31 Aug 2017

Arriving at the same conclusion, essentially at the same time, three research groups have independently mapped the site where proteases snip off the extracellular portion of TREM2. Two papers in the August 30 issue of EMBO Molecular Medicine and one under review and posted on the BioRiv preprint server report that the site coincides exactly with a mutation in the microglial receptor, H157Y, that boosts AD risk by as much as 11-fold. Its incredible that all three results are essentially identical, said Christian Haass of Ludwig-Maximilians-Universitt, Munich, the senior author on one of the EMBO Molecular Medicine papers. Peter St. George-Hyslop of the University of Toronto, who co-led the second published study with Damian Crowther and Iain Chessell of AstraZeneca in Cambridge, U.K., presented their findings earlier this year at the 13th International Conference on Alzheimers and Parkinsons Diseases in Vienna (Apr 2017 news). Ulf Neumann at Novartis in Basel, Switzerland, led the thirdgroup.

TREM2 binds anionic lipids released during neuronal and glial damage. It supports microglial metabolism and it promotes the migration, cytokine release, phagocytosis, proliferation, and survival of the cells (Aug 2017 news;Feb 2015 news). Evidence suggests that TREM2 spurs microglia to form a neuroprotective barrier around amyloid plaques and to clear A (May 2016 news; Jul 2016 news). Motivated by the discovery of roughly a dozen TREM2 genetic variants that increase the risk of frontotemporal dementia, AD, and possibly other neurodegenerative diseases, including amyotrophic lateral sclerosis and Parkinsons disease, researchers are searching for ways to bolster TREM2s protective function. Thinking that limiting ectodomain shedding might improve TREM2 signaling (see image below), the three research groups set out to find the cleavagesite.

Snip Site. ADAM10, or other proteases, may shed TREM2s extracellular ligand binding domain, abolishing TREM2-mediated signaling. (Courtesy of Yeh et al., 2017, Trends MolMed.)

In Haasss lab, first author Kai Schlepckow focused on the TREM2 C-terminal fragment (CTF) left over after shedding. Because -secretase quickly chews up the CTF in microglia, much as it does the C-terminal fragment of amyloid precursor protein in neurons, Schlepckow generated human embryonic kidney 293 (HEK293) cells expressing TREM2 and treated them with DAPT, a -secretase inhibitor. After immunoprecipitating the TREM2 CTF, he analyzed it by mass spectrometry. The result was crystal clear: a single major peak corresponding to a fragment with an N-terminus at serine 158. That amino acid lies in the extracellular domain of TREM2, 17 amino acids from the predicted transmembrane domain. Ive had experience mapping other cleavage sites and this one is super-precise. Ive never seen something like it, saidHaass.

Indeed, the result was so clean, Haass wondered if it was real. He had the researchers analyze the sequence on the other side of the break. Because sTREM2, the soluble extracellular domain, is too big to analyze by mass spectrometry, Schlepckow created a TREM2 construct with a tobacco etch virus protease cleavage site shortly before the proposed sheddase site. That way, the researchers could immunoprecipitate sTREM2 from the cell medium, shorten it with the TEV protease, and then determine its mass. Consistent with the CTF results, they obtained a single sharp peak corresponding to a peptide terminating at histidine 157. The researchers got the same results using human THP-1 cells, which are similar to monocytes, the cells that give rise tomicroglia.

Crowthers group also relied on mass spectrometry. First we used a library of peptide protease inhibitors to get a quick-and-dirty answer to where the site might be, said Crowther. First author Peter Thornton at AstraZeneca synthesized a set of D-amino acid polypeptides that overlapped TREM2 amino acids 140-176, where they thought metalloprotease likely cleaved. Then they used these peptides to compete with TREM2 for the protease in primary humanmacrophages.

All peptides that spanned TREM2 amino acids 158-160 reduced TREM2 ectodomain shedding, whereas peptides mapping to nearby regions did not. Interestingly, the most effective peptide worked equally well when its sequence was reversed, suggesting that the responsible metalloproteases recognize biophysical properties, such as charge, to target a specific sequence. To map that sequence more precisely, Thornton immunoprecipitated sTREM2 from the conditioned media of several cell types, isolated it by gel chromatography, digested it with trypsin and analyzed the fragments by mass spectrometry. From human macrophages, primary murine microglia, and HEK293 cells expressing human TREM2, H157 surfaced as the most likely sheddasesite.

