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. Diabetes's effect on the resting potential was limited to A0 and Cinf somas, shifting the potential from -55mV to -44mV in A0 and from -49mV to -45mV in Cinf. In Ainf neurons, diabetes caused a significant increase in the duration of action potentials and after-hyperpolarization durations (from 19 ms and 18 ms to 23 ms and 32 ms, respectively) and a decrease in dV/dtdesc (from -63 to -52 V/s). Diabetes exerted a dual effect on Cinf neurons, decreasing the action potential amplitude while enhancing the after-hyperpolarization amplitude, resulting in a shift from 83 mV and -14 mV to 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). For the DB1 group, diabetes exhibited no impact on this parameter, which remained constant at -58 pA pF-1. Despite failing to boost membrane excitability, changes in sodium current are potentially explicable by the diabetic-induced alterations in the kinetics of sodium current. Our observations on the impact of diabetes on membrane properties across diverse nodose neuron subpopulations imply potential pathophysiological relevance to diabetes mellitus.
Mitochondrial dysfunction in aging and diseased human tissues is underpinned by deletions within the mitochondrial DNA molecule. The multicopy nature of the mitochondrial genome results in mtDNA deletions displaying a diversity of mutation loads. Insignificant at low frequencies, molecular deletions, once exceeding a critical percentage, lead to functional impairment. The impact of breakpoint placement and deletion size upon the mutation threshold needed to produce oxidative phosphorylation complex deficiency differs depending on the specific complex. Beyond this, the amount of mutations and the loss of particular cell types can vary from cell to cell within a tissue, demonstrating a mosaic distribution of mitochondrial impairment. 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. We describe the protocols for laser micro-dissection and single-cell lysis of tissues, including the subsequent determination of deletion size, breakpoints, and mutation burden via long-range PCR, mtDNA sequencing, and real-time PCR.
Cellular respiration's fundamental components are encoded within the mitochondrial DNA (mtDNA). During the normal aging process, mtDNA (mitochondrial DNA) accumulates low levels of point mutations and deletions. Improper mitochondrial DNA (mtDNA) care, unfortunately, is linked to the development of mitochondrial diseases, which result from the progressive decline in mitochondrial function, significantly influenced by the rapid creation of deletions and mutations in the mtDNA. In order to acquire a more profound insight into the molecular mechanisms responsible for the emergence and spread of mtDNA deletions, a novel LostArc next-generation sequencing pipeline was developed to detect and quantify infrequent mtDNA variations in minuscule tissue samples. LostArc procedures are formulated to decrease PCR amplification of mitochondrial DNA, and conversely to promote the enrichment of mitochondrial DNA through the targeted demolition of nuclear DNA molecules. Cost-effective high-depth sequencing of mtDNA, achievable with this approach, provides the sensitivity required for identifying one mtDNA deletion per million mtDNA circles. We provide a detailed description of protocols for isolating genomic DNA from mouse tissues, enzymatically concentrating mitochondrial DNA after the destruction of linear nuclear DNA, and ultimately creating libraries for unbiased next-generation sequencing of the mitochondrial genome.
Pathogenic variants within both the mitochondrial and nuclear genomes are responsible for the varied clinical presentations and genetic makeup of mitochondrial disorders. Human mitochondrial diseases are now known to be associated with pathogenic variants in well over 300 nuclear genes. While a genetic basis can be found, diagnosing mitochondrial disease remains a difficult endeavor. Despite this, a range of strategies are now available to ascertain causative variants in patients with mitochondrial disorders. This chapter delves into the recent progress and diverse strategies in gene/variant prioritization, employing whole-exome sequencing (WES) as a key technology.
In the past decade, next-generation sequencing (NGS) has emerged as the definitive benchmark for diagnosing and uncovering novel disease genes linked to diverse conditions, including mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations encounters greater challenges than other genetic conditions, attributable to the specific complexities of mitochondrial genetics and the imperative for thorough NGS data management and analysis protocols. sandwich immunoassay We present a comprehensive, clinically-applied procedure for determining the full mtDNA sequence and measuring mtDNA variant heteroplasmy levels, starting from total DNA and ending with a single PCR amplicon product.
There are many benefits to be gained from the ability to transform plant mitochondrial genomes. Even though the introduction of exogenous DNA into mitochondria remains a formidable undertaking, mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) now facilitate the disabling of mitochondrial genes. The nuclear genome underwent a genetic modification involving mitoTALENs encoding genes, thus achieving these knockouts. Investigations conducted previously have showcased that double-strand breaks (DSBs) induced by mitoTALENs are repaired using the mechanism of ectopic homologous recombination. Due to homologous recombination-mediated DNA repair, a segment of the genome encompassing the mitoTALEN target site is excised. The mitochondrial genome's complexity is augmented by the processes of deletion and repair. We describe a process for identifying ectopic homologous recombination events, stemming from double-strand break repair mechanisms induced by mitoTALENs.
Presently, the two microorganisms, Chlamydomonas reinhardtii and Saccharomyces cerevisiae, are routinely employed for mitochondrial genetic transformation. Yeast provides a fertile ground for the generation of a wide range of defined alterations and the insertion of ectopic genes into the mitochondrial genome (mtDNA). Microprojectiles, coated in DNA and delivered via biolistic bombardment, successfully introduce genetic material into the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and Chlamydomonas reinhardtii cells thanks to the highly efficient homologous recombination mechanisms. The transformation rate in yeast, while low, is offset by the relatively swift and simple isolation of transformed cells due to the readily available selection markers. In marked contrast, the isolation of transformed C. reinhardtii cells remains a lengthy endeavor, predicated on the identification of new markers. The protocol for biolistic transformation, encompassing the relevant materials and procedures, is described for introducing novel markers or inducing mutations within endogenous mitochondrial genes. While alternative strategies for mtDNA editing are being established, gene insertion at ectopic loci is, for now, confined to biolistic transformation techniques.
Mouse models exhibiting mitochondrial DNA mutations show potential for optimizing mitochondrial gene therapy and generating pre-clinical data, a prerequisite for human clinical trials. The elevated similarity between human and murine mitochondrial genomes, and the augmenting access to rationally engineered AAV vectors that selectively transduce murine tissues, establishes their suitability for this intended application. read more Our laboratory's protocol for optimizing mitochondrially targeted zinc finger nucleases (mtZFNs) leverages their compactness, making them ideally suited for in vivo mitochondrial gene therapy employing adeno-associated virus (AAV) vectors. The murine mitochondrial genome's precise genotyping and the subsequent in vivo use of optimized mtZFNs are the focus of the precautions outlined in this chapter.
Utilizing next-generation sequencing on an Illumina platform, 5'-End-sequencing (5'-End-seq) provides a means to map 5'-ends across the entire genome. impedimetric immunosensor The mapping of free 5'-ends within fibroblast mtDNA is accomplished by this method. Employing this methodology, researchers can investigate the intricate relationships between DNA integrity, DNA replication mechanisms, priming events, primer processing, nick processing, and double-strand break processing throughout the entire genome.
Disruptions to mitochondrial DNA (mtDNA) maintenance, including problems with replication systems or insufficient deoxyribonucleotide triphosphate (dNTP) supplies, are causative in a range of mitochondrial disorders. Multiple single ribonucleotides (rNMPs) are a consequence of the ordinary replication process happening within each mtDNA molecule. Embedded rNMPs' modification of DNA stability and properties could have consequences for mtDNA maintenance, thereby contributing to the spectrum of mitochondrial diseases. They are also employed as a measurement instrument to quantify the intramitochondrial nucleotide triphosphate-to-deoxynucleotide triphosphate ratio. This chapter details a method for ascertaining mtDNA rNMP levels, employing alkaline gel electrophoresis and Southern blotting. For the examination of mtDNA, this process can be used with either total genomic DNA or purified samples. Beyond that, the procedure can be executed using equipment commonplace in the majority of biomedical laboratories, affording the concurrent analysis of 10-20 samples depending on the utilized gel system, and it is adaptable to the analysis of other mtDNA variations.