Serrano-Pozo A, Li H, Li Z, Muñoz-Castro C, Jaisa-Aad M, Healey MA, Welikovitch LA, Jayakumar R, Bryant AG, Noori A, Connors TR, Hu M, Zhao K, Liao F, Lin G, Pastika T, Tamm J, Abdourahman A, Kwon T, Bennett RE, Woodbury ME, Wachter A, Talanian RV, Biber K, Karran EH, Hyman BT, Das S. Astrocyte transcriptomic changes along the spatiotemporal progression of Alzheimer's disease. Nat Neurosci. 2024 Dec;27(12):2384-2400. Epub 2024 Nov 11 PubMed.
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Universidade Federal do Rio Grande do Sul
This is an impressive study by Serrano-Pozo, Hyman, Das and colleagues that explores astrocyte transcriptomic molecular programs in aging and Alzheimer’s Disease. Using single-nucleus RNA sequencing of more than 600,000 astrocytes from five brain regions, the authors characterized astrocyte phenotypes ranging from normal aging to severe AD. They found that astrocytes adopt multiple phenotypes, reinforcing the idea that we should move away from the simplistic resting/reactive astrocyte dichotomy. This was predicted and emphasized in our consensus paper in 2021 (Escartin et al., 2021).
This work reinforces previous findings in mouse models (Habib et al., 2020) and humans (Mathys et al., 2024), suggesting the existence of multiple astrocyte phenotypes and that they undergo dynamic changes as a function of pathophysiological alterations. We and others have shown that, for example, astrocytes respond differently to amyloid and tau pathology (Jiwaji et al., 2022; De Bastiani et al., 2023).
The central role of astrocytes has been gaining attention in recent years, mainly due to the idea of “neuroinflammation” as a key player in AD. However, it is important to keep in mind that astrocytes do much more than that; their role in energy metabolism, synaptic function, the blood-brain barrier, and other processes is vital. Thus, the coexistence of multiple astrocyte phenotypes suggests that we should address astrocyte heterogeneity, stratify astrocyte subtypes, and consider multiple astrocyte targets for developing different astrocyte biomarkers.
In summary, we should think about astrocytes the way we think about neurons. How many neuronal subtypes do we have? Glutamatergic, GABAergic, dopaminergic ... The same approach should be applied to astrocytes—it is time to characterize them!
References:
Escartin C, Galea E, Lakatos A, O'Callaghan JP, Petzold GC, Serrano-Pozo A, Steinhäuser C, Volterra A, Carmignoto G, Agarwal A, Allen NJ, Araque A, Barbeito L, Barzilai A, Bergles DE, Bonvento G, Butt AM, Chen WT, Cohen-Salmon M, Cunningham C, Deneen B, De Strooper B, Díaz-Castro B, Farina C, Freeman M, Gallo V, Goldman JE, Goldman SA, Götz M, Gutiérrez A, Haydon PG, Heiland DH, Hol EM, Holt MG, Iino M, Kastanenka KV, Kettenmann H, Khakh BS, Koizumi S, Lee CJ, Liddelow SA, MacVicar BA, Magistretti P, Messing A, Mishra A, Molofsky AV, Murai KK, Norris CM, Okada S, Oliet SH, Oliveira JF, Panatier A, Parpura V, Pekna M, Pekny M, Pellerin L, Perea G, Pérez-Nievas BG, Pfrieger FW, Poskanzer KE, Quintana FJ, Ransohoff RM, Riquelme-Perez M, Robel S, Rose CR, Rothstein JD, Rouach N, Rowitch DH, Semyanov A, Sirko S, Sontheimer H, Swanson RA, Vitorica J, Wanner IB, Wood LB, Wu J, Zheng B, Zimmer ER, Zorec R, Sofroniew MV, Verkhratsky A. Reactive astrocyte nomenclature, definitions, and future directions. Nat Neurosci. 2021 Mar;24(3):312-325. Epub 2021 Feb 15 PubMed.
Habib N, McCabe C, Medina S, Varshavsky M, Kitsberg D, Dvir-Szternfeld R, Green G, Dionne D, Nguyen L, Marshall JL, Chen F, Zhang F, Kaplan T, Regev A, Schwartz M. Disease-associated astrocytes in Alzheimer's disease and aging. Nat Neurosci. 2020 Jun;23(6):701-706. Epub 2020 Apr 27 PubMed.
