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 technologies that are currently available for single-cell analysis, 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. This allows isolation of mitochondria with unprecedented spatial precision with minimal disruption to mitochondria and their immediate cellular environment

No excess of mitochondrial DNA deletions within muscle in progressive multiple sclerosis

BACKGROUND: Mitochondrial dysfunction is an established feature of multiple sclerosis (MS). We recently described high levels of mitochondrial DNA (mtDNA) deletions within respiratory enzyme-deficient (lacking mitochondrial respiratory chain complex IV with intact complex II) neurons and choroid plexus epithelial cells in progressive MS. OBJECTIVES: The objective of this paper is to determine whether respiratory enzyme deficiency and mtDNA deletions in MS were in excess of age-related changes within muscle, which, like neurons, are post-mitotic cells that frequently harbour mtDNA deletions with ageing and in disease. METHODS: In progressive MS cases (n=17), known to harbour an excess of mtDNA deletions in the central nervous system (CNS), and controls (n=15), we studied muscle (paraspinal) and explored mitochondria in single fibres. Histochemistry, immunohistochemistry, laser microdissection, real-time polymerase chain reaction (PCR), long-range PCR and sequencing were used to resolve the single muscle fibres. RESULTS: The percentage of respiratory enzyme-deficient muscle fibres, mtDNA deletion level and percentage of muscle fibres harbouring high levels of mtDNA deletions were not significantly different in MS compared with controls. CONCLUSION: Our findings do not provide support to the existence of a diffuse mitochondrial abnormality involving multiple systems in MS. Understanding the cause(s) of the CNS mitochondrial dysfunction in progressive MS remains a research priority

A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders

\ua9 2024 The AuthorsMitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre

Challenges of caring for children with mental disorders: Experiences and views of caregivers attending the outpatient clinic at Muhimbili National Hospital, Dar es Salaam - Tanzania

It is estimated that world-wide up to 20 % of children suffer from debilitating mental illness. Mental disorders that pose a significant concern include learning disorders, hyperkinetic disorders (ADHD), depression, psychosis, pervasive development disorders, attachment disorders, anxiety disorders, conduct disorder, substance abuse and eating disorders. Living with such children can be very stressful for caregivers in the family. Therefore, determination of challenges of living with these children is important in the process of finding ways to help or support caregivers to provide proper care for their children. The purpose of this study was to explore the psychological and emotional, social, and economic challenges that parents or guardians experience when caring for mentally ill children and what they do to address or deal with them. A qualitative study design using in-depth interviews and focus group discussions was applied. The study was conducted at the psychiatric unit of Muhimbili National Hospital in Tanzania. Two focus groups discussions (FGDs) and 8 in-depth interviews were conducted with caregivers who attended the psychiatric clinic with their children. Data analysis was done using content analysis. The study revealed psychological and emotional, social, and economic challenges caregivers endure while living with mentally ill children. Psychological and emotional challenges included being stressed by caring tasks and having worries about the present and future life of their children. They had feelings of sadness, and inner pain or bitterness due to the disturbing behaviour of the children. They also experienced some communication problems with their children due to their inability to talk. Social challenges were inadequate social services for their children, stigma, burden of caring task, lack of public awareness of mental illness, lack of social support, and problems with social life. The economic challenges were poverty, child care interfering with various income generating activities in the family, and extra expenses associated with the child's illness. Caregivers of mentally ill children experience various psychological and emotional, social, and economic challenges. Professional assistance, public awareness of mental illnesses in children, social support by the government, private sector, and non-governmental organizations (NGOs) are important in addressing these challenges