The long noncoding RNA neuroLNC regulates presynaptic activity by interacting with the neurodegeneration-associated protein TDP-43

The cellular and the molecular mechanisms by which long noncoding RNAs (lncRNAs) may regulate presynaptic function and neuronal activity are largely unexplored. Here, we established an integrated screening strategy to discover lncRNAs implicated in neurotransmitter and synaptic vesicle release. With this approach, we identified neuroLNC, a neuron-specific nuclear lncRNA conserved from rodents to humans. NeuroLNC is tuned by synaptic activity and influences several other essential aspects of neuronal development including calcium influx, neuritogenesis, and neuronal migration in vivo. We defined the molecular interactors of neuroLNC in detail using chromatin isolation by RNA purification, RNA interactome analysis, and protein mass spectrometry. We found that the effects of neuroLNC on synaptic vesicle release require interaction with the RNA-binding protein TDP-43 (TAR DNA binding protein-43) and the selective stabilization of mRNAs encoding for presynaptic proteins. These results provide the first proof of an lncRNA that orchestrates neuronal excitability by influencing presynaptic function

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics

Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase. Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed. Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.Peer reviewe

Exploring the neuroblastoma DNA methylome: from biology to biomarker

Neuroblastoma (NB), a childhood tumor arising from immature sympathetic nervous system cells, is a heterogeneous disease with prognosis ranging from excellent long-term survival to high-risk with fatal outcome. In order to determine the most appropriate treatment modality, patients are stratified into risk groups at the time of diagnosis, based on combinations of clinical and biological parameters, namely age of the patient, tumor stage, histology, grade of differentiation, MYCN oncogene amplification, chromosome 11q aberration and DNA ploidy. However, use of this risk classification system has shown that accurate assessment of NB prognosis remains difficult and that additional prognostic markers are warranted. Therefore, we aimed to identify prognostic tumor DNA methylation biomarkers for NB. To find new biomarkers, we profiled the primary tumor DNA methylome using methyl-CpG-binding domain (MBD) sequencing, i.e. massively parallel sequencing of methylation-enriched DNA fractions, captured using the high affinity of MBD to bind methylated cytosines. As proof of principle, we applied this technology to 8 NB cell lines, and in combination with mRNA expression studies, this led to a first selection of 43 candidate biomarkers. Next, methylation-specific PCR (MSP) assays were designed, to allow candidate-specific methylation analysis in a primary tumor cohort of 89 samples. As such, we identified new prognostic DNA methylation biomarkers, and delineated the technological aspects and data analysis pipeline to set up a more extended biomarker study. In this follow-up study, the DNA methylome of 102 primary tumors, selected for risk classification and survival, was characterized by MBD sequencing. Differential methylation analyses between the prognostic patient groups put forward 78 top-ranking biomarker candidates, which were subsequently tested on two independent cohorts of 132 and 177 samples, adopting the high-throughput MSP pipeline of our pilot study. Multiple individual MSP assays were prognostically validated and through the implementation of a newly developed statistical framework, a robust 58-marker methylation signature predicting overall and event-free survival was established. This study represents the largest DNA methylation (biomarker) study in NB so far. The MBD sequencing data were shared with the research community through the format of a data descriptor. As such, these data are fully available to others, ensuring its reusability for other research purposes. To illustrate how these data can be applied to gain new insights into the NB pathology, we characterized the DNA methylome of stage 4S NB, a special type of NB found in infants with widespread metastases at diagnosis that paradoxically is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. More specifically, we compared promoter methylation levels between stage 4S, stage 1/2 (localized disease with favorable prognosis) and stage 4 (metastatic disease with dismal prognosis) tumors, and showed that specific chromosomal locations are enriched in stage 4S differentially methylated promoters and that specific subtelomeric promoters are hypermethylated in stage 4S. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S. In conclusion, by exploring the DNA methylome of NB, we have not only demonstrated that DNA methylation patterns are intimately related to NB biology, but also found additional clinically relevant prognostic biomarkers