Mathys H, Boix CA, Akay LA, Xia Z, Davila-Velderrain J, Ng AP, Jiang X, Abdelhady G, Galani K, Mantero J, Band N, James BT, Babu S, Galiana-Melendez F, Louderback K, Prokopenko D, Tanzi RE, Bennett DA, Tsai LH, Kellis M. Single-cell multiregion dissection of Alzheimer's disease. Nature. 2024 Aug;632(8026):858-868. Epub 2024 Jul 24 PubMed.
Jiwaji Z, Tiwari SS, Avilés-Reyes RX, Hooley M, Hampton D, Torvell M, Johnson DA, McQueen J, Baxter P, Sabari-Sankar K, Qiu J, He X, Fowler J, Febery J, Gregory J, Rose J, Tulloch J, Loan J, Story D, McDade K, Smith AM, Greer P, Ball M, Kind PC, Matthews PM, Smith C, Dando O, Spires-Jones TL, Johnson JA, Chandran S, Hardingham GE. Reactive astrocytes acquire neuroprotective as well as deleterious signatures in response to Tau and Aß pathology. Nat Commun. 2022 Jan 10;13(1):135. PubMed.
De Bastiani MA, Bellaver B, Brum WS, Souza DG, Ferreira PC, Rocha AS, Povala G, Ferrari-Souza JP, Benedet AL, Ashton NJ, Karikari TK, Zetterberg H, Blennow K, Rosa-Neto P, Pascoal TA, Zimmer ER, Alzheimer's Disease Neuroimaging Initiative. Hippocampal GFAP-positive astrocyte responses to amyloid and tau pathologies. Brain Behav Immun. 2023 May;110:175-184. Epub 2023 Mar 4 PubMed.
View all comments by Eduardo R. ZimmerWeizmann Institute of Science
The manuscript by Alberto Serrano-Pozo and colleagues elegantly describes the dynamic changes in astrocytes across various brain areas, and at different stages of Alzheimer’s disease. This study is extremely important as it highlights the dynamics of astrocyte changes in AD and emphasizes the notion that a treatment aimed at modifying the disease, based on pathological changes identified in astrocytes at a particular disease stage and in a specific brain region, may not be applicable to all stages of the pathology, and could even result in adverse effects. This aligns with our current understanding of microglia, particularly in relation to high Trem2-expressing microglia; elevated levels of Trem2 have been observed in activated microglia and in senescent microglia, which exhibit distinct signatures and trajectories.
View all comments by Michal SchwartzVIB-Center for Molecular Neurology
VIB-Center for Molecular Neurology
This is a very interesting and thorough study investigating astrocyte heterogeneity in Alzheimer’s disease. Astrocytes are highly diverse in the brain, with several transcriptional and morphological subtypes. Serrano-Pozo and colleagues elegantly profile the transcriptional states of astrocytes across distinct brain regions as well as along a temporal disease axis. The analysis of the data is cleverly designed and aims to provide new insights into how astrocyte diversity links to pathology.
It is important to note that, similar to the field of microglia, studies like this will help us move away from the very simplistic, dichotomous A1/A2 classification and build the foundation for a more comprehensive astrocyte subtype annotation. The changes reported in astrocytes over different pathological states are very interesting and pave the way for follow-up studies looking into the functional contribution of different astrocyte subsets in disease. It would also be very interesting to determine how genetics would alter the response of astrocytes to Alzheimer’s disease, for example by stratifying by APOE genotypes.
One interesting point is whether the underlying astrocytic heterogeneity could contribute to the differential susceptibility observed in different CNS regions across neurodegenerative disorders. The concept of neuronal selective susceptibility is well accepted and a topic of intense research. It is plausible that the same concept could be applied to glial cells, where glial susceptibility would consist of either intrinsic regional changes of glial subpopulations, or specific genetic alterations that selectively affect glial function, resulting in an increased local susceptibility to neurodegeneration.
Studies like the one presented here, as well as other large-scale transcriptomic studies published over the last few years, could serve as a roadmap to start exploring these exciting new research avenues.
View all comments by Baukje BijnensImperial College London
This report contributes substantively to an understanding of potential roles of astrocytes in Alzheimer’s disease and, in doing so, provides a stronger foundation for exploration of the astrocyte as a therapeutic target. Technically, it illustrates how far the field has come over the last few years.
