Expression Map of the Human Exome in CD34+ Cells and Blood Cells: Increased Alternative Splicing in Cell Motility and Immune Response Genes

International audienceBACKGROUND: Hematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC) residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS) to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells. PRINCIPAL FINDINGS: Using Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php). At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi), a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes. CONCLUSION: The relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function

Feasibility of β-D-glucuronidase gene transfer in the CNS of mucopolysaccharidosis type VII affected dogs.

La mucopolysaccharidose de type VII (MPS VII) est une maladie génétique de surcharge lysosomale provoquée par une déficience en β-D-glucuronidase (β-glu). β-glu est impliquée dans la cascade de dégradation et de recyclage des glycosaminoglycannes (GAGs). Sa déficience provoque une accumulation vésiculaire de GAGs non dégradés engendrant in fine la mort cellulaire. Notre but est de développer et de tester la pertinence des vecteurs adénoviraux canins helper-dépendant (HD-CAV-2) pour traiter la neurodégénération provoquée par la MPS VII dans le cadre d'une thérapie génique dans le SNC de chien. Parce que les vecteurs CAV-2 transduisent préférentiellement les neurones et qu'ils sont transportés de manière rétrograde le long des axones, leurs distributions dans tout le SNC permettraient de délivrer largement la β-glu dans tout le parenchyme cérébral. Nous avons alors étudié la sureté, la durée d'expression, l'efficacité et la possible réversion du phénotype après injections dans le SNC de chiens MPS VII d'un HD-CAV-2 exprimant le gène humain de la GUSB : HD-RIGIE. Des études préliminaires ont montré la faisabilité du transfert des vecteurs HD-CAV-2 dans le SNC, qu'ils induisaient une réponse immunitaire minimale et qu'ils transduisaient préférentiellement, efficacement et largement les neurones.Nous avons produit un HD-RIGIE de qualité pour les injections intracérébrales et nous avons analysé son efficacité sur l'accumulation de GAGs non dégradés. Les injections de HD-RIGIE montrent une augmentation générale de l'activité β-glu dans tout le SNC des chiens MPS VII (sites d'injections et structures éloignées comme le cortex) et ce 1 mois ou 4 mois après les injections. L'analyse de la GFP confirme une distribution globale de HD-RIGIE dans le SNC d'animaux de grande taille. De plus, grâce aux propriétés intrinsèques de la β-glu (transport rétrograde et phénomène de libération/recapture), nous avons observé une diminution générale de l'accumulation vésiculaire neuronale des GAGs non dégradés dans tout le parenchyme cérébral. D'autre part grâce à la stratégie d'isolement et de non vaccination des chiens MPS VII, nous n'observons ni de réponse immunitaire humorale, ni d'aggravation de l'inflammation du parenchyme.Mucopolysaccharidosis type VII (MPS VII) is a rare inherited lysosomal storage disease caused by β-D-glucuronidase (β-glu) deficiency. β-glu is involved in the physiological turnover of glycosaminoglycans (GAGs). Its deficiency causes accumulation of undegraded GAGs inside vesicles leading to cell death. Our goals are to develop and test the clinical relevance of helper-dependant (HD) canine adenovirus type 2 (CAV-2) vectors to treat neural degeneration caused by MPS VII in a dog model. Because CAV-2 targets preferentially neurons and traffics via axons, the distribution of the transgene throughout the CNS will allow widespread delivery of the missing lysosomal enzyme in these disorders with a minimum number of injections. We tested a HD-CAV-2 vector expressing the human GUS gene in the canine model of MPS VII for their safety, efficacy, duration of expression and possible reversion of the MPS VII induced symptoms.A previous study based on HD-CAV-EGFP vector injections in MPS VII-/- and healthy dogs showed that we are now able to inject HD-CAV-2 in the dog brain, have a minimal induction of the immune response, an efficient transduction of the neurons and an efficient biodistribution of transduced cells. After the production of a suitable vector (HD-RIGIE) for injections in the CNS of MPS VII dogs we analysed its efficiency on GAGS storage in neurons.Injections of HD-RIGIE showed after 1 month or 4 months post injections a widespread increase in general level of β-glu activity, in the sites of injections and in distant areas such as cortex. Analysis of GFP, also permit to observe a widespread biodistribution of the vector. Because of β-glu property of cross-correction we observed a global decreased in GAGs storage in the entire MPS VII brains. Finally, the dogs did not present humoral immune response and no aggravation of inflammatio

