Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Bibliography: leaves 123-137.

Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Bibliography: leaves 121-137.

Wolfram Syndrome (WFS) is a debilitating autosomal recessive neurodegenerative disorder characterized by juvenile onset insulin dependent diabetes mellitus (DM) and optic atrophy (OA) as well as a number of neurological and endocrine complications that result in early death due to respiratory complications. Previous research has mapped Wolfram syndrome to chromosome 4p16.1 and the disease has been attributed to mutations in the WFS1 gene affecting the WFS1 protein (wolframin), an ER membrane glycoprotein that plays an important role in the unfolded protein response (UPR) and in intracellular Ca2+ homeostasis. An additional locus for WFS on chromosome 4q22-24 was identified by linkage studies of four Jordanian Bedouin families with 16 affected individuals (El-Shanti et al., 2000). In this study, we attempted to identify the causative gene for the second WFS locus and identified a single missense mutation in a novel highly conserved iron-sulfur binding domain gene, CISD2, in the three consanguineous families of Jordanian descent from the El-Shanti et al. (2000) study (Amr et al., 2007). A G→C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the missense mutation was found to disrupt messenger RNA splicing by eliminating exon 2 which results in the introduction of a premature stop codon. CISD2 is expressed in a wide variety of tissues, including those affected in WFS, the brain and pancreas. The CISD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein) localizes to the endoplasmic reticulum but does not appear to interact directly with wolframin. Furthermore, lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium. To understand the underlying pathogenesis in WFS2 patients, we examined cell death as well as known stress pathways. Cisd2 was knocked down in three cell lines derived from tissues most affected by the disease, namely rat pancreatic insulinoma cells (INS1), mouse neuroblastoma cells (N1E115), and mouse embryonic stem cell like cells (P19) which could be differentiated into neuronal cells. Cisd2 knockdown in INS1 cells shows an increase in apoptotic cell death and in the expression of the apoptotic markers CHOP and BAX, but no increase in the autophagic marker LC3-II. Assessment of the UPR in CISD2 deficient cells shows no activation of the UPR response, while Cisd2 expression in wild-type INS1 and N1E115 cells did not increase under conditions of ER stress. These findings indicate that there is an increase in apoptosis in WFS2 similar to WFS1 but the pathogenesis involves a molecular mechanism that is different than that in WFS1. Investigation of markers of oxidative stress, another major contributor to diabetes and neurodegeneration, show an increase in expression of the antioxidant enzymes Sod1 and Sod2 as well as an increase in global tyrosine nitration in INS1 Cisd2 knockdown cells compared with controls. Cell death in those cells was exacerbated with addition of known oxidative stressors, thapsigargin and paraquat compared with controls. These findings indicate that oxidative stress is a contributor to WFS2 pathogenesis, but it is not clear whether it is the primary causative factor. A recent article implicated ERIS in the BCL-2 associated inhibition of autophagy (Chang et al., 2009) and showed an increase in levels of autophagy in response to starvation in Cisd2 knockdown in a human epithelial carcinoma cell line (H1299). Starvation of INS1 Cisd2 knockdown cells did not elicit a greater autophagic response compared with controls, but did show an increase in expression of Cisd2. P19 Cisd2 knockdown cells that were differentiated into neurons by retinoic acid treatment did not show an inhibition in differentiation markers, but Cisd2 levels returned to levels similar to pre-differentiation wildtype P19 cells, which indicates that Cisd2 is upregulated during neuronal differentiation. In conclusion, the pathogensis of WFS2 can be attributed to apoptotic death of cells in affected tissues, with oxidative stress and not endoplasmic reticulum stress contributing to the development of disease, while ERIS’ relationship with BCL-2-mediated autophagy and neuronal differentiation suggest its important role in cell differentiation and survival.

