Astrocytes display ultrastructural alterations and heterogeneity in the hippocampus of aged APP-PS1 mice and human post-mortem brain samples

Abstract

The past decade has witnessed increasing evidence for a crucial role played by glial cells, notably astrocytes, in Alzheimer’s disease (AD). To provide novel insights into the roles of astrocytes in the pathophysiology of AD, we performed a quantitative ultrastructural characterization of their intracellular contents and parenchymal interactions in an aged mouse model of AD pathology, as aging is considered the main risk factor for developing AD. We compared 20-month-old APP-PS1 and age-matched C57BL/6J male mice, among the ventral hippocampus CA1 strata lacunosum-moleculare and radiatum, two hippocampal layers severely affected by AD pathology. Astrocytes in both layers interacted more with synaptic elements and displayed more ultrastructural markers of increased phagolysosomal activity in APP-PS1 versus C57BL6/J mice. In addition, we investigated the ultrastructural heterogeneity of astrocytes, describing in the two examined layers a dark astrocytic state that we characterized in terms of distribution, interactions with AD hallmarks, and intracellular contents. This electron-dense astrocytic state, termed dark astrocytes, was observed throughout the hippocampal parenchyma, closely associated with the vasculature, and possessed several ultrastructural markers of cellular stress. A case study exploring the hippocampal head of an aged human post-mortem brain sample also revealed the presence of a similar electron-dense, dark astrocytic state. Overall, our study provides the first ultrastructural quantitative analysis of astrocytes among the hippocampus in aged AD pathology, as well as a thorough characterization of a dark astrocytic state conserved from mouse to human.

Investigation of microglial diversity in a mouse model of Parkinson’s disease pathology

“…As previous research has identified DM (and intermediate microglia, presenting features of both typical microglia and DM) as an abundant cell population in aging and neurodegenerative disease pathology (3,39,45,46), a quantitative approach to measuring the shade, or “darkness”, of microglia on electron micrographs was next employed. Using ImageJ, the integrated density of microglial cytoplasm and integrated density of nearby astrocytic cytoplasm (in either cell body or process) was measured.…”

“…for group-wise comparison. These ranges were chosen to match previous qualitative categorization of typical, intermediate, and dark microglia, respectively (3,39,45,46). In short, intermediate microglia can be distinguished from typical microglia based on their more electron-dense cytoplasm and heterochromatin pattern, accompanied by intermediate markers of cellular stress such as ER dilation (3,39,45,46).…”

“…These ranges were chosen to match previous qualitative categorization of typical, intermediate, and dark microglia, respectively (3,39,45,46). In short, intermediate microglia can be distinguished from typical microglia based on their more electron-dense cytoplasm and heterochromatin pattern, accompanied by intermediate markers of cellular stress such as ER dilation (3,39,45,46). DM can be further distinguished from typical and intermediate microglia based on their cytoplasm and nucleus, which appear even more electron-dense, and frequent absence of typical nuclear heterochromatin pattern, in addition to their various markers of cellular stress including ER dilation and mitochondrial alterations (3,4,39,41,45).…”

Microglia/macrophages are ultrastructurally altered by their proximity to spinal cord injury in adult female mice

“…Sections from two to four levels per animal (6-8 µm apart) were cut at a thickness of 73 nm and placed onto silicon wafers for SEM imaging using a Zeiss Crossbeam 350 SEM operating at a 1.4 kV voltage and 1.2 nA current. Images of the ultrathin sections were rst acquired at a resolution of 25 nm per pixel to allow for the identi cation of MG/MDM cell bodies with relation to the SCI site (25)(26)(27). MG/MDM located near the lesion area (directly contacting degraded myelinated axons or surrounded by parenchyma with clear signs of dystrophy or myelin alterations) and far (in proximity to the white matter of the dorsal column adjacent to the lesion surrounded by parenchyma without any signs of dystrophy or myelin alterations but observed in the same ultrathin section) were next imaged at a resolution of 5 nm per pixel for ultrastructural analyses.…”

“…All images were blinded to the experimental conditions. We analyzed a total sample size of 79 MG/MDM cell bodies which was determined to be su cient to obtain statistical power based on the software G*Power V3.1 (effect size of 0.9; power of 0.9 estimated at 60 MG/MDM) (25,27,28). We did not perform immunostaining to distinguish MG/MDM as we wanted to further investigate the presence of glycogen granules within their cytoplasm (27) and the possible presence of dark MG (27,29).…”