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Open Access Publications from the University of California

Scholarly Works (14 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2016)

Ocular Albinism (OA) is a disease characterized by hypopigmentation of the eyes and misrouting of the optic nerves. It is caused by mutations in the OA1 gene, which encodes a G-protein coupled receptor localized in the retinal pigment epithelium. Previously, microarrays carried out in the Farber lab comparing mRNAs from E15 control and Oa1-/- mouse eyes showed that doublecortin and Creb-binding protein (CBP) were down-regulated in Oa1-/- mice. To further explore if these proteins are involved in the Oa1 signaling cascade, I conducted a series of immunohistochemistry experiments to determine the localization of doublecortin, phosphorylated Creb (pCreb) and CBP in control and Oa1-/- retina/RPE. In addition to confirming their down-regulation in Oa1-/- retinas, my results suggest that doublecortin, pCreb and CBP are indeed involved in the Oa1 signaling cascade and may be related to the misrouting of the optic nerves in Oa1-/- mice and OA patients.

    Cover page: Study of Proteins Associated with Retinal Ganglion Cell Axon Misrouting in Ocular Albino Mouse Model
    • Thesis
    • Peer Reviewed
    UCLA Electronic Theses and Dissertations (2012)

    Retinal degenerative diseases are a leading cause of irreversible blindness. While the retina of amphibians and birds has a remarkable capacity for near complete regeneration - Müller cells now recognized as the likely source of this regeneration - no spontaneous regeneration has been observed in the mammalian retina. Therapies to augment retinal repair could improve the quality of life of millions of individuals.

    In my studies, I examined the possibility of augmenting the retina's endogenous regenerative capacity using embryonic stem cell-derived microvesicles (ESMVs) released by embryonic stem cells (ESCs). I found that ESMVs contain multiple mRNAs encoding the core transcription factors known to induce pluripotency in somatic cells and microRNAs of the 290 cluster, pivotal in the control of the ESC-specific cell cycle.

    In vitro, I demonstrated that ESMVs selectively transfer their mRNA and microRNA cargo to human Müller cells and induce in them morphological, mRNA, and microRNA expression changes. I observed upregulation of mRNAs involved in induction and maintenance of stem cell pluripotency, early ocular genes, and genes important for retinal protection and remodeling, as well as downregulation of inhibitory and scar-related genes; and upregulation of microRNAs promoting pluripotency and cell proliferation and downregulation of those involved in differentiation and cell cycle arrest. Moreover, a subset of the ESMV-treated Müller cells expressed markers of amacrine, ganglion, and rod photoreceptor cells. These results suggested that ESMVs induce dedifferentiation of Müller cells to a stem cell phenotype and differentiation towards other retinal lineages.

    I then conducted preliminary studies on the ability of ESMVs to augment retinal regeneration in vivo, injecting ESMVs into mouse retinas damaged with NMDA. I observed many proliferating cells in ESMV-treated and none in non-treated retinas. The majority of these cells expressed the Müller cell marker CRALBP, and a subset of them, the amacrine neuronal markers Syntaxin 1a and GAD67. ERGs demonstrated functional improvement in several ESMV-treated retinas. My findings suggest that ESMVs may have the potential to augment retinal endogenous regenerative capacity. Further studies of the effects of ESMVs on the regeneration of damaged retinas may lead to the discovery of novel therapeutic approaches for retinal degenerations.

      Cover page: Activation of the retina's regenerative potential by embryonic stem cell-derived microvesicles
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2011)

      Purpose

      ZBED4, a protein in cones and Müller cells of human retina, may play important functions as a transcriptional activator of genes expressed in those cells or as a co-activator/repressor of their nuclear hormone receptors. To begin investigating these potential roles of ZBED4, we studied the developmental expression and localization of both the Zbed4 mRNA and protein of mouse retina.

      Methods

      northern blots showed the presence of Zbed4 mRNA in retina and other mouse tissues, and western blots showed the nuclear and cytoplasmic expression of Zbed4 at different developmental times. Antibodies against Zbed4 and specific retinal cell markers were used for retinal immunohistochemistry.

      Results

      Zbed4 mRNA was present at different levels in all the mouse tissues analyzed. The Zbed4 protein was barely detectable at embryonic day (E)14.5 but was clearly seen at E16 at both retinal outer and vitreal borders and throughout the retina by E18 and postnatal day 0 (P0). Thereafter, Zbed4 expression was more restricted to the inner retina. While ZBED4 is localized in cones and endfeet of Müller cells of human retina, in adult mouse retina Zbed4 is only detected in Müller cell endfeet and processes. The same localization of Zbed4 was observed in rat retina. In early development, Zbed4 is mainly present in the nuclear fraction of the mouse retina, and in adulthood it becomes more enriched in the cytoplasmic fraction.

