FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins.

Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease.

Redefining microglia states: Lessons and limits of human and mouse models to study microglia states in neurodegenerative diseases.

Microglia are resident macrophages of the brain parenchyma and play an essential role in various aspects of brain development, plasticity, and homeostasis. With recent advances in single-cell RNA-sequencing, heterogeneous microglia transcriptional states have been identified in both animal models of neurodegenerative disorders and patients. However, the functional roles of these microglia states remain unclear; specifically, the question of whether individual states or combinations of states are protective or detrimental (or both) in the context of disease progression. To attempt to answer this, the field has largely relied on studies employing mouse models, human in vitro and chimeric models, and human post-mortem tissue, all of which have their caveats, but used in combination can enable new biological insight and validation of candidate disease pathways and mechanisms. In this review, we summarize our current understanding of disease-associated microglia states and phenotypes in neurodegenerative disorders, discuss important considerations when comparing mouse and human microglia states and functions, and identify areas of microglia biology where species differences might limit our understanding of microglia state.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegeneration.

Tauopathies are a class of neurodegenerative diseases associated with pathological tau. Despite many advances in our understanding of these diseases, the direct mechanism through which tau contributes to neurodegeneration remains poorly understood. Previously, our laboratory implicated the histone demethylase LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer's disease cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate tau-mediated neurodegeneration and tau-induced gene expression changes. Finally, we find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration and counteract tau-induced expression changes. These results suggest that inhibiting LSD1 via sequestration contributes to tau-mediated neurodegeneration. Thus, LSD1 is a promising therapeutic target for tauopathies such as Alzheimer's disease.