A retroviral link to vertebrate myelination through retrotransposon-RNA-mediated control of myelin gene expression.

Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.

Monitoring recovery after CNS demyelination, a novel tool to de-risk pro-remyelinating strategies.

In multiple sclerosis, while remarkable progress has been accomplished to control the inflammatory component of the disease, repair of demyelinated lesions is still an unmet need. Despite encouraging results generated in experimental models, several candidates favouring or promoting remyelination have not reached the expected outcomes in clinical trials. One possible reason for these failures is that, in most cases, during preclinical testing, efficacy was evaluated on histology only, while functional recovery had not been assessed. We have generated a Xenopus laevis transgenic model Tg(mbp:GFP-NTR) of conditional demyelination in which spontaneous remyelination can be accelerated using candidate molecules. Xenopus laevis is a classic model for in vivo studies of myelination because tadpoles are translucent. We reasoned that demyelination should translate into loss of sensorimotor functions followed by behavioural recovery upon remyelination. To this end, we measured the swimming speed and distance travelled before and after demyelination and during the ongoing spontaneous remyelination and have developed a functional assay based on the visual avoidance of a virtual collision. Here we show that alteration of these functional and clinical performances correlated well with the level of demyelination and that histological remyelination, assayed by counting in vivo the number of myelinating oligodendrocytes in the optic nerve, translated in clinical-functional recovery. This method was further validated in tadpoles treated with pro-remyelinating agents (clemastine, siponimod) showing that increased remyelination in the optic nerve was associated with functional improvement. Our data illustrate the potential interest of correlating histopathological parameters and functional-clinical parameters to screen molecules promoting remyelination in a simple in vivo model of conditional demyelination.

Immunoblot-based assays for assessing autophagy in the turquoise killifish Nothobranchius furzeri.

Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.

Physical models of infant mortality: implications for defects in biological systems.

Reliability engineering concerned with failure of technical inanimate systems usually uses the vocabulary and notions of human mortality, e.g., infant mortality vs. senescence mortality. Yet, few data are available to support such a parallel description. Here, we focus on early-stage (infant) mortality for two inanimate systems, incandescent light bulbs and soap films, and show the parallel description is clearly valid. Theoretical considerations of the thermo-electrical properties of electrical conductors allow us to link bulb failure to inherent mechanical defects. We then demonstrate the converse, that is, knowing the failure rate for an ensemble of light bulbs, it is possible to deduce the distribution of defects in wire thickness in the ensemble. Using measurements of lifetimes for soap films, we show how this methodology links failure rate to geometry of the system; in the case presented, this is the length of the tube containing the films. In a similar manner, for a third example, the time-dependent death rate due to congenital aortic valve stenosis is related to the distribution of degrees of severity of this condition, as a function of time. The results not only validate clearly the parallel description noted above, but also point firmly to application of the methodology to humans, with the consequent ability to gain more insight into the role of abnormalities in infant mortality.

Mosaic Heterochrony in Neural Progenitors Sustains Accelerated Brain Growth and Neurogenesis in the Juvenile Killifish N. furzeri.

Although developmental mechanisms driving an increase in brain size during vertebrate evolution are actively studied, we know less about evolutionary strategies allowing accelerated brain growth. In zebrafish and other vertebrates studied to date, apical radial glia (RG) constitute the primary neurogenic progenitor population throughout life [1]; thus, RG activity is a determining factor of growth speed. Here, we ask whether enhanced RG activity is the mechanism selected to drive explosive growth, in adaptation to an ephemeral habitat. In post-hatching larvae of the turquoise killifish, which display drastic developmental acceleration, we show that the dorsal telencephalon (pallium) grows three times faster than in zebrafish. Rather than resulting from enhanced RG activity, we demonstrate that pallial growth is the product of a second type of progenitors (that we term NGPs for non-glial progenitors) that actively sustains neurogenesis and germinal zone self-renewal. Intriguingly, NGPs appear to retain, at larval stages, features of early embryonic progenitors. In parallel, RGs enter premature quiescence and express markers of astroglial function. Altogether, we propose that mosaic heterochrony within the neural progenitor population might permit rapid pallial growth by safeguarding both continued neurogenesis and astroglial function.

