Dissecting surveying behavior of reactive microglia under chronic neurodegeneration

Reviewer #1 (Public review):

Summary:

In this manuscript, Subhramanian et al. carefully examined how microglia adapt their surveillance strategies during chronic neurodegeneration, specifically in prion-infected mice. The authors used ex vivo time-lapse imaging and in vitro strategies, finding that reactive microglia exhibit a highly mobile, "kiss-and-ride" behavior, which contrasts with the more static surveillance typically observed in homeostatic microglia. The manuscript provides fundamental mechanistic insights into the dynamics of microglia-neuron interactions, implicates P2Y6 signaling in regulating mobility, and suggests that intrinsic reprogramming of microglia might underlie this behavior. The conclusions are therefore compelling.

Strengths:

(1) The novelty of the study is high, in particular, the demonstration that microglia lose territorial confinement and dynamically migrate from neuron to neuron under chronic neurodegeneration.

(2) The possible implications of a stimulus-independent high mobility in reactive microglia are particularly striking. Although this is not fully explored (see comments below).

(3) The use of time-lapse imaging in organotypic slices rather than overexpression models provided a more physiological approach.

(4) Microglia-neuron interactions in neurodegeneration have broad implications for understanding the progression of other diseases that are associated with chronic inflammation, such as Alzheimer's and Parkinson's.

Weaknesses:

(1) The Cx3cr1/EGFP line labels all myeloid cells, which makes it difficult to conclude that all observed behaviors are attributable to microglia rather than infiltrating macrophages. The authors refer to this and include it as a limitation. Nonetheless, complementary confirmation by additional microglia markers would strengthen their claims.

(2) Although the authors elegantly describe dynamic surveillance and envelopment hypothesis, it is unclear what the role of this phenotype is for disease progression, i.e., functional consequences. For example, are the neurons that undergo sustained envelopment more likely to degenerate?

(3) Moreover, although the increase in mobility is a relevant finding, it would be interesting for the authors to further comment on what the molecular trigger(s) is/are that might promote this increase. These adaptations, which are at least long-lasting, confer apparent mobility in the absence of external stimuli.

(4) The authors performed, as far as I could understand, the experiments in cortical brain regions. There is no clear rationale for this in the manuscript, nor is it clear whether the mobility is specific to a particular brain region. This is particularly important, as microglia reactivity varies greatly depending on the brain region.

(5) It would be relevant information to have an analysis of the percentage of cells in normal, sub-clinical, early clinical, and advanced stages that became mobile. Without this information, the speed/distance alone can have different interpretations.

Reviewer #2 (Public review):

This is a nice paper focused on the response of microglia to different clinical stages of prion infection in acute brain slices. The key here is the use of time-lapse imaging, which captures the dynamics of microglial surveillance, including morphology, migration, and intracellular neuron-microglial contacts. The authors use a myeloid GFP-labeled transgenic mouse to track microglia in SSLOW-infected brain slices, quantifying differences in motility and microglial-neuron interactions via live fluorescence imaging. Interesting findings include the elaborate patterns of motility among microglia, the distinct types and duration of intracellular contacts, the potential role of calcium signaling in facilitating hypermobility, and the fact that this motion-promoting status is intrinsic to microglia, persisting even after the cells have been isolated from infected brains. Although largely a descriptive paper, there are mechanistic insights, including the role of calcium in supporting movement of microglia, where bursts of signaling are identified even within the time-lapse format, and inhibition studies that implicate the purinergic receptor and calcium transient regulator P2Y6 in migratory capacity.

Strengths:

(1) The focus on microglia activation and activity in the context of prion disease is interesting.

(2) Two different prions produce largely the same response.

(3) Use of time-lapse provides insight into the dynamics of microglia, distinguishing between types of contact - mobility vs motility - and providing insight into the duration/transience and reversibility of extensive somatic contacts that include brief and focused connections in addition to soma envelopment.

(4) Imaging window selection (3 hours) guided by prior publications documenting preserved morphology, activity, and gene expression regulation up to 4 hours.

(5) The distinction between high mobility and low mobility microglia is interesting, especially given that hyper mobility seems to be an innate property of the cells.

(6) The live-imaging approach is validated by fixed tissue confocal imaging.

(7) The variance in duration of neuron/microglia contacts is interesting, although there is no insight into what might dictate which status of interaction predominates.

(8) The reversibility of the enveloping action, that is not apparently a commitment to engulfment, is interesting, as is the fact that only neurons are selected for this activity.

(9) The calcium studies use the fluorescent dye calbryte-590 to pick up neuronal and microglial bursts - prolonged bursts are detected in enveloped neurons and in the hyper-mobile microglia - the microglial lead is followed up using MRS-2578 P2Y6 inhibitor that blunts the mobility of the microglia.

Weaknesses:

(1) The number of individual cells tracked has been provided, but not the number of individual mice. The sex of the mice is not provided.

(2) The statistical approach is not clear; was each cell treated as a single observation?

(3) The potential for heterogeneity among animals has not been addressed.

(4) Validation of prion accumulation at each clinical stage of the disease is not provided.

(5) How were the numerous captures of cells handled to derive morphological quantitative values? Based on the videos, there is a lot of movement and shape-shifting.

(6) While it is recognized that there are limits to what can be measured simultaneously with live imaging, the authors appear to have fixed tissues from each time point too - it would be very interesting to know if the extent or prion accumulation influences the microglial surveillance - i.e., do the enveloped ones have greater pathology>