Carolin Neu (PhD):The impact of glycosylation and glycation on the generation and uptake of vehicles in aging cells
Linda Kulka (PhD):Acetylation and glycosylation in protein folding and misfolding: Impact on ageing-related diseases
Acetylation and glycosylation are widespread posttranslational modifications (PTMs) involved in diverse cellular processes and can affect the structure of proteins. We have demonstrated that proteins can misfold and form amyloid structures upon inhibition of histone deacetylases (HDACs) in clinically relevant concentrations. Acetylation plays a role in the aetiology of ageing-related proteinopathies such as Alzheimer’s disease, Parkinson’s disease or Huntington’s disease. Furthermore, aggregate formation is fostered by mis-glycosylation of proteins; for instance, mutations in one key enzyme of terminal glycosylation (sialylation) are responsible for the age-dependent GNE (bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase) myopathy. We aim to investigate how acetylation and glycosylation influence protein folding and misfolding, in particular proteins involved in ageing-related diseases. We also analyse, how protein shuttling factors can ameliorate protein misfolding and how a different PTM patterns can influence the shuttling process.
Rebecca Rosenstengel (MD): Analysis of transcription factors regulating the expression of GNE throughout the progression of GNE-myopathy and their role during aging
GNE myopathy, previously termed hereditary inclusion body myopathy-HIBM, is a neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, and a typical muscle pathology. Today, it is recognized that GNE myopathy is a worldwide disorder since more than 150 different mutations in this same gene have been identified. The product of the GNE gene is UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase/ManNAc kinase, GNE). GNE contains 753 amino acids and is highly conserved in mammals. It consists of two functional domains, an N-terminal epimerase domain and a C-terminal kinase domain. The mutations related to GNE myopathy are dispersed along the entire gene, both in the epimerase and in the kinase coding sequences. GNE is the key enzyme in the metabolic pathway leading to the synthesis of N-acetylneuraminic acid (Neu5Ac). Neu5Ac is the biosynthetic precursor of virtually all of the naturally occurring sialic acids, the most abundant terminal monosaccharides of glycoconjugates in eukaryotic cells. The biological importance of GNE is reflected by the fact that a knockout of the gene in mice is lethal to the embryo at day 8.5.To date, the pathophysiological pathway leading from GNE mutations to the muscle phenotype in GNE myopathy is still very unclear. The most obvious hypothesis is that impaired sialylation of skeletal muscles is the cause of the disease. However, these lines of argumentation are still controversial, since marked GNE deficiency has not been observed in GNE Myopathy patients and no mis-localization of GNE in patients’ skeletal muscle could be documented. In addition, all clinical supplementation trials using sialic acid or sialic acid precursors failed so far. Because of the late onset, the role of aging is discussed in the progression of the myopathy. Advanced Glycation Endproducts (AGEs) increase with age and lead to modified proteins. If AGEs have an impact on GNE function is not fully understood. In contrast to biochemical aspects, very little is known on the promotors, splice variants and the genetic regulation of the GNE gene, which also might contain a non-coding RNA. Therefore, the aim of this project is to study the genetic organization of the GNE gene in vitro and in vivo. Here the focus will be on transcription factors and GNE promotor binding, before and after myotube differentiation in C2C12 cells and the role of AGEs in GNE gene regulation. We already have shown that Methylglyoxal (MGO) modify GNE and lead to AGE-GNE in vitro, reducing the activity of the enzyme. Therefore, we plan to analyze the expression of GNE after MGO treatment in C2C12 cells. In silico analysis will be used to identify potential transcription factors for the promotors of the two major isoforms of GNE. Potential transcription factors will be further analyzed using Electrophoretic Mobility Shift Assay (EMSA), CRISPR-Cas9 and overexpression experiments. Understanding the genetic regulation of the GNE is of general interest for the field and might also help to find new explanations and therapies for the treatment of GNE myopathy.