Aging is an intrinsic process that is independent of obvious disease. In contrast to normal aging, age-related diseases are conditions that typically manifest at advanced ages, are associated with explicit pathology and cause disability and premature death. We used Drosophila as a model to investigate the molecular-genetic mechanisms associated with aging and age-related disease. Age-related locomotor impairment (ARLI) is a serious condition for the elderly and greatly impacts their quality of life. Toward identifying genes and mechanisms that influence ARLI, we performed a forward genetic screen using Drosophila mutants. This screen identified a loss of function mutant in PDK1, a component of the insulin signaling pathway. Additional loss of function mutants in the insulin signaling pathway genes PI3K Dp110, and AKT also delayed ARLI. These results suggest a role for insulin signaling in ARLI. Wolfram Syndrome (WFS) is a progressive neurodegenerative disease that is caused by mutations in the genes WFS1 and CISD2. The function of CISD2, the most recently identified gene has not been fully resolved. We used RNAi to knockdown wfs2, the fly ortholog of CISD2 to identify genes and pathways associated with wfs2 that will provide insight into the normal function of this gene. Through a targeted genetic screen in the Drosophila eye we identified that wfs2 interacts with two lysosomal storage disease genes PPT1 and CLN3. These results suggest that WFS and lysosomal storage diseases may be influenced by common molecular-genetic mechanisms. Furthermore, wfs2 may play a role in the neurodegenerative pathways associated with lysosomal storage disease. Oxidative stress is associated with aging and age-related disease. To identify genes that can protect against endogenous oxidative stress we performed a candidate suppressor screen. This screen revealed that expression of wild-type Ataxin-3 suppressed the short lifespan of Sod2 knockdown flies. The ubiquitin associated function of Ataxin-3 was determined to be important for this suppression. Interestingly, Ataxin-3 expression also extended the short lifespan due to knockdown of thioredoxin reductase in muscle. These results suggest that Ataxin-3 expression may play a protective role against enhanced endogenous oxidative stress due to reduced function of a number of antioxidant enzymes.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration. Two causative genes have been identified so far, WFS1 and WFS2, both encoding endoplasmic reticulum (ER) localized transmembrane proteins. Since WFS1 is involved in the ER stress pathway, Wolfram syndrome is considered an ER disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome, the molecular mechanism linking ER to the death of β cells and neurons has not been elucidated. The endoplasmic reticulum (ER) is an organelle that forms a network of enclosed sacs and tubes that connect the nuclear membrane and other organelles including Golgi and mitochondria. ER plays critical functions in protein folding, protein transport, lipid metabolism, and calcium regulation. Dysregulation of ER function disrupts normal cell metabolism and activates an array of anti-survival pathways, eventually leading to disease state. Here we show that calpain is involved in both prototypes of Wolfram syndrome. Calpain 2 activity is negatively regulated by WFS2 protein, and hyper-activation of calpain 2 by WFS2-knockdown leads to cell death. Calpain hyper-activation is also present in WFS1 loss of function cells due to the high cytosolic calcium. Extensive calpain activation exists in the Wolfram syndrome mouse model as well as in patient cells. A compound screen targeting ER homeostasis reveals that dantrolene, a ryanodine receptor inhibitor, can prevent cell death in cell models of Wolfram syndrome. Our results demonstrate that the pathway leading to calpain activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Le Syndrome de Wolfram (SW; OMIM #222300, prévalence 1-9 / 1000 000) est une maladie neurodégénérative, qui se présente avec un début juvénile, intégrant le diabète insipide, diabète sucré, l’atrophie optique (AO), et la surdité. AO est généralement son premier symptôme neurologique, commençant à l’âge de 11 ans et se terminant par la cécité 8 ans plus tard. Malheureusement, un modèle murin du SW approprié aux symptômes ophtalmologiques n'a pas encore été trouvé, donc la recherche de la thérapie pour sauver la vision en est à ces débuts. Dans cette thèse j’ai étudié l’atteinte visuelle de deux modèles de souris mutantes pour le SW et succès d’une approche de thérapie génique (TG) avec le gène humain WFS1.Premièrement, les souris Wfs1exon8del sont été examinées à 3 et 6 mois pour l’acuité visuelle (AV) et la sensibilité aux contrastes (SC) via changements dans le reflexe optomoteur (ROM), la fonction rétinienne neurale par électrorétinogramme (ERG), ainsi que la physiologie de l’œil par la fondoscopie et tomographie par cohérence optique (TCO). De plus, la proportion des cellules ganglionnaire de la rétine (CGRs) et la perte axonale dans le nerf optique (NO) à 7 mois ont été examinés avec marquage anti-Brn3a et microscopie électronique, respectivement. Il y avait une perte progressive de l’AV et la SC chez les souris