The G-protein coupled receptor GPR34 promotes the homeostatic state of microglia and restrains the disease-associated microglial response in an AD model

Reviewer #1 (Public review):

Summary:

The authors sought to understand the impact of the decreased expression of the G-protein-coupled receptor GPR34 in Alzheimer´s disease (AD). They analyzed the transcriptional impact of GPR34 deficiency in mice and found that it induced a DAM-like phenotype in control mice and enhanced the DAM signature in the AD model 5xFAD, although it did not result in amyloid plaque clearance or gross changes in microglia or astrocytes. Next, the authors developed an in vitro model of GPR34 deficiency using a CRISPR/Cas9 strategy in human iPSCs to introduce functional mutations that resulted in GPR34 protein deficiency in induced microglial cells. In this model, the authors identified myelin as a ligand of GPR34 and showed that GPR34 deficiency resulted in reduced myelin debris engulfment and transcriptional changes related to lysosomal pathways.

Strengths:

The combined strategy of using in vivo and in vitro models of GPR34 depletion is robust, and the transcriptional analyses are thoroughly performed.

Weaknesses:

The paper´s two main findings related to the lack of GPR34 (enhancement of DAM signature in vivo and reduced myelin engulfment in vitro) are disconnected. At the very least, the authors should discuss what the relevance of myelin clearance in AD is, but the paper would strongly benefit from a more thorough assessment of the impact of GPR34 deficiency in vivo, particularly because no effects on amyloid clearance were observed. The authors could assess whether GPR34-deficient 5xFAD mice have reduced cognitive performance, which, based on their in vitro findings, could be related to the myelin pathology in AD (previously described: see PMID 36284351). The analysis showing reduced myelin content in GPR34-deficient microglia in vitro is superficial and does not allow for identifying whether GPR34 is related to reduced engulfment or increased degradation, which could be related to the changes in the lysosomal gene CD68 identified in vivo. In addition, it would be interesting to compare the transcriptional profile induced by myelin phagocytosis with that of 5xFAD or AD patients, to gain insight into the impact of the signature. Finally, the transgenic approach to delete GPR34 in vivo could have been complemented with experiments with the GPR34 antagonists (YL-365 or S-E49) or agonist (Compound 4B), possibly helping in identifying the source of discrepancy with previous papers showing that GPR34 promotes amyloid clearance.

Reviewer #2 (Public review):

Summary:

Using the 5xFAD model in combination with GPR34 mice, the authors explore the function of microglia in the context of neurodegeneration. Using a broad spectrum of methodology, they show that DAM signatures are increased in KO 5xFAD mice. Using several KO clones of GPR34 KO iMGLs and another set of broad methodologies, the authors show that GPR34 is important for microglia homeostasis,
phagocytosis, specifically of myelin. GPR34 KO iMGLs also show a distinct transcriptional response to myelin. Together, they propose that GPR34 limits microglial activation in neurodegeneration.

Strengths:

All methods are state-of-the-art, and the combination of mouse and human microglia responses is a particular strength.

Weaknesses:

No weaknesses were identified by this reviewer.

Author response:

We greatly thank the Reviewing Editor, Senior Editor and the reviewers for their constructive and thoughtful feedback, as well as for recognizing the significance and strengths of our study. We are very encouraged by overall positive assessment and appreciate these insights to strengthen the manuscript. Below, we outline our plans to address the key points raised in reviewer#1’s public review. We also note that reviewer#2 did not identify any weaknesses in the study and are thankful for this positive evaluation.

Point 1: Relating in vivo and in vitro findings and discussing the relevance of myelin clearance in AD. As suggested, we will elaborate our discussion to cover the relationship between our in vivo and in vitro findings and present a more unified picture of GPR34 function. We will also highlight the relevance of myelin clearance in Alzheimer’s disease independent of amyloid plaque burden.

Point 2: in vivo assessments and phenotypes. While we recognize the value of expanding our in vitro and cellular findings to in vivo and cognitive measures, we believe this additional assessment is beyond the scope of this current study. At least, we will expand our discussion to relate our findings of GPR34-medilated myelin pathology in the contexts of neurodegeneration and cognitive deficiency in AD.

Point 3: Myelin engulfment versus lysosomal degradation. We agree that the clear distinction between impaired myelin engulfment and altered lysosomal degradation is an important point. Although additional experiments to address these scenarios are beyond the scope of the study, we will perform targeted pathway analysis of existing transcriptomic datasets focusing on the phagosome and lysosomal degradation pathways along with the in vivo findings related to CD68, which could offer an interpretation.

Point 4: Comparison of transcriptional signatures. As suggested, we will compare the transcriptional signatures induced by myelin exposure in WT iMGLs with our 5xFAD RNA-seq datasets, as well as publicly available datasets from human Alzheimer’s disease microglia, to unravel the potential converged and distinct features.

Point 5: Reconciling our findings with previous GPR34 studies. We will expand our discussion to compare and clarify our current findings with the previous studies and cover potential reasons for those different observations. We will also cover the current limitations of existing GPR34 pharmacological tools compounds, including their limited blood-brain permeability, which precludes the effective use of those tools for proposed in vivo studies.