Etude de l'efficacité du transfert du gène de la beta-D-glucuronidase dans le SNC de chiens atteints de mucopolysaccharidose de type VII.

La mucopolysaccharidose de type VII (MPS VII) est une maladie génétique de surcharge lysosomale provoquée par une déficience en b-D-glucuronidase (b-glu). b-glu est impliquée dans la cascade de dégradation et de recyclage des glycosaminoglycannes (GAGs). Sa déficience provoque une accumulation vésiculaire de GAGs non dégradés engendrant in fine la mort cellulaire. Notre but est de développer et de tester la pertinence des vecteurs adénoviraux canins helper-dépendant (HD-CAV-2) pour traiter la neurodégénération provoquée par la MPS VII dans le cadre d'une thérapie génique dans le SNC de chien. Parce que les vecteurs CAV-2 transduisent préférentiellement les neurones et qu'ils sont transportés de manière rétrograde le long des axones, leurs distributions dans tout le SNC permettraient de délivrer largement la b-glu dans tout le parenchyme cérébral. Nous avons alors étudié la sureté, la durée d'expression, l'efficacité et la possible réversion du phénotype après injections dans le SNC de chiens MPS VII d'un HD-CAV-2 exprimant le gène humain de la GUSB : HD-RIGIE. Des études préliminaires ont montré la faisabilité du transfert des vecteurs HD-CAV-2 dans le SNC, qu'ils induisaient une réponse immunitaire minimale et qu'ils transduisaient préférentiellement, efficacement et largement les neurones.Nous avons produit un HD-RIGIE de qualité pour les injections intracérébrales et nous avons analysé son efficacité sur l'accumulation de GAGs non dégradés. Les injections de HD-RIGIE montrent une augmentation générale de l'activité b-glu dans tout le SNC des chiens MPS VII (sites d'injections et structures éloignées comme le cortex) et ce 1 mois ou 4 mois après les injections. L'analyse de la GFP confirme une distribution globale de HD-RIGIE dans le SNC d'animaux de grande taille. De plus, grâce aux propriétés intrinsèques de la b-glu (transport rétrograde et phénomène de libération/recapture), nous avons observé une diminution générale de l'accumulation vésiculaire neuronale des GAGs non dégradés dans tout le parenchyme cérébral. D'autre part grâce à la stratégie d'isolement et de non vaccination des chiens MPS VII, nous n'observons ni de réponse immunitaire humorale, ni d'aggravation de l'inflammation du parenchyme.Mucopolysaccharidosis type VII (MPS VII) is a rare inherited lysosomal storage disease caused by b-D-glucuronidase (b-glu) deficiency. b-glu is involved in the physiological turnover of glycosaminoglycans (GAGs). Its deficiency causes accumulation of undegraded GAGs inside vesicles leading to cell death. Our goals are to develop and test the clinical relevance of helper-dependant (HD) canine adenovirus type 2 (CAV-2) vectors to treat neural degeneration caused by MPS VII in a dog model. Because CAV-2 targets preferentially neurons and traffics via axons, the distribution of the transgene throughout the CNS will allow widespread delivery of the missing lysosomal enzyme in these disorders with a minimum number of injections. We tested a HD-CAV-2 vector expressing the human GUS gene in the canine model of MPS VII for their safety, efficacy, duration of expression and possible reversion of the MPS VII induced symptoms.A previous study based on HD-CAV-EGFP vector injections in MPS VII-/- and healthy dogs showed that we are now able to inject HD-CAV-2 in the dog brain, have a minimal induction of the immune response, an efficient transduction of the neurons and an efficient biodistribution of transduced cells. After the production of a suitable vector (HD-RIGIE) for injections in the CNS of MPS VII dogs we analysed its efficiency on GAGS storage in neurons.Injections of HD-RIGIE showed after 1 month or 4 months post injections a widespread increase in general level of b-glu activity, in the sites of injections and in distant areas such as cortex. Analysis of GFP, also permit to observe a widespread biodistribution of the vector. Because of b-glu property of cross-correction we observed a global decreased in GAGs storage in the entire MPS VII brains. Finally, the dogs did not present humoral immune response and no aggravation of inflammationMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Fatty acid transport protein 1 regulates retinoid metabolism and photoreceptor development in mouse retina

