RESUMO
Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of >20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.
Assuntos
Genômica , Biópsia , Humanos , Microdissecção e Captura a Laser/métodosRESUMO
In Parkinson disease (PD), mitochondrial dysfunction associates with nigral dopaminergic neuronal loss. Cholinergic neuronal loss co-occurs, particularly within a brainstem structure, the pedunculopontine nucleus (PPN). We isolated single cholinergic neurons from postmortem PPNs of aged controls and PD patients. Mitochondrial DNA (mtDNA) copy number and mtDNA deletions were increased significantly in PD patients compared to controls. Furthermore, compared to controls the PD patients had significantly more PPN cholinergic neurons containing mtDNA deletion levels exceeding 60%, a level associated with deleterious effects on oxidative phosphorylation. The current results differ from studies reporting mtDNA depletion in nigral dopaminergic neurons of PD patients. Ann Neurol 2017;82:1016-1021.
Assuntos
Neurônios Colinérgicos/metabolismo , DNA Mitocondrial/metabolismo , Doença de Parkinson/metabolismo , Núcleo Tegmental Pedunculopontino/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neurônios Colinérgicos/patologia , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Doença de Parkinson/genética , Doença de Parkinson/patologia , Núcleo Tegmental Pedunculopontino/patologiaRESUMO
In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .
Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Microdissecção e Captura a Laser , Mitocôndrias Musculares/genética , Músculo Esquelético/citologia , Reação em Cadeia da Polimerase/métodosRESUMO
Mitochondrial vitality is critical to cellular function, with mitochondrial dysfunction linked to a growing number of human diseases. Tissue and cellular heterogeneity, in terms of genetics, dynamics and function means that increasingly mitochondrial research is conducted at the single cell level. Whilst, there are several single-cell technologies that are currently available, each with their advantages, they cannot be easily adapted to study mitochondria with subcellular resolution. Here we review the current techniques and strategies for mitochondrial isolation, critically discussing each technology's limitations for future mitochondrial research. Finally, we highlight and discuss the recent breakthroughs in sub-cellular isolation techniques, with a particular focus on nanotechnologies that enable the isolation of mitochondria, from subcellular compartments, with unprecedented spatial precision with minimal disruption to mitochondria and their immediate cellular environment.