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Neurodegenerative diseases represent a large group of neurological disorders with heterogeneous clinical and pathological presentations--often affecting a specific subset of cells in particular anatomical regions. These complex and often overlapping features presents a challenge in identifying the cause behind the vulnerability of these cell populations and anatomical area. However, with recent advancements in omics technology, the complexity of the mammalian brain is being rapidly teased out. Two of these technologies offer a unique perspective and deeper resolution to study the heterogeneity in the brain: long-read sequencing and single-cell microfluidics. In this dissertation, I demonstrate the use of one or both technologies to further characterize the heterogeneity in human brain and its relationship to various neurodegenerative diseases. My initial work identifies a novel genomic structural variant in the gene SNCA, a causal gene in Parkinson's disease (PD) and other synucleinopathies. These intron-less sequences are known as gencDNAs (genomic cDNA) and are found in human cortical neurons of both PD and age-matched controls in a mosaic manner. Furthermore, it is unveiled that a small subset of copies contains point mutations in loci reported to cause monogenic PD, if mutated in its native counterpart, when using long-read amplicon sequencing. Though much is unknown about of SNCA gencDNAs, it is hypothesized to be created due to a reverse transcription event and re-inserted back into the genome. My subsequent work combines both single-cell RNA sequencing and long-read sequencing to examine and compare the transcriptome of various neurodegenerative diseases in the frontal cortex at a single cell resolution. Despite being a synucleinopathy, dementia with Lewy bodies (DLB) has more transcriptomic similarities to Alzheimer's disease (AD) than with PD. While profiling the isoforms of selected genes within these diseases, a vast number of novel isoforms are also identified, with some specific to a particular cell type, such as NRGN. Finally, some major isoform switching is observed in at a cell type resolution, even in genes that were not differentially expressed in our short-read RNA-seq data, such as BIN1 in DLB oligodendrocyte and CLU in AD and DLB excitatory neurons. This dissertation further illuminates the heterogeneity in the human brain at a genomic and transcriptomic layer in the context of various neurodegeneration using long-read sequencing and single cell microfluidics.
DNA copy number variations (CNVs) have previously been reported in human cortical neurons from non-diseased patients, but these alterations do not appear to be consistent from cell to cell and appear to be rare among neurons overall. Interestingly, Alzheimer's disease patients appear to have a higher prevalence of CNVs than non-diseased, although the biological significance of this observation is still largely unknown. Single-cell whole-genome next-generation sequencing holds promise to investigate these variations and the regions in which they occur in an unbiased manner. Unlike recent advances in single-cell RNA-seq, however, library preparation for single-cell DNA-seq suffers from extremely limited throughput. Furthermore, it is difficult to assess the significance of individual variations from whole-genome sequencing alone, particularly when control samples from non-diseased patients also show some variation at lower frequency. A potential solution is a multi-omics approach, in which information is collected about multiple species of biomolecules simultaneously from each sample, which taken together aid the interpretation of individual observations with respect to biological significance. This dissertation describes the design and development of a technology to physically separate DNA and RNA and to prepare sequencing libraries from each in parallel from limited starting samples without splitting, which we called Gel-seq. Thirty-two paired DNA and RNA sequencing libraries were successfully prepared from a variety of human and mouse cells lines and from mouse liver tissue using Gel-seq. Sample types could be clearly distinguished from each other based on either genomic copy number or transcriptomic profiles. This dissertation also describes the design and development of a technology to prepare a thousand single-cell whole-genome sequencing libraries in a single run. A proof-of-concept was performed with 87 cells from human and mouse lines. Copy number profiles agreed with bulk, and 96% and 92% of human and mouse cells, respectively, clustered correctly within their cell line based on copy number profile alone. These technologies will help to enable the unbiased characterization of genomic alterations not only in neurodegenerative disorders, but potentially also in other conditions associated with mosaic genomic backgrounds, such as cancer, microbiome disorders, or infectious diseases.
The human brain can be organized using various different layers of information about the cells: epigenetic, genomic, transcriptomic, proteomic, etc. Recent endeavors have put tremendous effort into mapping the brain cell-by-cell using these layers of information. A challenge associated with these multi-modal approaches is being able to parse through the giga- to terabyte scale amount of data that is generated. My thesis work has focused on investigating the diversity of the brain's genome (DNA) and transcriptome (RNA) and developing bioinformatic tools to make that possible. My work can be broken into two general categories, addressing the genome and the transcriptome. On the genomic side, I focused on identifying novel features known as gencDNAs (genomic cDNAs). gencDNAs are hypothesized to result from transcription of a highly expressed gene which is then spliced, reverse-transcribed, and inserted back into the genome at the site of a DNA strand break. These novel sequences are predicted to be functional, resulting in additional translation of a protein. APP, the amyloid precursor protein gene, was the first gene to be identified as a gencDNA and was determined to be more prevalent in neurons of Alzheimer's disease (AD) patient brains. I developed an unbiased approach to identify additional gencDNAs in the genome from short-read sequencing data. The transcriptome can be studied at various resolutions. Through several projects, I examined gene expression at the single-cell level, and I additionally characterized full-length isoforms using long-read sequencing technologies. Recent advances in sequencing have made it possible to sequence the entire lengths of mRNA transcripts. This technology is relatively new, and bioinformatic tools need to be developed to handle this type of data. While several packages and tools exist for quality control, alignment, reduction of redundancy, and annotation, a tool for comparing isoforms (known and novel) across multiple samples and groups is not available. I made a database-driven tool for this purpose that is compatible with current analysis pipelines. The applications of this software were demonstrated by examining a dataset from the 1000 Genomes Project in addition to a large single-cell dataset investigating gene and isoform expression changes in several neurodegenerative diseases.