Neumanns group tackled the question by generating a series of TREM2 constructs with deletions or amino acid replacements in the stalk region, located between the transmembrane and extracellular domains (see image above). They identified two regions, amino acids 169-172 and 156-164, in which mutations strongly reduced TREM2 cleavage induced by the protein kinase C activator PMA. To pinpoint the cleavage site, they designed a series of labeled peptides spanning the stalk region, incubated them with the metalloprotease ADAM17 in a test tube, and analyzed the resulting fragments using high-performance liquid chromatography and mass spectrometry. The researchers used ADAM17 because their prior data indicated it was a major TREM2sheddase.

Data from Dominik Feuerbach at Novartis also revealed the H157-S158 bond as the cleavage site. The researchers repeated the experiments using liquid chromatography and mass spec to analyze sTREM2 generated by HEK293 cells expressing human TREM2 and TYROBP, which forms a complex with the cytoplasmic portion of TREM2 (see image above). Feuerbach said they included TYROBP to mimic TREM2s physiological state as closely as possible. Again, they found cleavage occurred at the H157-S158site.

Researchers have reported that a histidine to tyrosine mutation at position 157 increases the risk for late-onset AD in Han Chinese and affects shedding (Ma et al., 2014). How might this change affect TREM2 processing? Haass and Crowthers groups compared shedding in cells expressing wild-type or the H157Y mutant. Crowther found that the extracellular domain of the wild-type protein has a half-life of less than one hour on the cells surface. Although the researchers didnt measure the mutant extracellular domain half-life directly, they found cells more rapidly pumped mutant sTREM2 into the conditioned medium. Shedding is fast in healthy cells, but it gets even faster with the variant, he said. Haass group obtained similar results. This was a surprise, said Haass, who was expecting the H157Y mutation to reduce sTREM2 production, just like other mutations that increase risk for neurodegeneration. The T66M and Y38C mutations associated with frontotemporal dementia, for example, preclude TREM2 from reaching the cell surface where shedding predominantly occurs, shutting down production of sTREM2. We got exactly the opposite of what we expected, saidHaass.

On further reflection, Haass and colleagues realized that increased shedding could have the same biological effect as reducing cell surface TREM2 because it reduced the amount of signaling-competent TREM2 on the cell surface. Indeed, Schlepckow found that monocytes expressing H157Y TREM2 phagocytosed a third less Escherichia coli than monocytes expressing the wild-type receptor. These findings support the idea that loss of function is key to the risk associated with H157Y TREM2, in line with most other TREM2 variants whose mechanisms have been dissected. Marco Colonna of Washington University in St. Louis noted that except for two TREM2 variants that increased ligand binding but were only weakly tied to AD risk, all other variants dampened TREM2 function, either by decreasing ligand binding, preventing TREM2 from reaching the cell surface, or, in this case, increasing TREM2 shedding. Feuerbach said the case for trying to therapeutically boost membrane-bound TREM2 is more compelling than ever. From a genetic point of view, this is one of the most attractive targets, said Feuerbach, first author of the BioRivpaper.

To identify the proteases responsible for wild-type and mutant TREM2 shedding, Thornton and colleagues used various protease inhibitors, as well as siRNA, to block or knock down the expression of ADAM17 or ADAM10,a.k.a. a-secretase, which process the A precursor protein (APP). Previous work implicated ADAM10 in TREM2 cleavage (Kleinberger et al., 2014). Lowering or blocking ADAM10, but not ADAM17, reduced TREM2 shedding. However, neither an inhibitor, nor ADAM10 siRNA, blocked shedding of the H157Y mutant as effectively as they blocked that of the wild-type. Crowther hypothesized the existence of a novel sheddase to account for the difference, but also acknowledged the mutant site might simply be more prone to ADAM10 cleavage and, thus, harder to fully block. It could just be that the H157Y conformation is so tasty, that ADAM10 just continues to nibble away, he said. Haass favors this latterpossibility.

Feuerbach, however, said his data points to ADAM17, rather than ADAM10, as the major TREM2 sheddase. In contrast to Crowthers group, which used HEK273 cells expressing human TREM2, but not TYROBP, Feuerbach used Chinese hamster ovary cells expressing both proteins, as well as human M2A macrophages, which are related to microglia and endogenously express TREM2 and TYROBP. To probe TREM2 cleavage, he used protease inhibitors, and ADAM10 or ADAM17 knockouts. His results indicate that TREM2 shedding depends much more on the activity of ADAM17. We would not exclude ADAM10 or other proteases from playing a role, but Id say ADAM17 probably accounts for 90 percent of the shedding, he said. Colonna and Thomas Brett, also at Washington University, thought that differences in cell types might account for the discrepancy. Different proteases might be more prevalent in different cellsprobably multiple proteases can do the job, said Colonna. Also, Feuerbach pointed out that TYROBP might affect the conformation of the cleavagesite.