A Semi-Automatic Dispenser for Solid and Liquid Food in Aquatic Facilities.

We present a novel, low-footprint and low-cost semi-automatic system for delivering solid and liquid food to zebrafish, and more generally to aquatic animals raised in racks of tanks. It is composed of a portable main module equipped with a contactless reader that adjusts the quantity to deliver for each tank, and either a solid food module or a liquid food module. Solid food comprises virtually any kind of dry powder or grains below 2 mm in diameter, and, for liquid-mediated food, brine shrimps (Artemia salina) and rotifers (Rotifera) have been successfully tested. Real-world testing, feedback, and validation have been performed in a zebrafish facility for several months. In comparison with manual feeding this system mitigates the appearance of musculoskeletal disorders among regularly-feeding staff, and let operators observe the animals' behavior instead of being focused on quantities to deliver. We also tested the accuracy of both humans and our dispenser and found that the semi-automatic system is much more reliable, with respectively 7-fold and 84-fold drops in standard deviation for solid and liquid food.

Conditional Demyelination and Remyelination in a Transgenic Xenopus laevis.

Multiple sclerosis (MS) is the first cause of acquired disability progression in the young adult. Pathology of MS associates inflammation, demyelination, and neurodegeneration. The development of immunotherapies, by reducing the relapse rate, has profoundly impacted short-term prognosis and patients' quality of life. These anti-inflammatory medications, however, have not proven to be sufficient to prevent long-term disability progression, resulting from axonal transection and neuronal damage, consequences of prolonged demyelination. Promoting remyelination is therefore a key therapeutic strategy to limit handicap progression, and represent the major therapeutic challenge in MS. Here we present a simple, rapid, and cost-effective experimental model developed in Xenopus laevis to screen in vivo molecules promoting remyelination.

The Xenopus tadpole: An in vivo model to screen drugs favoring remyelination.

BACKGROUND: In multiple sclerosis, development of screening tools for remyelination-promoting molecules is timely. OBJECTIVE: A Xenopus transgenic line allowing conditional ablation of myelinating oligodendrocytes has been adapted for in vivo screening of remyelination-favoring molecules. METHODS: In this transgenic, the green fluorescent protein reporter is fused to E. coli nitroreductase and expressed specifically in myelinating oligodendrocytes. Nitroreductase converts the innocuous pro-drug metronidazole to a cytotoxin. Spontaneous remyelination occurs after metronidazole-induced demyelinating responses. As tadpoles are transparent, these events can be monitored in vivo and quantified. At the end of metronidazole-induced demyelination, tadpoles were screened in water containing the compounds tested. After 72 h, remyelination was assayed by counting numbers of oligodendrocytes per optic nerve. RESULTS: Among a battery of molecules tested, siponimod, a dual agonist of sphingosine-1-phosphate receptor 1 and 5, was among the most efficient favoring remyelination. Crispr/cas9 gene editing showed that the promyelinating effect of siponimod involves the sphingosine-1-phosphate receptor 5. CONCLUSION: This Xenopus transgenic line constitutes a simple in vivo screening platform for myelin repair therapeutics. We validated several known promyelinating compounds and demonstrated that the strong remyelinating efficacy of siponimod implicates the sphingosine-1-phosphate receptor 5.

Remyelination by Resident Oligodendrocyte Precursor Cells in a Xenopus laevis Inducible Model of Demyelination.

We have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of the myelin basic protein regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by the green fluorescent protein reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Ablation of oligodendrocytes by MTZ treatment of the tadpole induced demyelination, and here we show that myelin debris are subsequently eliminated by microglial cells. After cessation of MTZ treatment, remyelination proceeded spontaneously. We questioned the origin of remyelinating cells. Our data suggest that Sox10+ oligodendrocyte precursor cells (OPCs), which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination, and this required only minimal (if any) cell division of OPCs. © 2015 S. Karger AG, Basel.

Opposing roles for Hoxa2 and Hoxb2 in hindbrain oligodendrocyte patterning.

