an-ex-vivo-explant-model-for-studying-glial-interactions-mouse

An <em>Ex Vivo</em> Explant Model for Studying Glial Interactions in the Mouse Retina

The role of glia in glaucoma is an increasingly prominent research topic, but much remains unknown about how populations of these support cells - namely ...

Harvard Medical School

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.

Rapid isolation of intact retinal astrocytes: a novel approach.

The role of glia, particularly astrocytes, in mediating the central nervous system's response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes - collectively referred to as 'reactivity' - in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression - both beneficially and detrimentally - and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic 'gray matter' astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous 'white matter' astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway - those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions - we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.

Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma.

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the "classical" gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina.

Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization.

The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5×10 viable cells/eye) into 3-6-month-old   knock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene  and an increase in expression of disease-associated genes including  and . Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.

Article	Year
Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes. iScience\| PubMed ID: 36465110	2022
Rapid isolation of intact retinal astrocytes: a novel approach. Acta neuropathologica communications\| PubMed ID: 37749651	2023
Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma. Frontiers in ophthalmology\| PubMed ID: 37829657	2023
Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization. Glia\| PubMed ID: 38785355	2024
Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation. bioRxiv : the preprint server for biology\| PubMed ID: 40475635	2025

Paul F. Cullen

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