RORA regulates neutrophil migration and activation in zebrafish

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting RORα, but not the closely related RORγ, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Rorα or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, RORα regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR- 99 and RORα in regulating neutrophil migration and anti-microbial defense.

E2F4-Based Gene Therapy Mitigates the Phenotype of the Alzheimer’s Disease Mouse Model 5xFAD

After decades of unfruitful work, no effective therapies are available for Alzheimer’s disease (AD), likely due to its complex etiology that requires a multifactorial therapeutic approach. We have recently shown using transgenic mice that E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, and controls gene networks affected in AD, represents a good candidate for a multifactorial targeting of AD. Here we show that the expression of a dominant negative form of human E2F4 (hE2F4DN), unable to become phosphorylated in a Thr-conserved motif known to modulate E2F4 activity, is an effective and safe AD multifactorial therapeutic agent. Neuronal expression of hE2F4DN in homozygous 5xFAD (h5xFAD) mice after systemic administration of an AAV.PHP.B-hSyn1.hE2F4DN vector reduced the production and accumulation of Aβ in the hippocampus, attenuated reactive astrocytosis and microgliosis, abolished neuronal tetraploidization, and prevented cognitive impairment evaluated by Y-maze and Morris water maze, without triggering side effects. This treatment also reversed other alterations observed in h5xFAD mice such as paw-clasping behavior and body weight loss. Our results indicate that E2F4DN-based gene therapy is a promising therapeutic approach against AD.

Functional Verification of Novel ELMO1 Variants By Live Imaging in Zebrafish

ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G) could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Acts as a dominant-negative form, p.R354X (c.1060C>T) which failed to rescue the elmo1 mutant and inhibited neutrophil movement in siblings. Based on those results, we identified that zebrafish elmo1 played conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo. Disclosures No relevant conflicts of interest to declare.

Unique functions for Notch4 in murine embryonic lymphangiogenesis

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

HIV-1 Vif protein is stabilized by AKT-mediated phosphorylation to enhance APOBEC3G degradation

HIV-1 virus has to counter anti-viral restriction factors for its successful replication after its entry in the cell. The host-pathogen dynamics operate as soon as HIV-1 interacts with the cell. HIV-1 Vif has been known for its role in degradation of APOBEC3G; a cytosine deaminase which leads to hyper mutations in the viral DNA leading to aberrant viral replication. The cellular proteins regulating the intracellular HIV-1 Vif protein levels can have profound impact on HIV-1 pathogenesis. MDM2 is known to induce degradation of Vif with subsequent effects on APOBEC3G. Here, we have identified AKT/PKB as one of the crucial regulators of HIV-1 Vif protein. The rationale for selecting Vif as a target substrate for AKT was the presence of RMRINT motif in it, which is similar to the AKT phosphorylation motif RxRxxS/T. Immunoprecipitation assay and Kinase assay revealed that AKT and Vif interact strongly with each other and Vif is phosphorylated at T20 position by AKT. This phosphorylation stabilizes HIV-1 Vif while Vif mutant T20A degrades faster. Moreover, use of dominant negative form of AKT (KD-AKT) and AKT inhibitors were found to destabilise Vif and increase its K48-ubiquitination profile. The consequences of this AKT-Vif interplay were also validated on APOBEC3G degradation, a target of Vif. AKT inhibition was found to restore APOBEC3G levels. This process can be interpreted as a strategy used by virus to prevent MDM2 mediated Vif degradation; AKT stabilises Mdm2, which then targets Vif for degradation but at the same time AKT stabilises Vif by phosphorylating it. Thus, AKT mediated stabilization of Vif might compensate for its degradation by MDM2. This study can have significant implications as HIV-1 Tat protein and growth factors like insulin activate PI3-K/AKT Kinase pathway and can potentially affect Vif and APOBEC3G protein levels and hence HIV-1 pathogenesis.