<div><p>In retinal pigment epithelium (RPE), RPE65 catalyzes the isomerization of all-<i>trans</i>-retinyl fatty acid esters to 11-<i>cis</i>-retinol in the visual cycle and controls the rhodopsin regeneration rate. However, the mechanisms by which these processes are regulated are still unclear. Fatty Acid Transport Protein 1 (FATP1) is involved in fatty acid uptake and lipid metabolism in a variety of cell types. FATP1 co-localizes with RPE65 in RPE and inhibits its isomerase activity <i>in vitro</i>. Here, we further investigated the role of FATP1 in the visual cycle using transgenic mice that overexpress human FATP1 specifically in the RPE (hFATP1TG mice). The mice displayed no delay in the kinetics of regeneration of the visual chromophore 11-<i>cis</i>-retinal after photobleaching and had no defects in light sensitivity. However, the total retinoid content was higher in the hFATP1TG mice than in wild type mice, and the transgenic mice also displayed an age-related accumulation (up to 40%) of all-<i>trans</i>-retinal and retinyl esters that was not observed in control mice. Consistent with these results, hFATP1TG mice were more susceptible to light-induced photoreceptor degeneration. hFATP1 overexpression also induced an ~3.5-fold increase in retinosome autofluorescence, as measured by two-photon microscopy. Interestingly, hFATP1TG retina contained ~25% more photoreceptor cells and ~35% longer outer segments than wild type mice, revealing a non-cell-autonomous effect of hFATP1 expressed in the RPE. These data are the first to show that FATP1-mediated fatty acid uptake in the RPE controls both retinoid metabolism in the outer retina and photoreceptor development.</p></div

Regulation of the visual retinoid cycle by FATP1.

<p>Schematic summarizing the effects of FATP1 overexpression in the mouse RPE. Rhodopsin, the light-sensitive protein in rods, is located in the disk membranes of rod outer segments (ROS). Absorption of a photon (hʋ) induces 11-<i>cis</i> to all-<i>trans</i> isomerization of retinaldehyde. All-<i>trans</i>-retinal then dissociates from rhodopsin and is reduced to all-<i>trans</i>-retinol, which is taken up by an RPE cell. Also in the RPE, FATP1 promotes long chain fatty acid (LCFA) uptake, thus providing LCFA-CoA for formation of phospholipids. All-<i>trans</i>-retinol is esterified with a phosphatidylcholine (PC)-derived LCFA to form all-<i>trans</i>-retinyl esters in a reaction catalyzed by LRAT. RPE65 then converts all-<i>trans</i>-retinyl esters to 11-<i>cis</i>-retinol. All-trans retinyl esters accumulate in the transgenic hFATP1TG RPE, suggesting increased levels of LCFAs and/or inhibition of RPE65. In ROS, the response to light remains unchanged, although rhodopsin expression increases. This increase is related to a greater number of PRs and their longer outer segments. Consequently, the rate of all-<i>trans</i> retinal formation is elevated, resulting in susceptibility to degeneration induced by light.</p

Light-induced retinal degeneration in hFATP1TG mice.

