Huber, Robert J

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Mutations in the CLN7 (MFSD8) gene, causes CLN7 disease, a subtype of neuronal ceroid lipofuscinosis. MFSD8 is a lysosomal transmembrane protein that transports chloride across membranes. Experimentation regarding Dictyostelium discoideum revealed that mfsd8 deficiency altered lysosomal enzyme activity. During starvation, the aggregation of mfsd8¬¬- cells was delayed, and cells formed more mounds that were smaller in size, phenotypes that were attributed to reduced cell-substrate adhesion and altered lysosomal enzymatic activities. This study examines the possible transcriptomic and secretomic basis for these phenotypes. This work generated new datasets for examining the effect of mfsd8 loss on the transcriptome and secretome. The validity of these datasets was supported by use of western blotting and RT-PCR along with a set of assays probing relevant biological processes. Together these results elucidate the biological mechanisms behind the observed phenotypes and lay the foundation for future studies to further study the cellular role of MFSD8.

Author Keywords: Battens disease, CLN7, Dictyostelium discoideum, MFSD8, Secretome, Transcriptome

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions.

Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses

Giardia intestinalis is a waterborne enteric parasite that lacks mitochondria and the capacity for heme biosynthesis. Despite this, Giardia encodes several heme proteins, including four cytochrome b5 isotypes (gCYTB5-I – IV) of unknown function. The aim of this thesis is to gain insight into the function of the Giardia cytochrome b5 isotype III (gCYTB5-III) that is found in the nucleus, as first reported by our laboratory using immunofluorescence microscopy experiments with an isotype-III specific antibody. Nuclear localization of isotype-III is supported by two of my experiments: i) immunoblot analysis of crude cytoplasmic and nuclear enriched fractions of Giardia trophozoites; ii) association of gCYTB5-III with the insoluble fraction of Giardia lysates crosslinked with formaldehyde is reversed by DNase I treatment. To gain an understanding of the possible roles of gCYTB5-III, I performed immunoprecipitation (IP) experiments on lysates from Giardia trophozoites to identify its protein partners. Mass spectroscopy analysis of the immunoprecipitate identified proteins localized to the nucleus (RNA polymerase, DNA topoisomerase, histones, and histone modifying enzymes). Intriguingly, over 40% of the known mitosomal proteome, which functions in iron-sulfur (Fe-S) cluster assembly was also associated with gCYTB5-III. One of these proteins, the flavoenzyme GiOR-1, has been shown to mediate electron transfer from NADPH to recombinant gCYTB5-III. These IP results provide evidence that GiOR-1 and gCYTB5-III interact in vivo, and furthermore, suggest that some proteins in the mitosome could interact with those in the nucleus. I also found that DNA stress, caused by low concentrations of formaldehyde (0.1 – 0.2%) resulted in the increased expression of gCYTB5-III. Collectively these findings suggest a role of gCYTB5-III in Giardia's response to DNA stress and perhaps the formation of Fe/S clusters.

Author Keywords: cluster, cytochrome, heme, iron, mitosome, nuclear