Through the analysis of the first derivative of the action potential's waveform, intracellular microelectrode recordings distinguished three distinct neuronal groups: A0, Ainf, and Cinf, each uniquely affected. The resting potential of A0 somas and Cinf somas were only depolarized by diabetes, changing from -55mV to -44mV and -49mV to -45mV, respectively. Within Ainf neurons, diabetes fostered a rise in action potential and after-hyperpolarization durations (increasing from 19 ms and 18 ms to 23 ms and 32 ms, respectively) alongside a decrease in dV/dtdesc, declining from -63 to -52 V/s. Diabetes-induced changes in Cinf neuron activity included a reduction in action potential amplitude and an elevation in after-hyperpolarization amplitude (from 83 mV to 75 mV and from -14 mV to -16 mV, respectively). Employing whole-cell patch-clamp recordings, we noted that diabetes induced a rise 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, exclusively in a cohort of neurons derived from diabetic animals (DB2). Within the DB1 group, diabetes' influence on this parameter was null, with the value persisting at -58 pA pF-1. Diabetes-induced changes in the kinetics of sodium current are a probable explanation for the observed sodium current shifts, which did not result in an increase in membrane excitability. Membrane properties of various nodose neuron subpopulations are demonstrably affected differently by diabetes, according to our data, suggesting pathophysiological consequences for diabetes mellitus.
The basis of mitochondrial dysfunction in human tissues, both in aging and disease, rests on deletions within the mitochondrial DNA (mtDNA). The presence of multiple copies of the mitochondrial genome leads to variable mutation loads of mtDNA deletions. Insignificant at low frequencies, molecular deletions, once exceeding a critical percentage, lead to functional impairment. Breakpoint locations and deletion extent affect the mutation threshold needed for deficient oxidative phosphorylation complexes, each complex exhibiting unique requirements. Moreover, mutation load and cell-type depletion levels can differ across contiguous cells in a tissue, presenting a mosaic pattern of mitochondrial dysfunction. For this reason, determining the mutation load, the locations of breakpoints, and the dimensions of any deletions present in a single human cell is often critical for advancing our understanding of human aging and disease. Detailed protocols for laser micro-dissection and single-cell lysis from tissue are described, followed by the analysis of deletion size, breakpoints, and mutation load using long-range PCR, mtDNA sequencing, and real-time PCR, respectively.
Cellular respiration's fundamental components are encoded within the 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. To develop a more profound insight into the molecular mechanisms governing the generation and progression of mtDNA deletions, we created the LostArc next-generation DNA sequencing platform, to detect and quantify uncommon mtDNA forms in small tissue specimens. LostArc's methodology is geared toward reducing mtDNA amplification during PCR, and instead facilitating mtDNA enrichment by strategically destroying the nuclear DNA. Cost-effective high-depth mtDNA sequencing is made possible by this method, exhibiting the sensitivity to identify one mtDNA deletion per million mtDNA circles. The following describes in detail the procedures for isolating genomic DNA from mouse tissues, enriching mitochondrial DNA by enzymatically eliminating linear nuclear DNA, and preparing libraries for unbiased next-generation mitochondrial DNA sequencing.
Varied clinical and genetic presentations in mitochondrial diseases are caused by pathogenic mutations present in both mitochondrial and nuclear genes. Over 300 nuclear genes linked to human mitochondrial diseases now harbor pathogenic variants. In spite of genetic testing's potential, diagnosing mitochondrial disease genetically is still an arduous task. Although, there are now diverse strategies which empower us to pinpoint causative variants within mitochondrial disease patients. Whole-exome sequencing (WES) is discussed in this chapter, highlighting recent advancements and various approaches to gene/variant prioritization.
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. Applying this technology to mtDNA mutations presents unique hurdles, distinct from other genetic conditions, due to the intricacies of mitochondrial genetics and the necessity of rigorous NGS data management and analysis. Developmental Biology This clinically-oriented protocol describes the process of sequencing the entire mitochondrial genome and quantifying heteroplasmy levels of mtDNA variants, from total DNA through the amplification of a single PCR product.
The power to transform plant mitochondrial genomes is accompanied by various advantages. Despite the present difficulties in the delivery of foreign DNA to mitochondria, mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) have enabled the elimination of mitochondrial genes. Genetic transformation of mitoTALENs encoding genes into the nuclear genome has enabled these knockouts. Earlier studies have revealed that double-strand breaks (DSBs) produced by mitoTALENs are mended through the process of ectopic homologous recombination. Following homologous recombination DNA repair, the genome experiences a deletion encompassing the location of the mitoTALEN target site. Processes of deletion and repair are causative factors in the rise of complexity within the mitochondrial genome. We describe a process for identifying ectopic homologous recombination events, stemming from double-strand break repair mechanisms induced by mitoTALENs.
Currently, in the microorganisms Chlamydomonas reinhardtii and Saccharomyces cerevisiae, mitochondrial genetic transformation is a routine procedure. 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). Through the application of biolistic techniques, DNA-coated microprojectiles are employed to introduce genetic material into mitochondria, with subsequent incorporation into mtDNA facilitated by the efficient homologous recombination systems in Saccharomyces cerevisiae and Chlamydomonas reinhardtii organelles. Yeast transformation, though occurring with a low frequency, enables the swift and facile isolation of transformants because of the substantial collection of selectable markers, both natural and synthetic. By contrast, the selection of transformants in C. reinhardtii is a protracted process, demanding the development of additional markers. Biolistic transformation techniques, including the materials and methods, are described to facilitate the process of inserting novel markers or inducing mutations in endogenous mitochondrial genes of the mtDNA. Even as alternative methods for mtDNA editing are being researched, the introduction of ectopic genes is presently subject to the constraints of biolistic transformation techniques.
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, in conjunction with the burgeoning availability of rationally designed AAV vectors capable of specifically transducing murine tissues, forms the basis for their suitability for this purpose. check details 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.
An Illumina platform-based next-generation sequencing assay, 5'-End-sequencing (5'-End-seq), permits the mapping of 5'-ends genome-wide. Technical Aspects of Cell Biology To ascertain the location of free 5'-ends in mtDNA isolated from fibroblasts, this method is utilized. This method provides the means to answer crucial questions concerning DNA integrity, replication mechanisms, and the precise events associated with priming, primer processing, nick processing, and double-strand break processing, applied to the entire genome.
A deficiency in mitochondrial DNA (mtDNA) maintenance, for example, due to issues with replication machinery or inadequate deoxyribonucleotide triphosphate (dNTP) levels, is a key factor in the development of numerous mitochondrial disorders. Multiple single ribonucleotides (rNMPs) are a consequence of the ordinary replication process happening within each mtDNA molecule. Embedded rNMPs, affecting the stability and nature of DNA, might thus affect mtDNA maintenance and have implications for mitochondrial disease. Correspondingly, they provide a detailed assessment of the intramitochondrial NTP/dNTP ratios. We detail, in this chapter, a method for quantifying mtDNA rNMP content through the use of alkaline gel electrophoresis and Southern blotting. For the examination of mtDNA, this process can be used with either total genomic DNA or purified samples. Furthermore, execution of this process is achievable with equipment present in most biomedical laboratories, facilitating concurrent evaluation of 10-20 samples based on the chosen gel method, and it can be adapted for the study of different mtDNA variations.