Biology subjects

El Kodsi, D. N.

Publications and source records attributed to El Kodsi, D. N..

3 recordsLinked to original sources

bioRxiv2024

Experimental and Computational Methods for Allelic Imbalance Analysis from Single-Nucleus RNA-seq Data

Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool for understanding gene function across diverse cells. Recently, this has included the use of allele-specific expression (ASE) analysis to better understand how variation in the human genome affects RNA expression at the single-cell level. We reasoned that because intronic reads are more prevalent in single-nucleus RNA-Seq (snRNA-Seq), and introns are under lower purifying selection and thus enriched for genetic variants, that snRNA-seq should facilitate single-cell analysis of ASE. Here we demonstrate how experimental and computational choices can improve the results of allelic imbalance analysis. We explore how experimental choices, such as RNA source, read length, sequencing depth, genotyping, etc., impact the power of ASE-based methods. We developed a new suite of computational tools to process and analyze scRNA-seq and snRNA-seq for ASE. As hypothesized, we extracted more ASE information from reads in intronic regions than those in exonic regions and show how read length can be set to increase power. Additionally, hybrid selection improved our power to detect allelic imbalance in genes of interest. We also explored methods to recover allele-specific isoform expression levels from both long- and short-read snRNA-seq. To further investigate ASE in the context of human disease, we applied our methods to a Parkinsons disease cohort of 94 individuals and show that ASE analysis had more power than eQTL analysis to identify significant SNP/gene pairs in our direct comparison of the two methods. Overall, we provide an end-to-end experimental and computational approach for future studies.

genomics↗

bioRxiv2020

Age-Associated Insolubility of Parkin in Human Midbrain is Linked to Redox Balance and Sequestration of Reactive Dopamine Metabolites

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

neuroscience↗

bioRxiv2020

Parkinson Disease-Linked Parkin Mediates Redox Reactions That Lower Oxidative Stress In Mammalian Brain

We recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H2O2) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H2O2 levels were unchanged in adult prkn-/- brain, a parkin-dependent reduction of cytosolic H2O2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn-/- mice. Nevertheless, this rise in oxidative stress led to changes in mitochondrial enzyme activities and the metabolism of glutathione in cells and mammalian brain. In parkins absence reduced glutathione concentrations were increased including in human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H2O2 reduction and glutathione regeneration. These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance.

neuroscience↗