Oligodendrocytes are the myelin-forming cells of the vertebrate CNS. Little is known about the molecular control of region-specific oligodendrocyte development. Here, we show that oligodendrogenesis in the mouse rostral hindbrain, which is organized in a metameric series of rhombomere-derived (rd) territories, follows a rhombomere-specific pattern, with extensive production of oligodendrocytes in the pontine territory (r4d) and delayed and reduced oligodendrocyte production in the prepontine region (r2d, r3d). We demonstrate that segmental organization of oligodendrocytes is controlled by Hox genes, namely Hoxa2 and Hoxb2. Specifically, Hoxa2 loss of function induced a dorsoventral enlargement of the Olig2/Nkx2.2-expressing oligodendrocyte progenitor domain, whereas conditional Hoxa2 overexpression in the Olig2(+) domain inhibited oligodendrogenesis throughout the brain. In contrast, Hoxb2 deletion resulted in a reduction of the pontine oligodendrogenic domain. Compound Hoxa2(-/-)/Hoxb2(-/-) mutant mice displayed the phenotype of Hoxb2(-/-) mutants in territories coexpressing Hoxa2 and Hoxb2 (rd3, rd4), indicating that Hoxb2 antagonizes Hoxa2 during rostral hindbrain oligodendrogenesis. This study provides the first in vivo evidence that Hox genes determine oligodendrocyte regional identity in the mammalian brain.

Live imaging of targeted cell ablation in Xenopus: a new model to study demyelination and repair.

Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the CNS. In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair.

Effect of a short-term HAART on SIV load in macaque tissues is dependent on time of initiation and antiviral diffusion.

BACKGROUND: HIV reservoirs are rapidly established after infection, and the effect of HAART initiated very early during acute infection on HIV reservoirs remains poorly documented, particularly in tissue known to actively replicate the virus. In this context, we used the model of experimental infection of macaques with pathogenic SIV to assess in different tissues: (i) the effect of a short term HAART initiated at different stages during acute infection on viral dissemination and replication, and (ii) the local concentration of antiviral drugs. RESULTS: Here, we show that early treatment with AZT/3TC/IDV initiated either within 4 hours after intravenous infection of macaques with SIVmac251 (as a post exposure prophylaxis) or before viremia peak (7 days post-infection [pi]), had a strong impact on SIV production and dissemination in all tissues but did not prevent infection. When treatment was initiated after the viremia peak (14 days pi) or during early chronic infection (150 days pi), significant viral replication persists in the peripheral lymph nodes and the spleen of treated macaques despite a strong effect of treatment on viremia and gut associated lymphoid tissues. In these animals, the level of virus persistence in tissues was inversely correlated with local concentrations of 3TC: high concentrations of 3TC were measured in the gut whereas low concentrations were observed in the secondary lymphoid tissues. IDV, like 3TC, showed much higher concentration in the colon than in the spleen. AZT concentration was below the quantification threshold in all tissues studied. CONCLUSIONS: Our results suggest that limited antiviral drug diffusion in secondary lymphoid tissues may allow persistent viral replication in these tissues and could represent an obstacle to HIV prevention and eradication.

Infection of macaques after vaginal exposure to cell-associated simian immunodeficiency virus.

BACKGROUND: The contribution of infected semen cells to sexual transmission of human immunodeficiency virus (HIV) is still debated. We addressed this issue in the model of experimental infection of macaques with simian immunodeficiency virus (SIV). METHODS: Frozen stocks of cells obtained from the spleen of macaques at the peak of SIVmac251 viremia were prepared. After being thawed and washed, cells were deposited at different concentrations in the vaginas of adult macaques treated with medroxyprogesterone acetate (Depo-Provera). To unravel mechanisms of infection, stock cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) were inoculated intravaginally. Follow-up testing of samples from the mucosa and different lymphoid tissues obtained 21 and 45 h later was performed by flow cytometry, immunohistochemical analysis, and in situ hybridization. RESULTS: Systemic and persistent infection was achieved after vaginal exposure of macaques to SIV-infected cells. The dose needed to infect 50% of females was 6.69 x 10(5)+/-2.08 x 10(5) viral DNA copies. At days 1 and 2 after exposure to cell-associated SIV labeled with CFSE, SIV-positive cells were detected in proximal and distal lymphoid tissues. CONCLUSIONS: Infection with SIV after exposure of vaginal and cervical mucosa to cell-associated virus represents a new mechanism of sexual transmission of HIV and SIV that may have significant impacts in the development of preventive approaches like microbicides.

