Characterizing the Huntington's disease, Parkinson's disease, and pan-neurodegenerative gene expression signature with RNA sequencing

Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative disorders that are characterized pathologically by degeneration of neurons in the brain and clinically by loss of motor function and cognitive decline in mid to late life. The cause of neuronal degeneration in these diseases is unclear, but both are histologically marked by aggregation of specific proteins in specific brain regions. In HD, fragments of a mutant Huntingtin protein aggregate and cause medium spiny interneurons of the striatum to degenerate. In contrast, PD brains exhibit aggregation of toxic fragments of the alpha synuclein protein throughout the central nervous system and trigger degeneration of dopaminergic neurons in the substantia nigra. Considering the commonalities and differences between these diseases, identifying common biological patterns across HD and PD as well as signatures unique to each may provide significant insight into the molecular mechanisms underlying neurodegeneration as a general process. State-of-the-art high-throughput sequencing technology allows for unbiased, whole genome quantification of RNA molecules within a biological sample that can be used to assess the level of activity, or expression, of thousands of genes simultaneously. In this thesis, I present three studies characterizing the RNA expression profiles of post-mortem HD and PD subjects using high-throughput mRNA sequencing data sets. The first study describes an analysis of differential expression between HD individuals and neurologically normal controls that indicates a widespread increase in immune, neuroinflammatory, and developmental gene expression. The second study expands upon the first study by making methodological improvements and extends the differential expression analysis to include PD subjects, with the goal of comparing and contrasting HD and PD gene expression profiles. This study was designed to identify common mechanisms underlying the neurodegenerative phenotype, transcending those of each unique disease, and has revealed specific biological processes, in particular those related to NFkB inflammation, common to HD and PD. The last study describes a novel methodology for combining mRNA and miRNA expression that seeks to identify associations between mRNA-miRNA modules and continuous clinical variables of interest, including CAG repeat length and clinical age of onset in HD

Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.Published versio

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

<p>Abstract</p> <p>Background</p> <p>Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. <it>Chlamydomonas</it>, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in <it>Chlamydomonas reinhardtii </it>using its recently completed genome sequence and all available ESTs and cDNAs.</p> <p>Results</p> <p>Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at <url>http://combi.cs.colostate.edu/as/chlamy</url>.</p> <p>Conclusions</p> <p>The extent of AS in <it>Chlamydomonas </it>that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in <it>Chlamydomonas</it>, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.</p

Large-Scale Discovery of ERK2 Substrates Identifies ERK-Mediated Transcriptional Regulation by ETV3

The mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase 2 (ERK2) is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2, a form of ERK2 engineered to use an analog of adenosine 5′-triphosphate (ATP), to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, mRNA processing, and regulation of the activity of the Rho family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E twenty-six family of transcriptional regulators), was extensively phosphorylated on sites within canonical and noncanonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.Massachusetts Institute of Technology (Eugene Bell Career Development Chair)David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)Massachusetts Institute of Technology (Whitaker Health Science Fellowship)National Science Foundation (U.S.) (Graduate Research Fellowship)Pfizer Inc.National Institutes of Health (U.S.) (grant ES002109)National Institutes of Health (U.S.) (grant R01DK42816)National Institutes of Health (U.S.) (grant R01CA118705)National Institutes of Health (U.S.) (grant U54CA112967

Differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative tauopathy found in individuals with a history of repetitive head impacts (RHI). Previous work has demonstrated that neuroinflammation is involved in CTE pathogenesis, however, the specific inflammatory mechanisms are still unclear. Here, using RNA-sequencing and gene set enrichment analysis (GSEA), we investigated the genetic changes found in tissue taken from the region CTE pathology is first found, the cortical sulcus, and compared it to neighboring gryal crest tissue to identify what pathways were directly related to initial hyperphosphorylated tau (p-tau) deposition. 21 cases were chosen for analysis: 6 cases had no exposure to RHI or presence of neurodegenerative disease (Control), 5 cases had exposure to RHI but no presence of neurodegenerative disease (RHI), and 10 cases had exposure to RHI and low stage CTE (CTE). Two sets of genes were identified: genes that changed in both the sulcus and crest and genes that changed specifically in the sulcus relative to the crest. When examining genes that changed in both the sulcus and crest, GSEA demonstrated an increase in immune related processes and a decrease in neuronal processes in RHI and CTE groups. Sulcal specific alterations were observed to be driven by three mechanisms: anatomy, RHI, or p-tau. First, we observed consistent sulcal specific alterations in immune, extracellular matrix, vascular, neuronal, and endocytosis/exocytosis categories across all groups, suggesting the sulcus has a unique molecular signature compared to the neighboring crest independent of pathology. Second, individuals with a history of RHI demonstrated impairment in metabolic and mitochondrial related processes. Finally, in individuals with CTE, we observed impairment of immune and phagocytic related processes. Overall, this work provides the first observation of biological processes specifically altered in the sulcus that could be directly implicated in CTE pathogenesis and provide novel targets for biomarkers and therapies

Predictors of Outcome in Aneurysmal Subarachnoid Hemorrhage Patients:Observations From a Multicenter Data Set

A table containing information on the qRT-PCR performed with seven novel miRNAs and two known miRNAs. Per miRNA, this information includes mean CT, range of CT, cDNA dilution, the number of samples (of 12) with CT < 40, the average read depth, and primer used. (XLSX 8 kb

Widespread perturbation of ETS factor binding sites in cancer.

Although \u3e90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted

Direct Recruitment of Polycomb Repressive Complex 1 to Chromatin by Core Binding Transcription Factors

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.Stem Cell and Regenerative Biolog

miR-10b-5p expression in Huntington’s disease brain relates to age of onset and the extent of striatal involvement

Background: MicroRNAs (miRNAs) are small non-coding RNAs that recognize sites of complementarity of target messenger RNAs, resulting in transcriptional regulation and translational repression of target genes. In Huntington’s disease (HD), a neurodegenerative disease caused by a trinucleotide repeat expansion, miRNA dyregulation has been reported, which may impact gene expression and modify the progression and severity of HD. Methods: We performed next-generation miRNA sequence analysis in prefrontal cortex (Brodmann Area 9) from 26 HD, 2 HD gene positive, and 36 control brains. Neuropathological information was available for all HD brains, including age at disease onset, CAG-repeat size, Vonsattel grade, and Hadzi-Vonsattel striatal and cortical scores, a continuous measure of the extent of neurodegeneration. Linear models were performed to examine the relationship of miRNA expression to these clinical features, and messenger RNA targets of associated miRNAs were tested for gene ontology term enrichment. Results: We identified 75 miRNAs differentially expressed in HD brain (FDR q-value <0.05). Among the HD brains, nine miRNAs were significantly associated with Vonsattel grade of neuropathological involvement and three of these, miR-10b-5p, miR-10b-3p, and miR-302a-3p, significantly related to the Hadzi-Vonsattel striatal score (a continuous measure of striatal involvement) after adjustment for CAG length. Five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-10b-3p, and miR-106a-5p) were identified as having a significant relationship to CAG length-adjusted age of onset including miR-10b-5p, the mostly strongly over-expressed miRNA in HD cases. Although prefrontal cortex was the source of tissue profiled in these studies, the relationship of miR-10b-5p expression to striatal involvement in the disease was independent of cortical involvement. Correlation of miRNAs to the clinical features clustered by direction of effect and the gene targets of the observed miRNAs showed association to processes relating to nervous system development and transcriptional regulation. Conclusions: These results demonstrate that miRNA expression in cortical BA9 provides insight into striatal involvement and support a role for these miRNAs, particularly miR-10b-5p, in HD pathogenicity. The miRNAs identified in our studies of postmortem brain tissue may be detectable in peripheral fluids and thus warrant consideration as accessible biomarkers for disease stage, rate of progression, and other important clinical characteristics of HD. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0083-3) contains supplementary material, which is available to authorized users