Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns

Generation of ρ0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (ρ0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK− the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria

Mapping Time-course Mitochondrial Adaptations in the Kidney in Experimental Diabetes

Abstract Oxidative phosphorylation drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homeostasis. In diabetic kidney disease, mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics, have not been previously documented. Here, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks) a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells of diabetic kidneys were clearly apparent, but no change urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary Kidney injury molecule (Kim)-1 excretion, where an increase in Complex I-linked oxygen consumption rate, in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of diabetic kidney disease. Summary statement We identified that dysfunction of cellular power stations, mitochondria, may precede the development of kidney disease in diabetes. This suggests that mitochondrial dysfunction is a primary cause of diabetic nephropathy, which could be targeted to improve the burden of this disease. Short title: Mitochondrial adaptations in diabetic nephropath

Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes

Abstract Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H 2 O 2 ) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP , indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD

Mitochondrial function as a determinant of life span

Average human life expectancy has progressively increased over many decades largely due to improvements in nutrition, vaccination, antimicrobial agents, and effective treatment/prevention of cardiovascular disease, cancer, etc. Maximal life span, in contrast, has changed very little. Caloric restriction (CR) increases maximal life span in many species, in concert with improvements in mitochondrial function. These effects have yet to be demonstrated in humans, and the duration and level of CR required to extend life span in animals is not realistic in humans. Physical activity (voluntary exercise) continues to hold much promise for increasing healthy life expectancy in humans, but remains to show any impact to increase maximal life span. However, longevity in Caenorhabditis elegans is related to activity levels, possibly through maintenance of mitochondrial function throughout the life span. In humans, we reported a progressive decline in muscle mitochondrial DNA abundance and protein synthesis with age. Other investigators also noted age-related declines in muscle mitochondrial function, which are related to peak oxygen uptake. Long-term aerobic exercise largely prevented age-related declines in mitochondrial DNA abundance and function in humans and may increase spontaneous activity levels in mice. Notwithstanding, the impact of aerobic exercise and activity levels on maximal life span is uncertain. It is proposed that age-related declines in mitochondrial content and function not only affect physical function, but also play a major role in regulation of life span. Regular aerobic exercise and prevention of adiposity by healthy diet may increase healthy life expectancy and prolong life span through beneficial effects at the level of the mitochondrion

Neck muscle cross-sectional area, brain volume and cognition in healthy older men; A cohort study

BACKGROUND: Two important consequences of the normal ageing process are sarcopenia (the age-related loss of muscle mass and function) and age-related cognitive decline. Existing data support positive relationships between muscle function, cognition and brain structure. However, studies investigating these relationships at older ages are lacking and rarely include a measure of muscle size. Here we test whether neck muscle size is positively associated with cognition and brain structure in older men. METHODS: We studied 51 healthy older men with mean age 73.8 (sd 1.5) years. Neck muscle cross-sectional area (CSA) was measured from T1-weighted MR-brain scans using a validated technique. We measured multiple cognitive domains including verbal and visuospatial memory, executive functioning and estimated prior cognitive ability. Whole brain, ventricular, hippocampal and cerebellar volumes were measured with MRI. General linear models (ANCOVA) were performed. RESULTS: Larger neck muscle CSA was associated with less whole brain atrophy (t = 2.86, p = 0.01, partial eta squared 17%). Neck muscle CSA was not associated with other neuroimaging variables or current cognitive ability. Smaller neck muscle CSA was unexpectedly associated with higher prior cognition (t = −2.12, p < 0.05, partial eta squared 10%). CONCLUSIONS: In healthy older men, preservation of whole brain volume (i.e. less atrophy) is associated with larger muscle size. Longitudinal ageing studies are now required to investigate these relationships further

Functional Changes in the Retina during and after Acute Intraocular Pressure Elevation in Mice MATERIALS AND METHODS Animals

PURPOSE. To examine retinal function using the full-field electroretinogram (ERG) during and after acute intraocular pressure (IOP) elevation in wild-type mice. METHODS. IOP was elevated by anterior chamber cannulation in wild-type C57/BL6 mice. The pressure-function relationship was determined by IOP elevation in steps from baseline to 80 mm Hg. The rate of functional recovery was assessed for 60 minutes after an IOP spike of 50 mm Hg for 30 minutes. During and immediately after IOP elevation, scotopic ERG signals were recorded in response to dim and bright flashes (Ϫ4.54, Ϫ2.23, and 0.34 log cd ⅐ s ⅐ m Ϫ2 ) and analyzed for photoreceptoral (a-wave), ON-bipolar (b-wave), oscillatory potentials (OPs), and scotopic threshold responses (positive [p]STR/negative [n] STR). A full ERG protocol was collected 2 days before and 7 days after the single 50-mm Hg IOP spike. RESULTS. The pSTR was most sensitive to IOP elevation with 50% amplitude loss () at 41 mm Hg (, 95% confidence limits (CL): 37.7, 45.6) followed by nSTR at 45 mm Hg (95% CL: 41.0, 49.1). pSTR was significantly more sensitive than the b-wave (95% CL: 41.4, 49.1), a-wave (95% CL: 47.6, 55.3), and OPs (95% CL: 49.6, 59.2). pSTR showed slower recovery immediately after the 50 mm Hg spike compared with the b-wave (P ϭ 0.02). One week after the 50-mm Hg spike, pSTR (Ϫ30% Ϯ 6%, P Ͻ 0.001) and OP (Ϫ27% Ϯ 2%, P Ͻ 0.001) amplitudes were reduced, whereas other components were unaffected. CONCLUSIONS. The STR in mice is more sensitive to acute IOP elevation and recovers slower than other ERG components. Reduction in pSTR and OP amplitude at 1 week suggests persistent impairment of inner retinal function can occur after a single IOP spike. (Invest Ophthalmol Vis Sci. 2009;50: 5732-5740) DOI:10.1167/iovs.09-3814 E xperimental mouse models are increasingly important in studies of neurodegeneration and glaucoma, largely due to an improved understanding of mouse genetics, the relative ease of genetic modifications and an ever-increasing number of murine lines with well-defined genotypes and phenotypes. Several models of intraocular pressure (IOP) elevation, both induced and spontaneously occurring, have been used to study retinal neuronal injury in mice. Investigators have examined the effect of acute IOP elevation on retinal function in humans and in some animal models. 6 -9 In general, their studies show that an IOP elevation of 30 to 35 mm Hg is needed to induce retinal dysfunction. Bui et al. 10 The effect of a single acute IOP elevation on retinal function and its recovery has been investigated in the DBA/2J mice, a strain predisposed to spontaneous glaucomatous optic neuropathy. Nagaraju et al. 11 showed that a 32% to 38% increase in IOP (ϳ5 mm Hg) could be achieved by a 60°head down position. Although this IOP change had no effect on the pattern ERG in 3-month-old DBA/2J mice, it produced a ϳ65% amplitude reduction in 10-month-old mice. The pressure-function relationship to a moderate IOP challenge has yet to be assessed in wild-type mice. In this study, we examined in detail the pressure (IOP)-function relationship in adult wild-type mice during a step-wise increase in IOP. In particular, we examined whether ERG components arising from the inner, middle, and outer retina show different sensitivity to IOP elevation. We also considered whether there was a difference in the immediate (60 minutes) and medium-term (7 days) recovery of ERG components after a single IOP spike

Pioglitazone and deoxyribonucleoside combination treatment increases mitochondrial respiratory capacity in m.3243A>G MELAS cybrid cells

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor &gamma; (PPAR&gamma;) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing &gt;90% of the m.3243A&gt;G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A&gt;G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A&gt;G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease