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Spinal Muscular Atrophy (SMA) is caused by mutation or deletion of the Survival Motor Neuron 1 (SMN1), which encodes cell-ubiquitous SMN protein. Although classified as a neuromuscular disease, a range of systemic pathologies is reported in SMA patients. Despite a clear understanding of the genetics, the role of SMN protein in SMA pathogenesis is somewhat unclear, especially in tissues outside the CNS. Here, we describe failed liver development in response to reduced SMN levels, in a Taiwanese mouse model of severe SMA. Molecular analysis revealed significant changes in proteins involved in cell cycling and blood homeostasis including coagulation prior to motor neuron pathology. With SMN being directly associated with some of these proteins, this indicates primary liver pathology in SMA. Study of livers obtained from two other mouse models of SMA; severe SMNΔ7 and intermediate 2B, which have slightly higher SMN levels than Taiwanese SMA mice, also revealed significant overlapping pathologies, suggestive of high intrinsic susceptibility of the liver to SMN decrease. Proteomic study of pre-symptomatic 2B/- liver revealed significant perturbations in mitochondrial bioenergetics, which could account for metabolic defects in SMA patients. Vascular changes can be observed in mouse models of SMA and even skeletal muscle of severe SMA patients. Although Taiwanese SMA liver showed no morphological changes to its vasculature, it does have impairments in several key vascular signaling molecules, including VEGF and Tie-2. Furthermore, we report for the first time significant vascular changes in a zebrafish model of SMA, that could be associated with defective neuronal-vascular signaling and is supported by preliminary findings in the Taiwanese SMA retina. This thesis uncovers perturbations in several clinically relevant signalling pathways directly linked to SMN decrease, independent of the motor neurone pathology. Taken together this work emphasises the importance of a systemic therapy in SMA.

Mitochondrial disorders are considered to be the most common cause of metabolic abnormalities in the paediatric neurology population (Zeviani et al., 1996). These authors reported that the phenotypes observed in 25-30% of the paediatric patients in their neurology clinics were due to a mitochondrial aetiology. The genetic aetiology in an equivalently affected paediatric population in South Africa is currently unknown. This study investigated the possibility that reported mutations could account for the mitochondrial phenotypes observed in the South African population. It focussed on the most frequent paediatric mitochondrial disorders namely: Leigh Syndrome (LS), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and myoclonic epilepsy and ragged red muscle fibres (MERRF). A clinically well characterised group of 25 patients with mitochondrial disorders was included in this study. The molecular analysis of the mitochondrial genome was initially based on a restriction fragment length polymorphism (RFLP) screening strategy for the ten most common mitochondrial DNA (mtDNA) mutations associated with the above¬mentioned three disorders. However, during the study the mutation analysis strategy was modified to a sequencing strategy as this provided more information than the RFLP approach. The modified sequencing strategy extended the study to incorporate fifteen additional mtDNA mutations, associated with other mitochondrial disorders, and individuals included in the study were thus investigated for the presence of 25 mtDNA mutations. Moreover, the modified strategy provided additional information of the regions encompassing the reported mutations. A single patient was observed to harbour the reported A3243G MELAS mutation. This mutation was noted to be heteroplasmic in the proband and two of her maternal relatives. None of the other 24 reported mutations were observed in this patient population. One novel mtDNA alteration in the tRNALeu(UUR) gene was observed in a single patient, although the pathogenicity of this mutation remains to be investigated. Novel and reported polymorph isms, some of which are associated with specific haplogroups, were also observed when comparing sequencing data against the Cambridge reference sequence. The data generated during this study contributed towards the understanding of the uniqueness of the South African population in the global context. This was apparent from the fact that only one of the reported mutations was observed in our patient population who were clinically well characterised and displayed phenotypes similar to those reported internationally. Results form this study underlined the complexity of mitochondrial disorders and argues in favour of whole mitochondrial genome sequence information to be used for diagnostic purposes. Moreover, the results confer with the hypothesis that novel mitochondrial mutations may account for the majority of mitochondrial phenotypes observed in the South African population. / Dissertation (MSc (Human Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

