Age-related mitochondrial DNA depletion and the impact on pancreatic beta cell function

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes

Secondary mtDNA defects do not cause optic nerve dysfunction in a mouse model of dominant optic atrophy

purpose. The majority of patients with autosomal dominant optic atrophy (DOA) harbor pathogenic OPA1 mutations and certain missense mutations, mostly within the GTPase domain, have recently been shown to cause multiple mitochondrial DNA (mtDNA) deletions in skeletal muscle. This raises the possibility that the optic neuropathy could be the result of secondary mtDNA defects accumulating within retinal ganglion cells (RGCs). To explore this hypothesis, the authors looked for evidence of mitochondrial dysfunction in a mouse model of DOA and documented the visual and neurologic progression in aging mutant mice. methods. Visual function was assessed with a rotating optokinetic (OKN) drum at ages 13 and 18 months and neurologic phenotyping was performed using the primary SHIRPA screen at age 13 months, comparing mutant Opa1+/− mice with wild-type C57Bl/6 mice. The presence of cytochrome c oxidase (COX) deficiency and multiple mtDNA deletions was investigated in gastrocnemius muscle and eye specimens harvested from 2- and 11-month-old Opa1+/+ and Opa1+/− mice. results. At age 13 months, Opa1+/− mice had a statistically significant reduction in OKN responses compared to C57Bl/6 controls with both 2° and 8° gratings (P < 0.001). At age 18 months, the difference between the two groups was significant for the 8° grating (P = 0.003) but not for the 2° grating (P = 0.082). Opa1+/− mice did not exhibit any significant neuromuscular deficits and no COX deficient areas or secondary mtDNA deletions were identified in skeletal muscle or the RGC layer. There was also no evidence of significant mtDNA depletion or proliferation in skeletal muscle from Opa1+/− mice. conclusions. COX deficiency and mtDNA abnormalities do not contribute to optic nerve dysfunction in pure DOA

Embryo-Derived Extracellular Vesicles and their Potential as Biomarkers for Embryo Quality during IVF

A systematic review looking at embryo-derived extracellular vesicles and their association with embryo quality; and their potential to be used for embryo quality assessment during IVF/IVP

Normal Levels of Wild-Type Mitochondrial DNA Maintain Cytochrome c Oxidase Activity for Two Pathogenic Mitochondrial DNA Mutations but Not for m.3243A→G

Mitochondrial DNA (mtDNA) mutations are a common cause of human disease and accumulate as part of normal ageing and in common neurodegenerative disorders. Cells express a biochemical defect only when the proportion of mutated mtDNA exceeds a critical threshold, but it is not clear whether the actual cause of this defect is a loss of wild-type mtDNA, an excess of mutated mtDNA, or a combination of the two. Here, we show that segments of human skeletal muscle fibers harboring two pathogenic mtDNA mutations retain normal cytochrome c oxidase (COX) activity by maintaining a minimum amount of wild-type mtDNA. For these mutations, direct measurements of mutated and wild-type mtDNA molecules within the same skeletal muscle fiber are consistent with the “maintenance of wild type” hypothesis, which predicts that there is nonselective proliferation of mutated and wild-type mtDNA in response to the molecular defect. However, for the m.3243A→G mutation, a superabundance of wild-type mtDNA was found in many muscle-fiber sections with negligible COX activity, indicating that the pathogenic mechanism for this particular mutation involves interference with the function of the wild-type mtDNA or wild-type gene products

Antiphospholipid antibodies increase the levels of mitochondrial DNA in placental extracellular vesicles: Alarmin-g for preeclampsia

The pathogenesis of preeclampsia remains unclear but placental factors are known to play a crucial role causing maternal endothelial cell dysfunction. One potential factor is placental micro-and nano-vesicles. Antiphospholipid antibodies (aPL) increase the risk of preeclampsia ten-fold, in part by damaging the mitochondria in the syncytiotrophoblast. Since mitochondrial DNA (mtDNA) is a danger-associated molecular pattern (DAMP/alarmin) that may activate endothelial cells, the aims of the current study were to investigate whether aPL affect the number of placental vesicles extruded, their mtDNA content and their ability to activate endothelial cells. Exposure of first trimester human placental explants to aPL affected neither the number nor size of extruded micro-and nano-vesicles (n = 5), however their levels of mtDNA were increased (n = 6). These vesicles significantly activated endothelial cells (n = 5), which was prevented by blocking toll-like receptor 9 (TLR-9), a receptor for extracellular DNA. Thus, aPL may increase the risk of preeclampsia in part by increasing the amount of mtDNA associated with placental vesicles. That mitochondrial DNA is recognised as a DAMP by TLR-9 to cause endothelial cell activation, raises the possibility that placental vesicles or TLR-9 might be a target for pharmaceutical intervention to reduce the consequences of aPL in pregnancy.Funding Agencies|University of Auckland Health Research Doctoral Scholarship; Freemasons Postgraduate Scholarship; Health Research Council of New Zealand [15/209]</p

The effect of glibenclamide on insulin secretion following <i>TFAM</i> silencing-induced mtDNA depletion.

<p>Seventy two hours post transfection cells were stimulated with 3 mM or 25 mM glucose, supplemented with or without 0.1 µM glibenclamide. Insulin secretion was determined by insulin ELISA and normalised to whole cell protein content. Data shown are from 4 separated experiments performed in triplicate, and are normalised to Scrambled negative control cells stimulated with 3 mM glucose without glibenclamide. Data presented are means ± SEM. * p&lt;0.05, ** p&lt;0.01.</p

TFAM mRNA silencing induces mtDNA depletion 72 h post transfection.

<p>MIN6 cells were transfected with TFAM-193, TFAM-429 or Scrambled siRNA probes, or with no siRNA (Shocked). TFAM mRNA expression was quantified relative to reference gene <i>B2M</i> by real-time PCR at 48 h (A) and 72 h (B) post transfection. mtDNA depletion was also measured by real-time PCR, using mitochondrial encoded <i>ND5</i> relative to nuclear encoded <i>GAPDH</i> at 48 h (C) and 72 h (D) post transfection. All results normalised to Scrambled negative control. Experiment repeated once (C), twice (A) or 4 times (B, D) in triplicate. Data presented are means ± SEM (SD in (C)). * p&lt;0.05, *** p&lt;0.001.B2M, β2 Microglobulin; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; ND5, NADH Dehydrogenase 5; TFAM, Mitochondrial Transcription Factor A.</p