Like earlier reports (Smith et al., 2022), the authors identify subtypes of astrocytes that may have relatively distinct functional phenotypes. However, the numbers of nuclei sequenced here allow more confidence in the expressed phenotypes. Important insights arose from their characterization in five brain regions chosen for their associations with AD pathological stages: the authors provide evidence for region-specific astrocyte phenotypes that may be analogous to those described in the mouse (Morel et al., 2017). Explorations of pseudo-temporal patterns of gene expression with disease progression show negative correlations of gene expression patterns with Aβ and tau pathology related to apparent loss of cell motility, impairments of energy and lipid metabolism, and loss of capacity for healthy neurotransmitter homeostasis.
The particular enrichment of microglia for expression of genes associated with AD has led to interest in microglial responses for understanding the genesis of disease. Studies like this rightly provoke a focus on the astrocyte for understanding disease progression. An intriguing hypothesis that arises from this work is that regional susceptibility to AD pathology may be determined (at least in part) by astrocytes, e.g., through the balance of trophic and adaptive reactive responses to pathology with down-regulation, particularly of glutaminergic regulatory capacity. Similar pathways have been proposed to contribute to progressive multiple sclerosis (Kaufmann et al., 2022). Augmentation of trophic, metabolic, or glutaminergic regulatory pathways in astrocytes may be a rational complement to clearance of toxic protein species for slowing disease progress ion in AD.
References:
Smith AM, Davey K, Tsartsalis S, Khozoie C, Fancy N, Tang SS, Liaptsi E, Weinert M, McGarry A, Muirhead RC, Gentleman S, Owen DR, Matthews PM. Diverse human astrocyte and microglial transcriptional responses to Alzheimer's pathology. Acta Neuropathol. 2022 Jan;143(1):75-91. Epub 2021 Nov 12 PubMed.
Morel L, Chiang MS, Higashimori H, Shoneye T, Iyer LK, Yelick J, Tai A, Yang Y. Molecular and Functional Properties of Regional Astrocytes in the Adult Brain. J Neurosci. 2017 Sep 6;37(36):8706-8717. Epub 2017 Aug 7 PubMed.
Kaufmann M, Schaupp AL, Sun R, Coscia F, Dendrou CA, Cortes A, Kaur G, Evans HG, Mollbrink A, Navarro JF, Sonner JK, Mayer C, DeLuca GC, Lundeberg J, Matthews PM, Attfield KE, Friese MA, Mann M, Fugger L. Identification of early neurodegenerative pathways in progressive multiple sclerosis. Nat Neurosci. 2022 Jul;25(7):944-955. Epub 2022 Jun 20 PubMed.
View all comments by Paul MatthewsHong Kong University of Science & Technology
This study presents a comprehensive analysis of transcriptomic changes in astrocytes across different parts of the neural network vulnerable to Alzheimer’s disease. Using single-nucleus RNA sequencing, the authors analyzed 628,943 astrocytes from 32 donors at the early, intermediate, late, and end stages of AD pathology based on CERAD neuritic plaque score and Braak staging.
Their findings first reveal spatial heterogeneity among astrocytes, particularly in the entorhinal cortex and visual cortex, highlighting how the surrounding microenvironment influences differential gene expression in astrocytes in aged human brains. The authors subsequently investigated how transcriptomic changes in astrocytes occur along the AD-vulnerable neural network and their relationship with AD pathologies. They identified distinct spatial transcriptomic expression patterns among astrocytes, which exhibit varying correlations with Aβ and/or tau and reveal a complex relationship between astrocytic responses and AD pathology. Notably, one of the identified patterns demonstrated that these changes are independent of AD-related pathologies, suggesting the presence of intrinsic astrocytic mechanisms that may be activated in response to aging itself. In addition to spatial patterns, the authors delineated distinct temporal trajectories of astrocytic transcriptomic changes that correspond to the stages of AD progression. Each trajectory is uniquely associated with AD pathology, underscoring the dynamic nature of astrocytic responses across disease progression.
