High Resolution Dissection of Reactive Glial Nets in Alzheimer’s Disease

Abstract

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

TREM2 is required for microglial instruction of astrocytic synaptic engulfment in neurodevelopment

“…Statistical results of two-way ANOVAs are indicated, where significant, above graphs. * p < .05, **p < .01, ***p < .001 has also previously been used to assess the regularity of microglial distribution (Bouvier et al, 2016). Interestingly, we found that in addition to occupying larger territories, Trem2 −/− microglia had more variability in territory size (Figure 3m and Figure S3b).…”

“…Using this method, we found that the average territory occupied by each microglia was significantly larger in Trem2 −/− mice across the cortex, hippocampus (Figure l), and other brain regions (Figure S3a). This method of analysis has also previously been used to assess the regularity of microglial distribution (Bouvier et al, ). Interestingly, we found that in addition to occupying larger territories, Trem2 −/− microglia had more variability in territory size (Figure m and Figure S3b).…”

MIC‐MAC: An automated pipeline for high‐throughput characterization and classification of three‐dimensional microglia morphologies in mouse and human postmortem brain samples

“…Immunostaining was performed as previously described (Bouvier et al, ; Quesseveur, Fouquier d'Hérouël, Murai, & Bouvier, ) with the modification that human brain sections were first irradiated with an ultraviolet lamp (Ushio, 30 W) for 18–24 hr in PBS solution to reduce residual autofluorescence background. In brief, 50 μm thick brain sections were rinsed three times for 10 min in PBS followed by a 30‐min permeabilization step with 0.3% Triton‐X 100 in PBS.…”

Human astrocytes are distinct contributors to the complexity of synaptic function

“…Progress in this field has been impeded due to the difficulty in collecting and processing human tissue. Recently, a method has been devised to process human brain tissue after long term storage while maintaining structural organization, allowing the study of three-dimensional anatomy of astrocytes in healthy and diseased tissue (Bouvier et al, 2016). Furthermore, methods to acutely purify astrocytes from human tissue have been improved using an immunopanning strategy with the cell surface protein HepaCAM and this led to the observation that human cellular uniqueness can be preserved in culture (Zhang et al, 2016).…”