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  1. Author Response:

    Reviewer #2:

    In this study, Jairaman et al used iPSC-derived microglia in which the AD-associated TREM2 gene has been knocked out to determine the impact of TREM2 loss of function on receptor-evoked Ca2+ signaling and chemotaxis. Cytoslic Ca2+ measurements were performed using the genetically-encoded ratiometric indicator Salsa6f previously developed by this laboratory. The authors made the critical discovery that loss of TREM2 leads to enhanced sensitivity and increased Ca2+ signaling of microglia to purinergic agonists, in particular to ADP. They showed that Store-operated Ca2+ entry in response to passive -maximal-store depletion by SERCA blockers was not altered in TREM2 KO cells. Rather, the enhanced sensitivity of the TREM2 KO cells was shown to originate from an upregulation of the purinergic receptors P2YR12 and P2YR13, leading to a left shift in EC50 of Ca2+ responses to ADP. The enhanced Ca2+ responses of TREM KO cells were associated with altered directional chemotaxis, whereby TREM2 KO cells showed enhanced displacement but reduced directionality. This phenotype was rescued with the application of P2YR antagonists in ADP-dependent chemotaxis assays. These results are novel, significant and of potentially broad impact to the pathology of AD. Although the molecular mechanisms of how lack of TREM2 leads to enhanced P2YRs is beyond the scope of this study, one moderate criticism of this manuscript pertains to lack of insights on how enhanced cytosolic Ca2+ leads to reduced directional chemotaxis and the potential effector proteins/pathways mediating this effect. Other relatively moderate issues and suggestions regarding controls have also been noted.

    We thank the reviewer for the positive comments and a thorough evaluation of the manuscript. We have addressed specific points related to the mechanism of purinergic Ca2+ signaling in TREM2 KO microglia. The issue of precisely how Ca2+ regulates chemotaxis differently in WT and TREM2 KO microglia, while no doubt a very important question, would involve a comprehensive evaluation of how Ca2+ affects different proteins involved in microglial motility and is best suited as part of a follow up study.

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  2. Evaluation Summary:

    Microglia are a key cell type in the brain that clear debris following tissue injury, infections, or in neurodegenerative diseases. This role is influenced strongly by directed migration of microglia towards the regions of brain injury or infection. TREM2 is a myeloid protein expressed in microglia that has been linked to Alzheimer's Disease, but the mechanisms of how TREM2 loss-of-function mutations affect microglial function is unclear. Here, Jairaman and colleagues address this question using CRISPR-based knockout of TREM2 in human iPSC-derived microglia. The study finds that TREM2 KO microglia have greatly exaggerated ADP/ATP evoked Ca signals, which is found to arise from increases in P2Y12 and P2Y13 receptor expression and enhanced receptor-evoked Ca signaling. TREM2 KO microglia show alterations in cell migration, which include, on the one hand, increased cell motility, but also reduced turning, and importantly, markedly reduced directed migration. The experiments and analysis are carefully performed using appropriate controls and the results are novel and add to our understanding of how loss-of-function TREM2 mutations impact microglial migration and the ensuing microglia-mediated clearance of plaques and damage seen in AD. Several weaknesses cloud the interpretation, but if appropriately addressed, this could be an important paper for the field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Jairaman and colleagues address the functional consequences of ablating the expression of TREM2, a myeloid protein that has been linked to Alzheimer's Disease. The study finds that TREM2 KO microglia derived from iPSC cells have exaggerated ADP/ATP evoked Ca signals, which is found to arise from increases in P2Y12 and P2Y13 receptor expression and enhanced receptor-evoked Ca signaling. Previous studies have shown that microglia migrate towards regions of brain damage or injury to clear the affected region of debris, infections, and plaques. Microglial migration is mediated in part by nucleotides released into the affected regions. Through detailed analysis of cell migratory patterns , the authors determine that the TREM2 KO microglia show alterations in cell migration that are manifested in several different ways. These changes include increase in the root mean square distance travelled, reduced turning, and markedly reduced directed migration in a chemotaxis assay. Directed migration is rescued by blocking P2Y12 receptors, confirming that the defects in ADP mediated cell migration is linked to enhanced P2Y12 receptor activity.

    The experiments and analysis are carefully performed using appropriate controls and the results are novel and add to our understanding of how loss-of-function TREM2 mutations impact microglial migration and the ensuing microglia-mediated clearance of plaques and damage seen in AD. That said there are some key issues that need to be addressed including whether increased displacement and track straightness in KO cells underlies the chemotaxis defect. Figure 7 I think shows a lot of promise of what could be, but there is no preliminary in vivo experiment for it to hold up which diminishes the significance of this in vitro study.

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  4. Reviewer #2 (Public Review):

    In this study, Jairaman et al used iPSC-derived microglia in which the AD-associated TREM2 gene has been knocked out to determine the impact of TREM2 loss of function on receptor-evoked Ca2+ signaling and chemotaxis. Cytoslic Ca2+ measurements were performed using the genetically-encoded ratiometric indicator Salsa6f previously developed by this laboratory. The authors made the critical discovery that loss of TREM2 leads to enhanced sensitivity and increased Ca2+ signaling of microglia to purinergic agonists, in particular to ADP. They showed that Store-operated Ca2+ entry in response to passive -maximal-store depletion by SERCA blockers was not altered in TREM2 KO cells. Rather, the enhanced sensitivity of the TREM2 KO cells was shown to originate from an upregulation of the purinergic receptors P2YR12 and P2YR13, leading to a left shift in EC50 of Ca2+ responses to ADP. The enhanced Ca2+ responses of TREM KO cells were associated with altered directional chemotaxis, whereby TREM2 KO cells showed enhanced displacement but reduced directionality. This phenotype was rescued with the application of P2YR antagonists in ADP-dependent chemotaxis assays. These results are novel, significant and of potentially broad impact to the pathology of AD. Although the molecular mechanisms of how lack of TREM2 leads to enhanced P2YRs is beyond the scope of this study, one moderate criticism of this manuscript pertains to lack of insights on how enhanced cytosolic Ca2+ leads to reduced directional chemotaxis and the potential effector proteins/pathways mediating this effect. Other relatively moderate issues and suggestions regarding controls have also been noted.

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  5. Reviewer #3 (Public Review):

    This study by Jairaman et al describes how iPSC-derived microglia exhibit exaggerated cytosolic Ca2+ responses to ADP stimulation in TREM2KO cells, and that this leads to a defect in turning behaviour and hence no directed migration to a chemotactic signal.

    The authors have used state-of-the-art molecular and cellular techniques to rigorously examine the inpact of TMEM2 KO on CA2_ signalling and astrocyte migration. Overall, the experiments are well conducted, carefully controlled and the findings are new and exciting. The authors nicely dissect out the underlying molecular basis for the larger Ca2+ responses to ADP and then extend their findings to cell movement and directed migration. Given the substantial body of evidence linking microglia to the pathogenesis of Alzheimer's disease, and the role for TREM2, the work by Jairaman et al. is of translational significance. As an aside, the introduction of the calcium-sensitive reporter Salsa6F is a welcome new tool in the arsenal for recording cytosolic calcium. The work will be of significant impact to the field because i) it identifies new roles for SOCE in the brain and ii) identifies CRAC channels as a target for altering microglia lol activity in Alzheimer's disease.

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