To further understand the transcriptomic dynamics within heterogeneous astrocytes in AD, the authors conducted cluster analysis. Consistent with previous studies (Green et al., 2024; Endo et al., 2022; Habib et al., 2020; Allen et al., 2023), they identified homeostatic, intermediate, and activated astrocyte subtypes. The authors specifically identified two unique astrocyte subpopulations: one enriched in trophic factors that declines with disease progression, and another that is activated in late-stage AD, indicating an exhausted response to neuropathology. This aligns with a previous study showing one astrocyte subcluster losing neuroprotective properties related to neurotransmitter recycling and synaptic signaling and another showing upregulation of chaperone-mediated responses in patients with AD (Lau et al., 2020). Together, these findings suggest the roles of astrocytes subclusters in neuroprotection and neurodegeneration as well as how these roles may change as AD progresses.
A unique aspect of this manuscript is its presentation of a continuous spectrum of astrocytes across aging and AD stages within individuals. The differential spatial and temporal responses of astrocytes to AD-related pathology suggest that these cells play multifaceted roles in the progression of AD and that they are influenced by alterations in the microenvironment. For instance, astrocytes may influence neurotransmission within the neural network and modulate the inflammatory responses of microglia, illustrating the intricate interplay between astrocytes and other cell types in the brain. A recent study published in Nature (Green et al., 2024) proposes causal relationships between specific subpopulation of astrocytes and microglia in relation to AD pathology, highlighting how their interactions may contribute to cognitive decline in individuals with AD. Understanding the specific biological processes and regulatory molecular mechanisms associated with each transcriptomic pattern of astrocytes could substantially advance our knowledge of the pathogenic mechanisms underlying AD.
In summary, this study provides a broad, comprehensive, spatiotemporal map of astrocytes, thereby facilitating a deeper understanding of their roles in specific stages of AD. Moreover, the findings help elucidate how astrocytic responses evolve and adapt in the context of neurodegeneration, offering valuable insights that can guide future research efforts aiming to understand the pathological mechanisms of AD and develop targeted therapeutic strategies.
References:
Green GS, Fujita M, Yang HS, Taga M, Cain A, McCabe C, Comandante-Lou N, White CC, Schmidtner AK, Zeng L, Sigalov A, Wang Y, Regev A, Klein HU, Menon V, Bennett DA, Habib N, De Jager PL. Cellular communities reveal trajectories of brain ageing and Alzheimer's disease. Nature. 2024 Sep;633(8030):634-645. Epub 2024 Aug 28 PubMed.
Endo F, Kasai A, Soto JS, Yu X, Qu Z, Hashimoto H, Gradinaru V, Kawaguchi R, Khakh BS. Molecular basis of astrocyte diversity and morphology across the CNS in health and disease. Science. 2022 Nov 4;378(6619):eadc9020. PubMed.
Habib N, McCabe C, Medina S, Varshavsky M, Kitsberg D, Dvir-Szternfeld R, Green G, Dionne D, Nguyen L, Marshall JL, Chen F, Zhang F, Kaplan T, Regev A, Schwartz M. Disease-associated astrocytes in Alzheimer's disease and aging. Nat Neurosci. 2020 Jun;23(6):701-706. Epub 2020 Apr 27 PubMed.
Allen WE, Blosser TR, Sullivan ZA, Dulac C, Zhuang X. Molecular and spatial signatures of mouse brain aging at single-cell resolution. Cell. 2023 Jan 5;186(1):194-208.e18. Epub 2022 Dec 28 PubMed.
Lau SF, Cao H, Fu AK, Ip NY. Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer's disease. Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25800-25809. Epub 2020 Sep 28 PubMed.
View all comments by Nancy IpUniversity of Pennsylvania
University of Pennsylvania
This was an ambitious study using single-nucleus RNA-Seq to characterize astrocyte heterogeneity across five brain regions in 32 donors who spanned the spectrum of Alzheimer’s disease neuropathology.
The authors’ analysis of normal brains revealed astrocyte heterogeneity across the neocortex. Astrocytes are known to exhibit morphological and functional diversity across brain regions, but how this heterogeneity may impact neurodegeneration is not well understood. Since astrocytes are integral to maintaining a healthy brain microenvironment, the observed transcriptional differences across cortical regions may contribute to those regions’ neuronal vulnerability versus resilience in AD. For example, the authors interestingly found genes involved in maintenance of the tripartite synapse (NRXN1) and glutamate uptake (SLC1A2) to be increased in visual cortex versus entorhinal cortex, which are relatively spared and vulnerable in AD, respectively. Baseline, more efficient glutamate management and responses to local stressors may underlie each region’s ability to cope with the development of excitotoxicity as well as with amyloid and tau pathology. Future studies will be needed to identify how regional astrocyte heterogeneity affects disease progression and whether these pathways can be appropriately modulated.
