Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Iontophoresis of dexamethasone in the treatment of endotoxin-induced-uveitis in rats.

The purpose of this study was to evaluate the efficacy of a Coulomb Controlled Iontophoresis system (CCI) in the local delivery of corticosteroids for the treatment of uveitis. The therapeutic efficacy of Dexamethasone (Dex) administered by CCI was compared to systemic injection and to topical application with the iontophoresis apparatus in the absence of electrical current. The evaluation was done in the treatment of the endotoxin-induced uveitis (EIU) model, and in the effect on TNF gene expression in the iris/ciliary body as well as in the retina and on TNF levels in aqueous humor and vitreous. Dex was administered either at the time of LPS injection or 5 hours later. For iontophoresis, we used a 1 ml reservoir-electrode covering the cornea, the limbus, and the first millimeter of the sclera. The applied electrical current was of 400 microA during four minutes with a total surface charge of 0.4 C cm-2. EIU was evaluated by clinical examination, by counts of intraocular inflammatory cells on histological sections, and by measuring the protein levels in the aqueous humor and in the vitreous. The TNF-alpha gene expression in the iris and ciliary body, and in the retina was evaluated by RT-PCR. The systemic effect of Dex delivered by CCI was evaluated on the level of serum TNF-alpha in EIU. Our results demonstrated that local administration of Dex by CCI inhibited anterior and posterior signs of intraocular inflammation as effectively as systemic administration, with no effect on systemic level of TNF. In the anterior and posterior segments of the eye, the protein exudation. TNF levels and the cellular infiltration were inhibited. The TNF-alpha gene expression was inhibited in the anterior as well as the posterior segment of the eye. No clinical nor histological damage were caused by the CCI apparatus. In conclusion, CCI administration of Dex allows for a therapeutic effect on the posterior as well as the anterior segment of the eye, and may present a viable alternative to systemic administration of glucocorticoids in severe ocular inflammations.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Developmental regulation of expression of schizophrenia susceptibility genes in the primate hippocampal formation

The hippocampal formation is essential for normal memory function and is implicated in many neurodevelopmental, neurodegenerative and neuropsychiatric disorders. In particular, abnormalities in hippocampal structure and function have been identified in schizophrenic subjects. Schizophrenia has a strong polygenic component, but the role of numerous susceptibility genes in normal brain development and function has yet to be investigated. Here we described the expression of schizophrenia susceptibility genes in distinct regions of the monkey hippocampal formation during early postnatal development. We found that, as compared with other genes, schizophrenia susceptibility genes exhibit a differential regulation of expression in the dentate gyrus, CA3 and CA1, over the course of postnatal development. A number of these genes involved in synaptic transmission and dendritic morphology exhibit a developmental decrease of expression in CA3. Abnormal CA3 synaptic organization observed in schizophrenics might be related to some specific symptoms, such as loosening of association. Interestingly, changes in gene expression in CA3 might occur at a time possibly corresponding to the late appearance of the first clinical symptoms. We also found earlier changes in expression of schizophrenia susceptibility genes in CA1, which might be linked to prodromal psychotic symptoms. A number of schizophrenia susceptibility genes including APOE, BDNF, MTHFR and SLC6A4 are involved in other disorders, and thus likely contribute to nonspecific changes in hippocampal structure and function that must be combined with the dysregulation of other genes in order to lead to schizophrenia pathogenesis.

Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

Comparison of Fundus Autofluorescence Between Fundus Camera (Topcon) and Confocal Scanning Laser Ophthalmoscope (HRA) in Various Pathologies

