1. Research Background:

By 2050, the global population over the age of 80 will triple. Thus, research for improving the quality of life at older age can be of enormous benefit for our ever-aging society. To address this challenge, we propose an innovative approach based on a combination of stem cell research with genetic experiments in C. elegans. Mechanisms that promote protein homeostasis (proteostasis) slow down aging and decrease the incidence of age-related disorders such as amyotrophic lateral sclerosis and Huntington’s disease. Since human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) replicate continuously in the absence of aging, we hypothesize that they can provide a novel paradigm to study proteostasis and its demise with age. We have found that hESCs exhibit increased proteasome activity. Moreover, we have uncovered that the proteasome subunit RPN-6 is required for this activity and sufficient to extend healthspan in C. elegans. However, the mechanisms by which the proteasome regulates hESC/iPSC function and organismal longevity remain unknown. Our first aim is to define how the proteasome regulates not only hESC/iPSC identity but also aging and the onset of age-related diseases. Moreover, one of the next challenges will be to define how other proteostasis pathways impinge upon hESC/iPSC function. Besides the proteasome system, we hypothesize that hESCs/iPSCs differentially regulate other stress response pathways designed to protect from any disequilibrium in the folding and degradation of the proteome. To this end, we will perform a comprehensive study of proteostasis of hESCs and iPSCs derived from patients. Then, we will mimic this network in somatic cells such as neurons to alleviate age-related diseases. Finally, we propose to determine whether loss of proteostasis promotes somatic stem cell (SC) exhaustion, which is one of the most obvious characteristics of the aging process and contributes to tissue degeneration. By using in vitro and in vivo models, we will examine whether sustained proteostasis delays neural SC exhaustion. Our research can have implications in several fields such as stem cell research, neurogenesis, proteostasis, aging and age-related diseases.

2. Research questions addresses by the group:

We seek to probe the requirements of proteostasis in hESC/iPSCs, healthspan and regenerative potential by addressing the following questions:

• Aim 1) What are the mechanisms by which proteasome activity regulates hESC/iPSC function and organismal aging?
• Aim 2) Do hESCs/iPSCs have increased proteome surveillance mechanisms? Can these mechanisms be adapted to slow down aging and age-related diseases?
• Aim 3) Can sustained increased proteasome activity ameliorate age-associated exhaustion of adult neural stem cells?

3. Possible projects:

• Defining the ubiquitin-proteasome system of pluripotent stem cells for disease intervention
  The ubiquitin-proteasome system (UPS) is a key node of the proteostasis network to terminate damaged and disease-related proteins. As such, alterations in the UPS hasten the neurodegeneration of Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD) and amyotrophic lateral sclerosis (ALS) disease models. Notably, we have found that iPSCs from patients exhibit a striking ability to prevent aggregation of disease-related proteins. Here, we propose to assess whether the intrinsic UPS of hESCs/iPSCs suppresses aggregation of disease-related proteins. In addition, we aim to mimic the UPS network of iPSCs in somatic cells (e.g., C. elegans, neurons derived from distinct patient-iPSCs) to define novel mechanisms that can correct proteostatic deficiencies and delay the onset of disease-related changes.
• Identifying novel networks for disease intervention by defining proteostasis of stem cells.
  Besides the proteasome system, we have found that hESCs/iPSCs also differentially regulate other subcellular stress response pathways designed to protect the cell from disequilibrium in the folding and degradation of disease-related proteins. Our goal is to determine whether these mechanisms impinge upon proteostasis, pluripotency and neurogenesis of hESCs/iPSCs. Finally, by using iPSC-derived neurons from patients and C. elegans models, we will assess whether intervention of these pathways ameliorates proteostatic deficits in distinct age-related neurodegenerative diseases.
• Regulation of neuronal proteostasis by the germline.
  Misfolded and aggregated proteins challenge cellular integrity, leading to loss of cell function and viability. The accumulation of protein aggregates in neurons is a hallmark of age-related diseases such as Huntington’s, Parkinson’s, Alzheimer’s, and amyotrophic lateral sclerosis (ALS). The late onset and heterogeneity of aggregates indicate that these neurodegenerative diseases are linked to converging cellular changes during aging. Indeed, post-mitotic cells such as neurons lose extensive control of the proteostasis equilibrium with age: widespread, aberrant changes in protein interactions, a loss of function in protein degradation machineries, and a generalized down regulation of chaperones often appear in somatic cells over time.
  Beyond intracellular changes, cumulative evidence indicates that proteostasis is also regulated by cell non-autonomous communication between tissues during aging. Thus, understanding cell non-autonomous mechanisms that modulate neuronal proteostasis is crucial to elucidate the links between aging and neurodegenerative diseases. In previous work, we demonstrated that inducing proteostasis deficits in the germline of young animals triggers pathological aggregation of Huntington’s and ALS- related proteins in the neurons of C. elegans. In this project, our overarching goal is to define cell non-autonomous mechanisms regulated by the germline and assess their potential application for disease-related neurodegeneration. To this end, we will combine genetics in C. elegans, state-of-the-art proteomics and disease modelling using iPSC-derived neurons and organoids.
• We have many other exciting open projects in the lab and we are very happy to discuss them with the applicants during the individual meetings.