      Conclusions

      The patterns of spatial and temporal expression of Zbed4 in the mouse retina suggest a possible involvement of this protein in retinal morphogenesis and Müller cell function.
        Cover page: ZBED4, a cone and Müller cell protein in human retina, has a different cellular expression in mouse.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2011)
        Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.
          Cover page: Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule CytoskeletonCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2012)
          Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.
            Cover page: Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina
            • Article
            • Peer Reviewed
            UC Santa Barbara Previously Published Works (2005)

            Retinal development in 3 strains of rd-3/rd-3 mutant mice, previously shown to have different rates of degeneration, was studied using light, electron, and immunofluorescence microscopy. The time course and phenotype of the degeneration as well as details on the mechanism of massive photoreceptor cell loss are compared with other known retinal degenerations in mice. Up until postnatal day (P) 10, the retinas of all three strains (RBF, 4Bnr, In-30) develop similarly to those of pigmented and nonpigmented controls. TUNEL-positive cells appear in the outer nuclear layer (ONL) by P14, and reach a maximum in all three mutant strains around P21. Scattered rods and cones form a loose, monolayered ONL by 8 weeks in the albino RBF strain, by 10 weeks in the albino 4Bnr strain, and by 16 weeks in the pigmented In-30 strain. Though the initial degeneration begins in the central retina, there is no preferred gradient of cell death between central and peripheral photoreceptors. Rods and cones are present at all ages examined. During development, stacks of outer segments (OS) form in all three strains though they never achieve full adult lengths, and often have disorganized, atypical OS. Rod opsin is expressed in the developing OS but is redistributed into plasma membrane as OS degeneration proceeds. Retinal pigment epithelial (RPE) cells of all mutant strains contain packets of phagocytosed OS, and their apical processes associate with the distal ends of the OS. At their synaptic sites, photoreceptor terminals contain ribbons apposed to apparently normal postsynaptic triads. As photoreceptors are lost, Muller cells fill in space in the ONL but they do not appear to undergo significant hypertrophy or migration, though during the degeneration, glial fibrillary acidic protein (GFAP) expression is gradually upregulated. Macrophage-like cells are found frequently in the subretinal space after the onset of photoreceptor apoptosis. As OS disappear, the RPE apical processes revert to simple microvilli. Late in the degeneration, some RPE cells die and neighboring cells appear to flatten as if to maintain confluence. In regions of RPE cell loss that happen to lie above retina where the ONL is gone, cells of the inner nuclear layer (INL), wrapped by Miller cell processes, may front directly on Bruch's membrane.

              Cover page: Morphological characterization of the retinal degeneration in three strains of mice carrying the rd-3 mutation
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2013)

              Purpose

              Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype.

              Methods

              Transgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1-/- mice.

              Results

              We found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice.

              Conclusions

              Our results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype.
                Cover page: A Constitutively Active Gαi3 Protein Corrects the Abnormal Retinal Pigment Epithelium Phenotype of Oa1−/− miceCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2016)
                Ocular albinism type 1 (OA), caused by mutations in the OA1 gene, encodes a G-protein coupled receptor, OA1, localized in melanosomal membranes of the retinal pigment epithelium (RPE). This disorder is characterized by both RPE macro-melanosomes and abnormal decussation of ganglion cell axons at the brain's optic chiasm. We demonstrated previously that Oa1 specifically activates Gαi3, which also signals in the Oa1 transduction pathway that regulates melanosomal biogenesis. In this study, we screened the human Gαi3 gene, GNAI3, in DNA samples from 26 patients who had all clinical characteristics of OA but in whom a specific mutation in the OA1 gene had not been found, and in 6 normal control individuals. Using the Agilent HaloPlex Target Enrichment System and next-generation sequencing (NGS) on the Illumina MiSeq platform, we identified 518 variants after rigorous filtering. Many of these variants were corroborated by Sanger sequencing. Overall, 98.8% coverage of the GNAI3 gene was obtained by the HaloPlex amplicons. Of all variants, 6 non-synonymous and 3 synonymous were in exons, 41 in a non-coding exon embedded in the 3' untranslated region (UTR), 6 in the 5' UTR, and 462 in introns. These variants included novel SNVs, insertions, deletions, and a frameshift mutation. All were found in at least one patient but none in control samples. Using computational methods, we modeled the GNAI3 protein and its non-synonymous exonic mutations and determined that several of these may be the cause of disease in the patients studied. Thus, we have identified GNAI3 as a second gene possibly responsible for X-linked ocular albinism.
                  Cover page: GNAI3: Another Candidate Gene to Screen in Persons with Ocular Albinism.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2012)
                  Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4's secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.
                    Cover page: Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2007)

                    Purpose

                    SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.

                    Methods

                    270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.

                    Results

                    Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the alpha-, beta- and gamma-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the beta-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.

                    Conclusions

                    If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.
                      Cover page: Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the beta-subunit of transducin with digenic disease.
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