Loss of ubiquitin-conjugating enzyme leads to impediment in growth of nerve cells/Link found between cellular machineries of protein degradation and regulation of the epigenetic landscape in human embryonic stem cells
A group of scientists from CECAD, the Cluster of Excellence ‘Cellular Stress Responses in Aging-Associated Diseases,’ have found a mechanism by which neurodevelopmental diseases concerning neurons can be explained: The loss of a certain enzyme, UBE2K, impeded the differentiation of stem cells by silencing the expression of genes important for neuronal differentiation and, therefore, the development and generation of neurons. More specifically, UBE2K regulates the levels and activation of histones, key proteins that pack and organize the DNA, regulating the expression of genes. Being part of the epigenetic landscape of the cell, the changes made to the histones are reversible and could provide a chance for future developments of treatments for neurodevelopmental diseases. The study is available in the current issue of Communications Biology.
Embryonic stem cells (ESCs) can replicate indefinitely while retaining their potential to differentiate into all other types of cells. Thus, nerve cells (neurons), muscle cells and all the other cells of the body are produced in a developing organism. Errors during this process can lead to congenital diseases. Degrading damaged proteins within the cell is an important factor in this process. Thus, the scientists studied the interaction between the proteasome, the main protagonist in terminating proteins, and the epigenetic landscape. Epigenetic landscapes are the heritable changes to an organism which are not determined by DNA but through changes to the chromatin, which can organize and silence DNA into tighter packages.
Azra Fatima from CECAD studied the interactions of the histones in immortal human embryonic stem cells (hESCs) which have a unique chromatin architecture and especially low levels of a certain histone called H3 which has undergone a chemical addition in the form of three methylgroups (H3K9me3).
Histones are proteins which are part of the chromatin in cell nuclei. They build up spools around which the DNA winds, shortening it by a ratio of 1:10 millions. They are also responsible for regulating the gene expression by which genes produce proteins in the organism. In addition, they play an important role in the process of cellular differentiation in which a cell, for example an embryonic stem cell, changes into another type of cell with a higher degree specialization.
They found that embryonic stem cells exhibit high expression of UBE2K (Ubiquitin-conjugating enzyme E2 K), a ubiquitin-conjugating enzyme. These enzymes are known for being important in the process of degradation of proteins. The loss of the enzyme in embryonic stem cells caused an increase in the levels of H3K9 trimethyltransferase SETDB1, resulting in higher trimethylation of H3K9, leading in turn to a repression of neurogenic genes during the differentiation of the stem cells. As a result, the loss of UBE2K impaired the ability of the stem cells to differentiate into neural progenitors a type of precursor cell that generate neurons and other cells of the nervous system.
Besides H3K9 trimethylation, the scientists found that UBE2K binds histone H3 to induce its polyubiquitination and degradation by the 26S proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates both histone H3 levels and H3K9 trimethylation in the germ cells of the model organism C. elegans. ‘Our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells like human embryonic stem cells and germline cells”, says Fatima.
‘We found a link between the ubiquitin-proteasome system and epigenetic regulation in immortal stem cells,’ Fatima concluded. ‘It would be also interesting to see if UBE2K regulates the epigenetic state in other cell types like cancer cells.’ David Vilchez, the corresponding author of the manuscript, added: ‘We believe that our findings can have important implications to understand the development of the human brain.’
By precisely regulating the levels of UBE2K it would be possible to determine the cell type specific epigenetic landscapes. Different diseases like Huntington’s disease are associated with alterations in epigenetic marks. Since the epigenetic marks are reversible, it will be interesting to study if the epigenetic status of pluripotent stem cells from patients can be modulated by controlling the proteasome system and UBE2K. In order to correct the disease phenotype, novel strategies could be designed to correct epigenetic alterations in early developmental stages and thus provide a potential treatment for diseases.
Dr. Azra Fatima
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Cold temperature delays the aging of the germline and extends fertility of C. elegans. The germline promotes the fitness of other tissues to induce longevity, as published in ‘Nature Metabolism’.
