Synapses are an early pathological target in a wide range of neurodegenerative conditions including adult-onset Alzheimer’s and Parkinson’s, and diseases of childhood such as spinal muscular atrophy and neuronal ceroid lipofuscinoses (NCLs). However, our understanding of the mechanisms regulating the stability of synapses and their exceptional vulnerability to neurodegenerative stimuli remains in its infancy. To address this, we have used the NCLs to model the molecular alterations underpinning synaptic vulnerability. Our primary objective is to identify novel regulators of synaptic stability as well as highlight novel therapeutic targets which may prove effective across multiple neurodegenerative conditions where synapses are an early pathological target. The NCLs, are the most frequent autosomal-recessive disease of childhood. There are currently 14 individual genes whose mutations result in similar phenotypes including blindness, cognitive/motor deficits, seizures and premature death. This suggests that despite the difference in the initiating mutation and the degenerative processes across this collective group are likely to impact on overlapping pathways. Focusing on two murine models of NCL; one with an infantile onset - CLN1 disease (Ppt1-/-) and one with a juvenile onset - CLN3 disease (Cln3-/-) we made use of the temporo-spatial synaptic vulnerability pattern in these mice to plan proteomic and in silico analyses. This pipeline was utilised to identify perturbed protein candidates and pathways correlating with differential regional synaptic vulnerability. This ultimately allowed the generation of a list of candidate proteins, some of which were relevant to human NCL as they were altered in post mortem brain samples. Interestingly, many of the correlative candidates also appear to show conserved alterations in both NCL forms examined and other neurodegenerative diseases. Next, candidates were genetically and/or pharmacologically targeted to study their modulatory effects on neuronal stability in vivo. This was done using CLN3 Drosophila as a rapid screening assay and led to the successful characterisation of a subset of candidates as either enhancers or suppressors of the CLN3-induced phenotype in vivo. As well as identifying regulators of neuronal stability, following a similar pipeline, we identified a set of putative biomarkers of disease progression in muscle and blood in the Ppt1- /- mice, a subset of which appeared conserved in Cln3-/- mice. One of these conserved candidates presented the same directionality of change in human post mortem brain samples, indicating its relevance to the human NCL. Following this workflow from spatio-temporal profiling of murine synaptic populations, to in silico analyses and in vivo phenotypic assessment, we demonstrate that we can identify multiple protein candidates capable of modulating neuronal stability in vivo and identified putative biomarkers that tracked disease progression.

Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Stella Yu-Chien Lee / Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative lysosomal storage disorders which is the most frequent group of inherited neurodegenerative disorders that affect children leading to severe pathological conditions such as progressive loss of motor neuron functions, loss of vision, mental retardation, epilepsy, ataxia and atrophy in cerebral, cerebella cortex and retina and eventually premature death. Among the many genes that cause NCL, mutations in CLN5 leads to different forms of NCL (infantile, late infantile, juvenile and adult) and mutations in CLN8 leads to progressive epilepsy with mental retardation (EPMR) and a variant late infantile form of NCL. The function(s) of both CLN5 and CLN8 proteins remain elusive. CLN5 is a glycosylated soluble protein that resides in the lysosome. We observed that endogenous CLN5 protein exist in two forms and identified a previously unknown C-terminal proteolytic processing event of CLN5. Using a cycloheximide chase experiment we demonstrated that the proteolytic processing of CLN5 is a post-translational modification. Furthermore treatment with chloroquine showed the processing occurs in low pH cellular compartments. After treatment with different protease inhibitors our results suggested the protease involved in the processing of CLN5 could be a cysteine protease. Using two glycosylation mutants of CLN5, retained in the endoplasmic reticulum (ER) or the Golgi we showed the proteolytic processing occurs in an organelle beyond the ER. This study contributes to understanding the characteristics of the CLN5 protein. CLN8 is an ER resident transmembrane protein that shuttles between the ER and the ER-Golgi intermediate compartment (ERGIC). In our study we identified a potential interaction between CLN8 and a PP2A holoenzyme complex consisting regulatory subunit A α isoform and regulatory subunit B α isoform. Using two CLN8 patient derived fibroblast cell lines we were able to show that the phosphorylated levels of PP2A target kinase Akt was reduced at both of its regulatory sites Ser473 and Thr308 and the activity of PP2A was increased. A delay of ceramide transport from ER to Golgi in CLN8 deficient patient cell lines was observed using BODIPY FL C5-Ceramide staining. Our results provide evidence for CLN8 protein being involved in the regulation of PP2A activity and trafficking of ceramide from ER to Golgi.

