagnosed with glioblastomas.

Rag1D600A, a novel catalytically inactive RAG mouse model

The RAG complex (RAG1 and RAG2) can bind to recombination signal sequences of antigen receptor loci gene segments and coordinate V(D)J recombination which is the primary method of generating antigen receptor diversity. Previous biochemistry studies discovered RAG1 D600, D708 and E962 residues as essential for catalytic DNA nicking and hairpin forming activity of the RAG complex. Neutralization of each of the acidic residues does not impair DNA binding to recombination signal sequence containing DNA substrates, but cleavage of the substrates is severely compromised. These three acidic residues are thought to comprise a DDE motif that is responsible for binding to a divalent cation that is necessary for cleavage activity. Although a Rag1-/-; RAG1-D708A transgenic mouse model system has been used to study dynamics of RAG activity, transgenic expression may not precisely mimic expression from the endogenous locus. In order to improve upon this model, we created Rag1D600A mice that lack B and T cells and demonstrate a developmental block at the pro-B and DN stages, respectively. Thus, Rag1D600A mice provide a novel mouse model system for studying the poorly understood noncanonical functions of RAG1.