WT Levels Research Articles

Abstract 626: Caveolin-1 (CAV1) Levels Determine the Cell Surface Expression of the Dopamine D1-like Receptors

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) increases cAMP production and negatively regulates sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein coupled receptor kinase type 4 (GRK4), which hyperphosphorylates and decreases D1R function, leading to sodium retention. We have previously shown that knockdown of CAV1 in proximal tubule cells impairs sodium transport. In this report we show that CAV1 reduction, knockdown or knockout leads to decreased dopamine D1-like receptor binding at the proximal tubule cell surface. Dopamine D1-like brush border and cell surface binding was measured using Bodipy-FL conjugated SKF83566 . WT and Caveolin 1 knockout (CAV1KO) kidney cortex brush border membrane isolation was performed by magnetic lotus tetragonolobus agglutinin affinity purification and fluorescent SKF83566 binding by microplate fluorometry. CAV1KO had a 35.8±3.3% decrease in binding vs WT (p<0.01, n=6 per group, WT level 18,492 RFU above background). In hTERT immortalized human renal proximal tubule cell lines, lentiviral shRNAi CAV1 stable knockdown cell lines had a 76.4±2.5%, p<0.001 n=4, reduction in CAV1 expression by western blot. Cell surface D1-like receptor binding was reduced by 34.0±1.3% (p<0.001, n=4) in the CAV1 knockdown cell lines (scrambled control levels 117193 RFU). We previously have shown that CAV1 expression is dramatically reduced in the hypertensive SHR proximal tubule cell line vs the normotensive WKY proximal tubule cell line. The SHR cell line had a 57.5±1.2% (p<0.001, n=7) reduction in D1-like receptor binding (WKY 116114 RFU). Re-expression of rat CAV1 alpha or CAV1 beta isoform in the SHR cell line by stable lentiviral infection increased binding by 51.6±2.1%, and 63.3±1.6% respectively (p<0.01, n=14). Sodium transport was decreased by CAV1 alpha and CAV1 beta expression in SHR cell lines. The average number of domes per 10x objective fields of view was 9.5±1.6 in SHR cell, 4.5±0.3 in SHR CAV1alpha (p<0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p<0.05, n=4 vs SHR). In mouse, rat, and human renal proximal tubule cells, CAV1 is necessary for dopamine D1-like receptor cell surface localization and for normal dopaminergic inhibition of sodium transport.

Transgenic Overexpression of Sox17 Promotes Oligodendrocyte Development and Attenuates Demyelination

We have previously demonstrated that Sox17 regulates cell cycle exit and differentiation in oligodendrocyte progenitor cells. Here we investigated its function in white matter (WM) development and adult injury with a newly generated transgenic mouse overexpressing Sox17 in the oligodendrocyte lineage under the CNPase promoter. Sox17 overexpression in CNP-Sox17 mice sequentially promoted postnatal oligodendrogenesis, increasing NG2 progenitor cells from postnatal day (P) 15, then O4+ and CC1+ cells at P30 and P120, respectively. Total Olig2+ oligodendrocyte lineage cells first decreased between P8 and P22 through Sox17-mediated increase in apoptotic cell death, and thereafter significantly exceeded WT levels from P30 when cell death had ceased. CNP-Sox17 mice showed increased Gli2 protein levels and Gli2+ cells in WM, indicating that Sox17 promotes the generation of oligodendrocyte lineage cells through Hedgehog signaling. Sox17 overexpression prevented cell loss after lysolecithin-induced demyelination by increasing Olig2+ and CC1+ cells in response to injury. Furthermore, Sox17 overexpression abolished the injury-induced increase in TCF7L2/TCF4+ cells, and protected oligodendrocytes from apoptosis by preventing decreases in Gli2 and Bcl-2 expression that were observed in WT lesions. Our study thus reveals a biphasic effect of Sox17 overexpression on cell survival and oligodendrocyte formation in the developing WM, and that its potentiation of oligodendrocyte survival in the adult confers resistance to injury and myelin loss. This study demonstrates that overexpression of this transcription factor might be a viable protective strategy to mitigate the consequences of demyelination in the adult WM.

