Indiana University-Purdue University Indianapolis (IUPUI) / Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.

No description available.

Preterm birth may lead to complications during the neonatal period that can cause visual dysfunctions. Retinopathy of prematurity (ROP) and neurological complications are well known reasons for visual dysfunctions, but preterm children with no or only mild ROP and no evident neurological problems may also be affected visually when they grow up. Retinal development starts early after gestation and continues long after birth. Major processes are underway during the second half of pregnancy when preterm children are born, and a preterm birth could possibly have a negative effect on normal retinal development. The aims of the studies were to evaluate retinal morphology and function in former preterm children and compare the results with children born at term. Former preterm children aged 5 to 17 years and born in a gestational age (GA) of 32 weeks or less were included in the different study groups. Children of similar ages who were born at term and with normal visual acuity (VA) acted as controls. Best corrected VA and refraction in cycloplegia were assessed in all children. Macular thickness and retinal nerve fiber layer (RNFL) thickness were measured with optical coherent tomography (OCT). Total retinal function was assessed with fullfield electroretinography (ffERG) and central macular function was assessed with multifocal electroretinography (mfERG). Preterm children had thicker central maculae than controls. There was a positive correlation between central macular thickness and GA at birth. RNFL thickness was reduced in the preterm children with severe ROP and treated ROP, but children with mild or no ROP did not differ from the fullterm children. The photoreceptor function measured with ffERG and the macular function measured with mfERG were reduced in the preterm group compared to controls. Preterm birth affects the retina both morphologically and functionally, and ROP has been suggested to be a reason for retinal changes. However, the results of this thesis indicate that children with no ROP also have retinal changes, suggesting an effect of prematurity itself. There were no correlations between any retinal changes and VA, but it is possible that larger studies using improved techniques may elucidate this further.

Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) have the ability to self renew indefinitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the differentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type affected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise differentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the definitive identification of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed significantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies.

Visual system homeobox 1 (Vsx1) is a paired-like:CVC homeodomain transcription factor that is expressed in a subset of retinal bipolar cells. Vsx1-null mice have previously been shown to have defects in bipolar cell terminal differentiation characterized by the reduced expression of four OFF bipolar cell-specific markers and electrophysiological defects in the OFF visual signaling pathway. The availability of recently identified bipolar cell markers enables a further characterization of the Vsx1-null mutant. I determined that Vsx1 is expressed in Type 7 ON bipolar cells and observed the upregulation of three cell markers: Cabp5, Chx10, and alpha-gustducin:GFP in this cell type in Vsx1-null mice. These data reveal a trend in which Vsx1 functions as a transcriptional repressor in Type 7 ON bipolar cells and as an activator in Type 2 OFF bipolar cells. Lastly, my data indicate that Vsx1 is required for the expression of two Type 3a bipolar cell markers, however, the mechanism by which it does so appears to be complex, as I was unable to detect Vsx1 protein or reporter gene expression in this cell type. / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.

