ItemFrom neurons to neuroblastoma: dietary flavonoids as regulators of life, death & differentiation(University College Cork, 2020-03-24) Alshangiti, Amnah M.; O'Keeffe, Gerard W.; Sullivan, Aideen M.; Hegarty, Shane; Government of Saudi ArabiaNeuroblastoma (NB) is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. Patients classified as stage 4 with aggressive tumors are considered to belong to a high-risk group. MYCN-amplification is detected as a hallmark of NB in 20% of high-risk cases. Based on the International Neuroblastoma Risk Group (INRG), the relapse survival rate of high-risk patients with MYCN-amplification is only 4%. Despite extensive studies, metastasis and tumor relapse lead to debilitating morbidity and frequently death, in high-risk NB patients. Moreover, NB chemotherapy can result in very debilitating long-term sequelae. Errors in the molecular mechanisms that control the development of cell of the sympathetic lineage have been linked to NB development. One group of signals, known as the bone morphogenetic proteins (BMPs), plays an important role in sympathetic neuritogenesis, by signaling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB, but it is unknown if the expression of BMPR1A or BMPR1B is altered. The use of neurotrophic factors such as nerve growth factor (NGF) and growth differentiation factor (GDF)5 to promote cell differentiation has been reported in postnatal day (P)1 mouse NC cultures and MYCN-amplified NB SK-N-BE(2), respectively. GDF5 induces cell differentiation in NB cells through activation of BMP-Smad signaling. However, recombinant BMPs are expensive to produce and their clinical use is limited, due to several factors. Therefore, there is an urgent need to identify novel therapeutic agents that could induce cell differentiation in post-chemotherapy tissue with the minimum side effects. Recently, several screening studies have identified small molecules that modulate the BMP pathway. These include natural dietary compounds, including flavonoids and in particular the chalcone subfamily. Interestingly, flavonoids have potential anti-tumor properties with selective cytotoxicity to cancer cells. Additionally, chalcones, which are the most potent flavonoid subclass, have also demonstrated anti-tumor activity in some contexts. This thesis first investigated, in chapter 2, the association of distinct type1 BMPRs with different molecular pathways and survival outcomes in NB. Lower expression of BMPR2 and BMPR1B, and higher BMPR1A expression, were observed in stage 4 and in MYCN-amplified NB, and they were associated with poor survival. In contrast, higher expression of BMPR2 and BMPR1B were associated with better survival. Regarding MYCN status, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This study therefore showed that expression of distinct BMPRs is associated with different survival outcomes in NB. In chapter 3, the effects of low doses of the chalcones isoliquiritigenin (ISLQ) and 4-hydroxychalcone (4HC), small molecule activator of BMP-Smad signaling, on the morphological differentiation of NB cells, as well as the molecular mechanism of their action, was investigated. ISLQ and 4HC promoted neurite growth in NB cells by activation of BMP-Smad signaling. Moreover, treatment with these chalcones lead to a significant reduction of cell aggregates (tumor-like aggregations) in SK-N-BE(2) cells. After these findings, in chapter 4, the cytotoxic effect of ISLQ in MYCN-amplified NB cells was investigated in detail. ISLQ induced cell death in MYCN-amplified NB cells SK-N-BE(2) and IMR-32 through an elevation of oxidative stress and necroptosis. Combined treatment of ISLQ with the chemotherapy agent cisplatin led to a greater cell toxicity in MYCN-amplified NB cells. In chapter 5, the cytotoxic effect of 4HC on NB cells, and its mode of action, was examined. 4HC demonstrated selective cytotoxicity on MYCN-amplified NB cells, which were more sensitive to 4HC than non-amplified SH-SY5Y cells. Cell death induced by 4HC was shown to result from glutathione (GSH) depletion, elevation of oxidative stress and mitochondrial impairment. This thesis, therefore, demonstrates the role of various types of BMPRs in different molecular pathways and survival outcomes in NB, It also highlights the potential of dietary chalcones for either the induction of NB cell differentiation at lower concentrations, through the activation of BMP-Smad signaling, or induction of selective NB cell death at higher concentrations, by elevation of oxidative stress. ItemA genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins(University College Cork, 2019) Baclaocos, Janinah; Atkins, John F.; Mackrill, John; Science Foundation IrelandAfter the genetic code was deciphered in the 1960s, Francis Crick formulated the ‘frozen accident’ hypothesis (Crick, 1968) to describe the origins of the genetic code as universal and resistant to change or evolution. Co-incidentally, evidence of the dynamic nature of genetic decoding emerged through a series of experimental observations which presented various cases of exceptions from what were known as the standard rules of decoding. There is now prevalent understanding and evidence that the genetic code is constantly evolving, and it can be altered by various organisms with possible implications for entire genomes or specific mRNAs. The incorporation of the 21st amino-acid selenocysteine in selenoproteins in response to the UGA translation ‘terminator’ codon is an example of a gene-specific expansion of the code. This thesis will deal primarily with two unique cases of UGA recoding. The first case is the synthesis of selenophosphate synthetase 1 (SPS1) (Chapter 2) whereby an unknown amino acid is inserted in response to a UGA codon in the hymenopteran honeybee, Apis mellifera, which lacks the machinery for Sec incorporation. The various attempts to characterize the amino acid inserted at this position by novel methods are described. In Chapter 3, the first extensive evolutionary analysis of the selenium