To delay the aging process of the entire organism at cold temperature, germ stem cells communicate with the rest of the body using the hormone prostaglandin, as David Vilchez and his team discovered. A moderate reduction of body temperature can induce a remarkable increase in lifespan. This phenomenon was first described in invertebrates such as worms, and later observed in fish and mice. Although the longevity effects of cold temperature were originally reported over a century ago, little is known about this process. ‘Neurons detect and process cold temperature to delay aging of the reproductive system and therefore extend fertility’, David Vilchez from the Cluster of Excellence CECAD reports. Their new research findings were published in Nature Metabolism.
The most important goal of the organism is to proliferate and keep the next generation coming. To assure this, the germline is especially protected. Living beings distribute most of their energetic resources to protect the germline, while less energy goes into protection of muscles, neurons, intestine and other somatic tissues. Therefore, when the germline is removed, organisms tend to live much longer as shown in worms and fruit flies. In these lines, distinct pathways that extend lifespan eventually affect fertility.
In their study, the researchers were able to show for the first time that under certain conditions such as cold temperature both goals – longevity and fertility – can be reached without sacrificing one of them. Specific neurons sense low temperature and communicate with the germ stem cells that rejuvenate the reproductive system, David Vilchez explains: “They say: don´t age, everything is alright, you can continue generating new germ stem cells.” Then, germ stem cells release the hormone prostaglandin E2 to maintain the quality of proteins in somatic tissues. In addition, prostaglandin E2 increases the levels of a protein called cbs-1 in the intestine. This protein produces the gas hydrogen sulphide – a substance which is known to prevent neurodegeneration and extend longevity in numerous organisms, including mammals. “High levels of this substance could kill you – but certain physiological levels make the worms and other organisms live longer,” as stated by Hyun Ju Lee and Alireza Noormohammadi, researchers involved in this study. Some mechanisms that extend lifespan lead to miserable-looking worms, laying less eggs and moving hardly. “That wasn´t the case here. Our worms lived long and prosper,” David Vilchez adds. Importantly, the researchers found that increasing hydrogen sulphide in the intestine alone was sufficient to extend lifespan even at high temperatures.
The new findings put aging in perspective, as Vilchez says: “Aging is not just a random process, where DNA damages, toxic proteins or other threats accumulate, but rather a regulated process.” Whereas current theories of aging and extensive evidence support that the reproductive system actively promotes the aging of other tissues, the findings indicate that this is not always the case. The researchers observed that increased maintenance of the germline is required for long lifespan at cold temperature. “Our findings provides a proof of regulatory systems that promote both germline and somatic fitness, without the need to sacrifice either fertility or lifespan,” David Vilchez summarizes.
Prostaglandin signals from adult germ stem cells delay somatic aging of C. elegans
Hyun Ju Lee, Alireza Noormohammadi, Seda Koyuncu, Giuseppe Calculli, Milos Simic, Marija Herholz, Aleksandra Trifunovic, David Vilchez
Nature Metabolism, DOI: https://doi.org/10.1038/s42255-019-0097-9
Mechanism that could reduce the toxic aggregation of huntingtin protein discovered / new therapies possible in the long run
The neuroscientist Dr David Vilchez and his team at CECAD, the University of Cologne’s Cluster of Excellence for Aging Research, have made an important step towards understanding the mechanisms that cause the neurodegenerative disorder Huntington’s disease. Particularly, they identified a system blocking the accumulation of toxin protein aggregates, which are responsible for neurodegeneration. The results have now been published in the journal ‘Nature Communications’.
Huntington’s disease is a neurodegenerative disorder that results in the death of brain cells, leading to uncontrolled body movement, loss of speech and psychosis. Mutations in the huntingtin gene cause the disease, resulting in the toxic aggregation of the huntingtin protein. The accumulation of these aggregates causes neurodegeneration and usually leads to the patient’s death within twenty years after the onset of the disease.
To examine the mechanisms underlying Huntington’s disease, Vilchez and his team used so-called induced pluripotent stem cells (iPSC) from Huntington’s disease patients, which are able to differentiate into any cell type, such as neurons. Induced pluripotent stem cells derived from patients with Huntington’s disease exhibit a striking ability to avoid the accumulation of toxic protein aggregates, a hallmark of the disease. Even though iPSCs express the mutant gene responsible for Huntington’s disease, no aggregates were found.