KO à partir du 1 mois. Elle était accompagnée d'une pâleur du disque optique (DO), d'amincissement de la rétine ainsi que des lésions axonales. Par contre, il n’avait pas de perte des CGRs ni stress du réticulum endoplasmique dans la rétine. Brièvement, les souris KO présentent un phénotype ophtalmique du SW significatif et peuvent servir comme modèle.Deuxièmement, à la recherche d'un autre modèle du SW, les fonctions visuelles de la lignée Wfs1E864K de la souris ont été étudiées. Déjà à 1 mois, les souris Wfs1E864K/E864K avait une perte drastique de la fonction des CGRs, mais en gardant le nombre de cellules à un niveau normal. Ceci a été accompagné par un amincissement de la rétine et d’un sévère dommage du NO, comme montré par le TCO et la fondoscopie, respectivement. En conséquence, les souris Wfs1E864K/E864K, avec leur fort phénotype ophtalmique, pourraient servir comme modèle du SW classique.Enfin, pour enquêter sur les futures options de traitement contre le SW, les souris de la lignée Wfs1exon8del à 1 mois ont subi une TG intravitréenne avec AAV-2/2-CMV-WFS1. Les examens à 3 et 6 mois ont montré une amélioration de l’AV, ainsi que le sauvetage de la pâleur du DO et réduction des lésions axonales chez les souris KO. En outre, aucun effet indésirable lié à des injections TG n’ont été noté. Suivant cette idée, les souris Wfs1E864K/E864K ont également été soumis à la TG intravitréenne, délivrée à P14, mais sans succès.En conclusion, la lignée Wfs1exon8del de la souris est un modèle fiable du SW, y compris les aspects visuels. Je propose le modèle Wfs1E864K/E864K comme une alternative, en particulier pour enquêter sur la fonction de Wfs1 dans l'œil. Enfin, la GT intravitréenne avec WFS1 a un potentiel pour sauver partiellement le phénotype ophtalmique, ouvrant la voie vers le traitement pour les patients du SW / Wolfram Syndrome (WS; OMIM #222300, prevalence 1-9 / 1 000 000) has a juvenile onset and incorporates diabetes insipidus, diabetes mellitus, optic atrophy (OA), and deafness; leading to death in middle age. OA is its first neurological symptom, starting in adolescence and ending with blindness within 8 years. Unfortunately, a suitable WS mouse model comprising ophthalmologic symptoms has not yet been found, therefore the search for its treatment is delayed. In this thesis, I studied visual impairment in two WS mouse models along with a success of a gene therapy (GT) approach with the human WFS1 gene.Firstly, 3 and 6 months old Wfs1exon8del mice were examined for the visual acuity (VA) and contrast sensitivity via changes in the opto-motor reflex (OMR), the neural retinal function via electroretinogram (ERG), as well as the eye physiology via fundoscopy and optic coher-ence tomography (OCT). Also, the proportion of retinal ganglion cells (RGC) and the axonal loss at the age of 7 months were determined with anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve (ON) sections, respectively. There was a progressive loss of VA and contrast sensitivity in Wfs1exon8del-/- mice, starting already at 1 month of age. It was accompanied by optic disc pallor, retinal thinning as well as axonal damage. However, there was no RGC loss and the endoplasmic reticulum (ER) stress in the retina was at a normal level. It suggested a presence of another cause for the reported degeneration in KO mice; in opposition to what was proposed in the literature. I brief, KO mice exhibit significant WS ophthalmic phenotype.Secondly, in search for another model, visual functions of Wfs1E864K mouse line were investigated. This line was originally a model of Wolfram-like Syndrome, characterized by dominant mutations in WFS1 leading to congenital progressive hearing impairment, diabetes mellitus and OA. Only homozygous mutants, however, showed expected visual impairment. Already at 1 month of age, Wfs1E864K/E864K mice had drastic loss of RGC function, albeit keeping the cell number at a normal level. This was accompanied by retinal thinning and a severe ON damage, as shown with OCT and fundoscopy, respectively. In contrast, the RGC function in Wfs1E864K/+ mice dropped slightly only at the age of 7 and 12 months, showing that the pathology of the E864K mutation-driven disease in mice is different than in humans. Therefore, Wfs1E864K/E864K mice, with their strong ophthalmic phenotype, could potentially serve as a model of the classical WS.Finally, to investigate future treatment options, 1 month old Wfs1exon8del+/+ (WT) and Wfs1exon8del-/- (KO) mice underwent a uni- and bi-lateral intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1. Exams at 3 and 6 months of age showed improved VA, as well as optic pallor and axonal damage rescue in KO mice. Also, no adverse effects related to either GT or sham injections were noted. Following this idea, the Wfs1E864K/E864K mice were also subjected to intravitreal GT, delivered at P14, but without success.In conclusion, Wfs1exon8del mouse line is a reliable model of WS, including the visual aspects. I propose the Wfs1E864K/E864K model as an alternative, especially to investigate Wfs1 function in the eye. Finally, the intravitreal AAV-driven GT with WFS1 has a potential to partially rescue the ophthalmic phenotype, paving the wave towards the treatment for WS patients.