The mammalian central nervous system (CNS) has a high degree of complexity and cell type diversity that enables sophisticated processing of sensory information, circuit formation, and behaviour. While much is known about the patterning and specification of the major neuronal classes in the CNS, through processes such as morphogen gradient signaling and transcription factor combinatorial coding, much less is known about how subtypes within each cell class are specified. Bipolar cells are one of the main classes of interneurons in the vertebrate retina and consist of fifteen different subtypes based on their physiological function, morphology, and unique gene expression. The cellular mechanisms behind the specification of these subtypes are not fully known. In this thesis, I examine these mechanisms by investigating the role of extrinsic and intrinsic factors on the specification and differentiation of bipolar cell subtypes. We hypothesize that the specification of bipolar cell subtypes occurs in a multi-step manner and is dependent on non-cell autonomous (extrinsic) signals. To test this hypothesis, I conducted a series of experiments on the early postnatal mouse retina, which is the period when bipolar cells are generated. First, I examined whether bipolar cell marker onset was temporally ordered as would be predicted in a multi-step model. Postnatal day 3 (P3) mice were injected with EdU (5-ethynyl-2’-deoxyuridine), a thymidine analog that labels proliferating cells and then dissociated and fixed the retinal cells 24-120 hours after injection. My results show that Vsx2-5.3-PRE-Cre, a marker of pan-bipolar cells specification, is first detected 36 hrs after cell cycle exit, whereas specialized bipolar subtype-specific markers are expressed 48-60 hrs post-EdU injection. This observation is consistent with the idea that bipolar cells develop in a stepwise manner, first as an unspecified, pan-bipolar cell intermediate and then into one of the 15 subtypes. To further investigate this possibility, I developed a novel dissociated retinal culture assay that enabled me to accurately track retinal progenitor cells and postmitotic precursor cells and determine the requirement of cell autonomous and non-cell autonomous mechanisms during bipolar cell subtype specification. This assay involves culturing dissociated retinal cells from P3 EdU-injected mice at high density (abundant cell contact) or low density (scarce cell contact) at various timepoints, thereby allowing me to probe the role of these mechanisms in RPCs, early postmitotic cells, and late postmitotic cells. My findings revealed the first 24-48 hrs post cell cycle exit to be a critical, cell contact-dependent period for the specification of bipolar cell subtypes. This assay also allowed us to test the effect of blocking or activating the Notch and the Sonic Hedgehog (Shh) signal transduction pathways by using pharmacological compounds and recombinant ligands. Co-activation of Notch and Shh pathways increased the specification of Vsx1+ subtypes suggesting they play a role in their specification. Altogether, our results suggest that bipolar cell subtype specification follows a multi-step model, through an undifferentiated bipolar cell intermediate, and that cell contact plays a role in the specification mechanisms of bipolar cell subtype development. This is a novel finding that provides insight into the mechanisms underlying retinal neuronal subtype development and possibly in other neuronal cell types throughout the CNS. / Graduate / 10000-01-01