The researchers identified a protein called UBR5 as a protective mechanism for the cells, promoting the degradation of mutant huntingtin. These findings can contribute to a better understanding of Huntington’s disease and could be a step stone to developing further treatment in patients.
The researchers screened immortal iPSCs from patients and derived neurons for differences in their ability to avoid mutant huntingtin aggregation. They found that huntingtin can be degraded by the cellular disposal system known as the proteasome. However, this system is defective in the neurons, which leads to the aberrant aggregation of the mutant huntingtin protein. Vilchez and his team found that UBR5 is increased in pluripotent stem cells to accelerate the degradation of huntingtin in the cells. To examine the role of UBR5 in the regulation of the mutant huntingtin gene (HTT), they reduced the levels of UBR5 and could immediately see an accumulation of aggregated proteins in iPSCs. ‘This was striking to see’, says Vilchez. ‘From nothing, the cells went to huge amounts of aggregates.’
The authors went a step further and examined whether UBR5 also controls mutant huntingtin aggregation in Huntington’s disease organismal models. Indeed, they found that dysregulation of UBR5 results in a massive increase in the aggregation and neurotoxic effects in neurons. On the other hand, promoting UBR5 activity blocks mutant huntingtin aggregation in the Huntington’s disease models.
To test the specificity of the results, the researchers also kept an eye on other illnesses. ‘We also checked the mechanism in other neurodegenerative diseases like amyotrophic lateral sclerosis’, says Seda Koyuncu, a doctoral student working in Vilchez’s lab and a main author of the publication. ‘Our result is very specific to Huntington’s disease’, adds Dr Isabel Saez, another main author working with Vilchez at CECAD. Even though the results could be important for treatment and drug development, there is no therapy yet. ‘It’s not like you discover something new and then there is a cure, it’s more difficult – but in some years there might be a therapy’, Saez comments. Until then, more research needs to be done.
CECAD is the Cluster of Excellence at the University of Cologne and Cologne University Hospital conducting research on diseases associated with aging. Its six research areas cover a wide range of basic research on the causes and mechanisms of these diseases. David Vilchez is a Principal Investigator in Research Area B: ‘Disruptions in Protein Metabolism Cause Aging-Associated Diseases’. His research mainly focuses on protein homeostasis regulation in stem cells and aging.
‘The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington’s disease patients’. Seda Koyuncu, Isabel Saez, Hyun Ju Lee, Ricardo Gutierrez-Garcia, Wojciech Pokrzywa, Azra Fatima, Thorsten Hoppe and David Vilchez. Nature Communications, volume 9, 23 July 2018, article number: 2886 (2018)
Since their discovery in 2006, induced pluripotent stem cells are a glimmer of hope for many diseases. But further research of the complex regulation of pluripotent stem cell identity revealed unexpected difficulties. A team of researchers at the Cluster of Excellence CECAD has now found an efficient way to produce neurons from pluripotent stem cells. Their research was published in Nature Communications.
As the origin of multicellular organisms, pluripotent stem cells can differentiate into all the cell types of the body. These cells can replicate indefinitely in culture and, therefore, are considered immortal. The gold standard of pluripotency is the embryonic stem cell (ESC). Somatic cells such as skin cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) that share similar characteristics with ESCs. As such, pluripotent stem cells hold a great promise for regenerative medicine as a potential source of healthy differentiated cells, including neurons. Moreover, these cells represent an invaluable resource to investigate human development and disease in the relevant cells (neurons) affected in disorders such as Alzheimer´s, Huntington’s or Parkinson´s.
Neuronal differentiation protocols of pluripotent stem cells are usually expensive and generate a mixture of different neuronal cells and other cell types. By knocking down a single gene, the team led by David Vilchez, was able to produce neurons with 100% efficiency: “By silencing one single protein with the gene-editing method CRISPR, the cells spontaneously start to differentiate into neurons! That´s a great and much faster way to increase neurogenesis.” In natural conditions, this factor called CSDE1 prevents differentiation and keeps the cells in a pluripotent state. “This could be a very powerful mechanism to have pure populations of neurons and to facilitate a better understanding of neurodegenerative diseases.”
