The dysregulation of cellular metabolism is a hallmark of ageing. To understand the metabolic changes that occur as a consequence of the ageing process and to find biomarkers for age-related diseases, we conducted a metabolomic analysis of brain, heart, kidney, liver, lung and spleen in young (9-10 weeks) and old (96-104 weeks) wild type (mixed genetic background of 129/J and C57BL/6) mice using NMR spectroscopy. We found differences in metabolic fingerprints of all tissues and identified several metabolites to be altered in most tissues, suggesting that they may be universal biomarkers of ageing. In addition, we found distinct tissue-clustered sets of metabolites throughout the organism. The associated metabolic changes may reveal novel therapeutic targets for the treatment of ageing and age-related diseases. Moreover, the identified metabolite biomarkers could provide a sensitive molecular read-out to age determine the age of biologic tissues and to validate the effectiveness and potential off-target effects of senolytic drug candidates on both a systemic and tissue-specific level.

Abstract: The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the presence of poly-PR/GR dipeptide repeats which are encoded by the C9orf72 gene. Recently, it was shown that poly-PR/GR alters chromatin accessibility which results in stabilization and enhancement of transcriptional activity of the tumor suppressor p53 in several neurodegenerative disease models. Reduction of p53 protein levels in cell and model organisms protects against neurotoxicity of poly-PR, and partially protects against neurotoxicity of poly-GR. Here, we aimed to study the detailed molecular mechanisms how p53 contributes to poly-PR/GR mediated neurodegeneration. Using a combination of biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, fluorescence polarization, turbidity assays and differential interference contrast (DIC) microscopy, we found that p53 physically interacts with poly-PR/GR and triggers liquid-liquid phase separation of p53. We identified p53 transactivation domain 2 (TAD2) as the main binding site for PR25/GR25 and show that binding of poly-PR/GR to p53 is mediated by a network of electrostatic and/or hydrophobic interactions. Our findings might help to understand the mechanistic role of p53 in poly-PR/GR - associated neurodegeneration.

Energy metabolism, including alterations in energy intake and expenditure, is closely related to aging and longevity. Metabolomics studies have recently unraveled changes in metabolite composition in plasma and tissues during aging and have provided critical information to elucidate the molecular basis of aging process. However, the metabolic changes in tissues responsible for food intake and lipid storage have remained unexplored. In this study, we aimed to investigate aging-related metabolic alterations in these tissues. To fill this gap, we employed NMR-based metabolomics in several tissues, including different parts of the intestine (duodenum, jejunum, ileum) and brown/white adipose tissues (BAT, WAT) of young (9-10 weeks) and old (96-104 weeks) wild-type (mixed genetic background of 129/J and C57BL/6) mice. We further included plasma and skeletal muscle of the same mice to verify previous results. Strikingly, we found that duodenum, jejunum, ileum, and WAT do not metabolically age. In contrast, plasma, skeletal muscle, and BAT show a strong metabolic aging phenotype. Overall, we provide first insights into the metabolic changes of tissues essential for nutrient uptake and lipid storage and have identified biomarkers for metabolites that could be further explored to study the molecular mechanisms of aging.

Lipoproteins are important cardiovascular (CV) risk biomarkers. This study aimed to investigate the associations of lipoprotein subclasses with micro- and macrovascular biomarkers to better understand how these subclasses relate to atherosclerotic CV diseases. One hundred fifty-eight serum samples from the EXAMIN AGE study, consisting of healthy individuals and CV risk patients, were analyzed by nuclear magnetic resonance (NMR) spectroscopy to quantify lipoprotein subclasses. Microvascular health was quantified by measuring retinal arteriolar and venular diameters. Macrovascular health was quantified by measuring carotid-to-femoral pulse wave velocity (PWV). Nineteen lipoprotein subclasses showed statistically significant associations with retinal vessel diameters and nine with PWV. These lipoprotein subclasses together explained up to 26% of variation (R²=0.26, F(29,121)=2.80, p<0.001) in micro- and 12% (R²=0.12, F(29,124)=1.70, p=0.025) of variation in macrovascular health. High-density (HDL-C) and low-density lipoprotein cholesterol (LDL-C) as well as triglycerides together explained up to 13% (R²=0.13, F(3,143)=8.42, p<0.001) of micro and 8% (R²=0.08, F(3,145)=5.46, p=0.001) of macrovascular variation. Lipoprotein subclasses seem to reflect micro- and macrovascular end organ damage more precisely as compared to only measuring HDL-C, LDL-C and triglycerides. Further studies are needed to analyse how the additional quantification of lipoprotein subclasses can improve CV risk stratification and CV disease prediction.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated to longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as brain and kidney during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage and energy consumption, we employed NMR-based metabolomics to study liver, plasma, intestine (duodenum, jejunum, ileum), brown/white adipose tissues (BAT, WAT), and skeletal muscle of young (9–10 weeks) and old (96–104 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. Deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to WT mice, whereas the metabolome of jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of metabolome during aging of liver, WAT, duodenum and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously unexplored critical role in both aging and energy metabolism.

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic method-ologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides suration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, like storage conditions and duration. It has been discussed that improper dehydration of tissue during processing influences quality of NAs and shelf-life of fixed tissue. Here we report on establishing a method to determine the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin, or a non-crosslinking fixative) and its correlation to the performance of NAs in qRT-PCR analyses. The amount of residual water depended primarily on the fixative type and the dehydration pro-tocol and, to a lesser extent on storage conditions and time. Moreover, we found that these pa-rameters were associated with the qRT-PCR performance of extracted NAs. Besides cross-linking of NAs and modification of nucleobases by formalin, hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research, but must also be taken into account in clinical diagnostics where reanalysis of archived tissue from a primary tumour may be required, e.g., after disease recurrence. We conclude that improving shelf-life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative in-fluence of residual water on NAs.