Cellular senescence promotes endothelial activation through epigenetic alteration, and consequently accelerates atherosclerosis

Senescent vascular cells are detected in atherosclerotic lesion, and its involvement in the development of atherosclerosis has been revealed; however, whether and the mechanism by which endothelial cell (EC) senescence is causally implicated in atherosclerosis remains unclear. We here investigate a role of EC senescence in atherosclerosis by utilizing EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 under the control of the Tie2 or vascular endothelial cadherin promoter. EC-specific progeria accelerated atherosclerosis in mice with target deletion of ApoE. Mechanistically, senescent ECs were markedly sensitive for inflammation-mediated VCAM-1 induction, leading to enhanced monocyte adhesion. Inhibition of NF-κB signaling abolished the enhanced inflammatory responses in senescent ECs, while NF-κB nuclear translocation in response to TNF-α were similar between young and senescent ECs. We found a higher association of VCAM-1 gene with active histone H3 trimethylated on lysine 4, leading to increased NF-κB accessibility in senescent ECs. Our data revealed that EC cellular senescence causes endothelial hyper-inflammability through epigenetic alteration, which consequently accelerates atherosclerosis. Therefore, EC senescence is a promising therapeutic target for the prevention and/or treatment of atherosclerotic disease in elderly population.

Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

EPEN-18. CROSS-SPECIES GENOMICS IDENTIFIES GLI2 AS AN ONCOGENE OF C11orf95 FUSION-POSITIVE SUPRATENTORIAL EPENDYMOMA

The majority of supratentorial ependymomas (ST-EPN) are driven by fusions between RELA and a zinc finger containing gene, C11orf95. Apart from fusions to the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs is unclear. Aiming at refining the molecular classification of ST-EPNs, we analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 617 ST-EPNs. Unsupervised clustering analysis of DNA methylation data revealed four distinct clusters that formed in addition to the known molecular groups ST-EPN-RELA and –YAP1. Tumors within these additional clusters were characterized by fusions of C11orf95 to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, suggesting a general role of C11orf95 in tumorigenesis of ST-EPN. Transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology. All fusion genes were sufficient to drive malignant transformation in the cerebral cortex of mice and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of C11orf95, were essential to exert tumorigenesis. When exploring genes commonly upregulated in C11orf95 fusion-expressing tumors of human and murine origin, the Sonic Hedgehog effector gene Gli2 was identified as a promising downstream target. Subsequent co-expression of C11orf95:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. We thus propose GLI2 as a potential therapeutic downstream target of C11orf95 fusion-dependent oncogenic signaling in ST-EPN.

Modulation of flight and feeding behaviours requires presynaptic IP3Rs in dopaminergic neurons

Innate behaviours, although robust and hard wired, rely on modulation of neuronal circuits, for eliciting an appropriate response according to internal states and external cues. Drosophila flight is one such innate behaviour that is modulated by intracellular calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs). Cellular mechanism(s) by which IP3Rs modulate neuronal function for specific behaviours remain speculative, in vertebrates and invertebrates. To address this, we generated an inducible dominant negative form of the IP3R (IP3RDN). Flies with neuronal expression of IP3RDN exhibit flight deficits. Expression of IP3RDN helped identify key flight-modulating dopaminergic neurons with axonal projections in the mushroom body. Flies with attenuated IP3Rs in these presynaptic dopaminergic neurons exhibit shortened flight bouts and a disinterest in seeking food, accompanied by reduced excitability and dopamine release upon cholinergic stimulation. Our findings suggest that the same neural circuit modulates the drive for food search and for undertaking longer flight bouts.

Hypoglycemia sensing neurons of the ventromedial hypothalamus require AMPK-induced Txn2 expression but are dispensable for physiological counterregulation

The ventromedial nucleus of the hypothalamus (VMN) is involved in the counterregulatory response to hypoglycemia. VMN neurons activated by hypoglycemia (glucose inhibited, GI neurons) have been assumed to play a critical, although untested role in this response. Here, we show that expression of a dominant negative form of AMP-activated protein kinase (AMPK) or inactivation of AMPK <i>α1</i> and <i>α2</i> subunit genes in Sf1 neurons of the VMN selectively suppressed GI neuron activity. We found that <i>Txn2</i>, encoding a mitochondrial redox enzyme, was strongly down-regulated in the absence of AMPK activity and that reexpression of <i>Txn2</i> in Sf1 neurons restored GI neuron activity. In cell lines, <i>Txn2</i> was required to limit glucopenia-induced ROS production. In physiological studies, absence of GI neuron activity following AMPK suppression in the VMN had no impact on the counterregulatory hormone response to hypoglycemia nor on feeding. Thus, AMPK is required for GI neuron activity by controlling the expression of the anti-oxidant enzyme Txn2. However, the glucose sensing capacity of VMN GI neurons is not required for the normal counterregulatory response to hypoglycemia. Instead, it may represent a fail-safe system in case of impaired hypoglycemia sensing by peripherally located gluco-detection systems that are connected to the VMN.