Specificity of human rhinovirus 2Apro is determined by combined spatial properties of four cleavage site residues

The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

Suppressed Retinal Degeneration in Aged Wild Type and APPswe/PS1ΔE9 Mice by Bone Marrow Transplantation

Alzheimer's disease (AD) is an age-related condition characterized by accumulation of neurotoxic amyloid β peptides (Aβ) in brain and retina. Because bone marrow transplantation (BMT) results in decreased cerebral Aβ in experimental AD, we hypothesized that BMT would mitigate retinal neurotoxicity through decreased retinal Aβ. To test this, we performed BMT in APPswe/PS1ΔE9 double transgenic mice using green fluorescent protein expressing wild type (wt) mice as marrow donors. We first examined retinas from control, non-transplanted, aged AD mice and found a two-fold increase in microglia compared with wt mice, prominent inner retinal Aβ and paired helical filament-tau, and decreased retinal ganglion cell layer neurons. BMT resulted in near complete replacement of host retinal microglia with BMT-derived cells and normalized total AD retinal microglia to non-transplanted wt levels. Aβ and paired helical filament-tau were reduced (61.0% and 44.1% respectively) in BMT-recipient AD mice, which had 20.8% more retinal ganglion cell layer neurons than non-transplanted AD controls. Interestingly, aged wt BMT recipients also had significantly more neurons (25.4%) compared with non-transplanted aged wt controls. Quantitation of retinal ganglion cell layer neurons in young mice confirmed age-related retinal degeneration was mitigated by BMT. We found increased MHC class II expression in BMT-derived microglia and decreased oxidative damage in retinal ganglion cell layer neurons. Thus, BMT is neuroprotective in age-related as well as AD-related retinal degeneration, and may be a result of alterations in innate immune function and oxidative stress in BMT recipient mice.

SWAP-70 Restricts Spontaneous Maturation of Dendritic Cells

Spontaneous maturation observed in dendritic cell (DC) cultures has been linked to their capacity to induce immune responses. Despite several recent studies, the mechanisms and signals triggering spontaneous maturation of DCs are largely unknown. We found that the absence of SWAP-70 causes spontaneous maturation of spleen- and bone marrow-derived DCs and, in vivo, of spleen-resident CD11c(+)CD11b(+)CD8α(-) DCs. Activation markers, cross-presentation of exogenous Ags, and activation of CD8(+) T cells are much increased in Swap-70(-/-) DCs. Spontaneous maturation of Swap-70(-/-) DCs depends on cell-cell contact and does not involve β-catenin signaling. SWAP-70 is known to regulate integrin activity. Signaling through the integrin CD11b (αM) subunit increases spontaneous maturation of wild-type (wt), but not of Swap-70(-/-) DCs. Signaling through the CD18 (β2) subunit decreases spontaneous maturation of wt and Swap-70(-/-) DCs. Constitutive activation of RhoA in Swap-70(-/-) DCs was determined as a key mechanism causing the increased spontaneous maturation. Inhibition of RhoA early, but not late, in the activation process reduces spontaneous maturation in Swap-70(-/-) DCs to wt levels. Inhibition of RhoA activation during CD11b integrin activation had a significant effect only in Swap-70(-/-) but not in wt DCs. Together, our data suggest that integrin-mediated spontaneous maturation of wt DCs does not depend on active RhoA, whereas the increase in spontaneous maturation of Swap-70(-/-) DCs is supported by integrin CD11b and by hyperactive RhoA. Thus, SWAP-70 deficiency reveals two pathways that contribute to spontaneous maturation of DCs.

The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization

C3a is a key complement activation fragment, yet its neutrophil-expressed receptor (C3aR) still has no clearly defined role. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to explore the role of C3aR in acute tissue injuries. C3aR deficiency worsened intestinal injury, which corresponded with increased numbers of tissue-infiltrating neutrophils. Circulating neutrophils were significantly increased in C3aR(-/-) mice after intestinal ischemia, and C3aR(-/-) mice also mobilized more circulating neutrophils after granulocyte colony-stimulating factor infusion compared with WT mice, indicating a specific role for C3aR in constraining neutrophil mobilization in response to intestinal injury. In support of this role, C3aR(-/-) mice reconstituted with WT bone marrow reversed IR pathology back to WT levels. Complement C5a receptor (C5aR) antagonism in C3aR(-/-) mice also rectified the worsened pathology after intestinal IR injury but had no effect on circulating neutrophils, highlighting the opposing roles of C3a and C5a in disease pathogenesis. Finally, we found that using a potent C3a agonist to activate C3aR in vivo reduced neutrophil mobilization and ameliorated intestinal IR pathology in WT, but not C3aR(-/-), mice. This study identifies a role for C3aR in regulating neutrophil mobilization after acute intestinal injury and highlights C3aR agonism as a potential treatment option for acute, neutrophil-driven pathologies.

