Biology subjects

Sabharwal, A.

Publications and source records attributed to Sabharwal, A..

6 recordsLinked to original sources

bioRxiv2026

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

bioRxiv2025

Non-Destructive Larval Genotyping of Danio rerio for Mitochondrial DNA Genetics

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, non-lethal larval genotyping methods in zebrafish (Danio rerio) research. This study optimizes and validates a non-destructive proteinase K digestion method for mitochondrial and nuclear DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial and nuclear genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA. The method successfully detected C-to-T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. This optimized method enables researchers to correlate genotypes with phenotypes in longitudinal studies while maintaining specimen viability particularly valuable for investigating early-onset mitochondrial diseases and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.

genetics↗

bioRxiv2025

VEGF-B/NRP1 Signaling Modulates Mitochondrial Homeostasis and Cardiac Function After Myocardial Infarction

BackgroundMyocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. And secondly, we further validated the protective role of VEFG-B in ischemic heart disease, shedding light on their roles not only in cardiac function but also in its therapeutic potential. MethodsIn this study, we utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, using either 1% oxygen or 200{micro}M cobalt chloride (CoCl2) to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo. ResultsOur findings indicate that both VEGF-B and NRP1 are predominantly expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. Our results demonstrate that VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Furthermore, we found that VEGF-B promotes cardiomyocyte survival by improving mitochondrial function, as evidenced by reduced oxidative stress and ROS accumulation, decreased oxidative stress, preserved mitochondrial membrane potential, and increased ATP levels. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. ConclusionOur study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. Importantly, we also demonstrated that VEGF-B is protective against ischemic injury in vivo using a novel zebrafish model Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMyocardial infarction (MI) results in mitochondrial dysfunction, cardiomyocyte death, and adverse ventricular remodeling. C_LIO_LIVascular Endothelial Growth Factor B (VEGF-B) is traditionally associated with cardiac metabolism, survival, vascular biology, particularly in angiogenesis and lipid metabolism. C_LIO_LIThe neurophilin-1 (NRP1) receptor, a co-receptor for VEGF-B, is expressed in cardiomyocytes and implicated in mitochondrial signaling pathways. C_LIO_LIEmerging evidence suggests that VEGF-B may influence mitochondrial integrity and function under stress conditions. C_LI What New Information Does This Article Contribute?O_LIVEGF-B exerts a protective effect on cardiomyocyte mitocondria following ischemic injury by preserving mitochondrial function and reducing oxidative stress. C_LIO_LINRP1 receptor as a key mediator of VEGF-B modulates mitochondrial homeostasis in cardiomyocytes. C_LIO_LIVEGF-B signaling can attenuate apoptosis and enhance mitochondrial bioenergetics post-MI, suggesting a novel cardioprotective mechanism. C_LIO_LIClinical Implication: These findings suggest that therapeutic strategies aimed at enhancing VEGF-B/NRP1 signaling may improve mitochondrial function and cardiac recovery after MI, offering a novel avenue for limiting heart failure progression in ischemic heart disease. C_LI

molecular biology↗

bioRxiv2021

The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

genetics↗

bioRxiv2020

A Genetic Model Therapy Proposes a Critical Role for Liver Dysfunction in Mitochondrial Biology and Disease

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Here we present a new animal model that recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. LRPPRC has native functions related to mitochondrial mRNA polyadenylation and translation as well as a role in gluconeogenesis. We used the Gene-Breaking Transposon (GBT) cassette to create a revertible, insertional mutant zebrafish line in the LRPPRC gene. lrpprc zebrafish homozygous mutants displayed impaired muscle development, liver function and lowered levels of mtDNA transcripts and are lethal by 12dpf, all outcomes similar to clinical phenotypes observed in patients. Investigations using an in vivo lipidomics approach demonstrated accumulation of non-polar lipids in these animals. Transcript profiling of the mutants revealed dysregulation of clinically important nuclearly encoded and mitochondrial transcripts. Using engineered liver-specific rescue as a genetic model therapy, we demonstrate survival past the initial larval lethality, as well as restored normal gut development, mitochondrial morphology and triglyceride levels functionally demonstrating a critical role for the liver in the pathophysiology of this model of mitochondrial disease. Understanding the molecular mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical diagnostic and therapeutic developments for patients suffering from these devastating disorders.

genetics↗

bioRxiv2019

A primer genetic toolkit for exploring mitochondrial biology and disease using zebrafish

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37 gene set and overall genomic structure ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also implicated to be a common component of a wide variety of other human illnesses ranging from neurodegenerative disorders like Huntingtons disease and Parkinsons disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system from genetics to imaging. This collection includes 22 mutant lines in 18 different genes created by locus-specific gene editing to induce frameshift or splice acceptor mutations leading to predicted protein truncation during translation. Also included are 6 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All of these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study of many different aspects of mitochondrial function with the goal of widening our understanding of their role in biology and human disease.

molecular biology↗