<p>(A) H&E and safranin staining of the retinas of aged wild type (WT) and hFATP1TG mice (n = 3 per group) after dark adaptation (control) or bright light exposure (3 h, 20,000 lux) followed by darkness for 5 days. (B) Quantification of photoreceptor loss measured as the ratio of outer and inner nuclear layer thickness (ONL/INL) under the conditions shown in (A). The INL thickness did not change significantly with age and was used as the reference. ***p < 0.001.</p

Validation of hFATP1 overexpression in the RPE of hFATP1TG mice.

<p>(A) qPCR analysis of FATP1 mRNA expression (human and mouse) in RPE of young (1–3-month-old, n = 4) and aged (6–9-month-old, n = 6) wild type (WT) and hFATP1 transgenic (TG) mice. Results are normalized to Mertk mRNA expression. (B) Western blot of hFATP1 protein expression in RPE of young and aged WT and hFATP1TG mice. GAPDH was probed as a loading control. (C) Kinetics of Bodipy C12 uptake, expressed as a percentage of total fluorescence, in flat-mounted RPE from young hFATP1TG and WT mice. The data are from n = 4–5 mice. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Confocal fluorescence microscopy of Bodipy C12 uptake in flat mounts of RPE from young WT and hFATP1TG mice. DAPI and ZO-1 labeling permitted visualization of nuclei and tight junctions, respectively, of individual RPE cells.</p

Visual function is not impaired by hFATP1 overexpression.

<p>(A) Full-field electroretinogram (ERG) analysis to determine light sensitivity of aged wild type (WT, n = 10) and hFATP1TG (n = 17) mice show no differences in a-wave and b-wave amplitudes or latencies. (B) Maximal b-wave amplitudes of rod and cone photoreceptors in aged WT (n = 6) and hFATP1TG (n = 8) mice. White light was used as stimulus for total rod and/or cone responses. Blue and green lights activate S and M cone photopigments respectively. No statistically significant differences were observed in any of the parameters measured.</p

Kinetics of the recovery of retinoids during dark adaptation of hFATP1TG mice.

<p>(A) Kinetics of b-wave amplitude recovery in aged wild type (WT, n = 14) and hFATP1TG (n = 16) mice kept in darkness for the indicated times after photobleaching. Values are expressed as the percentage of the b-wave amplitude in dark-adapted (DA) mice. (B) HPLC quantification of total retinoids during dark adaptation of young (n = 3 per group) and aged (n = 6 per group) WT and hFATP1TG mice. (C) Kinetics of the recovery of 11<i>c</i>RAL (top), <i>at</i>RAL (middle), and <i>at</i>RE (bottom) contents in the retina of young (2-month-old, n = 3) and aged (4–6-month-old, n = 6) WT and hFATP1TG mice. Measurements were performed after overnight dark adaptation (DA), immediately after photobleaching (PB), or after being kept in darkness for the indicated times after PB. *p < 0.05, **p < 0.01, ***p < 0.001.</p

Non-cell-autonomous effects of hFATP1 in the neural retina.

<p>(A) H&E and safranin staining of 5 μm sagittal sections of eyecups of young (3-month-old) and aged (6–15-month-old) hFATP1TG and wild type (WT) mice. (B) Quantification of the ratio of outer and inner nuclear layer thickness (ONL/INL) of young and aged WT and transgenic (TG) and mice (n = 20 sections per mouse, 5 mice per group). (C) Quantification of photoreceptor outer segment (POS) length in young and aged WT mice and transgenic (TG) (n = 20 measures of length throughout the retina per mouse, 5 mice per group). (D) Spectral domain-optical coherence tomography (SD-OCT) measurements of retinal thickness of aged hFATP1TG and WT mice (n = 4 per group). o.n., optic nerve. (E) Quantification of rhodopsin mRNA (qPCR) and protein (western blot) levels in the neural retina of WT (n = 7) and hFATP1TG (n = 8) mice. (F) qPCR quantification of FATP1 mRNA levels in the neural retina of WT (n = 8) and transgenic (n = 7) mice. Data were normalized to actin mRNA and tubulin protein levels. *p < 0.05, **p < 0.01, ***p < 0.001.</p