Mitochondrial diversity probed in mouse cerebellum elucidates cell type-specific fine-tuning of mitochondrial biology

Mitochondria house a variety of cellular functions, including catabolic and anabolic pathways, apoptosis and Ca2+ handling. These functions critically depend on nuclear-encoded proteins given that mitochondrial DNA only encodes for 13 proteins, which are incorporated into the respiratory chain. While mitochondria differ in morphology and functions among tissues in vivo, mitochondrial diversity among cell types is less well understood – especially in heterogeneous tissues such as the nervous system. Here, I present an in vivo tool for the characterization of cell type-specific mitochondria in mouse. Via the MitoTag mouse model, mitochondria from the cell type of interest are tagged in a Cre recombinase-dependent manner with GFP, which is localized to the outer mitochondrial membrane (GFP-OMM). This tagging allows for the immunocapture of organelles and their subsequent investigation through functional assays and omics-based screenings. We applied the MitoTag approach to the cerebellum and profiled the mitochondrial proteome of Purkinje cells, granule cells and astrocytes. Among these cell types, we found 196 proteins differentially enriched, of which 19 candidates were independently confirmed as cell type-enriched mitochondrial ‘markers’. Further analysis revealed functional specializations that we corroborated in independent assays using immunocaptured mitochondria. Specifically, astrocytic mitochondria superiorly oxidized long-chain fatty acids, while neuronal mitochondria demonstrated enhanced Ca2+ uptake via the mitochondrial calcium uniporter in granule cells and enhanced contact sites with the endoplasmic reticulum via regulator of microtubule dynamics protein 3 in Purkinje cells. In studies across species, I confirmed that neural mitochondrial diversity is conserved in the nervous system of mammals, aves and amphibian. Hence, we used neuronal and astrocytic mitochondrial ‘markers’ to show mitochondrial pathology in mouse models and human cases of Alzheimer’s disease and amyotrophic lateral sclerosis. The MitoTag approach enables mitochondrial research in a defined cellular context in vivo. Future applications will reveal the cell type-specific fine-tuning of mitochondria in many contexts, such as development, aging and diseases, as well as their contribution to the selective vulnerability of certain cell types

Development and application of novel bioinformatics tools for protein function prediction

Pearson Correlation Coefficient and provides a value between -1 to 1, with -1 being a total negative correlation, 0 is no correlation and 1 is a total positive correlation based on the observed and predicted ligand-binding site residues. Scores of 0.40 to 0.69 are strong positive relationships and 0.70 and higher are strong positive relationships. The downside of MCC is that it does not take into consideration the overall 3D structure of the protein model. Therefore, BDT will also be utilised as this score, which is also scored from -1 to 1, to take into consideration the 3D structure. Both MCC and BDT are only possible to produce when there is an observed (actual) structure available with bound ligands to compare against the predicted structure and hence why MCC and BDT are objective measures of ligand-binding site prediction. The average MCC and BDT score from CASP11 was 0.42 and 0.51, respectively. CASP12 saw the prediction of ligands for low annotation level proteins with no known ligands, demonstrating the potential use of FunFOLD3 in novel protein prediction. The average MCC and BDT score from CASP13 was 0.47 and 0.53. CAFA3 showed FunFOLDQ can be used in the prediction of GO terms, however further refinements are needed to increase specificity of the term predictions. The development option this thesis has explored is the use of docking (preferred orientation of interacting partners) with AutoDock Vina to improve the accuracy of ligand-binding residues by FunFOLD3, as the problem with TBM methods can be that predicted ligand(s) from a similar template will be forced to fit within the ligand-binding pocket. However, with docking, the aim of this method is to predict the preferred orientation of the ligand within the ligand-binding space. Utilisation of docking has also added to the novelty of this research, as different grid box calculations around the ligand-binding space was explored, with varying degrees of success with each grid box calculation. Examples of two CASP targets which had improvements in MCC and BDT score following docking were CASP11 target T0783 (2-C-methyl-D-erythritol 4- phosphate cytidylyltransferase) the MCC and BDT scores by FunFOLD3 were 0.17 and 0.21, respectively. Following docking the MCC and BDT scores increased to 0.63 and 0.45, respectively. CASP13 target T1016 (alpha-ribazole-5'-P phosphatase) had MCC and BDT scores of 0.556 and 0.646 by FunFOLD3, respectively. Following docking the MCC and BDT increased to 0.85 and 0.91, respectively. Lastly, CASP_Commons, a community-wide experiment to find the consensus structures, explored the role of FunFOLD3 with predicting ligands and ligand-binding sites for the novel protein and proteins domains of SARS-CoV-2. The protein domains were non-structural proteins 2, 4 and 6, open reading frames 3a, 6, 7b, 8 and 10, membrane protein and papain�like protease. FunFOLD3 predicted ligands for ten of the protein domains, of which there were a total of 32 targets due to domains being split into smaller residues and subsequent rounds of 3D modelling improvement. Increased understanding of protein structures can provide further insight into a protein’s function, particularly if ligands are bound and identified, an example in this thesis is the prediction of chlorophyll A for non-structural protein 4 (nsp4). Chlorophyll A, like haemoglobin is a porphyrin ring and templates related to nsp4 show a role in blood clotting. Therefore, whilst chlorophyll A might not be the exact ligand, similarities between haemoglobin and chlorophyll A can clearly be determined and assist in understanding the role of nsp4 in the pathology of COVID-19. Identification of GO terms can provide more detailed understanding into the function or functions of proteins and, in proteins with limited annotation information this can assist with comprehending their role. This thesis has focused on improving and developing a function prediction method, FunFOLD3, to better understand the role and function of proteins. The new method of FunFOLD3 which utilises docking will be integrated into the McGuffin group prediction servers and will be benchmarked in subsequent CASP competitions, to critically assess the performance of the developed method