Hyun Ju Lee, first author of the study was most excited about the fast changes observed in her assays: “We could visualize the changes and really see it happening, the differentiation goes really fast. We also double-checked in multiple stem cell lines from different donors and induced pluripotent stem cells and got the same results.” For the study, human embryonic stem cells, induced pluripotent stem cells and mouse stem cells were used.
By using the new approach, it would be possible to facilitate the generation of neurons from samples of different patients and study the disease or test pharmaceuticals on it. Even though those results are another step to clinical application, there is still a long way to go, says David Vilchez: “New neurons from the dish could be important for studying diseases like Parkinson´s, Alzheimer´s or Huntington´s, but we are still at the starting point of this exciting research.” The research was a collaboration between CECAD, the Center for Molecular Medicine Cologne and the University Clinic Cologne.
A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells
Hyun Ju Lee, Deniz Bartsch, Cally Xiao, Santiago Guerrero, Gaurav Ahuja, Christina Schindler, James J. Moresco, John R. Yates III, Fátima Gebauer, Hisham Bazzi, Christoph Dieterich, Leo Kurian and David Vilchez
Nature Communications, DOI: 10.1038/s41467-017-01744-5
With age, somatic cells such as neurons lose their ability to maintain the quality of their protein content. Pluripotent stem cells, on the contrary, do not age and have increased mechanism to maintain the integrity of their proteins. Researchers from CECAD, the Cluster of Excellence on Aging Research based at the University of Cologne, defined the mechanisms underlying increased protein quality control of pluripotent stem cells. Then, the researchers mimicked these mechanisms in somatic tissues of model organisms to extend lifespan and delay age-related diseases. Their research was published in Nature Communications on November 28.
The survival of an organism is linked to its ability to maintain the quality of the cellular proteins. A group of proteins called chaperones facilitate the folding of proteins and are essential to regulating the quality of the cellular protein content. This ability declines during the aging process, inducing the accumulation of damaged and misfolded proteins that can lead to cell death or malfunction. Several neurodegenerative age-related disorders such as Alzheimer’s, Parkinson’s or Huntington’s disease are linked to a decline in protein quality control.
Human pluripotent stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity necessarily demands avoidance of any imbalance in the integrity of their protein content. “There is one chaperone system, the TRiC/CCT-complex that is responsible for folding about 10% of all the cellular proteins. By studying how pluripotent stem cells maintain the quality of their proteome, we found that this complex is regulated by the subunit CCT8,” says David Vilchez, senior author of the study. “Then, we discovered a way to increase the assembly and activity of the TRiC/CCT complex in somatic tissues by modulating this single subunit, CCT8. The increase resulted in prolonged lifespan and delay of age-related diseases of the model organism Caenorhabditis elegans,” he adds.
“For this study we combined the results from human pluripotent stem cells and C. elegans, to have both in vitro and in vivo models, providing a more convincing approach. Our results show that expressing CCT8 as the key subunit of the complex is sufficient to boost the assembly of the whole system,” says Alireza Noormohammadi, one of the first authors of the paper. “It is very interesting that expressing this single subunit is enough to enhance protein quality and extend longevity, even in older animals,” adds Amirabbas Khodakarami, the other main author.
“One of our next steps will be to test our findings in mice,” outlines David Vilchez. “We hope to make further progress in understanding aging diseases and to get closer to finding therapies against diseases like Huntington’s or Alzheimer’s. CCT8 could be a candidate to correct deficiencies in age-related diseases associated with protein dysfuntions.”
Alireza Noormohammadi*, Amirabbas Khodakarami*, Ricardo Gutierrez-Garcia, Hyun Ju Lee, Seda Koyuncu, Tim Konig, Christina Schindler, Isabel Saez, Azra Fatima, Christoph Dieterich & David Vilchez. Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan. NATURE COMMUNICATIONS | 7:13649 | DOI: 10.1038/ncomms13649
* These authors contributed equally to this work.