Antiretroviral treatment start-time during primary SIV(mac) infection in macaques exerts a different impact on early viral replication and dissemination.

BACKGROUND: The time of infection is rarely known in human cases; thus, the effects of delaying the initiation of antiretroviral therapy (ART) on the peripheral viral load and the establishment of viral reservoirs are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Six groups of macaques, infected intravenously with SIV(mac251), were given placebo or antiretroviral therapy to explore reservoir establishment; macaques were treated for 2 weeks, with treatment starting 4 hours, 7 or 14 days after infection. Viral replication and dissemination were measured in the gut (rectum), in the lung and in blood and lymphoid tissues (peripheral lymph nodes), by quantifying viral RNA, DNA and 2LTR circles. We used immunohistochemistry (CD4 and CD68) to assess the impact of these treatments on the relative amount of virus target cells in tissue. Treatment that was started 4 hours post-infection (pi) decreased viral replication and dissemination in blood and tissue samples, which were assessed on day 14 (RNA/DNA/2LTR circles). The virus remained detectable and lymphoid tissues were activated in LN and the gut in both placebo- and ART-treated animals. Viral RNA in plasma continued to be lower in macaques treated seven days after infection; however, this was not the case for viral DNA in peripheral blood mononuclear cells. There was a small but significant difference in RNA and DNA levels in tissues between placebo- and ART-treated animals on day 21. When started 14 days after infection, treatment resulted in a limited decrease in the plasma viral load. CONCLUSIONS: Treatment that was started 4 hours after infection significantly reduced viral replication and dissemination. When started 7 days after infection, it was of slight virological benefit in peripheral blood and in tissues, and treatment was even less effective if started 14 days pi. These data favor starting ART no longer than one week after intravenous SIV(mac251) exposure.

Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.

Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques.

BACKGROUND: Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. RESULTS: The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. CONCLUSION: We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.

Human immunodeficiency virus type 1 KK26-27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import.

We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26-27 in MA reported nuclear localization signal (NLS)-1 impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.

Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation.

We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.

Persistent infection with primate foamy virus type 1 increases human immunodeficiency virus type 1 cell binding via a Bet-independent mechanism.

We report that human T cells persistently infected with primate foamy virus type 1 (PFV-1) display an increased capacity to bind human immunodeficiency virus type 1 (HIV-1), resulting in increased cell permissiveness to HIV-1 infection and enhanced cell-to-cell virus transmission. This phenomenon is independent of HIV-1 receptor, CD4, and it is not related to PFV-1 Bet protein expression. Increased virus attachment is specifically inhibited by heparin, indicating that it should be mediated by interactions with heparan sulfate glycosaminoglycans expressed on the target cells. Given that both viruses infect similar animal species, the issue of whether coinfection with primate foamy viruses interferes with the natural course of lentivirus infections in nonhuman primates should be considered.

CD40-activated macrophages become highly susceptible to X4 strains of human immunodeficiency virus type 1.

Activating cells of the immune system may stimulate human immunodeficiency virus type 1 (HIV-1) replication and contribute to select pathogenic variants in vivo. Here, we examined the possible effect of a major pathway of immune activation, CD40 interaction with its ligand (CD40L), on the susceptibility of monocyte-derived macrophages (MDMs) to various HIV-1 strains. Stimulation of MDMs with CD40L led to reduced replication of R5 HIV-1(Ba-L), whereas this strongly enhanced the replication of X4 HIV-1(Lai) as well as of X4 primary isolates, and this was associated with strong cytopathic effects. The replication of X4 strains was inhibited by stromal cell-derived factor 1, an indication of the restricted usage of CXCR4 as virus coreceptor in this case. CD40L induced the activation of mitogen-activated protein kinases ERK1/ERK2 and stimulated MDMs to secrete RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, interleukin 6 (IL-6), IL-1beta, and tumor necrosis factor alpha. From this data, it may be hypothesized that activated macrophages represent a favorable environment for the replication of classically T lymphocyte-tropic X4 variants and, thus, may contribute significantly to the selection of such variants at late stages of clinical HIV-1 infection.