From a therapeutic standpoint, researchers agreed the proteases matter less than the location of the TREM2 cleavage site. No one is interested in developing an ADAM10 inhibitor for AD, said Crowther, noting it would have too many undesirable secondary effects. Indeed, ADAM10s cutting of APP prevents the generation of A, noted Terrence Town, University of Southern California, Los Angeles. Hoping to more specifically target TREM2 clipping, Haass has begun generating antibodies against the TREM2 cleavage site. Not only could such antibodies help people with the rare H157Y mutation, but they might also help nearly anyone with AD, he believes. Antibodies against the TREM2 cleavage site could have a very potent effect, said Crowther. A boost in membrane-bound TREM2 that would rev up A phagocytosis would be beneficial. Indeed, evidence suggests people with sporadic and familial AD have increased levels of freewheeling sTREM2 in their spinal fluid, which may reflect an uptick in TREM2 shedding (Jan 2016 news;Suarez-Calvet et al., 2016). Nonetheless, Haass cautioned that it will be complicated to develop such a therapy because excessive TREM2 activity could beharmful.

Furthermore, any therapy that keeps TREM2 from casting off its extracellular domain will also cause a drop in sTREM2 levels. Brett wondered about the consequences, noting that, at least according to one study, sTREM2 causes inflammation, which is harmful, but also promotes microglial survival (Feb 2017 news). Colonna agreed. At the end of the day, we dont know if blocking sTREM2 production will have a positive, negative, or neutral effect, hesaid.

Regardless of whether regulating TREM2 processing has a future in the clinic, the new studies offer important basic insights, said Colonna. By looking at every single mutation, well understand TREM2 better.MarinaChicurel

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TREM2 Cleavage Site Pinpointed: A Gateway to New Therapies? - Alzforum

The virtual embryo offers predictions which cells express -- for example -- the genes even skipped (red) and twist (green). To appreciate the spatial distribution, researchers can look at the fly embryo from all angles. Credit: Drosophila Virtual Expression eXplorer, BIMSB at the MDC

After 13 rapid divisions a fertilized fly egg consists of about 6,000 cells. They all look alike under the microscope. However, each cell of a Drosophila melanogaster embryo already knows by then whether it is destined to become a neuron or a muscle cellor part of the gut, the head, or the tail. Now, Nikolaus Rajewsky's and Robert Zinzen's teams at the Berlin Institute of Medical Systems Biology (BIMSB) of the Max Delbrck Center for Molecular Medicine in the Helmholtz Association (MDC) have analyzed the unique gene expression profiles of thousands of single cells and reassembled the embryo from these data using a new spatial mapping algorithm. The result is a virtual fly embryo showing exactly which genes are active where at this point in time. "It is basically a transcriptomic blueprint of early development," says Robert Zinzen, head of the Systems Biology of Neural Tissue Differentiation Lab. Their paper appears as a First Release in the online issue of Science.

"Only recently has it become possible to analyze genome-wide gene expression of individual cells at a large scale. Nikolaus recognized the potential of this technology very early on and established it in his lab," says Zinzen. "He started to wonder whethergiven a complex organized tissueone would be able to compute genome-wide spatial gene expression patterns from single-cell transcriptome data alone." BIMSB combines laboratories with different backgrounds and expertise, emphasizing the need of bringing computing power to biological problems. It turns out the institute had not only the perfect model systemthe Drosophila embryoto address Rajewsky's question, but also the right people with the right expertise, from physics and mathematics to biochemistry and developmental biology.

"The virtual embryo is much more than merely a cell mapping exercise," says Nikolaus Rajewsky, head of the Systems Biology of Gene Regulatory Elements Lab, who enjoyed returning to fly development 15 years after studying gene regulatory elements in Drosophila embryos during his post-doctoral time at the Rockefeller University. Using the interactive Drosophila Virtual Expression eXplorer (DVEX) database, researchers can now look at any of about 8,000 expressed genes in each cell and ask, "Gene X, where are you expressed and at what level? What other genes are active at the same time and in the same cells?" It also works with the enigmatic long non-coding RNAs. "Instead of time-consuming imaging experiments, scientists can do virtual ones to identify new regulatory players and even get ideas for biological mechanisms," says Rajewsky. "What would normally take years using standard approaches can now be done in a couple of hours."

Breaking the synchronicity of the first cell divisions

In their paper, the MDC researchers describe a dozen new transcription factors and many more long non-coding RNAs that have never been studied before. Also, they propose an answer to a question that has puzzled scientists for 35 years: How does the embryo break synchronicity of cell divisions to develop more complex structures?

