Investigations into the contributions of mitochondrial dynamics and function to platelet ageing and reactivity

PhD ThesisPlatelets are essential for the physiological process of haemostasis, but also drive pathological thrombosis. Platelet lifespan is a tightly controlled process through which platelets exist for approximately 10 days within the circulation of healthy individuals. However, in a number of disease states this process is dysregulated leading to an accelerated platelet turnover. Indeed, there are a number of reports suggesting that newly formed platelets are hyper-reactive and their presence has been associated with a higher risk of thrombosis. Whilst there are these indications of hyper-reactivity in young platelets, there are few systematic studies. Here I have used proteomics coupled with functional studies and immunofluorescence to show that there is a progressive decline in mitochondrial and cytoskeletal proteins as platelets age and an increase in apoptotic pathways. Given the apparent importance of mitochondria in supporting the predetermined platelet lifespan, it raised the question as to whether mitochondria are important for other platelet functional processes. Therefore, I sought to elucidate the impact of platelet activation on mitochondrial function and dynamics. Physiological stimulation causes an increase in mitochondrial respiration, consistent with an increase in energy demand. Interestingly, P2Y12 receptor inhibition causes a reduction in basal oxygen consumption, suggesting a dysregulation in mitochondrial function. Furthermore, this work highlights a role for mitochondria beyond energy production, with indications that stimulation causes platelets to package and release their mitochondria into microvesicles. Interestingly, these mitochondria-containing microvesicles have high P-selectin expression suggesting they may be more likely to interact with neutrophils than the rest of the microvesicle population. Indeed, incubation of neutrophils with mitochondria-positive microvesicles but not mitochondria-negative microvesicles causes alterations in the expression of surface markers; CD11b, CD66b and CXCR2, indicative of neutrophil activation potentially as a result of phagocytosis. This work highlights an important role of mitochondria in both platelet ageing and activation

Incretin dysregulation of lysyl oxidase: a novel mechanism for diabetic osteopenia

Incretins are gastric hormones released by intestinal K-cells in response to food consumption and stimulate insulin secretion from pancreatic beta cells. One of these hormones, glucose-dependent insulinotropic peptide (GIP) is also anabolic in bone. Individuals with diabetes experience diminished bone quality caused by a low bone formation osteopenia. The present study seeks to identify a mechanism for diabetic osteopenia in which diabetes interferes with GIP-stimulated increases in the collagen cross-linking enzyme lysyl oxidase (LOX) in osteoblasts, leading to decreased collagen integrity and the trabecular abnormalities seen in diabetic bone. Micro-CT analysis and picrosirius red histology of long bones from LOX +/- and wild type mice made diabetic by low dose streptozotocin induction revealed a profound exacerbation of the decreased bone volume, impaired trabecular structure, and disorganized collagen matrix seen in diabetic mice when the mice were also haploinsufficient for LOX. Furthermore, qPCR of RNA isolated from diabetic long bones revealed a more than 20 fold decrease in LOX expression in diabetic bone from wild type mice. Treatment of wild type osteoblasts in culture with GIP results in a significant increase in LOX transcript and protein levels. Interestingly in our diabetic mice there is a decrease in osteoblast derived LOX and an abnormal increase in serum levels of the anti-incretin gut-derived dopamine, which is known to inhibit the effects of GIP in the pancreas. Therefore the ability of dopamine to inhibit GIP-stimulated signaling in osteoblasts was examined. Data indicate a strong dose-dependent inhibition of GIP-stimulated LOX expression when primary osteoblast cultures are pretreated with dopamine. Finally, pretreatment of primary osteoblasts with the dopamine receptor inhibitor amisulpride restored the impaired GIP stimulated increases in LOX expression in osteoblasts isolated from diabetic mice. This study defines a potential mechanism for diabetic bone disease and suggests that interference with dopamine signaling would likely restore bone health in diabetes

Spina bifida

Spina bifida is a birth defect in which the vertebral column is open, often with spinal cord involvement. The most clinically significant subtype is myelomeningocele (open spina bifida), which is a condition characterized by failure of the lumbosacral spinal neural tube to close during embryonic development. The exposed neural tissue degenerates in utero, resulting in neurological deficit that varies with the level of the lesion. Occurring in approximately 1 per 1,000 births worldwide, myelomeningocele is one of the most common congenital malformations, but its cause is largely unknown. The genetic component is estimated at 60-70%, but few causative genes have been identified to date, despite much information from mouse models. Non-genetic maternal risk factors include reduced folate intake, anticonvulsant therapy, diabetes mellitus and obesity. Primary prevention by periconceptional supplementation with folic acid has been demonstrated in clinical trials, leading to food fortification programmes in many countries. Prenatal diagnosis is achieved by ultrasonography, enabling women to seek termination of pregnancy. Individuals who survive to birth have their lesions closed surgically, with subsequent management of associated defects, including the Chiari II brain malformation, hydrocephalus, and urological and orthopaedic sequelae. Fetal surgical repair of myelomeningocele has been associated with improved early neurological outcome compared with postnatal operation. Myelomeningocele affects quality of life during childhood, adolescence and adulthood, posing a challenge for individuals, families and society as a whole. For an illustrated summary of this Primer, visit: http://go.nature.com/fK9XNa