Στόχος: Ο υποδοχέας του γλουταμινικού οξέος ΝMDA (N-methyl-D-aspartate), αποτελεί μόριο-κλειδί που διαμεσολαβεί πολλούς τύπους συναπτικής πλαστικότητας στο κεντρικό νευρικό σύστημα. Στο οπτικό σύστημα η πλαστικότητα ξεκινά στο επίπεδο του αμφιβληστοειδούς χιτώνα. Κατά την ανάπτυξη του αμφιβληστροειδούς οι υποδοχείς NMDA συμμετέχουν σε φαινόμενα πλαστικότητας εξαρτώμενα από την εμπειρία όπως ο λειτουργικός διαχωρισμός των ON και OFF μονοπατιών. Η ανάπτυξη και η οπτική αποστέρηση επηρεάζουν επίσης τις ηλεκτροφυσιολογικές ιδιότητες των υποδοχέων NMDA στον αμφιβληστροειδή του επίμυος καθώς και την έκφραση των υπομονάδων του NR1 και NR2. Η βασική υπομονάδα NR1 υφίσταται εναλλακτικό μάτισμα με αποτέλεσμα να εμφανίζεται σε οκτώ διαφορετικές ισομορφές που προσδίδουν μοριακή ποικιλότητα στον υποδοχέα. Το ερώτημα που ετέθη ήταν εάν η έκφραση των ισομορφών της υπομονάδας NR1 ρυθμίζεται κατά την ανάπτυξη του αμφιβληστροειδούς χιτώνα του επίμυος και εάν μεταβάλλεται από την οπτική εμπειρία. Μέθοδος: Χρησιμοποιήθηκαν αμφιβληστροειδείς επίμυων (Wistar) που μεγάλωσαν είτε σε φυσιολογικό ημερήσιο κύκλο 12 ώρες φως/12 ώρες σκοτάδι [normal-reared (NR)], είτε σε διαρκές σκοτάδι [dark-reared (DR)] από την 9η έως την 60η ημέρα μετά τη γέννηση. Η μελέτη της έκφρασης των ισομορφών του αμινοτελικού (NR1a, NR1b) και του καρβοξυτελικού (NR1-1, NR1-2, NR1-3, NR1-4) άκρου της υπομονάδας NR1 έγινε με τη μέθοδο της real-time PCR. Αποτελέσματα: Το αναπτυξιακό προφίλ όλων των ισομορφών εμφάνισε διαφορετική αύξηση κατά τη διάρκεια της δεύτερης και τρίτης εβδομάδας, με μέγιστη έκφραση στο τέλος της τρίτης εβδομάδας. Μεταξύ των ισομορφών του αμινοτελικού άκρου, η NR1b εκφραζόταν σταθερά σε υψηλότερα επίπεδα σε σχέση με την NR1a, ενώ μεταξύ των ισομορφών του καρβοξυτελικού άκρου, η NR1-2 εκφραζόταν σε υψηλότερα επίπεδα από την NR1-4, ενώ τόσο η NR1-1 όσο και η NR1-3 εκφράζονταν σε χαμηλά επίπεδα. Η ανάπτυξη στο σκοτάδι μείωσε την έκφραση όλων των ισομορφών σε πολλά αναπτυξιακά στάδια και στο ενήλικο ζώο. Σημαντική αλληλεπίδραση μεταξύ ηλικίας και οπτικής εμπειρίας προέκυψε για τις ισομορφές NR1a, NR1-2 και NR1-4. Συμπεράσματα: Η έκφραση όλων των ισομορφών της υπομονάδας NR1 μεταβάλλεται κατά την ανάπτυξη του αμφιβληστροειδούς του επίμυος, ενώ ορισμένες (NR1a, NR1-2 and NR1-4) ρυθμίζονται τόσο από την ηλικία όσο και από την οπτική εμπειρία. Τέτοιες μεταβολές μπορεί να παίζουν σημαντικό ρόλο σε φαινόμενα πλαστικότητας που λαμβάνουν χώρα στον αμφιβληστροειδή χιτώνα. / Purpose: The N-methyl-D-aspartate (NMDA) type of glutamate ionotropic receptor is a key molecule mediating plasticity related processes in the central nervous system. Visual system plasticity begins in the retina. During postnatal retinal development NMDA receptor has been shown to be involved in experience dependent plasticity such as the functional segregation of ON and OFF pathways. Development and visual deprivation have been found to affect the kinetics of NMDA receptor in rat retina and the expression of its main subunits NR1 and NR2. The NR1 fundamental subunit of NMDA receptor exists in eight distinct splice isoforms. Knowing that alternative splicing of the NR1 subunit offers a further molecular diversity to the receptor, we have addressed the question of whether the alternative splicing of NR1 subunit of the NMDA receptor is regulated during postnatal retinal development and whether this regulation is altered by visual experience. Methods: Retinas were dissected from eyes of Wistar rats raised either in normal 12-hour light/12-hour dark cycle [normal-reared (NR)], or in complete darkness [dark-reared (DR)] at postnatal days 9 to 60. Real-time PCR was performed in order to assess the mRNA expression of NR1 isoforms using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). Results: The developmental profiles of mRNA expression levels of both N- and C-terminal NR1 isoforms showed differential increases during the second and third postnatal weeks, while their expression peaked at the end of the third week. Among N-terminal isoforms NR1b was constantly expressed at higher levels compared to NR1a and among the C-terminal isoforms, NR1-2 was expressed at higher levels than NR1-4, while both NR1-1 and NR1-3 were expressed at low levels. Dark-rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and in adult retina. A significant age and experience interaction was observed at NR1a N-terminal isoform, and at the most abundant C-terminal isoforms NR1-2 and NR1-4. Conclusions: Our results have demonstrated that all NR1 splice isoforms are developmentally regulated in rat retina and some of them (NR1a, NR1-2 and NR1-4) are also bidirectionally regulated by age and visual experience. Such changes may play an important role in the plastic and activity-dependent events taking place in retina.

