Curtis L. Nordgaard

Mitochondrial Proteomics of the Retinal Pigment Epithelium at Progressive Stages of Age-Related Macular Degeneration

PURPOSE Age-related macular degeneration (AMD) is the leading cause of vision loss in individuals over the age of 65. Histopathological changes become evident in the retinal pigment epithelium (RPE), a monolayer that provides metabolic support for the overlying photoreceptors, even at the earliest stages of AMD that precede vision loss. In a previous global RPE proteome analysis, changes were identified in the content of several mitochondrial proteins associated with AMD. In this study, the subproteome of mitochondria isolated from human donor RPE graded with the Minnesota Grading System (MGS) was analyzed. METHODS Human donor eye bank eyes were categorized into one of four progressive stages (MGS 1-4) based on the clinical features of AMD. After dissection of the RPE, mitochondrial proteins were isolated and separated by two-dimensional gel electrophoresis based on their charge and mass. Protein spot densities were compared between the four MGS stages. Peptides from spots that changed significantly with MGS stage were extracted and analyzed by using mass spectrometry to identify the protein. RESULTS Western blot analyses verified that mitochondria were consistently enriched between MGS stages. The densities of eight spots increased or decreased significantly as a function of MGS stage. These spots were identified as the alpha-, beta-, and delta-ATP synthase subunits, subunit VIb of the cytochrome c oxidase complex, mitofilin, mtHsp70, and the mitochondrial translation factor Tu. CONCLUSIONS The results are consistent with the hypothesis that mitochondrial dysfunction is associated with AMD and further suggest specific pathophysiological mechanisms involving altered mitochondrial translation, import of nuclear-encoded proteins, and ATP synthase activity.

Mitochondrial DNA Damage as a Potential Mechanism for Age-Related Macular Degeneration

PURPOSE Increasing evidence suggests a central role for mitochondrial (mt) dysfunction in age-related macular degeneration (AMD). Previous proteomic data from the retinal pigment epithelium (RPE) revealed significant changes to mt proteins, suggesting potential functional defects and damage to mitochondrial DNA (mtDNA) with AMD progression. The present study tests the hypothesis that mtDNA damage increases with aging and AMD. METHODS Genomic DNA was isolated from the macular region of human donor RPE graded for stages of AMD (Minnesota Grading System [MGS] 1-4). Region-specific mtDNA damage with normal aging was evaluated in 45 control subjects (ages 34-88 years, MGS 1) and AMD-associated damage in diseased subjects (n = 46), compared with that in age-matched control subjects (n = 26). Lesions per 10 kb per genome in the mtDNA and nuclear DNA were measured with long-extension polymerase chain reaction (LX PCR). The level of deleted mtDNA in each donor was measured with quantitative real-time PCR (qPCR). RESULTS With aging, an increase in mtDNA damage was observed only in the common deletion region of the mt genome. In contrast, with AMD, mtDNA lesions increased significantly in all regions of the mt genome beyond levels found in age-matched control subjects. mtDNA accumulated more lesions than did two nuclear genes, with total damage of the mt genome estimated to be eight times higher. CONCLUSIONS Collectively, the data indicate that mtDNA is preferentially damaged with AMD progression. These results suggest a potential link between mt dysfunction due to increased mtDNA lesions and AMD.

Human Retinal Pigment Epithelium Proteome Changes in Early Diabetes

Aims/hypothesisDiabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE.MethodsThe RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n = 6) and from eyes of age-matched control donors (n = 17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry.ResultsAnalysis of 325 spots on 2-D gels identified 31 spots that were either up- or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism.Conclusions/interpretationChanges identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.

Use of a Computer Decision Support System and Antimicrobial Therapy Appropriateness

OBJECTIVE  To determine whether antimicrobial (AM) courses ordered with an antimicrobial computer decision support system (CDSS) were more likely to be appropriate than courses ordered without the CDSS. DESIGN  Retrospective cohort study. Blinded expert reviewers judged whether AM courses were appropriate, considering drug selection, route, dose, and duration. SETTING  A 279-bed university-affiliated Department of Veterans Affairs (VA) hospital. PATIENTS  A 500-patient random sample of inpatients who received a therapeutic AM course between October 2007 and September 2008. Intervention. An optional CDSS, available at the point of order entry in the VA computerized patient record system. RESULTS CDSS courses were significantly more likely to be appropriate (111/254, 44%) compared with non-CDSS courses (81/246, 33%, P = .013). Courses were more likely to be appropriate when the initial provider diagnosis of the condition being treated was correct (168/273, 62%) than when it was incorrect, uncertain, or a sign or symptom rather than a disease (24/227, 11%, P < .001. In multivariable analysis, CDSS-ordered courses were more likely to be appropriate than non-CDSS-ordered courses (odds ratio [OR], 1.83; 95% confidence interval [CI], 1.13-2.98). Courses were also more likely to be judged appropriate when the initial provider diagnosis of the condition being treated was correct than when it was incorrect, uncertain, or a sign or symptom rather than a disease (OR, 3.56; 95% CI, 1.4-9.0). CONCLUSIONS  Use of the CDSS was associated with more appropriate AM use. To achieve greater improvements, strategies are needed to improve provider diagnoses of syndromes that are infectious or possibly infectious.