The Molecular and Cellular Basis of Neurodegenerative Diseases: Underlying Mechanisms presents the pathology, genetics, biochemistry and cell biology of the major human neurodegenerative diseases, including Alzheimer’s, Parkinson’s, frontotemporal dementia, ALS, Huntington’s, and prion diseases. Edited and authored by internationally recognized leaders in the field, the book's chapters explore their pathogenic commonalities and differences, also including discussions of animal models and prospects for therapeutics. Diseases are presented first, with common mechanisms later. Individual chapters discuss each major neurodegenerative disease, integrating this information to offer multiple molecular and cellular mechanisms that diseases may have in common. This book provides readers with a timely update on this rapidly advancing area of investigation, presenting an invaluable resource for researchers in the field. Covers the spectrum of neurodegenerative diseases and their complex genetic, pathological, biochemical and cellular features Focuses on leading hypotheses regarding the biochemical and cellular dysfunctions that cause neurodegeneration Details features, advantages and limitations of animal models, as well as prospects for therapeutic development Authored by internationally recognized leaders in the field Includes illustrations that help clarify and consolidate complex concepts
This volume provides an introduction to the essential techniques required for studying the molecular biology of brain disease. The approaches and strategies for investigations of gene structure and regulation are described with reference to the molecular genetics of prion and Alzheimer's disease. The effects of aberrant gene regulation can also be examined at the protein level by immunocytochemistry and autoradiography. Improved understanding of basic biology has resulted in new approaches to animal models using transgenic techniques and new therapeutic approaches. The volume is structured to illustrate all these approaches and demonstrate the practice and promise of molecular neuropathology.
Cyclin Dependent Kinase 5 provides a comprehensive and up-to-date collection of reviews on the discovery, signaling mechanisms and functions of Cdk5, as well as the potential implication of Cdk5 in the treatment of neurodegenerative diseases. Since the identification of this unique member of the Cdk family, Cdk5 has emerged as one of the most important signal transduction mediators in the development, maintenance and fine-tuning of neuronal functions and networking. Further studies have revealed that Cdk5 is also associated with the regulation of neuronal survival during both developmental stages and in neurodegenerative diseases. These observations indicate that precise control of Cdk5 is essential for the regulation of neuronal survival. The pivotal role Cdk5 appears to play in both the regulation of neuronal survival and synaptic functions thus raises the interesting possibility that Cdk5 inhibitors may serve as therapeutic treatment for a number of neurodegenerative diseases.
This book represents the third in a series of International Conferences related to Alzheimer's (AD) and Parkinson's (PD) diseases. The first one took place in Eilat, Israel, in 1985; and the second one in Kyoto, Japan, in 1989. This book contains the full text of oral and poster presentations from the Third International Conference on Alzheimer's and Parkinson's Diseases: Recent Developments, held in Chicago, Illinois, U.S.A. on November 1-6, 1993. The Chicago Conference was attended by 270 participants. The Scientific Program was divided into nine oral sessions, a keynote presentation, and a poster session. The conference culminated in a Round Table Discussion involving all of the participants in the conference. The four and one-half day meeting served as an excellent medium for surveying the current status of clinical and preclinical developments in AD and PD. There were 59 oral presentations and 93 posters. This book incorporates a majority of both.
This monograph describes the progress in neuropathological HD research made during the last century, the neuropathological hallmarks of HD and their pathogenic relevance. Starting with the initial descriptions of the progressive degeneration of the striatum as one of the key events in HD, the worldwide practiced Vonsattel HD grading system of striatal neurodegeneration will be outlined. Correlating neuropathological data with results on the functional neuroanatomy of the human brain, subsequent chapters will highlight recent HD findings: the neuronal loss in the cerebral neo-and allocortex, the neurodegeneration of select thalamic nuclei, the affection of the cerebellar cortex and nuclei, the involvement of select brainstem nuclei, as well as the pathophysiological relevance of these pathologies for the clinical picture of HD. Finally, the potential pathophysiological role of neuronal huntingtin aggregations and the most important and enduring challenges of neuropathological HD research are discussed.
Motivated by the explosion of molecular data on humans-particularly data associated with individual patients-and the sense that there are large, as-yet-untapped opportunities to use this data to improve health outcomes, Toward Precision Medicine explores the feasibility and need for "a new taxonomy of human disease based on molecular biology" and develops a potential framework for creating one. The book says that a new data network that integrates emerging research on the molecular makeup of diseases with clinical data on individual patients could drive the development of a more accurate classification of diseases and ultimately enhance diagnosis and treatment. The "new taxonomy" that emerges would define diseases by their underlying molecular causes and other factors in addition to their traditional physical signs and symptoms. The book adds that the new data network could also improve biomedical research by enabling scientists to access patients' information during treatment while still protecting their rights. This would allow the marriage of molecular research and clinical data at the point of care, as opposed to research information continuing to reside primarily in academia. Toward Precision Medicine notes that moving toward individualized medicine requires that researchers and health care providers have access to very large sets of health- and disease-related data linked to individual patients. These data are also critical for developing the information commons, the knowledge network of disease, and ultimately the new taxonomy.