Purpose: To investigate the differences between the Fundus Camera (Topcon TRC-50X) and Confocal Scanning Laser Ophthalmoscope (Heidelberg retina angiogram (HRA)) on the fundus autofluorescence (FAF) imaging (resolution and FAF characteristics). Methods: Eighty nine eyes of 46 patients with various retinal diseases underwent FAF imaging with HRA (488nm exciter / 500nm barrier filter) before fluorescein angiography (FFA) and Topcon Fundus Camera (580nm exciter / 695nm barrier filter) before and after FFA. The quality of the FAF images was estimated, compared for their resolution and analysed for the influence of fixation stability and cataracts. Hypo- and hyper-FAF behaviour was analysed for the healthy disc, healthy fovea, and a variety of pathological features. Results: HRA images were found to be of superior quality in 18 eyes, while Topcon images were estimated superior in 21 eyes. No difference was found in 50 eyes. Both poor fixation (p=0.009) and more advanced cataract (p=0.013) were found to strongly increase the likelihood of better image quality by Topcon. Images acquired by Topcon before and after FFA were identical (100%). The healthy disc was usually dark on HRA (71%), but showed mild autofluorescence on Topcon (88%). The healthy fovea showed in 100% Hypo-FAF on HRA, while Topcon showed in 52% Iso-FAF, in 43% mild Hypo-FAF, and in 5% Hypo-FAF as on HRA. No difference of FAF was found for geographic atrophy, pigment changes, and drusen, although Topcon images were often more detailed. Hyper-FAF due to exudation showed better on HRA. Pigment epithelium detachment showed identical FAF behaviour on the border, but reduced FAF with Topcon in the center. Cystic edema was visible only on HRA in a petaloid pattern. Hard exsudates caused Hypo-FAF only on HRA, hardly visible on Topcon. Blocage phenomenon by blood however was identical. Conclusions: The filter set of Topcon and the single image acquisition appear to be an advantage for patients with cataract or poor fixation. Preceding FFA does not alter the Topcon FAF image. Regarding the FAF behaviour, there are differences between the two systems which need to be taken into account when interpreting the images.

Inhibition of Notch Pathway Enhances Photoreceptor Commitment From Cultured Retinal Stem Cells

Purpose: Consequently to the principle that photoreceptors have to be at a very precise development stage to be successfully transplanted (MacLaren 2006), we are trying to mimic this development stage in vitro using retinal stem cells. The latter one isolated from the newborn mouse retina, derived from the radial glia population, which were previously isolated and characterized in our laboratory. We developed a protocol to commit these cells to the photoreceptor fate, but even if the percentage of cells expressing photoreceptor markers is high (30%), the differentiation process is incomplete so far (Merhi-Soussi 2006). Methods: In order to ameliorate photoreceptor differentiation, we hypothesized that the Notch pathway may interfere with this process by either promoting glia commitment, or maintaining an undifferentiated state. We are thus using a gamma-secretase inhibitor (DAPT), which inhibits Notch receptor cleavage and thus Notch activation. DAPT was used either during the whole differentiation stimulation, or only during a restricted period in two various retinal stem cell lines (RSC AA and RSC MP1). Results: RT-PCR performed during cell proliferation, showed the same positive expression in both cell lines for the following genes: Math3, Six3, Hes1, NeuroD, Pax6 and Notch1. Additionally, Mash1, Hes5, Prox1, Crx and Otx2 were detected in both cell lines but with a stronger expression in RSC MP1. Opposite results were obtained for Chx10. Nrl, Peripherin/RDS, GFAP and Math5 were detected neither in RSC AA, nor in RSC MP1. The constant presence of DAPT i) leads to a 233% (RSC AA) or 900% (RSC MP1) increase in peripherin/RDS-positive (photoreceptor marker) cells, compared to controls (no DAPT, n=3, P<0.02) along with a 68% (RSC AA) or 80% (RSC MP1) decrease in GFAP- positive cells (n=3, P<0.04), ii) modifies the ratio between uni-/bi- (23%) and multi- (77%) polar peripherin/RDS-positive cells to 45% and 55%, respectively, for both cell lines and iii) reduces by 50% the total cell number during the whole differentiation process for both cell lines. Conclusions: We are now exploring whether this reduction in total cell number is due to inhibition of cell proliferation or to cell death and whether photoreceptor differentiation is promoted instead of glial induction. We also want to confirm the results obtained with DAPT with RSCs isolated from Notch1-loxP mice. Such protocol may help to better mimic photoreceptor development, but this needs to be confirmed by genomic and proteomic profile analyses.

