Intracellular microelectrode recordings, focusing on the first derivative of the action potential's waveform, categorized neurons into three groups (A0, Ainf, and Cinf), demonstrating varied responses to the stimulus. The resting potential of A0 somas and Cinf somas were only depolarized by diabetes, changing from -55mV to -44mV and -49mV to -45mV, respectively. In Ainf neurons, diabetes led to an increase in action potential and after-hyperpolarization durations, rising from 19 and 18 milliseconds to 23 and 32 milliseconds, respectively, and a decrease in dV/dtdesc, dropping from -63 to -52 volts per second. The amplitude of the action potential in Cinf neurons decreased, while the amplitude of the after-hyperpolarization increased, a consequence of diabetes (originally 83 mV and -14 mV; subsequently 75 mV and -16 mV, respectively). Using the whole-cell patch-clamp technique, we observed that diabetes produced an elevation in the peak amplitude of sodium current density (from -68 to -176 pA pF⁻¹), and a shift in steady-state inactivation towards more negative transmembrane potentials, solely in neurons from the diabetic animal group (DB2). The diabetes-affected DB1 group displayed no change in this parameter, showing a sustained value of -58 pA pF-1. An increase in membrane excitability did not occur despite the changes in sodium current, likely owing to modifications in sodium current kinetics brought on by diabetes. Analysis of our data indicates that diabetes's effects on membrane properties differ across nodose neuron subpopulations, suggesting pathophysiological consequences for diabetes mellitus.
Deletions in mitochondrial DNA (mtDNA) are a foundation of mitochondrial dysfunction observed in aging and diseased human tissues. Mitochondrial genome's multicopy nature results in a variation in the mutation load of mtDNA deletions. Despite having minimal effect at low levels, deletions accumulate to a critical point where dysfunction inevitably ensues. The size of the deletion and the position of the breakpoints determine the mutation threshold for oxidative phosphorylation complex deficiency, which differs for each complex type. Moreover, the mutation burden and the depletion of specific cellular species can differ significantly from cell to cell within a tissue, leading to a pattern of mitochondrial malfunction resembling a mosaic. It is often imperative, for the study of human aging and disease, to be able to accurately describe the mutation load, the breakpoints, and the extent of any deletions from a single human cell. Laser micro-dissection and single-cell lysis protocols from tissues are presented, along with subsequent analysis of deletion size, breakpoints and mutation burden via long-range PCR, mitochondrial DNA sequencing, and real-time PCR, respectively.
Essential components of cellular respiration are specified by mitochondrial DNA (mtDNA). In the course of normal aging, mitochondrial DNA (mtDNA) undergoes a gradual accumulation of low-level point mutations and deletions. Poor mtDNA maintenance, however, is the genesis of mitochondrial diseases, originating from the progressive loss of mitochondrial function caused by the rapid accumulation of deletions and mutations in the mtDNA. With the aim of enhancing our understanding of the molecular underpinnings of mtDNA deletion formation and transmission, we designed the LostArc next-generation sequencing pipeline to detect and quantify rare mtDNA populations within small tissue samples. LostArc techniques are engineered to minimize polymerase chain reaction amplification of mitochondrial DNA and, in contrast, to enrich mitochondrial DNA through the selective destruction of nuclear DNA. The sensitivity of this approach, when applied to mtDNA sequencing, allows for the identification of one mtDNA deletion per million mtDNA circles, achieving high depth and cost-effectiveness. This document outlines comprehensive procedures for extracting genomic DNA from mouse tissues, enriching mitochondrial DNA through enzymatic removal of linear nuclear DNA, and preparing libraries for unbiased next-generation mitochondrial DNA sequencing.
The clinical and genetic spectrum of mitochondrial diseases arises from the interplay of pathogenic variations in both mitochondrial and nuclear genes. In excess of 300 nuclear genes associated with human mitochondrial diseases now bear the mark of pathogenic variants. Nonetheless, the genetic determination of mitochondrial disease presents significant diagnostic obstacles. Yet, a multitude of strategies are now available for identifying causative variants in individuals with mitochondrial disease. This chapter details the recent advancements and approaches to gene/variant prioritization, using the example of whole-exome sequencing (WES).