transporting protein, selenoprotein P (SELENOP) in invertebrates is described with focused characterization in the mollusc, Pacific oyster, Crassostrea gigas. This unique case presented an unprecedentedly high Sec content (46 Sec) in the C-terminal domain of its SELENOP highlighting an extreme case of deviation from the standard genetic code read-out. It was shown that a supplemented heterologous system, was able to facilitate translation of oyster SelenoP mRNA up to the third or fourth Sec codon position of the distal region but was inadequate to produce the full-length protein. Further, the Sec-dedicated protein factor, the oyster SECIS binding protein 2 (SBP2) was characterized and its potential tested for processive Sec-incorporation. Specific mRNA structures in the 3’UTR, termed Selenocysteine Insertion Sequence (SECIS), are essential for the recoding of UGA to specify selenocysteine instead of termination. While previously known multi-Sec codon SelenoP genes have two functionally distinct SECISes, the two in C. gigas showed no distinction in-vitro. In Chapter 4, in-vivo selenium regulation of selenoproteins in C.gigas was investigated by ribosome profiling. Total selenium levels in oyster tissues were found to increase up to 50-fold with supplementation, also resulting to an increase in mRNA abundance and translation. The translation of the full-length Pacific oyster SelenoP demonstrates an inefficient selenocysteine specification at UGA 1 (> 6%) and very high efficiency at the distal UGAs (UGAs 2 to 46). Additional genetic elements relevant to SelenoP translation include a leader ORF, and the RNA structure, termed Initiation Stem Loop (ISL) which were found to potentially modulate ribosome progression in a selenium-dependent manner. It was further validated that selenocysteines were metabolically incorporated in response to UGAs during the synthesis of oyster SELENOP as indicated by 75Selenium labelling experiments. These findings highlight the increasing understanding of the plasticity of the genetic code, as well as the ecological importance of selenium and its diverse utilization across species. ItemRedox biology in retinal degeneration(University College Cork, 2019) Ruiz López, Ana M.; Cotter, Thomas G.; Foundation Fighting Blindness; Science Foundation IrelandRetinitis pigmentosa (RP) involves a group of hereditary diseases that cause progressive and severe visual impairment, with a prevalence of about 1 in 4,000 individuals worldwide. Unfortunately, there is currently no cure for RP and most patients become legally blind by age 40 due to the loss of retinal photoreceptors. The difficulty in finding a treatment relies on the complexity of its genetics. Although more than 3,000 mutations have been described to cause it, about 40-50% of RP cases still correspond to unknown patterns. In most cases, mutations affecting rods, which degenerate first, subsequently produce the death of the remaining photoreceptor cells, the cones. Several therapeutic approaches have been studied during the last decades. In 2011, our group discovered the neuroprotective properties of ‘Norgestrel’, a synthetic progestin used in the female oral contraceptive pill, in the retina. Norgestrel was shown to protect against retinal cell death in three different models: in vitro, in vivo and ex vivo, using retinal explants. In fact, two mouse models were used in order to demonstrate such protection, the balb/c induced light damage model and the genetic rd10 (Pde6b rd10– /rd10– ) model of RP. Since then, some components of its mechanism of action have been elucidated, as is the case of the receptor through which it works, the progesterone receptor membrane component 1 (PGRMC1); the neurotrophic factor basic fibroblast growth factor (bFGF) and its ability to reduce inflammation and gliosis in the diseased retina. Reactive oxygen species (ROS), have been traditionally associated with cellular damage, and have been discovered to participate in signalling responses, including cellular responses that are protetective. The number of studies about their protective properties of ROS have increased in the last decades. Nevertheless, the accumulation of ROS and/or their persistance during time within cells have detrimental consequences. Antioxidant machinery is the defense mechanism that cells possess against harmful ROS. However, this system is not infallible. Worsening of several diseases including RP is known to be produced by the disregulation of intracellular ROS levels, which is known as ‘oxidative stress’. In 2016, we demonstrated that Norgestrel effectively reduces the damaging ROS levels in the balb/c light damage mouse model. In the current study, using the 661W cone photoreceptor-like cell line and retinal explants from rd10 mice, we demonstrated that ROS are used by Norgestrel to enhance cell survival. We found that such stimulation of pro-survival ROS levels occurs very rapidly, and is both PGRMC1 and bFGF-dependent (Chapter 3). Interestingly, we demonstrated that treatment with some antioxidants that prevent the up-regulation of ROS molecules, abrogates the Norgestrel-mediated neuroprotection and therefore, indicates that ROS are a crucial part of this survival response. However, little was known about the subsequent downstream mechanism in the Norgestrel-mediated signalling response that prevents cell death and thus, this study aimed to elucidate other processes that could be implicated in such a response (Chapter 4). Using the genetic rd10 mouse model of RP we additionally demonstrated that Norgestrel was able to reduce the levels of damaging ROS due to its antioxidant properties via stimulating the transcription factor nuclear factor erythroid 2 (NF-E2)-related factor-2 (Nrf2) and its effector protein, superoxide dismutase 2 (SOD2) (Chapter 5). Taken together, this study highlights a dual nature of reactive oxygen species and we have demonstrated for the first time the implication of redox biology in the Norgestrel-mediated neuroprotection against retinal degeneration.