Properdin is increased after allergen challenge of asthmatic patients and contributes to OVA-induced airway inflammation and AHR at the effector phase by enhancing C3a generation in mice (P1355)

Abstract The complement alternative pathway (AP) and anaphylatoxins have been implicated in allergic asthma, but the significance and mechanisms remain to be fully characterized. We hypothesized that factor properdin (fP), the only known positive regulator of AP contributes to allergen-induced airway inflammation. In human asthmatic patients, BALF collected after allergen challenge contained higher fP levels than before challenge. Mice were sensitized and challenged with OVA and examined for fP levels and airway inflammation. High levels of fP were detected in the BALF of OVA-sensitized and challenged but not naïve mice. Compared with WT mice, fP-deficient (fP-/-) mice had significantly reduced cell counts in BALF. fP-/- mice also had significantly attenuated AHR to methacholine stimulation. Antibody blocking of fP at both sensitization and challenge or at challenge phase alone, but not at sensitization phase alone, reduced airway inflammation. Conversely, intranasal reconstitution of fP to fP-/- mice at the challenge phase restored airway inflammation to WT levels. Notably, we found C3a levels in the BALF of OVA-challenged fP-/- mice to be significantly lower than in challenged WT mice, and intranasal co-administration of anti-C3a mAb with fP to fP-/- mice prevented restoration of airway inflammation. Collectively, our data suggest a detrimental role of fP in allergen-induced airway inflammation via C3a production and therapeutic targeting of fP may be beneficial in human asthma.

Role for subgenomic mRNA in host translation inhibition during Sindbis virus infection of mammalian cells

Sindbis virus subgenomic mRNA is efficiently translated in infected vertebrate cells whereas host translation is shut-off. Deletions in the 5′UTR of the subgenomic mRNA were made to investigate its role in viral gene expression. Deletion of nucleotides 1–10 and 11–20 caused a small plaque phenotype, reduced levels of subgenomic mRNA and structural proteins, and increased expression of nonstructural proteins. Whereas deletion 1–10 virus inhibited cellular protein synthesis, deletion 11–20 did so inefficiently. A large plaque revertant of deletion 11–20, possessing a duplication of the subgenomic promoter region, produced subgenomic mRNA at WT levels and restored inhibition of host protein synthesis. Further analysis of the mutant and revertant 5′UTR sequences showed the ability to shut-off host cell translation correlated with the efficiency of translation of subgenomic mRNA. We propose that the translational efficiency and quantity of the subgenomic mRNA play a role in inhibition of host cell translation.

Cross‐talk between central ACE/ACE2 and RhoA/ROCKII Pathways in Chronic Heart Failure.

A hallmark of chronic heart failure (CHF) is an overactivation of the renin‐angiotensin system (RAS) and an imbalance of angiotensin converting enzyme (ACE) and angiotensin converting enzyme 2 (ACE2). This leads to increased Angiotensin II (AngII) and contributes to sympathetic hyperactivity and oxidative stress. The RhoA/ROCKII pathway is activated by AngII and is hyperactive in CHF contributing to a pro‐oxidant state. We hypothesized that there is an interaction between central RhoA/ROCKII and RAS pathways in CHF. Preliminary studies from our lab indicated in a rabbit model of CHF, central Fasudil (a ROCKII inhibitor) normalized ACE and AngII type 1 Receptor (AT1R) expression to sham levels and increased ACE2 expression in the RVLM. To further investigate the connection between these two pathways, we induced CHF in both wild type C57Bl/6 (WT) mice and in transgenic human ACE2 overexpression mouse model selectively in the brain (synhACE2). In the brainstem of CHF WT mice, there was a significant (~10 fold, p=0.03) increase in pRhoA and a 10% increase in ROCKII protein compared to sham. However in CHF synhACE2 mice, pRhoA and ROCKII were blunted compared to CHF WT (pRhoA: 3.6 ±0.5 vs 10.6 ± 1.8, p=0.06; ROCKII: 0.3±0.1 vs 1.3±0.1, p<0.05). Silencing the mas Receptor by siRNA in the CHF synhACE2 mice returned pRhoA and ROCKII to CHF WT levels. Taken together, these data suggest a cross‐talk between central RAS and RhoA/ROCKII pathways in CHF. Increasing ACE2 and Angiotensin (1–7) may provide a strategy to limit the negative central effects of AngII.

