Screening of ATP5D, a candidate for Amyotrophic Lateral Sclerosis (ALS)

Background: Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease, characterized by progressive death of upper and lower motor neurons (MN), leading to muscle atrophy and eventually death. ALS occurs in both sporadic and familial forms, which are clinically indistinguishable. Approximately 10% of cases are familial, and 20% of these have been linked to a dominant mutant form of the Cu/Zn superoxide dismutase 1 (SOD1) enzyme1. Previous studies have highlighted the complexity of this disease and its multifactorial origin. Several reports suggest that alterations in the metabolism occur at very early stages in human ALS patients. It is well documented that ALS patients are hypermetabolic 2-4, despite the reduction in muscle mass that occurs during the course of the disease. Our own findings indicate activation of the fatty acid metabolism along with the carbohydrate metabolism to satisfy a higher demand for ATP synthesis. Although several components of the respiratory chain and of the ATP synthase are upregulated in our microarray study, the ATP synthase subunit (Atp5d) was downregulated. This subunit plays a key role in the coupling between the energy produced by proton translocation within the F0 subunit of the ATP synthase and ATP production. Decreased expression of this gene has also been reported in human ALS7. The transcription factors and the mechanisms directly involved in the transcriptional regulation of this subunit are still unknown. Nevertheless, one report showed that Atp5d is copper sensitive8 and a low concentration of mitochondrial copper can lead to its downregulation without altering the expression level of other subunits of the respiratory chain. This characteristic might affect ATP5D regulation in cases of ALS, in which altered affinity of mutant SOD1 for copper has been described9. In yeast, 30% reduction of the ATP synthase subunit lead to a massive proton leak across the mitochondrial inner membrane, without affecting the ATP synthase complex structure and stoichiometry10. This change represents a potential mechanism for the early generation of motor neuronal oxidative stress and identifies Atp5d as a good candidate in the pathophysiology of ALS.

Hypothesis: Our hypothesis is that dysfunction or dysregulation of the subunit delta of the ATP synthase might be involved in the cascade of events leading to the development of ALS. ATP5D dysfunction can cause proton leak across the mitochondrial membrane, partially uncoupling the ATP synthesis process and affecting the mitochondrial membrane potential. This process would constitute an additional source of oxidative stress beyond the physiological level. The cellular antioxidant response could compensate for this unbalance in young age, but time would lead to accumulation of reactive oxygen species (ROS) and oxidative damage, especially with age, when the cellular antioxidant response becomes lower. Oxidised are ubiquitinated and targeted for proteasomal degradation. Over time, the accumulation of damaged proteins and ROS is likely to cause a general collapse in cellular functioning, leading to downregulation of compensatory pathways previously activated and leaving the cell with decreased energy and turnover. Motor neurones would be among the cells most sensitive to this process because of the amount of energy they need in order to maintain an efficient axonal transport and compartment functionality.

Aims: Our aim is to sequence the gene encoding for ATP5D in ALS sporadic and familial cases and controls in order to screen for potential or functional polymorphisms that might affect the structure or function of this subunit of the ATP synthase machinery.

Research plan: ATP5D is a small gene, containing 5 exons. These will be sequenced in DNA samples isolated from central nervous system (CNS) tissue of 141 MND cases (11 familial, 130 sporadic) in the Sheffield Brain Tissue Bank. All samples are negative for SOD1 mutations. These Tissue Bank donations accrued from patients attending an MND clinic between 1989 and 2006. 200 control samples from the Sheffield DNA bank are also available. All control individuals were neurologically normal and matched to the disease cohort by age, sex and geographical location. Ethnicity of cases and controls is UK Caucasian. Primers will be designed to amplify the 5 exons of ATP5D, including 101bp of the 5’UTR immediately upstream of the translation start site, intron/exon boundaries and 387bp of the 3’UTR. Following clean-up with ExoSAP-IT (GE Healthcare, UK), PCR products will be bidirectionally sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosciences) as per manufacturer’s instructions. Samples will be run on a 3730 capillary analyser (Applied Biosciences) at the Core Genetics facility at the University of Sheffield. Electropherograms will be then analysed using Finch TV software and sequences aligned using Sequencher. Potential mutations will be then verified using restriction digestion where possible. During the first week of the project, the student will be taken through the primer design process and will familiarise with the PCR technique. From week two until week five the 141 samples will be sequenced and sequences analysed. ATP5D is highly conserved and only three non-synonymous polymorphisms have been identified so far, but frequency data are not available. If genetic variants are identified, the 200 control samples will be screened for comparison. During week six the samples that need to be repeated or sequence changes verified will be re-analysed using restriction enzyme digestion or TaqMan Genotyping Assays (Applied Biosystems) when possible, otherwise sequencing will be repeated.

Research training which will be provided by the project.

The student will learn basic techniques of molecular biology, such as primer design, PCR, sequencing and single nucleotide polymorphism (SNP) analysis, which have wide application in several research fields. Moreover, the candidate will have the opportunity to use software and databases of fundamental importance in scientific research, i.e. Primer Express, Ensembl, Blast, PubMed. This experience will also provide skills and experience in planning, executing, recording and reporting on laboratory-based research. In addition, the student will familiarise him/her self with the environment of a research lab and with the simple but fundamental rules that this kind of experience requires, i.e. health and safety measures, respect for common spaces and instruments, punctuality. The student will be in a scientific research environment for six weeks during which he/she will attend lab meetings, talks, lectures and journal clubs, providing further opportunities to increase his/her knowledge and understanding of the pathophysiology of ALS, as well as stimulate his/her interest towards wider topics in the field of Neuroscience.