In a process called gastrulation, distinct germ layers form and cells become restricted with regard to which tissues and organs they may differentiate into. "We believe that the Hippo signaling pathway is at least partly responsible for setting up gastrulation," says Rajewsky. The pathway controls organ size, cell cycles and cell proliferation, but had never been implicated in the development of the early embryo. "We not only showed that Hippo is active in the fly, but we could even predict in which regions of the embryo this would lead to a different onset of mitosis and therefore break synchronicity. And that is just one example for how useful our tool is to understand mechanisms that have escaped traditional science."

Project underwent a tough gestation period

When the researchers started creating the virtual embryo, they did not know whether it would be possible. A key pillar of their eventual success is the Drop-Seq technology, a droplet-based, microfluidic method that allows the transcriptional profiling of thousands of individual cells at low cost. This technique had been newly set up in the Rajewsky lab by Jonathan Alles, a summer student.

However, the fly embryos needed to be selected precisely at the onset of gastrulation. Philipp Wahle, a PhD student in Robert Zinzen's lab, hand-picked about 5,000 of them before dissociating them into single cells. "I was convinced this would give us a large and completely unique data set. This was a great motivation for me," says Wahle. That laborious process created a new challenge. "You need to collect over several sessions to have enough material for a sequencing run," says Christine Kocks, who led the single-cell sequencing team. It was composed of Jonathan Alles, Salah Ayoub and Anastasiya Boltengagen, who jointly with computational scientist Nikos Karaiskos optimized the droplet-based sequencing. "So we had to find a way to stabilize the transcriptomes in the cells," added Kocks. "Finally, based on his earlier work with C. elegans embryos, Nikolaus suggested using methanol." The new single-cell fixation method was published in BMC Biology in May 2017.

As the data got better and better, Nikos Karaiskos, a theoretical physicist and computational expert in Rajewsky's lab, took on the challenge of spatially mapping such a large number of cells to their precise embryonic position. None of the existing approaches in the field of spatial transcriptomics was suitable to reconstruct the Drosophila embryo. "It was a reiterative process to filter the data, see what is inside and try to map it. It changed many times along the way," says Karaiskos. There was a lot of back and forth between members of the computer lab and wet labexchanges that are a defining characteristic of the BIMSB. "I had to question my work all the time, see where it was lacking and develop something better." He came up with a new algorithm called DistMap that can map transcriptomic data of cells back to their original position in the virtual embryo.

Navigating unchartered territory

The construction of the virtual embryo allowed Karaiskos to readily predict the expression of thousands of genes, an almost impossible task by traditional experimental means. Philipp Wahle, supported by Claudia Kipar, validated these predictions by visualizing the gene expression profiles at the bench with a traditional approach: In situ hybridization allows visualizing patterns of gene expression with colorful dyes that are visible under the microscope. "At this stage, a single layer of cells surrounds the entire fly embryo," says Wahle. "This makes it very accessible, thus enabling you to compare the computational data with imaging."

It is the first time that it has been possible to look at the about 6,000 cells of the embryo individually, assess their gene expression profilesand understand what determines their behavior in the embryo. "The most important technological advance of this study is that we don't lose the spatial information that is required to understand how embryonic cells act in concert," say the scientists. "This really is unchartered territory and requires new bioinformatics approaches to make sense of the collected data. This worked beautifully in our collaboration, not least because of the unique make-up of the Rajewsky lab, which integrates wet lab and computational approaches." One major advantage is that both groups are not only interested in technology but have specific biological questions that motivate them, says Rajewsky. "Robert has a deep understanding of early development. We can do single-cell sequencing runs and have the computational power to develop the tools that help us actually understand the underlying gene regulatory interactions."

The groups are already planning follow-up projects. One example would be to map the cells at different time points to see how they work together to form organs and tissues. Another would be to check whether the mapping approaches are applicable to more complex tissues.

Explore further: Lockdown genes to reduce IVF failure rates

More information: "The Drosophila Embryo at Single Cell Transcriptome Resolution" Science (2017). science.sciencemag.org/lookup/ 1126/science.aan3235

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Reconstructing life at its beginning, cell by cell - Phys.Org

Newswise New York (August 31, 2017) Scientific innovator and physician Dr. Pawel Muranski has joined NewYork-Presbyterian and Columbia University Medical Center (CUMC) as director of cellular immunotherapy at the newly established Good Manufacturing Practices (GMP) cell production lab and assistant director of Transfusion Medicine and Cellular Therapy. He will also serve on the faculty of CUMC as Assistant Professor of Medicine, Pathology and Cell Biology, a principal investigator at Columbia Center for Translational Immunology (CCTI) and a member of Columbias Herbert Irving Comprehensive Cancer Center.