Structural basis of human cannabinoid CB1 G protein‐coupled receptor activation

Transmembrane signal transduction is achieved by activation of G protein‐coupled receptors (GPCRs) like the human cannabinoid CB1 receptor. These receptors exist in an ensemble of conformations, each of which might bind to different signaling molecules. Mutating a single residue, Thr 210, to Ile in the third transmembrane (TM3) domain, makes it more active then WT, whereas mutating it to Ala makes it fully inactive. We used the Gensemble method to predict 3D structures of these receptors. We find conformational differences that explain the CB1 receptor's activation mechanism. These predictions were validated by designing double mutants that were expected to switch the inactive T210A back to WT levels of activation. The 2nd mutation for T210A is predicted to cause an important salt‐bridge between TMs 2 and 6 to break. GTPδS binding assays show a large increase in G protein‐coupling for the double mutants indicating increased activation. We docked known agonists to these receptors and then performed 50 ns of molecular dynamics. The inactive T210A receptor with the docked agonist WIN55212‐2 maintains two stable interhelical salt‐bridges. Similarly, the WT receptor maintains a salt‐bridge between TMs 3 and 6, which suggests that a G protein is necessary to stabilize the active conformation.This work was funded by gifts from PharmSelex/Accelerator and NIH grants (R01NS071112, R01NS073115, R01AI040567, and DA020763).

MSPCA with KDE thresholding to support QC in electrical motors production line

In this paper a Fault Detection and Isolation (FDI) procedure is applied for the defects detection and analysis of electrical motors at the end of production line in hood factories. The objective consists of developing a fast and robust methodology to detect defective motors and to identify defects for quality analysis on production line. Using a signal based FDI procedure, an end of a line bench system is designed, which is able to analyze the defects of produced motors. Multi-Scale Principal Component Analysis (MSPCA) is used for defect detection and a Kernel Density Estimation (KDE) algorithm is used for fault isolation on the PCA residual contributions. Also a method to choose the WT levels is adopted. MSPCA with KDE thresholding advantage is demonstrated by experimentations on test bench, using vibration measurements. Experiments show that the stochastic method used to compute thresholds on PCA residuals is robust and at the same time accurate.

Nox2-Dependent Redox Regulation of Calcium Influx in Dystrophic Skeletal Muscle

Duchenne Muscular Dystrophy (DMD) is an X-linked, muscle-wasting disease caused by deletions in the gene that encodes dystrophin, an integral muscle membrane protein that links the contractile proteins to the muscle membrane. Recent studies have suggested that increased Ca2+ influx into the muscle and increased production of free radicals (or reactive oxygen species, ROS) are essential for increased susceptibility of mdx muscle to damage. However, the source of the ROS, the Ca2+ channels affected, and the mechanism(s) of how mechanical stress results in altered regulation of these signaling pathways have yet to be determined. We hypothesis that NADPH oxidase (Nox2) drives excessive ROS production, increased Ca2+ influx, and muscle damage. Manganes (Mn2+) quench was used to assess the role of Nox2 dependent ROS on sarcolemmal Ca2+ influx in response to a physiological stretch and depolarization. Our results show that stretch-activated Ca2+ entry in mdx is significantly increased 4.2-fold (p<0.001) compared to WT, while K+-induced depolarization results in 20-fold (p<0.001) increase in Mn2+ quench in mdx skeletal muscle. Administration of either gp91ds-TAT, an inhibitor of Nox2, or reduced glutathione ethyl ester, a glutathione analogue, in mdx muscle reduces the Mn2+ quench rates back to the WT levels for both passive stretch and K+depolarization. Addition of BTP2, a calcium release activated calcium channel inhibitor, also significantly attenuates Mn2+ quench rates in mdx compared to WT. Our data supports a model in which Nox2 dependent redox modifications of stretch and depolarization activated Ca2+ channels leads to exuberant Ca2+ influx. Our results identify Nox2 as a potential therapeutic approach to minimize or delay the progressive DMD disease.