4. Applied Methods and model organisms:

• hESC culturing and differentiation into distinct lineages (including specific neuronal types such as striatum neurons and motorneurons)
• iPSC derived from amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD) patients
• Brain organoids from hESCs/iPSCs
• C. elegans: ageing and proteotoxic stress
• ALS and HD C. elegans models
• RNAi
• CRISPR/Cas9
• Proteomics
• Metabolomics
• Co-Immunoprecipitation and interactome assays
• Western blot
• Quantitative PCR
• RNA sequencing
• ChIP-sequencing
• Ribosome profiling

5. Desirable skills and qualifications:

We seek to find enthusiastic and highly motivated students interested in stem cell and aging research. Experience in stem cell research or C. elegans is welcome but not necessary. If you are motivated and eager to learn, it will be easy to get familiarized with all the techniques in the lab.

6. References:

1. H. Alirzayeva, R. Loureiro, S. Koyuncu, F. Hommen, Y. Nabawi, W.H. Zhang, T.T.P. Dao, M. Wehrmann, H.J. Lee, and D. Vilchez (2024). ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes. Cell Reports 43:114626.
2. H.J. Lee, H. Alirzayeva, S. Koyuncu, A. Rueber, A. Noormohammadi, and D. Vilchez (2023). Cold temperature extends longevity and prevents disease- related protein aggregation through PA28γ-induced proteasomes. Nature Aging 3: 546-566.
3. E. Llamas, S. Koyuncu, H.J. Lee, M. Wehrmann, R. Gutierrez-Garcia, N. Dunken, N. Charura, S. Torres-Montilla, E. Schlimgen, A.M. Mandel, E. Boelen- Theile, J. Grossbach, P. Wagle, B. Schermer, T. Benzing, A. Beyer, P. Pulido, M. Rodriguez-Concepcion, A. Zuccaro, and D. Vilchez (2023). In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation. Nature Aging 3: 1345-1357 (cover).
4. R. Gutierrez-Garcia, S. Koyuncu, F. Hommen, S. Bilican, H.J. Lee, A. Fatima, and D. Vilchez (2023). G3BP1-dependent mechanism suppressing protein aggregation in Huntington’s models and its demise upon stress granule assembly. Human Molecular Genetics 32: 1607-1621.
5. Koyuncu, R. Loureiro, H.J. Lee, P. Wagle, M. Krueger and D. Vilchez (2021). Rewiring of the ubiquitinated proteome determines ageing in C. elegans. Nature. DOI:10.1038/s41586-021-03781-z
6. G. Calculli, H.J. Lee, K. Shen, U. Pham, M. Herholz, A. Trifunovic, A. Dillin and D. Vilchez (2021). Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans. Science Advances 7: eabg3012.
7. E. Llamas, S. Torres-Montilla, H.J. Lee, M.V. Barja, E. Schlimgen, N. Dunken, P. Wagle, W. Werr, A. Zuccaro, M. Rodriguez-Concepcion and D. Vilchez. The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress. In Press in Ageing Cell. BiorXiv preprint:https://www.biorxiv.org/content/10.1101/2021.01.19.427268v1
8. E. Llamas, H. Alirzayeva, R. Loureiro and D. Vilchez (2020). The intrinsic proteostasis network of stem cells. Current Opinion in Cell Biology 67: 46-55.
9. A. Fatima, D. Irmak, A. Noormohammadi, M.M. Rinschen, A. Das, O. Leidecker, C. Schindler, V. Sánchez-Gaya, P. Wagle, W. Pokrzywa, T. Hoppe, A. Rada-Iglesias and D. Vilchez (2020). The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells. Communications Biology3: 262.
10. A. Thiruvalluvan, E. P. de Mattos, J.F. Brunsting, R. Bakels, D. Serlidaki, L. Barazzuol, P. Conforti, A. Fatima, S. Koyuncu, E. Cattaneo, D. Vilchez, S. Bergink, E. HWG Boddeke, S. Copray and H.H. Kampinga (2020). DNAJB6, a key factor in neuronal sensitivity to amyloidogenesis. Molecular Cell78: 346-358.
11. C. Kumsta, J.T. Chang, R. Lee, E.P. Tan, Y. Yang, R. Loureiro, E.H. Choy, S.H.Y. Lim, I. Saez, A. Springhorn, T. Hoppe, D. Vilchez and M. Hansen (2019). The autophagy receptor p62/SQST-1 promotes proteostasis and longevity in C. elegans by inducing autophagy. Nature Communications 10: 5648.
12. H.J. Lee, A. Noormohammadi, S. Koyuncu, G. Calculli, M. Simic, M. Herholz, A. Trifunovic and D. Vilchez (2019). Prostaglandin signals from adult germline stem cells delay somatic ageing of Caenorhabditis elegans. Nature Metabolism1: 790-810 (cover).