Were thrilled to have Dr. Muranski joining us to continue his innovative work, said Dr. Gary Schwartz, chief of the Division of Hematology/Oncology at NewYork-Presbyterian/CUMC and the Clyde 56 and Helen Wu Professor of Oncology (in medicine) at CUMC. His approach to T cell-based therapy holds so much potential and could revolutionize care for cancer patients, transplant patients and others.

Dr. Muranski is a hematologist who specializes in bone marrow transplantation and in developing adoptive T cell therapies, in which white blood cells called T lymphocytes are removed from a patient or a donor and then programmed to target viral infections, leukemic cells and solid tumors. Adoptive transfer of T cells, including Chimeric Antigen Receptor (CAR)-T therapy has shown great promise in early trials of patients with leukemia, lymphoma and several solid cancersin some cases leading to a complete remission.

Dr. Muranskis research will continue to focus on exploiting and enhancing the capability of engineered T cells to recognize and target cancerous cells or dangerous viruses. He has a particular interest in developing CD4+ T helper cellsthe master orchestrators of immune responseas a potentially powerful weapon against cancer. His T cells can also target viral infections in patients whose immune systems have been weakened by bone marrow or organ transplantation, cancer treatment, or autoimmune diseases.

Despite recent spectacular advances in the field of cancer immunotherapy, very few institutions have GMP laboratories with the capacity to grow and manipulate T cells, said Dr. Muranski. NewYork-Presbyterian and Columbia University Medical Center are now positioned to become leaders in cutting-edge cellular immunotherapies. Im excited to work with the team here on developing a comprehensive program that brings these innovative treatments to our patients.

In addition to his work in the GMP lab, Dr. Muranski will be working with Dr. Prakash Satwani, a pediatric hematologist and oncologist at NewYork-Presbyterian and associate professor of pediatrics at CUMC, on an upcoming major CAR-T cell initiative. He will also work closely with Dr. Markus Mapara, director of the Adult Blood and Marrow Transplantation Program at NewYork-Presbyterian/Columbia and professor of medicine at CUMC.

Dr. Muranski trained as a fellow at the Surgery Branch, National Cancer Institute (NCI), National Institutes of Health (NIH) in Bethesda, Maryland, where he performed innovative studies aimed at understanding of the role of CD4+ T cells as mediators of curative anti-tumor immunity. Most recently, he served in Hematology Branch, National Heart, Lung and Blood Institute (NHLBI) at the NIH, where his research focused on using T cell-based therapies to prevent viral infections in patients undergoing donor-based stem cell transplantation for blood cancers.

He earned his medical degree from the Medical University of Warsaw in Poland before completing a research fellowship at the Institute for Molecular Medicine and Genetics, Medical College of Georgia and a residency at St. Francis Hospital in Evanston, Illinois. He completed a clinical fellowship in hematology and oncology at the National Institutes of Health in Bethesda, Maryland.

NewYork-Presbyterian

NewYork-Presbyterian is one of the nations most comprehensive, integrated academic healthcare delivery systems, whose organizations are dedicated to providing the highest quality, most compassionate care and service to patients in the New York metropolitan area, nationally, and throughout the globe. In collaboration with two renowned medical schools, Weill Cornell Medicine and Columbia University Medical Center, NewYork-Presbyterian is consistently recognized as a leader in medical education, groundbreaking research and innovative, patient-centered clinical care.

NewYork-Presbyterian has four major divisions:

Columbia University Medical Center

Columbia University Medical Centerprovides international leadership in basic, preclinical, and clinical research; medical and health sciences education; and patient care. The medical center trains future leaders and includes the dedicated work of many physicians, scientists, public health professionals, dentists, and nurses at the College of Physicians and Surgeons, the Mailman School of Public Health, the College of Dental Medicine, the School of Nursing, the biomedical departments of the Graduate School of Arts and Sciences, and allied research centers and institutions. Columbia University Medical Center is home to the largest medical research enterprise in New York City and State and one of the largest faculty medical practices in the Northeast. The campus that Columbia University Medical Center shares with its hospital partner, NewYork-Presbyterian, is now called the Columbia University Irving Medical Center. For more information, visit cumc.columbia.eduorcolumbiadoctors.org.

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Dr. Pawel Muranski to Head New Cellular Immunotherapy Laboratory at NewYork-Presbyterian/Columbia University ... - Newswise (press release)