SP7: Metabolic memory of cells as posttranslational modifications (PTMs) - PTMs as key regulators of metabolic ageing in adult stem cells
Principal Investigator: Anne Navarrete Santos (Institute of Anatomy and Cell Biology, MLU Halle-Wittenberg)
Adipose derived stem cells (ASC) reduce their adipogenic capacity when they become old, which leads to aging and increase the risk of obesity related diseases. How does the metabolic environment affect the capacity of differentiation of ASC? Our hypothesis is that gerometabolites and APOE affects the production of NAD+/NADH and AMPK activity, in consequence Sirtuin 1 —a deacetylase enzyme related with transcription factors regulation— and the peroxisome proliferator activated receptor gamma —essential for the plasticity and differentiation of mesenchymal stem cell— diminish their actions. The methodological approach of this project bases on two biological models - the New Zealand white APOE-/- rabbit and human mesenchymal stem cell lines from patients. ASCs from the different donors (grouped by age and metabolic status) will be analysed for AMPK-Sirt1-PPARy pathway activation, PPARy modification and adipogenic differentiation capacity.
Alicia Toto Nienguesso (PhD):Metabolic memory of cells as posttranslational modifications (PTMs) - PTMs as key regulators of metabolic ageing in adult stem cells
Our interest focuses on changes in histone modifications of stem cells, which were shown to depend on age and metabolic changes in the cell environment in a previous study. We are investigating the potential molecular mechanisms based on the crosstalk of two important posttranslational modifications - histone methylation and O-GlcNAcylation (addition of N-acetylglucosamine (GlcNac) to serine/threonine residues) of the methylase EZH2. The O-GlcNAcylation of EZH2 at several serine and threonine moieties such as serine 75 seems to be required for EZH2 protein stability in tumor cells and therefore may facilitate the histone H3 trimethylation at K27 to form H3K27me3. Both O-GlcNAcylation and EZH2-mediated H3K27me3 formation play a pivotal role in adipogenic development and stem cell maintenance. Our hypothesis is that specific O‑GlcNAcylation of EZH2 regulates stem cell properties and provides a new mechanism of cellular ageing in embryonic and adult stem cells.
SP10: Glycosylation in the aging brain
Principal Investigator: Christian Hübner (Institute of Human Genetics, University Hospital Jena)
Ageing is associated with a functional decline of the nervous system. This comes along with an overall decrease of brain mass and an altered synaptic function. Many proteins involved in synaptic function are glycosylated. Glycosylation plays a prominent role in protein stability and conformation, cell-to-cell communication, cell matrix interaction, adhesion, protein targeting and folding. Abnormal glycosylation of proteins can induce deleterious effects as observed in congenital disorders of glycosylation, which often result in serious, sometimes fatal malfunctions of different organ systems such as brain and muscle. While changes in protein glycosylation in the diseased brain are well characterized, it is yet unclear whether alterations in protein glycosylation may contribute to age-dependent changes in synaptic function and thus brain function. Therefore, we will assess the glycoproteome of the aging brain. For selected candidates we will then address how age-associated changes in glycosylation might contribute to the age-dependent decline of brain function.
SP14: The role of Nit1 in aging-associated stress response
Principal Investigator: Otmar Huber (Institute for Biochemistry II, Jena University Hospital)
In this project we want to identify effectors and pathways that are regulated by Nit1 and how these effectors affect aging-associated stress responses. Using a mass spectrometry-based approach to compare cells with a stable shRNA-mediated knock-down of Nit1 with scrambled shRNA-treated control cells, we want to screen for differentially regulated proteins. After bioinformatic analysis and verification of candidates we will study the role of Nit1 in the regulation of candidates specifically in the context of our previously observed crosstalk with FOXO3a and b-catenin signaling and how Nit-dependent regulation of these factors may affect senescence and aging.
Ageing-associated changes in tissue homeostasis are a consequence of multiple molecular and cellular changes induced by alterations of signalling and metabolic pathways and accumulation of damaged molecules due to impaired repair mechanisms. We recently identified Nit1 as a new binding partner of b-catenin and repressor of the canonical Wnt-pathway disrupting the b-catenin/TCF-complex. Stress conditions were reported to redirect b-catenin from TCF to FOXO3a thereby activating FOXO3a transcriptional activity. Our results indicate that Nit1 is differentially regulated in stress responses and thereby could contribute to this effect. Interestingly, Nit1 has a second role as a metabolite repair enzyme acting as a dGSH (deamino-glutathione)-hydrolase. dGSH is generated as a side product of transaminase reactions. We here want to assess this dual role of Nit1 in stress response and investigate which posttranslational modifications modulate this activity during ageing and senescence.
SP16: Proteostasis at the old blood-brain barrier: Implications for late-onset Alzheimer’s disease
Maira Tariq (PhD): Proteostasis at the old blood-brain barrier: Implications for late-onset Alzheimer’s disease
Ageing of the blood-brain barrier (BBB) results from an accumulation of deficiencies with contributions of senescence, increased inflammation, and oxidative stress. Though age related changes in BBB function may also represent an adaptation for healthy ageing, but results in consequence in a dysfunctional BBB, which often correlate with the progressive cause of brain diseases like the development of neural diseases, including late-onset Alzheimer's disease (LOAD). Ubiquitination and autophagy target proteins via post-translational modifications for regulating the protein homeostasis of amyloid precursor protein (APP) and its harmful byproduct amyloid beta (Aβ) in neurons, but also in other cells of the neurovascular unit including the BBB. Further, APP expression is considered as a neuroprotective response to stress factors with impact on healthy aging. Therefore, we ask how ubiquitination and autophagy in the BBB do change during ageing and how do these changes contribute to the development of LOAD. We study ubiquitination and autophagy in induced pluripotent stem cell-derived brain-capillary endothelial cells, pericytes, astrocytes, microglia, and neurons.
Chaudhry Luqman Abid (PhD): Glycosylation of Alzheimer-associated proteins in the context of microglia-mediated inflammation and disturbed blood-brain barrier functions
Age-dependent disturbed immune plasticity of microglia and disturbed blood-brain barrier BBB functionality may provide early phenotypes of late-onset Alzheimer's disease (LOAD). LOAD is age-related and the number of patients grows steadily not only in Germany. We published mutations (TREM2, ABCA7) and described an association (CD33) of microglia- and BBB-related genes that confer a risk for LOAD. These proteins undergo LOAD-associated posttranslational modifications (PTMs) including glycosylation and particularly sialylation. We therefore ask how glycosylation of CD33, TREM2, and ABCA7 contribute to LOAD and how are alterations in these PTMs relate to cellular ageing mechanisms. In this project, we will analyse the impact of mutations on PTMs of the proteins. We established LOAD-specific induced pluripotent stem (iPS) cell models for microglia and BBB cultures. We will apply these in vitro models for analysing the impact of PTMs on the functionality of TREM2, ABCA7, and CD33 and their contribution to the onset of LOAD.
Hendrik Schlüter (MD):The impact of protein homeostasis in the clearance of amyloid beta peptide at the aged blood-brain barrier
The blood-brain barrier (BBB) forms a selective gateway for the entrance and exit of all factors and molecules in and out of the brain. It is not yet fully understood why impaired BBB function is associated with various neurodegenerative and immunological diseases and why old age has a negative impact on BBB function in this context. Nevertheless, the respective trigger proteins must cross the BBB at a certain point in time. In Alzheimer’s disease, the trigger protein is amyloid beta (Aß), a polypeptide of 37-49 amino acids, which is generated by proteolysis from the Amyloid-beta precursor protein (APP). A major pathway of protein degradation is executed by lysosomes, which are organelles containing a huge number of hydrolases including peptidases. Lysosomes are mainly involved in the degradation of extracellular material that enter the cell by endocytosis but are also involved in the homeostasis of other organelles and cellular proteins, including mitophagic and autophagic processes in an age dependent manner. Thus, we will address the question, whether in our induced pluripotent stem cell-derived BBB models these processes are affected by age and/or genetic preposition that are associated with Alzheimer’s disease.
SP17: Role of PTMs in senescent-induced transformation of the secretory pathway
One of the hallmarks of senescent cells is the senescence-associated secretory phenotype (SASP). The SASP-secretome is very well characterized, but the basis for the SASP is a rearrangement of the whole secretory pathway, indicated by morphological changes like dispersal of the Golgi, increase in lysosomal volume, increase in lysosomal lipofuscin aggregation and increased expression of the lysosomal enzyme b-galactosidase. Not surprisingly endoplasmatic reticulum (ER)-stress pathways including the UPR are up-regulated in senescent cells. Little is known about the molecular mechanisms mediating the rearrangement of the secretory pathway that ultimately results in SASP.