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.

Next generation sequencing technologies are changing the way scientific experiments and diseases diagnostics are performed and thus will allow what is called personalized medicine. The sense of presented thesis is to make survey of new approaches to DNA sequencing and demonstrate usage and constraints of bioinformatic analytical tools available to day. Discussed techniques are then applied to the case study of finding molecular basis for rare hereditary disease. Introductory part deals with overview of commercially available sequencing techniques (454 Life Science, Applied Biosystems, Illumina, Helicos). Fundamentals of each method are described and possible further development is outlined. Post sequencing data analysis is than discussed in details. In practical section we demonstrate genome analysis techniques successfully used to reveal causal mutation in the gene responsible for adult form of autozomal neuronal ceroid lipofuscinosis (ANCL). Combination of linkage analysis (Merlin), copy number variant analysis (Genome-Wide Human SNP Array 6.0), analysis of expression profiles (HumanRef-8 v2 Expression BeadChips) and exome sequencing (SOLiD™ 4 System) has been applied to members of one ANCL family. We also paid attention to comparison, evaluation and selection of available mapping algorithms used in...

Master of Science / Division of Biology / Stella Y Lee / Neuronal Ceroid Lipofuscinoses (NCLs) are a group of lysosomal storage diseases that are characterized by accumulating autofluorescent lipopigments in cells. NCLs are a form of progressive neurodegenerative diseases with symptoms ranging from blindness, loss of speech and motor activities to ataxia and seizures. Patients do not live to adulthood in most cases, making it prevalent in children. Among the many genes that cause NCL, CLN5 leads to different forms of NCL (infantile, late infantile, juvenile, and adult). CLN5 protein resides in the lysosomes but its function has not been established. It is predicted to contain eight N-glycosylation sites, but the role of N-glycosylation on its function and trafficking has not been assessed. We analyzed the role of N-glycosylation on the transport and stability of human CLN5. We created N-glycosylation mutants of each site by changing the Asn to Gln and our analysis of these mutants show that all the eight N-glycosylation sites are used in vivo. We also report effects of abolishing individual N-glycosylation sites on the trafficking of CLN5. While the lack of glycosylation at some sites results in CLN5 being retained in the ER or Golgi, others do not affect CLN5 trafficking. Cycloheximide chase experiments show that one of the mutants (N401Q) in CLN5 leads to lower protein levels in cell pellets with an increased secretion compared to CLN5 wild type, while other mutations show differential stability in cell pellets. These results demonstrate that each N-glycosylation site plays a different role(s) in the stability, transport and/or function of CLN5.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 112-129.

No description available.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases predominantly affecting infants and children, which in some cases can present into adulthood. There are fourteen genes comprising the 13 known subtypes of NCLs (CLN1-CLN8, CLN10-CLN14; CLN9 has been reclassified as CLN4). The NCL diseases share common molecular and clinical features, including cellular accumulation of autofluorescent storage material, characteristic histological findings (curvilinear inclusions, fingerprint profiles, and granular osmophilic deposits), markedly low brain weight, seizures, blindness, motor dysfunction and behavioral disabilities. Though the functions of the CLN proteins are not fully understood, they are mainly localized to the lysosomal compartment and autophagic pathway. Previous works have focused on understanding the individual functions of the CLN proteins. However, there is little research examining the interactions between CLN proteins and their involvement in neurogenesis. The CLN proteins also show involvement in various other signaling pathways, notably the mTOR and p53 pathways, and may therefore have implication as important signaling molecules during development and aging. In this thesis, I outline a variety of interactions between CLN proteins, as well as their role in lysosome formation and autophagy. I further examine the involvement of these proteins in lysosomes of microglia, and potential functions of microglia during neurogenesis in childhood and adulthood. I hypothesize that the CLN proteins are likely involved in a common pathway which is highly regulated during neurogenesis through microglial release of pro-inflammatory molecules. Though these diseases are incurable, enzyme replacement shows promise as a treatment for NCL; cerliponase alpha (BioMarin Pharmaceuticals) is the first and only FDA-approved enzyme replacement treatment for CLN2 disease. Future in-depth investigation of protein-protein interactions as well as their involvement in signaling pathways during development is necessary in order to find a cure for these devastating diseases.