Investigating the biology of mitochondrial DNA disease transmission to enable affected families to have healthy children

Mitochondria are critically important for generating the energy that is essential for life. Despite the importance of mitochondria, one in 400 people has a maternally-inherited mutation in mitochondrial DNA (mtDNA), the blue print for some vital mitochondrial components. While most women transmitting these mtDNA mutations will have children who only develop mild symptoms, such as deafness in old age, they may be severely affected. The mtDNA mutations can cause a range of illnesses, including deafness, blindness, diabetes, loss of skills, heart and liver failure and there are no curative treatments.

Some centres are developing techniques for replacing disabled mitochondria with healthy ones. In this technique (so-called "nuclear transfer"), embryos of the couple at risk of having an affected child are generated in vitro. At an early stage, the nucleus that contains all of the genetic material apart from the mitochondria is removed from the egg (oocyte) and placed into a healthy egg or embryo from which the nucleus has been removed. The resulting embryo can then be placed in the mother's womb where it becomes a baby. While experiments on monkeys and mice suggest that such babies will probably be healthy, they have not been done in humans. Replacing the nucleus does not prevent development into a baby, but it causes damage that probably requires radical re-organisation of the cell. Because the embryo is already highly organised at this stage, such manipulations could have consequences later in life.

We aim to improve the genetic management of these diseases using less radical techniques. Current approaches are hampered by our poor understanding of the underlying mechanisms. MtDNA is inherited via the female line only. The chances that a woman carrying mtDNA mutations will pass them on to her child, and that child develops symptoms, are exceptionally difficult to predict. This is because both mutant and normal mtDNA are found in the same individual. The severity of the disease depends on the proportion of abnormal mtDNAs in particular cells of the body. However, this proportion varies from one generation to the next and cannot be predicted. The variability in the number of abnormal mtDNAs inherited is caused by an event known as the mitochondrial bottleneck which takes place in the female germline.

The biological processes that we plan to study determine the effectiveness of medical interventions. These treatments include preimplantation genetic diagnosis where eggs are fertilized in a test tube, tested, and low risk embryos selected to start a pregnancy. We will investigate existing cellular mechanisms that might be able to eliminate the unhealthy mitochondria, without such drastic interventions. In the long term we may determine whether nuclear transfer damages the cellular processes for maintaining healthy mitochondria. We may also develop less radical procedures, to generate embryos that have healthy mitochondria, without causing damage to the cell. Our ultimate aim is to enable affected families to have healthier children.

Either mitophagy (recycling of mitochondria) or clonal proliferation of mtDNA subpopulations may underlie the unpredictable transmission of mutant mtDNA.

We propose that mtDNA turnover by mitophagy can be modulated to enhance purifying selection of normal mtDNA during early development.

We will determine whether:
1) mitochondrial function influences mtDNA content of:
a) "spare" human preimplantation embryos becoming available from families with mtDNA disease undergoing in vitro fertilisation
b) mouse oocytes with impaired mitochondrial function

2) mtDNA synthesis occurs in:
a) mouse and human oocytes maturating in vitro
b) mouse and donated human preimplantation embryos
using BrdU pulse labelling.

This may reveal any contribution of clonal proliferation of mtDNA to "purifying selection"

3) Modulators of mitochondrial recycling by mitophagy can be developed to enhance purifying selection. We will investigate and quantitate mitophagy in mouse pre-implantation embryos using transgenics with fluorescent organelles. We will cross mice with GFP targeted to LC3 in the autophagosomes against mice with DS-red targeted to mitochondria. Mitophagy is apparent by co-localisation of red and green signals in offspring (and this can be quantitated in cell lines using Imagestream, a technique combining flow cytometry and fluorescence microscopy, fig1).

We will use this model to determine whether mitophagy:
a) occurs in mouse oocytes and pre-implantation embryos
b) can be driven by exogenous treatments
c) is exacerbated in mouse models expected to modulate mitophagy, such as OPA1 knock down (required for mitochondrial fusion).

4) Purifying selection of wild type mtDNA in pluripotent cell cultures, heteroplasmic for pathogenic mtDNA mutants, can be driven by agents identified in (3).

Ultimately we will use the mouse with fluorescent autophagosomes to investigate mitophagy in:
a) nuclear and cytoplasmic transfer
b) multiple disease model

One in 400 Caucasians carries a pathogenic mitochondrial DNA (mtDNA) mutation. Nevertheless, prenatal diagnosis to prevent transmission is poorly developed, because the unique segregation patterns resulting from heteroplasmy (co-existing normal and mutant mtDNA) during development are not understood. Even pre-implantation genetic diagnosis, where low risk embryos are preferentially transferred to the uterus, is poorly developed for mtDNA disease.

Immediate beneficiaries from this research are therefore those who suffer from these conditions, their progeny, and those who treat them. Longer-term beneficiaries include: both public-sector and commercial providers of fertility treatments; policymakers and regulators (including specifically the HFEA and its successor), who will gain information on the clinical possibilities; charities in the area of infertility treatment and counselling; and the general public whose understanding of science will be enlarged.

How will they benefit from this research?

The research will impact on the nation's health and wealth as follows. Understanding the biological mechanisms of mtDNA transmission will improve both prenatal and preimplantation diagnostic techniques for mtDNA diseases. As well as informing genetic management our study may also enable us to develop a new strategy for reducing transmission risks. Indeed, we intend to determine whether the existing cellular mechanisms for mitochondrial quality control can be used to destroy mutant mtDNA and reduce the transmission risk. This may be a safer alternative to genetic manipulation of mtDNA mutant load, or may be usable in conjunction with pre-implantation diagnosis. Furthermore the research required to develop safe nuclear transfer requires very many donated human oocytes. If our work superceeded nuclear transfer, oocytes would become available for other purposes. For instance, donor oocytes provide a simple alternative to affected women who want to reduce their transmission risk by being the birth mother but not the genetic mother. Available oocytes may also be donated for developing stem cell therapies.

Through this project, we will increase understanding of early embryo development by determining whether mitophagy (autophagy that specifically recycles spent mitochondria) occurs during preimplantation development, is important and/or affects segregation of mtDNA mutant populations. As nutrient availability affects autophagy, mitophagy could be an important determinant of metabolic programming. This will promote transfer of new, translational technology from laboratory to bedside and relieve the pressure on the donor oocytes needed for other applications.

The mouse model with red mitochondria and green autophagic vacuoles will facilitate future studies of mitophagy. A mouse model with down-regulated mitophagy would allow future studies of (i) potential ways to influence the mitochondrial transmission (ii) developmental programming of diabetes risks.

Public engagement
The principal applicant (JP) has experience explaining research to the general public. In 2009 she organised, chaired and lectured at an information evening for Oxford mitochondrial patients. This was highly rated by families attending ("Relevant to my family" 85%, "Overall satisfaction" 91). Patient support organisations presented posters at this meeting, including a woman who had a healthy child as a result of oocyte sampling (she described her experiences in JP's article Poulton et al, BMJ 338, 345 (2009)). A similar event is scheduled for 2011 and then every 1-2 years. In 2010 JP organised and chaired an international workshop on mtDNA transmission, with the European Neuromuscular Centre, to which she invited a patient representative. He presented lay views to the participating scientists and ethicists, including his experience of counselling, pre-implantation genetic diagnosis and the birth of his healthy child.