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Quantitative Large Scale Proteomics of Lysosomal Storage Disease

Final Report Summary - STORAGE PROTEOMICS (Quantitative Large Scale Proteomics of Lysosomal Storage Disease)

Lysosomal storage diseases (LSD) are a group of ~50 inherited disorders with a cumulative incidence of 1 in 5.000 new-borns. The common feature of this group of diseases is an alteration or loss of function of enzymes which are responsible for the degradation of macromolecules (mainly lipids, proteins and glycosaminoglycans) in cells. The lack of degradation leads to the accumulation of these substances resulting in numerous effects in patients. Among these are frequently neurological symptoms, for example neuronal cell death in the neuronopathic form of Gaucher disease or demyelination in Metachromatic Leukodystrophy. When glycosaminoglycans are affected, patients present in addition to their neurologic symptoms also with severe skeletal abnormalities.
So far, the majority of research projects dealing with LSDs were targeted at specific effects in certain cell types, which reflect late stages of lysosomal storage, investigating single proteins and their role in the diseased state. These studies provide pieces of a greater picture which is not yet completely understood.
My group is addressing this question using quantitative proteomics approaches utilizing liquid chromatography tandem mass spectrometry. This allows to investigate the entity of proteins from a given cell type within one experiment. In order to accurately compare the amount of proteins present in different samples, I make use of stable isotopes. The underlying principle is that different samples are chemically modified in a way that alters their molecular weight but not their chemical properties. The samples are combined and in the following mass spectrometric analysis, molecules originating from the individual samples can be distinguished based on their mass. This enables us to identify proteins from a sample and to perform accurate relative quantification.
Using this technology, I am comparing many thousands of proteins between diseased and healthy cells and investigate which changes are occurring due to lysosomal storage. Proteins which are regulated between the normal and the diseased state are further investigated using software tools searching for protein function, subcellular localization or known roles of the protein in the cell in order to draw a global picture of processes impaired by LSDs. I am using different cell culture models for these investigations dealing with immortalized cell lines as well as primary cells originating from the central nervous system.
The Marie Curie fellow is currently heading a research group located at the Institute for Biochemistry and Molecular Biology at the University of Bonn which consists of 5 Ph.D. and 1 Master student as well as one technician.
The proposal consisted of 3 main objectives: 1. The investigation of Gaucher diseae by CBE treatment in Cell culture; 2. The investigation of NG2 cell differentiation in Metachromatic Leukodystrophy and 3. The analysis of PNS alterations in Metachromatic Leukodystrophy.
For objective 1, my group performed cell culture studies in CBE treated B35 neuroblastoma cells and in vivo studies on brain tissue of CBE treated mice. Both approaches did not result in reasonable results as the data in the cell culture model were unexpectedly variable and in the brain samples no significant changes could be identified. We therefore extended our strategy to the analyses of primary neuronal cultures using hippocampal and cortical neurons. From these analyses we obtained first promising results from label free mass spectrometry based quantification experiments and are now performing biological replicates on neuronal cultures. In parallel, as an alternative model system, we established the treatment of mouse embryonic fibroblasts (MEFs) with the inhibitor U18666A which mimics the cellular phenotype of the lipid storage disorder Niemann Pick Disease Type C (NPC). Initial analyses of whole protein level abundances showed strong regulation of the cholesterol synthetic pathway but no pronounced changes of lysosomal or lysosome associated proteins. We extended our studies to phosphoproteomic analyses of whole cell lysates and lysosome enriched fractions. These experiments yielded more promising results and we are currently working on selected target proteins. For objective 2, we performed experiments for the generation of NG2 cells and finally oligodendrocytes from mouse using the neurosphere assay. In this assay, neuronal stem cells from mouse are cultured as free floating spheres and differentiated to NG2 cells. We succeeded in generating mouse NG2 cells using this assay, however, we were not able to differentiate them to oligodendrocytes. It was not possible, however, to keep the generated NG2 cells for more than a limited period of time in culture. In parallel we performed the same differentiation experiments successfully in rat NG2 cells. However, it was not possible to obtain a rat model for the disease investigated. Therefore we characterized only the differentiation processes of the cells in vitro but did not apply this strategy to cells reflecting the desired disease phenotype as proposed. From these studies we selected a kinase and a transcription factor which we suspected to be implicated in the differentiation process. We are currently investigating their role in this process. For the 3rd objective, we analyzed sciatic nerves of wild type and metachromatic leukodystrophy mice using quantitative comparative mass spectrometric measurements. We were able to identify ~2700 proteins including 54 upregulated and 14 down-regulated proteins. Manual analysis of these proteins did not result in concrete working hypothesis why we decided not to continue with this project at this point as we had more promising target proteins from the other projects.
The most promising project currently seems to be the investigation of Niemann Pick Disease Type C using U18666A treated MEFs. We therefore dedicate the work of 2 students on this project. Furthermore, for the analysis of Gaucher disease we are investigating primary neurons and are establishing a method which will allow us to analyze specific cell populations isolated from mice in order to circumvent the problems we are currently facing with the cell culture models. We expect these experiments to provide promising results on Gaucher disease in the near future.