Doctor of Philosophy

dissertationHuman retinitis pigmentosa (RP) typically involves decades of progressive vision loss before some patients become blind, and prospective therapies target patients who have been blind for substantial time, even decades. Evaluations of molecular and cellular therapies have primarily employed short-lived mouse models lacking the scope of remodeling common in human RP. The Rho Tg P347L transgenic rabbit offers a unique opportunity to evaluate the primary degeneration event and subsequent progressive remodeling that ensues over a timespan that recapitulates the human disease phenotype. Retinas from a TgP347L rabbit model of human dominant RP and wild-type litter mates were harvested over an 8-year span and processed for transmission electron microscope connectomics, immunocytochemistry for a range of macromolecules, and computational molecular phenotyping for small molecules, including transport tracing with D-Asp. Early time points in the TgP347L rabbit recapitulate the established sequence of photoreceptor loss, retinal remodeling, and reprogramming, and also reveal progressive disruptions in Müller cell metabolism, where rather than observing a homogeneous glial population, chaotic metabolic signatures emerge. By 4 years, virtually all remnants of photoreceptors are gone and the neural retina manifests severe cell loss and near complete loss of glutamine synthetase, though glial glutamate transport persists. By 6 years, there is a global >90% neuronal loss. In some regions the retina is devoid of identifiable cells and replaced by unknown debris-like assemblies. Though the 6-year retina does have locations with recognizable neurons, all cell types are drastically reduced in number and some have altered metabolic phenotypes. These results are never seen in wt littermates, including rabbits which are 8 years old. Electron microscopic analysis using wide-field connectomics imaging of the 6-year TgP347L sample demonstrates some structurally normal synapses, indicating that survivor neurons in these regions are not quiescent despite the lack of sensory input for a substantial period of time. These results indicate that, although photoreceptor degeneration is the trigger, retinal remodeling ultimately gives way to neurodegeneration, which is a separate unrelenting disease process independent of the initial insult, closely resembling slow progressive CNS neurodegenerations. Indeed, both metabolic disruption and debris-related degeneration predicts the existence of a persistent neuropathy, and increases in ?-synuclein levels support a proteinopathy component. Remodeling and neurodegeneration progress until the retina is devoid of recognizable cells. There is no stable state into which the retina settles and no cell type is spared. This has profound implications for current therapeutics. There will likely be critical windows for implementation but, ultimately, suspension of neurodegenerative remodeling will be required for long-term success

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Retinal Remodeling And Metabolic Alterations in Human AMD

Age-related macular degeneration (AMD) is a progressive retinal degeneration resulting in central visual field loss, ultimately causing debilitating blindness. AMD affects 18% of Americans from 65 to 74, 30% older than 74 years of age and is the leading cause of severe vision loss and blindness in Western populations. While many genetic and environmental risk factors are known for AMD, we currently know less about the mechanisms mediating disease progression.The pathways and mechanisms through which genetic and non-genetic risk factors modulate development of AMD pathogenesis remain largely unexplored. Moreover, current treatment for AMD is palliative and limited to wet/exudative forms. Retina is a complex, heterocellular tissue and most retinal cell classes are impacted or altered in AMD. Defining disease and stage-specific cytoarchitectural and metabolic responses in AMD is critical for highlighting targets for intervention. The goal of this paper is to illustrate cell types impacted in AMD and demonstrate the implications of those changes, likely beginning in the retinal pigment epithelium (RPE), for remodeling of the the neural retina.Tracking heterocellular responses in disease progression is best achieved with computational molecular phenotyping (CMP), a tool that enables acquisition of a small molecule fingerprint for every cell in the retina. CMP uncovered critical cellular and molecular pathologies (remodeling and reprogramming) in progressive retinal degenerations such as retinitis pigmentosa (RP). We now applied these approaches to normal human and AMD tissues mapping progression of cellular and molecular changes in AMD retinas, including late-stage forms of the disease.Major findings: 1) Evidence of metabolic instability in RPE in dry-AMD.2) Photoreceptors show clear indications of stress prior to cell death.3) Cone opsin processing by the RPE in AMD retinas may be differentially compromised vs. rod opsin.4) Müller cells in AMD exhibit alterations in metabolism and morphology consistent with other retinal degenerative diseases.5) Inner retinal neurons in AMD remodel by sprouting neurites and projecting to aberrant locations.6) Retinal remodeling occurs underneath drusen.7) Retinal remodeling occurs in the presence of cone and rod photoreceptors

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function