DATA-DRIVEN BAYESIAN APPROACH TO THE ANALYSIS OF CELL SIGNALLING NETWORKS IN SYNERGISTIC LIGAND-INDUCED NEURITE OUTGROWTH IN PC12 CELLS

Ph.DDOCTOR OF PHILOSOPH

Application of bioinformatics in studies of sphingolipid biosynthesis

The studies in this dissertation demonstrate that the gene expression pathway maps are useful tools to notice alteration in different branches of sphingolipid biosynthesis pathway based on microarray and other transcriptomic analysis. To facilitate the integrative analysis of gene expression and sphingolipid amounts, updated pathway maps were prepared using an open access visualization tool, Pathvisio v1.1. The datasets were formatted using Perl scripts and visualized with the aid of color coded pathway diagrams. Comparative analysis of transcriptomics and sphingolipid alterations from experimental studies and published literature revealed 72.8 % correlation between mRNA and sphingolipid differences (p-value < 0.0001 by the Fisher's exact test).The high correlation between gene expression differences and sphingolipid alterations highlights the application of this tool to evaluate molecular changes associate with sphingolipid alterations as well as predict differences in specific metabolites that can be experimentally verified using sensitive approaches such as mass spectrometry. In addition, bioinformatics sequence analysis was used to identify transcripts for sphingolipid biosynthesis enzyme 3-ketosphinganine reductase, and homology modeling studies helped in the evaluation of a cell line defective in sphingolipid metabolism due to mutation in the enzyme serine palmitoyltransferase, the first enzyme of de novo biosynthesis pathway. Hence, the combination of different bioinformatics approaches, including protein and DNA sequence analysis, structure modeling and pathway diagrams can provide valuable inputs for biochemical and molecular studies of sphingolipid metabolism.Ph.D.Committee Chair: Alfred H Merrill Jr; Committee Member: Cameron Sullards; Committee Member: I King Jordan; Committee Member: Marion B. Sewer; Committee Member: Stephen C Harve

Multiomic analyses implicate a neurodevelopmental program in the pathogenesis of cerebral arachnoid cysts

Cerebral arachnoid cysts (ACs) are one of the most common and poorly understood types of developmental brain lesion. To begin to elucidate AC pathogenesis, we performed an integrated analysis of 617 patient-parent (trio) exomes, 152,898 human brain and mouse meningeal single-cell RNA sequencing transcriptomes and natural language processing data of patient medical records. We found that damaging de novo variants (DNVs) were highly enriched in patients with ACs compared with healthy individuals (P = 1.57 × 10-33). Seven genes harbored an exome-wide significant DNV burden. AC-associated genes were enriched for chromatin modifiers and converged in midgestational transcription networks essential for neural and meningeal development. Unsupervised clustering of patient phenotypes identified four AC subtypes and clinical severity correlated with the presence of a damaging DNV. These data provide insights into the coordinated regulation of brain and meningeal development and implicate epigenomic dysregulation due to DNVs in AC pathogenesis. Our results provide a preliminary indication that, in the appropriate clinical context, ACs may be considered radiographic harbingers of neurodevelopmental pathology warranting genetic testing and neurobehavioral follow-up. These data highlight the utility of a systems-level, multiomics approach to elucidate sporadic structural brain disease

Structural analysis of carbohydrate – protein interactions under special consideration of lipopolysaccharide fragments, hyaluronic acid, sulfated polysaccharides and sialic acids

Carbohydrate – protein interactions play very important roles in many biological functions. For example, cell-cell recognition, cell adhesion, signal transduction, fertilization, infection and inflammation etc. The better understanding of these binding activities could help to develop more cutting-edge treatment strategies. With the progress of science and technology, more and more new structural analyses of carbohydrate-protein interactions are published. Within this thesis, the focus was on analyzing carbohydrate – protein interactions under special consideration of LPS fragments, hyaluronic acid, sulfated saccharides and sialic acids, with biophysical (NMR, X-ray, SPR), biochemical and cell-biological methods

PRION CHARACTERIZATION USING CELL BASED APPROACHES

Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources