Nanobiopsy investigation of the subcellular mtDNA heteroplasmy in human tissues.

Mitochondrial function is critical to continued cellular vitality and is an important contributor to a growing number of human diseases. Mitochondrial dysfunction is typically heterogeneous, mediated through the clonal expansion of mitochondrial DNA (mtDNA) variants in a subset of cells in a given tissue. To date, our understanding of the dynamics of clonal expansion of mtDNA variants has been technically limited to the single cell-level. Here, we report the use of nanobiopsy for subcellular sampling from human tissues, combined with next-generation sequencing to assess subcellular mtDNA mutation load in human tissue from mitochondrial disease patients. The ability to map mitochondrial mutation loads within individual cells of diseased tissue samples will further our understanding of mitochondrial genetic diseases.

A subcellular cookie cutter for spatial genomics in human tissue.

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.

The Isolation and Deep Sequencing of Mitochondrial DNA.

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 .

Mitochondrial isolation: when size matters.

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.

Mitochondrial DNA changes in pedunculopontine cholinergic neurons in Parkinson disease.

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.

An animal model mimicking pedunculopontine nucleus cholinergic degeneration in Parkinson's disease.

A rostral brainstem structure, the pedunculopontine nucleus (PPN), is severely affected by Parkinson's disease (PD) pathology and is regarded a promising target for therapeutic deep-brain stimulation (DBS). However, understanding the PPN's role in PD and assessing the potential of DBS are hampered by the lack of a suitable model of PPN degeneration. Rats were rendered Parkinsonian through a unilateral substantia nigra pars compacta (SNpc) stereotaxic injection of the proteasome inhibitor Lactacystin, to investigate whether the lesion's pathological effects spread to impact the integrity of PPN cholinergic neurons which are affected in PD. At 5 weeks post-surgery, stereological analysis revealed that the lesion caused a 48 % loss of dopaminergic SNpc neurons and a 61 % loss of PPN cholinergic neurons, accompanied by substantial somatic hypotrophy in the remaining cholinergic neurons. Magnetic resonance imaging revealed T2 signal hyper-/hypointensity in the PPN of the injected hemisphere, respectively at weeks 3 and 5 post-lesion. Moreover, isolated PPN cholinergic neurons revealed no significant alterations in key autophagy mRNA levels, suggesting that autophagy-related mechanisms fail to protect the PPN against Lactacystin-induced cellular changes. Hence, the current results suggest that the Lactacystin PD model offers a suitable model for investigating the role of the PPN in PD.

Behavioral testing regimens in genetic-based animal models of Parkinson's disease: cogencies and caveats.

Although the onset and progression of Parkinson's disease (PD) is fundamentally sporadic, identification of several of the genes implicated in the disease has provided significant insight concerning patho-physiological mechanisms potentially underlying sporadic PD. Moreover, such studies have caused a revolution in the way researchers view the disease. Since single genes responsible for rare familial forms of the disease have only been identified within the past few years, animal models based on these defects have only recently been generated, thereby not leaving a lot of time for their evaluation and subsequent improvement. The current article provides an extensive review of the major motor and non-motor behavioral tests used in genetically-induced Parkinsonian animals. Moreover, we assess the insights concerning the etiopathogenesis of PD generated from use of such tests and how these have improved available treatment strategies for alleviating aspects of sporadic and non-sporadic parkinsonism.