The prenatal period of development is uniquely susceptible to lasting harmful health effects from exposure to environmental toxicants. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants which have a wide variety of associated health effects, including impaired neurodevelopment when exposure occurs in-utero. While a handful of mechanisms have been implicated in PAH neurodevelopmental toxicity, none fully resolve the intricate biological processes that contribute to these outcomes. Mitochondria are increasingly being studied as sensitive targets of many environmental toxicants including PAHs. Despite the known mitochondrial sensitivity to PAHs, and the role of mitochondrial functions in neurodevelopment, little research has been done to evaluate mitochondrial dysfunction as mechanism of PAH neurodevelopmental toxicity. The work of this dissertation seeks to investigate a number of questions on this topic using a wide variety of techniques. We study exposure sources of individual PAHs, the relationship between prenatal PAH exposure constituents and mitochondrial functional outcomes and mitochondrial DNA copy number (mtDNAcn) in multiple biospecimens, including windows of exposure. We employ both epidemiological and experimental techniques, leveraging the advantages of one approach against the weakness of another to draw robust conclusions. Chapter 2 begins by comprehensively studying the demographic and behavioral variables predictive of personal PAH exposure. We combined a significant amount of personal exposure data collected using silicone wristbands with prenatal questionnaires to identify variables most predictive of both exposure to individual PAH compounds and total exposure. This work revealed complex relationships between multiple parameters in the prediction of each individual PAH. We found demographic and socioeconomic variables to be the most common predictors of exposure, followed by behavior variables. This work provides the foundation to identify pathways to reduce exposure and protecting the most vulnerable populations. In chapter 3, we describe two epidemiological studies conducted in Northern Manhattan birth cohorts. The first study uses data from the Columbia Center for Children’s Environmental Health (CCCEH) Fair Start cohort. We measured mitochondrial DNA copy number (mtDNAcn) in umbilical cord tissue, a novel biospecimen with unique utility due to its ease of acquisition and homogenous cellular composition. We measured individual exposure to 63 PAH compounds using silicone wristband samplers and analyze this data using both individual models and quantile G computation to estimate the overall mixture effect. We identified three compounds associated with mtDNAcn in individual models, and a positive association between the mixture of 19 compounds and mtDNAcn. In the second study we expanded upon previous analyses in the CCCEH Mothers and Newborns cohort which had demonstrated an association between summed-total prenatal exposure to 8 carcinogenic PAHs and scores on the Bayley Scale of Infant Development-II at age 3. We used measures of mtDNAcn in umbilical cord blood to evaluate the role of mitochondrial toxicity in PAH neurodevelopment and improved upon prior studies by including adjustment for cell type composition. We utilized both traditional linear model approaches as well as quantile G computation to evaluate the mixture both as a sum-total and using newly developed mixture methods. We determined that while prenatal PAH exposure was negatively associated with umbilical cord blood mtDNAcn using mixtures methods mtDNAcn was not associated with neurodevelopment. The bidirectional effect of prenatal PAH exposure on mtDNAcn between these two studies reveals the complexity of mtDNAcn as a biomarker and the need for more direct measures of mitochondrial functions in the study of PAH neurodevelopmental toxicity. Chapter 4 seeks to complement the epidemiological research with an experimental system. Using mouse preimplantation embryos, we measured the effect of exposure to an environmentally relevant mixture of PAHs on morphological development, superoxide production, mitochondria membrane potential, and mtDNAcn. We found exposure to low levels of a PAH mixture from days 2.5-3.5 post fertilization caused a significant decrease in healthy embryo morphology and a reduction in mtDNAcn. PAH exposure increased mitochondrial membrane potential under several dosing regimens while the effect on superoxide levels was variable and potentially mediated by changes in mitochondrial mass. As a whole these results indicate mitochondrial dysfunction as a result of low-level PAH exposure during the earliest periods of development with a window of heightened susceptibility immediately prior to implantation. In chapter 5 we evaluate the relative mitochondrial potency of the 8 commonly studied carcinogenic PAHs and an environmental relevant mixture of those 8 compounds. Using human umbilical cord mesenchymal stem cells, we specifically study these effects in the context of prenatal development. Superoxide production, mitochondrial membrane potential, mitochondrial mass, cell death and mtDNAcn was quantified at 9 doses for each exposure. This data was used to fit dose response curves and determine relative potency of each exposure/outcome endpoint. We identified benzo[k]fluoranthene and chrysene among the most toxic compounds analyzed and noted differences between relative mitochondrial toxicity and carcinogenicity of these constituents emphasizing the need for continued research into the non-cancer endpoints of PAH exposure. With the intentional comparable exposures and outcomes utilized in these studies comes the opportunity to make connections and draw conclusions across chapters to arrive at four major conclusion: (1) Demographic variables, not behavior, are most predictive of exposure to many PAH compounds (2) prenatal PAH exposure affects multiple measures of mitochondrial functions, (3) there is variability in the susceptibility during early development, and (4) the developmental mitochondrial toxicity of previously studied PAH compounds does not follow the same patterns of relative potency seen in carcinogenesis. This work provides significant insight into the impact of prenatal PAH exposure on mitochondrial functions while highlighting critical areas for further research. More studies are needed to fully understand the mechanisms and long-term effects of PAH exposure on early development, as well as to identify effective interventions to mitigate these risks.

The vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of visual impairment worldwide. The only treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms that regulate the development and lifelong protection of the lens is critical toward the development of future therapies to prevent or treat cataract. To determine those molecules and mechanisms that may be important for these lens requirements we employed high-throughput RNA sequencing of microdissected differentiation statespecific lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified differentiation state-specific molecules that regulate mitochondrial populations between lens epithelial cells that require the maintenance of a functional population of mitochondria and lens fiber cells that must eliminate their mitochondria for their maturation. In addition, we discovered a novel mechanism for how lens epithelial cells clear apoptotic cell debris that could arise from damage to the lens and found that UVlight likely compromises this system. Moreover, the data herein provide a framework to determine novel lens cell differentiation state-specific mechanisms. Future studies are required to determine the requirements of the identified molecules and mechanisms during lens development, lens defense against damage, and cataract formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Diabetic retinopathy is an ischemic retinal neovascular disease causing vision loss among adults. The studies presented involve the design and testing of a gene therapy vector to inhibit retinal revascularization, similar to that found in diabetic retinopathy. Gene therapy has proven to be an effective method to introduce therapeutic proteins to treat retinal diseases. Targeting a specific cell type and expression of therapeutic proteins according to the tissue microenvironment should have an advantage over traditional gene therapy by avoiding unwanted transgene expression. Hypoxia plays a significant role in the pathophysiology of many retinal ischemic diseases. Retinal Mèuller cells provide structural and functional support to retinal neurons, as well as playing a significant role in retinal neovascularization. Targeting Mèuller cells may be an effective strategy to prevent retinal neovascularization under pathological conditions. ... The hypoxia regulated, glial specific vector successfully reduced the abnormal neovascularization in the periphery by 93% and reduced the central vasobliterated area by 90%. A substantial amount of exogenous endostatin was produced in the retinas of P17 OIR mice. A significant increase in human endostatin protein and reduced vascular endothelial growth factor (VEGF) were identified by Western blot and ELISA, respectively. These findings suggest hypoxia-regulated, glial cell-specific scAAV mediated gene expression may be useful to prevent blindness found in devastating retinal diseases involving neovascularization. / by Manas Ranjan Biswal. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed. / by Kori Mulholland. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A&ltG). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.

Current approaches to treat neurodegenerative disease provide only mild symptomatic relief but do not modify the natural history of these conditions. A large body of evidence suggests that mitochondrial dysfunction is a key event in the pathophysiology of neurodegeneration. Supporting and improving mitochondrial function has a big potential as a strategy for neuroprotection. The goal of this dissertation was to test whether interventions that target mitochondrial function are effective at preventing neurodegeneration induced by mitochondrial failure in vivo. A rodent model of optic neuropathy induced by the mitochondrial toxin rotenone was used to test the neuroprotective effects of methylene blue (MB) and near-infrared light (NIL), two interventions with mechanisms of action localized to mitochondria. This work also tested the effects of memantine, an NMDA receptor blocker, to further characterize the relationship between excitotoxicity and mitochondrial dysfunction. Neuroprotective effects were evaluated via behavioral testing of visual function and histopathological analysis of the retina. The neurochemical effects of MB, NIL and memantine were analyzed in vitro and in vivo with indicators of oxidative stress, cell respiration and catalytic activity of respiratory enzymes, including NADH dehydrogenase and cytochrome oxidase. MB, a diaminophenothiazine with potent antioxidant and unique redox properties, prevented the changes in visual function and the retinal histopathology induced by rotenone. In vitro, MB increased oxygen consumption and prevented the increases in oxidative stress in brain tissue induced by rotenone. NIL prevented the behavioral impairment and the decrease in retinal and visual pathway metabolic activity, retinal nerve fiber layer thickness and ganglion cell layer cell density induced by rotenone in a dose-dependent manner. Whole-brain cytochrome oxidase and superoxide dismutase activities were also increased in NIL-treated subjects in a dose-dependent manner, suggesting an in vivo transcranial effect of NIL. Finally, uncompetitive NMDA receptor blockade with memantine displayed neuroprotection against rotenone-induced neurodegeneration in a dose-response manner, and this effect was associated with a decrease in retinal oxidative stress and a long-term increase in neuronal energy metabolism capacity. These data constitute a proof-of-principle that interventions that target the mitochondria and support the function of the respiratory chain are effective at preventing neurodegeneration in vivo. / text