Hands-on Science. Advancing Science. Improving Education

The book herein aims to contribute to the advancement of Science to the improvement of Science Education and to an effective implementation of a sound widespread scientific literacy at all levels of society. Its chapters reunite a variety of diverse and valuable works presented in this line of thought at the 15th International Conference on Hands-on Science “Advancing Science. Improving Education

Telomere, DNA Methylation and Gene Expression changes caused by exercise training

Exercise training is one of the few therapeutic interventions that improves health span by delaying the onset of age-related diseases and preventing early death. Despite the clear benefits to health conferred by exercise training, our understanding of the underlying molecular mechanisms remain crude. The primary purpose of this thesis is to determine and analyse the molecular biology changes that occur with strenuous aerobic exercise. Specifically, the main objectives were to investigate the impact of strenuous aerobic exercise training on structural DNA modifications, measured in context with cardiovascular health and fitness adaptations. In the first part of this thesis I investigated the influence of endurance exercise training on leukocyte telomere length and cardiovascular health. Leukocyte telomere length reflects biological age. Indeed, excessively short leukocyte telomeres are associated with age-related chronic diseases. Epidemiological studies indicate endurance athletes live longer than people from the general public who do not engage in extensive aerobic exercise training. In Chapter 2, my literature review on the subject of exercise and telomere biology suggested that, at the time of this study, the impact of exercise training on leukocyte telomere length was equivocal. Therefore, to determine whether strenuous aerobic exercise training influences biological ageing (assessed by leukocyte telomere length), I conducted two cross-sectional studies on leukocyte telomere length differences between endurance athletes and healthy controls. The first study (Chapter 3) was a cross-sectional analysis of leukocyte telomere length between athletes and controls, determined by quantitative polymerase chain reaction (qPCR). This is a relative measurement of telomere length expressed as a telomere (T) to single copy gene (S) ratio. Relative to the healthy controls (n = 56), the ultra-marathon runners (n = 67) possessed 11% longer leukocyte telomeres in age-adjusted analysis (ultra-marathon runners vs controls; average T/S ratio: 3.56 vs 3.16, p = 1.4 × 10-4) and the difference was not explained by the favourable cardiovascular health profile exhibited by the athletes (p = 2.2 × 10-4). The difference in leukocyte telomere length indicated the athletes had reduced their biological age by 16.2 years. To elucidate the potential mechanism for the longer leukocyte telomeres observed in endurance athletes, I recruited another cohort of athletes and controls and measured leukocyte telomere length and gene expression of genes involved in telomere length regulation. In the second study (Chapter 4), I describe data replicating the finding that endurance athletes possess longer leukocyte telomeres compared to healthy controls (athletes v controls mean T/S ratio ± SE: 3.64 ± 0.06 vs 3.38 ± 0.06, p = 0.002). This difference was associated with a concomitant increased activity of two important telomere regulating genes, telomerase reverse transcriptase (TERT) and adrenocortical dysplasia homolog (TPP1) (2- fold and 1.3-fold, respectively, both p < 0.05). The difference in leukocyte telomere length and leukocyte telomere-regulating gene (TERT and TPP1 mRNA) expression was ameliorated after adjusting for maximal oxygen uptake and resting heart rate (all p > 0.05). This finding indicates that cardiorespiratory fitness is an important determinant of telomere biology. Together, these two cross-sectional studies suggest that regular endurance exercise training is associated with longer leukocytes telomeres and that this is likely achieved through higher TPP1 and TERT mRNA expression gained through improved cardiorespiratory fitness. The findings in Chapters 3 and 4 provide evidence for extensive endurance exercise training as an effective lifestyle strategy to attenuate biological ageing. In parallel to telomere length changes, epigenetic modifications (e.g. DNA methylation) caused by environmental factors alter the transcriptomic milieu of cells. My thorough literature review (Chapter 5) revealed that exercise training seems to rearrange chromatin by modifying the DNA methylome in a variety of cells and that the extent is dictated by exercise duration and intensity. Therefore, in the second part of my thesis, I investigated the DNA methylation changes in leukocytes (which are somatic cells) and sperm (male germ cells) from healthy men before and after sprint interval training (SIT). Unlike traditional, long duration training at moderate intensity training, SIT involves short, intense (>85% VO2max to supra-maximal) efforts followed by periods of rest (3–4 min), typically repeated 3–8 times. It is an effective type of training that improves cardiorespiratory fitness quicker than traditional long slow distance training. Thus, to establish the DNA methylome changes associated with SIT, I conducted two training studies and analysed the leukocyte and