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, São Bernardo do Campo, 2018. / A determinação do destino das células neuronais é um processo dinâmico regulado pela expressão de centenas de genes simultaneamente. A modificação pós-transcricional das caudas N-terminais das histonas é uma forma dinâmica de regulação gênica. Várias evidências sugerem que a modulação das modificações das histonas desempenha um papel importante na regulação da determinação do destino neuronal e atuam em muitos processos do desenvolvimento. Entre os diferentes moduladores, as enzimas modificadoras de histonas, que possuem como alvos as caudas das histonas, estão no centro da atenção. As histonas demetilases (HDMs) são uma grande família de enzimas que possuem atividade catalítica seletiva contra sítios específicos de metilação de histonas. Jmjd3 é uma HDM específica para histona H3K27 cuja atividade enzimática torna o ambiente propício para aumentar a taxa de transcrição gênica. Para investigar o provável papel da Jmjd3 no desenvolvimento da retina em ratos na fase pós-natal, realizamos o bloqueio desta enzima com o composto farmacológico GSK-J1. Como primeira abordagem, determinamos a localização de Jmjd3 na retina de ratos neonatos e adultos, o que foi consistente com a localização em neurônios diferenciados, incluindo células ganglionares na retina de ratos neonatos. Nesta idade do desenvolvimento, também observamos a presença de Jmjd3 em células indiferenciadas. Injeções subretinianas de GSK-J1 causaram a diminuição do nível proteico de H3k27me3 em retinas de ratos neonatos. Curiosamente, a injeção de GSK-J1 aumentou simultaneamente o número de células proliferativas e apoptóticas. Além disso, mais células imaturas foram detectadas na camada plexiforme externa, com processos neuronais mais longos. Finalmente, a influência da GSK-J1 na citogênese retiniana pós-natal foi examinada. Fomos capazes de determinar que a GSK-J1 especificamente causou uma diminuição significativa no número de células PKC-positivas que, quando localizadas na parte externa da camada nuclear interna, é um marcador confiável de células bipolares de bastonete. Estes dados fornecem as primeiras evidências dos efeitos do bloqueio farmacológico in vivo das histonas demetilases durante o desenvolvimento inicial da retina pós-natal, com impacto sobre processos como proliferação celular, maturação, indução de apoptose e determinação celular específica. / Neuronal cell fate determination is dynamic process regulated by expression of hundreds of genes simultaneously. The posttranslational modification of the N-terminal tails of the histone proteins is dynamic way of gene regulation. Countless numbers of the evidences propose that regulation of histone modification play principal role in various developmental process such as neuronal fate determination. Among different modulators the histone modifying enzymes that are targets histone tails are in the center of attraction. The histone demethylases (HDMs) family is comprised of several enzymes that have selective catalytic activity against specific sites of histone methylation. The enzymatic activity of the histone H3K27-specific demethylase Jmjd3 leads to transcriptionally permissive chromatin environments. To investigate the probable role of Jmjd3 in early postnatal rat retinal development, we tried to block this enzyme with pharmacological compound GSK-J1. As a first approach, we determined the localization of Jmjd3 in neonate and adult rat retina, which is consistent with localization in differentiated neurons, including ganglion cells in the retina of neonate rats. At this developmental age, we also observed the presence of Jmjd3 in undifferentiated cells. Subretinal injection of GSK-J1 caused the decrease of the global level of H3k27me3 in retinas of neonate rats. Interestingly, injection of GSK-J1 simultaneously increased the number of proliferative and apoptotic cells. In addition, more immature cells were detected in outer plexiform layer, with longer neuronal processes. Finally, the influence of GSK-J1 on postnatal retinal cytogenesis was examined. We were able to determine that GSK-J1 specifically caused significant decrease in the number of PKCa-positive cells, which when located in the outer part of the inner nuclear layer is a reliable marker of rod-on bipolar cells. These data provide the first evidence of in vivo pharmacological blocking of histone demethylases during early postnatal retinal development. In summary, we were able to show that application of GSK-J1 can influence on cell proliferation, maturation, apoptosis induction, and specific cell determination.

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>