Specific aims of this project:
(1) To establish a comprehensive atlas of senescent-induced PTMs in the secretory pathway. (2) To mechanistically understand the impact on individual PTMs on proteins of the secretory pathway and their role in senescence and SASP-development. (3) To identify targets and prepare for screening assays for the development of future therapeutic interventions.
The anti-aging protein Klotho is strongly expressed in kidney and choroid plexus (CP). In kidney, full length Klotho functions as a co-receptor for FGF23. In addition, Klotho is shed from the surface and, together with a secreted splice form of Klotho, supplies the periphery with soluble Klotho (Klothos). In the CP, the function of Klotho is not known, but Klothos is a constituent of cerebrospinal fluid (CSF) produced by the CP. In mice and humans, Klotho is involved in cognitive performance and is down-regulated in ageing. Klotho is heavily glycosylated, which could affect its stability or activity. In our project, we want to test whether Klotho activity and/or stability is modulated by enzymatic and non-enzymatic PTM. We also analyse the role of Klotho in brain ageing using specific mouse knock-out models. Finally, we want to analyse the role of mammalian Klotho in IGF1/FOXO signalling and its modulation by PTMs.
During ageing and in models of premature ageing the number and functionality of muscle stem cells decreases dramatically. Different signalling pathways have been reported to be altered in aged muscle stem cells. Therefore, we are investigating which intrinsic changes in muscle stem cells occur during ageing and impair their functionality. Furthermore, we are examining the interaction of the muscle stem cell with its niche and how changes in the posttranslational modifications of membrane receptors such as glycosylation are changing in muscle stem cells during ageing. Additionally, we are analysing how these changes are affecting downstream signalling pathways.
SP20: Identification of novel O-GlcNAcylated substrates in endothelial cells and investigation of their role in senescence and ageing
Principal Investigator: Florian Meier (Functional Proteomics, Jena University Hospital) & Daria Zibrova (Institute of Molecular Cell Biology, Jena University Hospital)
Felix Schneidmadel (PhD):Survey of the Post-Translational Modification Landscape in Ageing via Trapped Ion Mobility Mass Spectrometry
Our current knowledge about biological processes involving protein post-translational modifications (PTMs) has been fueled by the widespread use of mass spectrometry-based proteomics. However, interactions between different PTMs remain largely elusive because state-of-the-art strategies typically analyze only one PTM at a time and involve tailored workflows to enrich a specific PTM of interest based on chemical or biochemical affinity. Conversely, all other peptides are discarded and, more critically, efficient enrichment strategies are lacking for most of the several hundreds of known in vivo modifications. Here, we propose to use an ‘open search’ bioinformatics approach combined with deep peptide fractionation to survey the PTM landscape of model systems in aging. We build on the recent development of rapid and sensitive trapped ion mobility mass spectrometry (TIMS-MS), which adds an additional dimension of separation as compared to conventional liquid chromatography – mass spectrometry.
Andreas Will (PhD):Identificatio of novel O-GlNAcylated substrates in endothelial cells and investigation of their role in senescence and ageing
The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification that regulates protein function. Increased O-GlcNAcylation underlies the aetiology of age-related vascular pathologies via unknown mechanisms. So far, O-GlcNAcylated substrates in vascular endothelium are barely studied and the identification of O-GlcNAc modification sites remains a technical challenge. We aim to establish a novel mass spectrometry-based approach for the identification of O-GlcNAcylated proteins in vascular endothelial cells using ion mobility spectrometry. With this workflow, we will study the role of O-GlcNAcylation in endothelial cell function and senescence.
SP21: The role of GMPPA in the developing and aging brain
Glycosylation is the most common posttranslational modification of proteins and lipids. It plays a prominent role in protein stability and conformation, cell-to-cell-communication, cell-matrix-interaction, adhesion, protein targeting and folding. We recently identified mutations in GDP-mannose-pyrophosphorylase-A (GMPPA) in patients suffering from muscle weakness, gait abnormalities, achalasia, alacrima and mental retardation (AAMR syndrome). We could subsequently show that GMPPA acts as an allosteric feedback inhibitor of GMPPB, which catalyzes the production of GDP-mannose, a key substrate for glycosylation. As a consequence, GMPPA defects result in the hyperglycosylation of several proteins. Here, we will use our GMPPA knockout mouse model and different in vitro approaches to assess the consequences of GMPPA defects for brain development and ageing.