Mutated CaV2.1 Channels Dysregulate CASK/P2X3 Signaling in Mouse Trigeminal Sensory Neurons of R192Q Cacna1a Knock-in Mice

BackgroundATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1.ResultsKI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents.ConclusionsWe propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine.

462: Dysregulation of the peripheral circadian regulator Npas2 leads to altered fetal metabolic pathways in a knockout model

regulator Npas2 leads to altered fetal metabolic pathways in a knockout model Derek O’Neil, Hector Mendez-Figueroa, Toni-Ann Mistretta, Chunliu Su, Robert Lane, Kjersti Aagaard Baylor College of Medicine, Translational Biology and Molecular Medicine, Houston, TX, Baylor College of Medicine, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Houston, TX, Texas Children’s Hospital, Pathology, Houston, TX, University of Utah, Pediatrics, Salt Lake City, UT OBJECTIVE: In our non-human primate model of maternal high fat diet exposure, we previously identified the peripheral circadian domain gene Npas2 as a mediator of fetal metabolism which is epigenetically reprogrammed. As circadian activity and metabolic reprogramming is not well understood, we aimed to employ a murine gene knockout model to understand the role of Npas2 in regulating hepatic function throughout the lifespan of mice. STUDY DESIGN: Npas2 KO mice ( / ), Npas2 HET mice ( / ) and Npas2 WT mice ( / ) livers were harvested at day 2 (newborn) and at 25 weeks (adult) of life. Total RNA was extracted from hepatic tissue and converted to cDNA. Labeled cDNA was loaded onto the GeneChip® Mouse Exon 1.0 ST array (n 3/cohort). First pass analysis was performed using the Partek Genomics Suite software and GeneSpring GX 11. Genes meeting statistical and expression thresholds after controlling for multiple comparisons were then processed through Ingenuity Pathway Analysis, GSEA, and GeneSpring pathway analysis software. QPCR was performed on cDNA from newborn and adult Npas2 KO and wt mice to determine gene expression levels of genes pertaining to affected metabolic pathways. RESULTS: We identified 2891 genes that were dysregulated in KO mice compared to wt (F .05, fold-change 1.5 or -1.5). The primary dysregulated pathway in the Npas2 KO was the lipid and fatty acid metabolism pathway. Gene expression of Adra2a, Lpin1, Scd1, Sds, and Upp2 demonstrate significant (p .05) neonatal alterations that do not persist into adult life and revert to wt levels. Fgf1, Gpd1, Grb14, Gulo, and Hmgcs1 demonstrated significant (p .05) alterations in neonatal life that reversed expression levels in adult life. CONCLUSION: Npas2 primarily regulates the lipid and fatty acid metabolism pathway during early murine development. However, compensatory pathways are enacted in adult life, indicating a crucial role of Npas2 during fetal/neonatal development which had previously not been definitively described.

Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Herpesvirus entry mediator is a serotype specific determinant of pathogenesis in ocular herpes

Infection with herpes simplex virus type 1 (HSV-1) and HSV-2 is initiated by viral glycoprotein D (gD) binding to a receptor on the host cell. Two receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate entry in murine models of HSV-1 and HSV-2. HVEM is dispensable for HSV-2 infection of the vagina and brain, but is required for WT pathogenesis of HSV-1 infection of the cornea. By challenging WT and HVEM KO mice with multiple strains of HSV-1 and HSV-2, we demonstrate that without HVEM, all HSV-1 strains tested do not replicate well in the cornea and infection does not result in severe symptoms, as observed in WT mice. In contrast, all HSV-2 strains tested had no requirement for HVEM to replicate to WT levels in the cornea and still cause severe disease. These findings imply that HSV-2 does not require HVEM to cause disease regardless of route of entry, but HVEM must be present for HSV-1 to cause full pathogenesis in the eye. These findings uncover a unique role for HVEM in mediating HSV-1 infection in an area innervated by the trigeminal ganglion and may explain why the presence of HVEM can lead to severe inflammation in the cornea. Thus, the dependence on HVEM is a dividing point between HSV-1 and HSV-2 that evolved to infect areas innervated by different sensory ganglia.