sperm methylomes using the Infinium HumanMethylation450 BeadChip (Illumina). My third study (Chapter 6) provides the first evidence showing an association between DNA methylation changes paralleled with improvements to lipid profile and cardiorespiratory fitness in humans. Twelve young men (18–24 years) undertook SIT (thrice weekly) for four weeks. Resting blood samples were obtained and whole-blood leukocytes were isolated by red blood cell lysis. Genome-wide DNA methylation was assessed using the 450K BeadChip (Illumina). Cardiorespiratory fitness, determined by maximal oxygen uptake, was improved by 2.1 ml.kg-1.min-1 and low-density lipo-protein cholesterol was decreased by 3.9% after SIT (p < 0.05). Notably, the leukocyte methylome was significantly affected by SIT, in regions throughout the genome in relation to CpG islands – CpG islands, North shores, N shelves, South shores and South shelve – and the nearest genes – 3’ untranslated region (UTR), 5’ UTR, exonic, intergenic, intronic, non-coding and promoter regions (all p < 0.001). Genes with differentially methylated CpG sites (q < 0.005) after SIT were enriched for cardiovascular gene ontology (GO) terms that included metabolic activity, biological adhesion and antioxidant activity. Similarly, pathway analysis revealed genes involved in focal adhesion, calcium signaling and mitogen activated protein kinase were modulated by SIT-induced DNA methylation changes. Amongst the 205,987 probes relating 32,445 transcripts differentially methylated after SIT (q < 0.05), with methylation changes between 0.1 – 62.8%, the largest and most statistically significant demethylated site was in the epidermal growth factor (EGF) gene, causing decreased mRNA expression. As with EGF, the microRNA-21 and microRNA-210 genes (MIR21 and MIR210, respectively), known for their roles in cardiovascular disease (ischemic heart disease and coronary atherosclerosis), had modest but consistently statistically significant DNA methylation changes at numerous CpG sites, which altered mature microRNA abundance. Together, these data suggest that genome-wide DNA methylation changes occur after short-term intense exercise training concurrently with improvements to blood cholesterol profile and cardiorespiratory fitness. The data presented in this thesis provided evidence that the epigenome of somatic cells is malleable to exercise. There is mounting evidence supporting the premise that environmental perturbations cause DNA methylation changes and these are subsequently transgenerationally inherited, altering phenotypes of future generations. In the current study I also asked the question; can exercise training reconfigure the DNA methylome of male germ cells (sperm)? Therefore, my next study (Chapter 7) entails an analysis of the impact that three months of SIT has on genome-wide DNA methylation of sperm in healthy men. Thirteen subjects undertook twice-weekly SIT for three months, while the controls were asked not to change their current physical activity habits (if any). Sperm samples were donated before and after the three-month intervention. Mature sperm were isolated using density gradient centrifugation and DNA was extracted using the Purelink Genomic DNA Mini Kit (Life Technologies). Global and genome-wide DNA methylation was assessed using an enzyme-linked immunosorbent assay-based kit and the 450K BeadChip (Illumina), respectively. Relative to controls, the cases decreased their resting heart rate and had a higher maximal treadmill speed during exercise testing (both p < 0.05). Cases had decreased global DNA methylation after SIT compared to controls (p < 0.05). Genome-wide DNA methylation analysis revealed numerous modest (0.3 – 6%) methylation changes to 7509 CpG sites, relating to 4602 transcripts (q ≤ 0.1). Differentially methylated CpG sites were in genes associated with developmental biology, which included GO terms, such as developmental process, anatomical structure, embryonic morphogenesis and organ development, together with known pathways regulated by exercise training (MAPK, ErbB and PI3K-Akt signalling). Genes with increased methylation were associated with numerous human diseases, with most overrepresented being psychiatric disorders (schizophrenia, Parkinson’s disease and autism). Notably, paternally imprinted genes associated with other diseases were also differentially methylated after SIT. Therefore, exercise training is associated with the modifications to genome-wide DNA methylation of both somatic and germ cells. In conclusion, the studies presented as a series of peer-reviewed publications, outlines investigations that describe an influence of strenuous exercise training on leukocyte telomere length regulation and the DNA methylome of both leukocytes and germ cells. Both of these molecular changes in leukocytes and sperm provide evidence for novel molecular mechanisms by which exercise improves cardiovascular health and fitness. Future investigations should focus on longitudinal studies determining whether these changes are required for improved health and fitness, and should establish whether exercise-induced DNA methylation changes are transgenerationally inherited, and if so, what impact this has to future generations. Such discoveries could change national physical activity guidelines and policies, by emphasising the benefit of regular exercise both in the present and to future offspring.Doctor of Philosoph