Abstract
Purpose:
Purpose: Diabetic retinopathy (DR) remains the leading cause of blindness among working age adults. Although the pathophysiology of DR is not known, early deficits in retinal neuronal structure, function and gene expression occur in diabetic patients and these deficits have been recapitulated in experimental rat models. Mitochondria, cellular organelles responsible for ATP generation through oxidative phosphorylation as well as calcium regulation, are implicated in neural deficiencies with DR in the retina. Mitochondria contain multiple circular ~16.5 kb genomes (mtDNA), which encode necessary subunits of the respiratory chain. Maintenance of the mtDNA is important for mitochondrial health and function. We hypothesize that mtDNA copy number and maintenance are decreased with DR in retinal neuronal cells.
Methods:
Methods: We have developed novel assays to quantify mtDNA copy numbers and assess mtDNA mutations and deletions, or degree of heteroplasmy, in retinal tissue from STZ-induced diabetic rats. Absolute mtDNA copy number quantitation from retinal genomic DNA was achieved by using digital PCR (dPCR) with multiplexed custom TaqMan probe sets specific for mtDNA and nuclear DNA. Heteroplasmy was measured through mtDNA enrichment, rapid transposome-mediated library generation and next-generation sequencing of retinal genomic DNA. Sequencing reads were aligned to an intra-sample mtDNA consensus reference genome, and variant detection analysis was carried out to identify low-frequency point mutations and deletions, respectively.
Results:
Results: Blood glucose and HbA1c levels were monitored throughout the study. There were no differences in mtDNA copy number between ND and D rats at 2, 12, or 21 weeks uncontrolled diabetes. However there was an increase in mtDNA copies with age (p < 0.05, 2-way ANOVA, n=8-10/group). In D animals, there was a significant increase in the number of mutations per animals compared to controls at (p < 0.05, parametric t-test, n=8-10/group).
Conclusions:
Conclusions: Contrary to our hypothesis, whole retinal mtDNA copy number is not altered significantly with uncontrolled diabetes. However, retinal mtDNA heteroplasmy did increase in D animals significantly compared to ND controls. These methods provide a powerful assessment of mtDNA maintenance in neuronal cell populations, which can be applied to any tissue or cell type, with metabolic dysregulation seen in diabetes.