13. I. Saez, J. Gerbracht, S. Koyuncu, H.J. Lee, M. Horn, V. Kroef, M. Denzel, C. Dieterich, N. Gehring and D. Vilchez (2019). The E3 ubiquitin ligase UBR5 interacts with the H/ACA ribonucleoprotein complex and regulates ribosomal RNA biogenesis in embryonic stem cells. FEBS Letters594: 175-188
14. A. Fatima, R. Gutierrez-Garcia and D. Vilchez (2019). Induced pluripotent stem cells from Huntington's disease patients: a promising approach to define and correct disease-related alterations. Neural Regeneration Research 14: 769-770.
15. D. Irmak, A. Fatima, R. Gutierrez-Garcia, M. Rinschen, P. Wagle, J. Altmüller, L. Arrigoni, B. Hummel, C. Klein, C.K. Frese, R. Sawarkar, A. Rada-Iglesias and D. Vilchez (2018). Mechanism suppressing H3K9 trimethylation in pluripotent stem cells and its demise by polyQ-expanded huntingtin mutations. Human Molecular Genetics 27: 4117-4134.
16. S. Koyuncu, I. Saez, H.J. Lee, R. Gutierrez-Garcia, W. Pokrzywa, A. Fatima, T. Hoppe and D. Vilchez (2018). The ubiquitin ligase UBR5 suppresses proteostasis collapse in immortal pluripotent stem cells from Huntington’s disease patients. Nature Communications 9: 2886.
17. I. Saez, S. Koyuncu, R. Gutierrez-Garcia, C. Dieterich and D. Vilchez (2018). Insights into the ubiquitin-proteasome system of human embryonic stem cells. Scientific Reports 8: 4092.
18. H.J. Lee, D. Bartsch, C. Xao, S. Guerrero, G. Ahuja, C. Schindler, J.M. Moresco, J.R. Yates III, F. Gebauer, H. Bazzi, C. Dieterich, L. Kurian and D. Vilchez (2017). A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells. Nature Communications 8: 1456.
19. A. Noormohammadi, G. Calculli, R. Gutierrez-Garcia, A. Khodokarami, S. Koyuncu and D. Vilchez (2017). Mechanisms of protein homeostasis (proteostasis) maintain stem cell identity in mammalian pluripotent stem cells. Cellular and Molecular Life Sciences 75: 275-290.
20. S. Koyuncu, A. Fatima, R. Gutierrez-Garcia and D. Vilchez (2017).Proteostasis of huntingtin in health and disease. International Journal of Molecular Sciences 18: E1568.
21. A. Noormohammadi, A. Khodakarami, R. Gutierrez-Garcia, H.J. Lee, S. Koyuncu, T. König, C. Schindler, I. Saez, A. Fatima, C. Dieterich and D. Vilchez (2016). Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan. Nature Communications 7: 13649.
22. H.J. Lee, R. Gutierrez-Garcia and D. Vilchez (2016). Embryonic stem cells: A novel approach to study proteostasis? FEBS Journal 284: 391-398.
23. S. Koyuncu, D. Irmak, I. Saez and D. Vilchez (2015).Defining the general principles of stem cell ageing: lessons from organismal models. Current Stem Cell Reports25: 162-169.
24. A. Khodakarami, I. Saez, J. Mels and D. Vilchez (2015).Mediation of organismal ageing and somatic proteostasis by the germline. Frontiers in Molecular Biosciences 2: 1-7
25. I. Saez and D. Vilchez (2014).The mechanistic links between proteasome activity, ageing and age-related diseases. Current Genomics 15: 38-51.
26. D. Vilchez, I. Saez andA. Dillin (2014).The role of protein clearance mechanisms in organismal ageing and age-related diseases. Nature Communications 5: 5659.
27. D. Vilchez^#, M. Simic^# and A. Dillin (2014). Proteostasis and ageing of stem cells. Trends in Cell Biology24: 161-170. ^#These authors contributed equally to this work.
28. D. Vilchez, L. Boyer, M. Lutz, C. Merkwirth, I. Morantte, C. Tse, B. Spencer, L. Page, E. Masliah, W.T. Berggren, F.H. Gage and A. Dillin (2013). FOXO4 is necessary for neural differentiation of human embryonic stem cells. Ageing Cell 12: 518-522.
29. D. Vilchez, I. Morantte, Z. Liu, P.M. Douglas, C. Merkwirth, A.P. Rodrigues, G. Manning and A. Dillin (2012). RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature489: 263-268.
30. D. Vilchez, L. Boyer, I. Morantte, M. Lutz, C. Merkwirth, D. Joyce, B. Spencer, L. Page, E. Masliah, F.H. Gage and A. Dillin (2012). Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature489: 304-308.