Robust vaccine-elicited cellular immune responses in breast milk following systemic Simian Immunodeficiency Virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

3D culture of postnatal retinal stem cells using self assembling polymers

PURPOSE We have previously shown that retinal stem cells (RSCs) can be isolated from the radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have a great capacity to renew and to generate a large number of neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, recent published results from our lab revealed that such cells have a poor integration and survival rate after grafting. The uncontrolled environment of a retina seems to prevent good integration and survival after grafting in vivo. To bypass this problem, we are evaluating the possibility of generating in vitro a hemi-retinal tissue before transplantation. METHODS RSC were expanded and cells passaged <10 were seeded in a solution containing poly-ethylene-glycol (PEG) polymer based hydrogels crosslinked with peptides that are chosen to be substrates for matrix metalloproteinases. Various doses of cross linkers peptides allowing connections between PEG polymers were tested. Different growth factors were studied to stimulate cell proliferation and differentiation. RESULTS Cells survived only in the presence of EGF and FGF-2 and generated colonies with a sphere shape. No cells migrated within the gel. To improve the migration and the repartition of the cells in the gels, the integrin ligand RGDSP was added into the gel. In the presence of FGF-2 and EGF, newly formed cell clusters appeared by cell proliferation within several days, but again no outspreading of cells was observed. No difference was even seen when the stiffness of the hydrogels or the concentration of the integrin ligand RGDSP were changed. However, our preliminary results show that RSCs still form spheres when laminin is entrapped in the gel, but they started to spread out having a neuronal morphology after around 2 weeks. The neuronal population was assessed by the presence of the neuronal marker b-tubulin-III. This differentiation was achieved after successive steps of stimulations including FGF-2 and EGF, and then only FGF-2. Glial cells were also present. Further characterizations are under process. CONCLUSIONS RSC can be grown in 3D. Preliminary results show that neuronal cell phenotype acquisition can be instructed by exogenous stimulations and factors linked to the gel. Further developments are necessary to form a homogenous tissue containing retinal cells.

Intraocular implants for extended drug delivery: therapeutic applications.

An overview of ocular implants with therapeutic application potentials is provided. Various types of implants can be used as slow release devices delivering locally the needed drug for an extended period of time. Thus, multiple periocular or intraocular injections of the drug can be circumvented and secondary complications minimized. The various compositions of polymers fulfilling specific delivery goals are described. Several of these implants are undergoing clinical trials while a few are already commercialized. Despite the paramount progress in design, safety and efficacy, the place of these implants in our clinical therapeutic arsenal remains limited. Miniaturization of the implants allowing for their direct injection without the need for a complicated surgery is a necessary development avenue. Particulate systems which can be engineered to target specifically certain cells or tissues are another promising alternative. For ocular diseases affecting the choroid and outer retina, transscleral or intrasscleral implants are gaining momentum.

Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy.

PURPOSE: The aim of this study was to characterize oligonucleotide-polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo. METHODS: The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)-ODN/PEI complexes specifically directed at transforming growth factor beta (TGFbeta)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC-ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections. RESULTS: Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core-shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFbeta-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity. CONCLUSIONS: Specific down-regulation of TGFbeta-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation.

Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.

Cibles rétiniennes d'action des médicaments [Retinal drug targets].

Retinal effects of systemically administered drugs are rare due to the hematoretinal barriers that protect the retina from circulating active principles. However, some compounds may have direct or indirect toxic effects on the retina through direct interaction with a specific receptor or due to their accumulation within pigment of uveal cells. In the latter case, toxicity is dose-dependent and may be observed years after cessation of medication, as observed with antimalarial drugs. Anti-infective and anti-inflammatory agents, particularly glucocorticoids, are currently injected peri- or intraocularly. The mechanisms and the exact toxicity of glucocorticoids on the retina remain poorly understood. More recently, anti-VEGF has been specifically developed for the treatment of retinal diseases. However, the long-term blockade of VEGF on normal retinal physiology should be determined taking into account VEGF and VEGF receptors expression in the normal and pathologic retina. Whilst enormous advances are made in the treatment of retinal diseases, basic research is still required to define more accurately the molecular targets of drugs to improve their benefits and reduce their potential side effects.