Introdução: O diabetes monogênico corresponde de 1% a 2% de todos os casos de diabetes mellitus, sendo causado por variantes em um único gene. Dentre esses, o mais comum é o MODY (Maturity Onset Diabetes of the Young), havendo, porém, diversas formas mais raras, como diabetes neonatal e sindrômico que podem estar associadas a outras comorbidades além do diabetes, bem como às lipodistrofias hereditárias. O diagnóstico genético permite a adequação do tratamento, seguimento clínico e aconselhamento familiar. Objetivos: (1) desenvolver e implantar um painel customizado de sequenciamento em larga escala para o diagnóstico genético-molecular das formas raras de diabetes monogênico e lipodistrofias hereditárias; (2) estabelecer o diagnóstico genético de casos com suspeita clínica; (3) realizar a correlação genótipo-fenótipo. Métodos: Os pacientes foram selecionados de acordo com os critérios de inclusão para cada tipo de diabetes monogênico raro. A pesquisa genética dos casos-índice foi feita por sequenciamento em larga escala. A segregação familiar e confirmação dos achados foram feitas pelo método de Sanger. A análise de bioinformática foi realizada considerando o tipo de variante, frequência em bancos de dados populacionais, predição in silico e segregação familiar. Resultados: Foram analisados 42 casos, sendo que em 23 foram encontradas variantes candidatas: 6/16 com diagnóstico de diabetes neonatal, 2/2 com quadro clínico de Síndrome de Wolfram, 11/11 com suspeita de lipodistrofia congênita generalizada, 4/13 com lipodistrofia parcial familiar. Conclusões: Um painel customizado de sequenciamento em larga escala permitiu o diagnóstico genético-molecular de diabetes monogênico, com positividade de 22/42 casos analisados, sendo possível realizar a correlação genótipofenótipo. O diagnóstico genético possibilitou o aprimoramento do seguimento clínico dos pacientes e suas famílias. Permitiu, ainda, aumentar o número de diagnósticos de casos anteriormente subdiagnosticados / Introduction: Monogenic Diabetes accounts for 1 to 2% of all cases of diabetes mellitus. It is caused for variants in a single gene. Among these, the most common is MODY (Maturity Onset Diabetes of the Young), but there are several other rare forms, which may be associated with other comorbidities besides diabetes. Genetic diagnosis can lead to appropriate treatment and follow-up, besides and family counseling. Objectives: (1) to develop a customized targeted massively parallel sequencing panel to sequence the rare types of monogenic diabetes; (2) To establish the genetic diagnosis of probands with clinical suspicion of monogenic diabetes; (3) To correlate their phenotype with genetic findings, leading to a better understanding of rare monogenic diabetes subtypes. Methods: Patients were selected according to the inclusion criteria for each type of rare monogenic diabetes. Genetic research of index cases was done by massively parallel sequencing. Family segregation and confirmation of the variants findings were done by Sanger method. Bioinformatics analysis was performed considering the type of variant, frequency in population databases, in silico prediction and family segregation. Results: We analyzed a total of 42 cases. We found 22 candidate causal variants: 6/16 in probands with Neonatal Diabetes, 2/2 with Wolfram Syndrome, 11/11 with suspected Generalized Congenital Lipodystrophy, and 4/13 with Familial Partial Lipodystrophy. Conclusions: A customized targeted massively parallel sequencing panel allowed genetic diagnosis of rare types of monogenic diabetes, with a positivity of 23/42 cases analyzed, being possible to perform the genotype-phenotype correlation. The genetic diagnosis allowed the improvement of the clinical follow-up of the patients and their families. It also increased the number of diagnoses of previously underdiagnosed cases

The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis. Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK). In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis. This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress. This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.