The last ten years have seen next-generation sequencing (NGS) ascend to the position of the definitive diagnostic and investigative technique for novel disease genes, including those contributing to heterogeneous conditions such as mitochondrial encephalomyopathies. In contrast to other genetic conditions, the deployment of this technology to mtDNA mutations necessitates overcoming additional obstacles, arising from the specific characteristics of mitochondrial genetics and the requirement for appropriate NGS data management and analysis. learn more A complete, clinically sound protocol for whole mtDNA sequencing and heteroplasmy quantification is presented, progressing from total DNA to a single PCR amplicon.
Modifying plant mitochondrial genomes offers substantial benefits. The delivery of foreign DNA to mitochondria faces current difficulties, but the use of mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) allows for the disabling of mitochondrial genes. The introduction of mitoTALENs encoding genes into the nuclear genome facilitated the achievement of these knockouts. Earlier research indicated that double-strand breaks (DSBs) formed by mitoTALENs are fixed via the mechanism of ectopic homologous recombination. Homologous recombination DNA repair results in the deletion of a chromosomal segment that includes the target site for the mitoTALEN. The intricate processes of deletion and repair are responsible for the increasing complexity of the mitochondrial genome. Here, we present a method to ascertain ectopic homologous recombination events following repair of double-strand breaks that are provoked by mitoTALENs.
Mitochondrial genetic transformation is a standard practice in the two micro-organisms, Chlamydomonas reinhardtii and Saccharomyces cerevisiae, presently. Yeast cells are notably suitable for both the generation of a diverse range of defined alterations and the insertion of ectopic genes into their mitochondrial genome (mtDNA). DNA-coated microprojectiles, launched via biolistic methods, integrate into mitochondrial DNA (mtDNA) through the highly effective homologous recombination systems present in Saccharomyces cerevisiae and Chlamydomonas reinhardtii organelles. Despite the low frequency of transformation events in yeast, the isolation of successful transformants is a relatively quick and easy procedure, given the abundance of selectable markers. However, achieving similar results in C. reinhardtii is a more time-consuming task that relies on the discovery of more suitable markers. To mutagenize endogenous mitochondrial genes or introduce novel markers into mtDNA, we detail the materials and methods employed in biolistic transformation. While alternative methods for modifying mitochondrial DNA are developing, the current approach for inserting foreign genes still predominantly utilizes biolistic transformation.
Mitochondrial gene therapy technology benefits significantly from mouse models exhibiting mitochondrial DNA mutations, offering valuable preclinical data before human trials. The high degree of similarity between human and murine mitochondrial genomes, combined with the expanding availability of rationally designed AAV vectors for the selective transduction of murine tissues, is the reason for their suitability in this context. Conditioned Media For downstream AAV-based in vivo mitochondrial gene therapy, the compactness of mitochondrially targeted zinc finger nucleases (mtZFNs) makes them highly suitable, a feature routinely optimized by our laboratory. Robust and precise genotyping of the murine mitochondrial genome, and the optimization of mtZFNs for subsequent in vivo use, are addressed in this chapter's precautions.
Mapping of 5'-ends across the entire genome is accomplished via the 5'-End-sequencing (5'-End-seq) assay, utilizing next-generation sequencing on an Illumina platform. Ponto-medullary junction infraction Our method targets the identification of free 5'-ends in mtDNA extracted from fibroblasts. Utilizing this method, researchers can investigate crucial aspects of DNA integrity, including DNA replication mechanisms, priming events, primer processing, nick processing, and double-strand break repair, across the entire genome.
Defects in mitochondrial DNA (mtDNA) maintenance, including flaws in replication mechanisms or inadequate dNTP provision, are fundamental to various mitochondrial disorders. In the typical mtDNA replication process, multiple individual ribonucleotides (rNMPs) are incorporated into each mtDNA molecule. Embedded rNMPs impacting the stability and characteristics of DNA, in turn, might affect the maintenance of mtDNA and thus be implicated in mitochondrial diseases. They are also a reflection of the intramitochondrial NTP/dNTP concentration. This chapter details a method for ascertaining mtDNA rNMP levels, employing alkaline gel electrophoresis and Southern blotting. This procedure is designed to handle mtDNA analysis within the context of total genomic DNA preparations, and independently on purified mtDNA. Besides, the process is performable using equipment frequently encountered in most biomedical laboratories, permitting the concurrent study of 10-20 specimens based on the employed gel system, and it can be modified for the examination of other mitochondrial DNA alterations.