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Inflammation and AD: modulating microglia function focussing on TREM2 and CD33 - Sofia ref.: 115976

Periodic Reporting for period 4 - PHAGO (Inflammation and AD: modulating microglia function focussing on TREM2 and CD33 - Sofia ref.: 115976)

Reporting period: 2019-11-01 to 2020-10-31

Alzheimer’s disease (AD) is an age-related chronic neurodegenerative disease with progressive loss of nerve cells and their connectivity in the brain. Today, over 46 million people live with dementia worldwide and this number is estimated to increase to 131.5 million by 2050.
Phagocytes accumulate around amyloid plaques in the brains of AD patients and show a dysfunctional activation profile. Recent systems biology approaches have identified two innate immune receptor genes TREM2 and CD33/SIGLEC3 as disease relevant players in AD. These findings present novel and attractive targets for treatment. However, their exact role and underlying cellular mechanisms are still unclear.
PHAGO aims to fill this knowledge gap and provide tools and assays for targeting these innate immune receptors, namely TREM2 and CD33/SIGLEC3, to pave the way for the future development of drugs that delay the progression of Alzheimer’s disease. To achieve this, PHAGO brings together experts from industry and academia in a powerful collaboration.
Furthermore, the PHAGO consortium is actively reaching out to other IMI-collaborative undertakings in order to maximally utilize synergisms throughout the European Research landscape. Particularly, PHAGO is collaborating with the IMI-initiative NEURONET ( to increase the outreach and making assets and knowledge more easily accessible and available to the Scientific Community.
PHAGO has regularly project discussion through reports, teleconferences within and between work packages and remote meetings via videoconferences. In total, five General Assembly Meeting have been organized to discuss the status of the project within the whole consortium.
In response to the Covid-19 restrictions, PHAGO partners have managed to shift work from the laboratory into remote computer-related tasks, but will face a delay in several work packages of approximately 6 months.
PHAGO has generated and characterized more than 40 iPSC lines, that will be deposited in the EBiSC2 iPSC repository to become available to the scientific community in the course of the project. Further, partner Life & Brain has established an up-scalable technique for high yield production of iPSC-derived microglia that is amenable to bioreactor formats. Life & Brain also filed a patent application on the cryopreservation of human iPSC derived microglia (reference EP20162230.5).
PHAGO has shown that antibodies against the stalk region of TREM2 can enhance the protective clearance function of microglia and might have possible future potential for a clinical application:
• Schlepckow K, et al. “Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region”, EMBO Mol Med. DOI 10.15252/emmm.201911227

PHAGO has contributed to elucidate the role of sialylation for regulation of microglial function and for protecting the brain against inflammatory age-related loss of neurons:
• Klaus C, et al. “Reduced sialylation triggers homeostatic synapse and neuronal loss in middle-aged mice”, Neurobiology of Aging.
• Allendorf DH, et al. "Lipopolysaccharide activates microglia via neuraminidase 1 desialylation of Toll-like Receptor 4”, JNC. DOI: 10.1111/jnc.15024
• Allendorf DH, et al. “Activated microglia desialylate their surface, stimulating complement receptor 3-mediated phagocytosis of neurons”, Glia.

PHAGO partners have identified a new rare TREM2 variant that is associated with dementia and shown that the AD associated TERM2 mutation R47H is leading to a locked immunometabolic phenotype in human microglia:
• Karsak M, et al. “A rare heterozygous TREM2 coding variant identified in familial clustering of dementia affects an intrinsically disordered protein region and function of TREM2.”. Hum Mutat. DOI: 10.1002/humu.23904
• Piers TM, et al. “A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia”, FASEB DOI: 10.1096/fj.201902447R

PHAGO has established highly sensitive biomarkers for detecting soluble TREM2 in the CSF of patients and monitored the levels of sTREM2 in carriers with rare TREM2 variants in relation to levels of neurofilament light chain:
• Ashton NJ, et al. “Plasma levels of soluble TREM2 and neurofilament light chain in TREM2 rare variant carriers”, Alzheimer Res Ther. DOI: 10.1186/s13195-019-0545-5

PHAGO has started several training sessions, hackatons and test runs of the knowledge platform, which focuses on knowledge and omics data from PHAGO partners that are enriched by literature and omics data from public resources. PHAGO plans to further optimize this database and is planning to release this helpful tool to the scientific public within the next year.
Overall, PHAGO is well within its established work plan by taking into account the Covid-19 related delay of 6 months.
Partners of PHAGO are pushing forward the knowledge on our understanding of TREM2/CD33 in respect to development of AD, that have been summarized in several concise reviews:
• Lewcock JW. et al. “Emerging Microglia Biology Defines Novel Therapeutic Approaches for Alzheimer’s Disease”, Neuron. DOI: 10.1016/j.neuron.2020.09.029
• Liao H, et al. “Control of Innate Immunity by Sialic Acids in the Nervous Tissue”, Int J Mol Sci. doi:10.3390/ijms21155494
• Puigdellivol M. et al. “Sialylation and Galectin-3 in Microglia-Mediated Neuroinflammation and Neurodegeneration”, Front Cells Neurosc. doi: 10.3389/fncel.2020.00162
• Klaus C, et al. “Sialylation acts as a checkpoint for innate immune responses in the central nervous system”, GLIA DOI: 10.1002/glia.23945
In addition, PHAGO has generated a strong platform of iPSC and characterized microglial cells from iPSC lines with TREM2/CD33 polymorphism, that is unique source for follow-up research and will becomes available worldwide via the cell bank of EBiSC. PHAGO continues to validate differentiation procedures of these iPSC lines across sites with the goal to make available a publicly available SOP.

Image description: Schematic illustration of the TREM2-TYROBP expressing HEK reporter system and the detection of phosphorylated Syk kinase by the AlphaLISA system. The TREM2 expressing cells were plated on 96-well plates and treated for 5 minutes at 37 oC with a TREM2-specific antibody. Cells were lysed and the lysates were incubated with acceptor beads (Incubation Acc. Mix) for one hour and subsequently with donor beads (Incubation Don. Mix) for another hour. For detection of phosphorylated Syk kinase levels (pSYK), samples were measured with a plate reader using standard AlphaLISA settings (AlphaLISA Readout).
TREM2 signalling detected by phosphorylation of the Syk kinase.