The authors also identified astrocytic transcriptional responses to increasing neuropathology. Interestingly, they found that the inflammatory response and upregulation of genes related to stress responses peaked in intermediate- and late-stage AD, respectively, before declining by end stage. This latter response could be related to the subpopulations of exhausted astrocytes identified in the study; it could also be secondary to severe neurodegeneration and loss of provoking stimuli necessary to continue generating the responses. It is not well understood how the different facets of reactive astrocytosis are more helpful or harmful in Alzheimer’s disease, and this study provided important spatial-temporal data on the progression of astrocytic transcriptomic responses over the course of neuropathology. Further investigations are needed to understand the mechanisms underlying these astrocytic responses and their contributions to neurodegeneration. All early and intermediate-stage donors were negative for the APOE e4 allele, while 50 percent of the end-stage donors were positive for the APOE e4 allele, so it will be interesting to see if future work identifies APOE allele-specific effects as well.
Using clustering analyses, the authors identified populations of homeostatic protoplasmic astrocytes, reactive astrocytes, and intermediate populations as well as the novel “exhausted” populations whose number decline in end-stage disease. Interestingly, they observed a reactive astrocyte population that increased in proportion with worsening pathology, while also decreasing in proportion in entorhinal versus visual cortex, suggesting complex anatomic-pathologic relationships that may impact disease progression. Two different reactive astrocyte clusters showed enrichment for genes that were positively correlated with the degree of p-tau pathology, suggesting that these astrocyte phenotypes may be responding to or developing alongside the buildup of tau pathology. Further studies are needed to better understand the heterogeneity underlying reactive astrocytosis, and their relative contributions to AD pathogenesis. The authors isolated gray matter for their study, but even within the gray matter, there are populations of astrocytes (perivascular, interlaminar, and subpial etc.) that exhibit increased expression of GFAP, CD44, and other cytoskeletal proteins that are upregulated in reactive astrocytes, so it is also possible that these populations may be reflected in one of the identified non-homeostatic clusters.
Using pseudotime analyses, the authors mapped the progression of astrocyte transcriptional response from homeostatic, through intermediate, and ending in reactive phenotypes. The different astrocyte populations demonstrated a gradient of transcriptional change, rather than stepwise distinct differences, suggesting that astrocyte reactivity may not progress in distinct states but may exist as a spectrum of reactive change. Furthermore, pseudotime analysis interestingly suggested that the homeostatic, protoplasmic astrocytes may also progress into terminal, nonreactive astrocytic populations that increase in proportion in late-stage disease but decrease in end-stage disease, which they dubbed the exhausted phenotype. These astrocytes demonstrated increased expression of genes associated with glucose metabolism and stress responses but not senescent markers. They also identified a population of astrocytes that were enriched for growth factors that decreased in proportion as pathology worsened, suggesting a possible loss of homeostatic function in the diseased brain.
Interestingly, we also identified protoplasmic astrocytes in a spectrum of reactive change in brains with Alzheimer’s disease neuropathologic change, and loss of homeostatic function was an important feature of this reactivity spectrum (Dai et al., 2023). Additional studies are needed to assess if loss of homeostatic function is isolated to certain populations of non-homeostatic astrocytes, or if it is a general feature of astrocyte reactivity. Furthermore, it will be necessary to understand the different natures of these astrocyte populations and their respective contributions to protecting against or worsening neurodegeneration.
Overall, this single-nucleus RNA-Seq study of 628,943 astrocytes across five cortical regions and 32 donors of varying degrees of Alzheimer’s disease neuropathology demonstrated important facets of normal astrocyte heterogeneity as well as their heterogeneous responses in disease. This study opens the door to future investigations to understand different astrocyte populations’ contributions to Alzheimer’s disease pathogenesis.
References:
Dai DL, Li M, Lee EB. Human Alzheimer's disease reactive astrocytes exhibit a loss of homeostastic gene expression. Acta Neuropathol Commun. 2023 Aug 2;11(1):127. PubMed.
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