Rescue of the HSC Maintenance Defects in Ku70-Deficient Mice by Overexpression of Bcl2 Reveals a Novel Role of Bcl2 in HSC

AbstractAbstract 1235Ku70 is a key component of the non-homologous end joining (NHEJ) pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. We find that HSC from Ku70-deficient mice are severely impaired in maintenance, defective in self-renewal, competitive repopulation and BM hematopoietic niche occupancy. HSCs from Ku70-deficient mice are not prone to spontaneous apoptosis, however, they display more active proliferation, and fewer cells in a quiescent state than HSC obtained from WT mice. Further, gene expression profiling showed that multiple HSC quiescence- related genes such as c-MPL and p57 were significantly downregulated in Ku70-deficient HSCs. These data suggested that loss of quiescence results in the dramatic defect in the maintenance of Ku70-deficient HSC.Bcl2 plays important roles in both anti-apoptosis and anti-proliferation, while the anti-apoptosis function of Bcl2 has been extensively studied and well established in various settings, including HSCs, the anti-proliferation function of Bcl2 in HSC has yet to be investigated. To exam whether overexpression of Bcl2 can rescue the HSC defect in Ku70-deficient mice, H2K-Bcl2 transgenic/Ku70-deficient double mutant mice were generated. Though overexpression of Bcl2 does not rescue the SCID phenotype in Ku70-deficient mice, overexpression of Bcl2 in Ku70-deficient HSCs almost completely rescued the impaired HSC quiescence, repopulation and BM hematopoietic niche occupancy capacities. At the transcriptional level, overexpression of Bcl2 restored the expression of c-MPL and p57 comparable to WT levels. However, additional deletion of Bax, a pro-apoptosis protein in the Bcl2 family, in Ku70-deficient mice could not rescue the HSC repopulation defect, suggesting that apoptosis is not the main mediator for the impaired HSC maintenance in Ku70-deficient mice.Together, our data indicate that the HSC maintenance defect of Ku70-deficient mice is due to the loss of HSC quiescence populations, whereas overexpression of Bcl2 rescues the HSC defect in Ku70-deficient mice by restoration of quiescence. Our study uncovers a novel role of Bcl2 in hematopoietic stem cell quiescence regulation. Disclosures:No relevant conflicts of interest to declare.

Allele-Specific Gene Silencing in Two Mouse Models of Autosomal Dominant Skeletal Myopathy

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4–6 month old heterozygous Ryr1Y524S/+ (YS/+) and Ryr1I4895T/+ (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca2+ release that occurred in the absence of a change in the magnitude of electrically-evoked Ca2+ release. Compared to the difference between the caffeine sensitivity of Ca2+ release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC50: 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC50: 2.8 mM) and 4 week (EC50: 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca2+ observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways(1,2). However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time(3-5). Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein(6). Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured(7-9). The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Heterologous Expression of sahH Reveals That Biofilm Formation Is Autoinducer-2-independent in Streptococcus sanguinis but Is Associated with an Intact Activated Methionine Cycle

Numerous studies have claimed deleterious effects of LuxS mutation on many bacterial phenotypes, including bacterial biofilm formation. Genetic complementation mostly restored the observed mutant phenotypes to WT levels, leading to the postulation that quorum sensing via a family of molecules generically termed autoinducer-2 (AI-2) is essential for many phenotypes. Because LuxS mutation has dual effects, this hypothesis needs to be investigated into the details for each bacterial species. In this study we used S. sanguinis SK36 as a model biofilm bacterium and employed physiological characterization and transcriptome approaches on WT and luxS-deficient strains, in combination with chemical, luxS, and sahH complementation experiments. SahH enables a direct conversion of SAH to homocysteine and thereby restores the activated methionine cycle in a luxS-negative background without formation of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione. With this strategy we were able to dissect the individual contribution of LuxS and AI-2 activity in detail. Our data revealed that S. sanguinis biofilm formation is independent from AI-2 substance pools and is rather supported by an intact activated methyl cycle. Of 216 differentially transcribed genes in the luxS mutant, 209 were restored by complementation with a gene encoding the S-adenosylhomocysteine hydrolase. Only nine genes, mainly involved in natural competence, were directly affected by the AI-2 quorum-sensing substance pool. Cumulatively, this suggested that biofilm formation in S. sanguinis is not under control of AI-2. Our study suggests that previously evaluated LuxS mutants in other species need to be revisited to resolve the precise contribution of AI-2 substance pools and the methionine pathways.