hiPSC-based models to decipher the contribution of human astrocytes to Alzheimer's disease and potential therapeutics

Perspective for Molecular Neurodegeneration Next-generation hiPSC-based models to decipher the contribution of human astrocytes to Alzheimer’s disease and potential targeting therapeutics Julia TCW1 and Amaia M Arranz2,3 1 Department of Pharmacology & Experimental Therapeutics, Boston University Chobanian & Avedisian School of Medicine, Boston, USA. 2 Achucarro Basque Center for Neuroscience, Leioa, Spain 3 Ikerbasque Basque Foundation for Science, Bilbao, Spain Correspondence: Julia TCW: julia[email protected] Amaia M Arranz: [email protected] Astrocytes constitute a large part of the brain cell mass and play essential functions in the central nervous system (CNS). They maintain brain homeostasis, provide trophic and metabolic support to neurons, regulate synapse formation, neurotransmission and calcium homeostasis, and control immune response and blood flow. In Alzheimer’s disease (AD), astrocytes undergo profound molecular, morphological and functional alterations that arise at early stages and exacerbate as disease evolves, indicating that astrocytes transition from homeostatic to dysfunctional disease-associated states and pointing to these cells as critical contributors to AD progression. Genome-wide association studies (GWAS) support this idea as many putative genes increasing the risk for developing late-onset AD (LOAD) are primarily expressed in glial cells, among which the major AD risk genes APOE, CLU and FERMT2 are predominantly expressed in astrocytes. While our current knowledge of astrocyte contribution to AD is mainly coming from studies in mouse models, critical species-specific differences highlight the importance of studying astrocyte (dys)function in human-based systems. The ability to generate human induced pluripotent stem cells (hiPSCs) from patients and differentiate them into astrocytes is providing exciting opportunities to explore their functions in AD. We summarize below recent studies in 2D and 3D cultures, co-culture systems and organoids giving essential clues on the impact of human astrocytes on AD, and propose potential astrocyte-targeting therapeutics. Earlier studies in 2D models revealed that healthy hiPSC-derived astrocytes play important roles in APP processing, secrete β-amyloid (Aβ) (1), and therefore have the potential to contribute to Aβ accumulation in AD brains. Astrocytes derived from AD patients carrying the PSEN1ΔE9 mutation display AD hallmarks including increased Aβ and reactive oxygen species (ROS) production, altered inflammatory responses and dysregulated calcium homeostasis compared to isogenic control astrocytes (2). When co-cultured with PSEN1 mutant astrocytes in a 3D system, isogenic control neurons also display alterations in calcium signaling (2), which highlights a major impact of human astrocytes on human neuron physiology and functionality. Interestingly, activation of NF-E2-related factor 2 (NRF2), key regulator of antioxidant defense pathways, reduces Aβ secretion and modulates cytokine release and oxidative stress in PSEN1 mutant astrocytes (3), suggesting that targeting NRF2 in AD astrocytes could be a potential therapeutic strategy. Astrocytes are the major cell type expressing and producing the protein apolipoprotein E (apoE) in the brain, although microglia, particularly in an activated state, also produce apoE. ApoE facilitates the transport of lipids and cholesterol to neurons, microglia and oligodendrocytes, and binds to Aβ plaques. The APOE gene has three polymorphic alleles (ε2, ε3 and ε4), being APOE4 the one conferring the greatest risk for LOAD with an earlier age of disease onset in a gene dose-dependent manner. Recent studies have started to elucidate how APOE affects human astrocyte functions in the context of AD. hiPSC-derived astrocytes carrying the APOE4 variant produce and secrete less apoE protein compared to APOE3 astrocytes, are less efficient at clearing the extracellular Aβ, and show impaired lipid/cholesterol metabolism (4,5). Astrocytes are one of the major cell types producing cholesterol, and increased cholesterol biosynthesis has also been found in organoids from individuals carrying Tau mutations (6), suggesting that perturbed cholesterol metabolism is a common pathway and early event in the etiology of AD. Besides, APOE4 astrocytes show increased inflammatory responses compared to APOE3 astrocytes (5). A recent study describes an exacerbated proinflammatory state on APOE4 astrocytes associated with Transgelin 3 (TAGLN3) downregulation and NF-kB activation (7). Interestingly, this state can be pharmacologically reverted by TAGLN3 supplementation, highlighting TAGLN3 as a target to modulate inflammation in APOE4 astrocytes. Importantly, imbalances at cellautonomous level also affect astrocyte-neuron communication. When co-cultured with neurons, APOE4 astrocytes provide less support for neuronal survival and synaptogenesis (8). Moreover, in organoids composed by either APOE3 or APOE4 neurons and astrocytes there is increased APOE4-dependent synapse loss, neurodegeneration and Tau pathology (4,9). Astrocyte-microglia crosstalk also has an impact on AD-associated inflammatory processes. In tri-culture systems with healthy hiPSC-derived astrocytes, neurons and microglia, the complement protein C3, which is elevated in brains of AD patients and involved in neurodegeneration, increases under inflammatory conditions due to astrocyte-microglia reciprocal signaling that induces them to overproduce C3. Astrocytic production of C3 induced by microglia, as well as microglial production of C3 re-induced by astrocytes are further enhanced in AD tri-cultures derived from hiPSCs harboring the APPSWE mutation (10). Another 3D tri-culture AD model that develops Aβ pathology and Tau accumulation shows that astrocyte-secreted interleukin-3 (IL-3) reprograms microglia at molecular, morphological and functional levels. Reprogrammed microglia acquire an acute immune response, increased motility and enhanced capacity to cluster and clear Aβ and Tau aggregates restricting AD pathology (11). Astrocytes secrete other inflammatory factors and molecules potentially involved in their crosstalk with microglia, neurons and other brain cells. hiPSC-based in vitro systems are best suited for modeling the inflammatory axis between these cells, dissecting the role of astrocytes in complex AD cellular environments and their impact on neurodegeneration in AD. Moreover, these models constitute a powerful approach for identifying key compounds and main molecular players enabling the development of astrocyte targeting therapeutic strategies. In summary, human astrocytes get dysfunctional at early stages of AD and aggravate the pathology affecting major processes including Aβ production/clearance, cholesterol/lipid metabolism, oxidative stress and calcium dynamics. Moreover, in AD, human astrocytes lose their homeostatic functions providing less support to synapses and neurons, while closely interacting with microglia, being key mediators of neuroinflammation. Novel therapeutic strategies targeting astrocytes should aim at enhancing their neurotrophic/neuroprotective properties, modulating their immune responses, and reversing their lipid/cholesterol metabolic alterations, oxidative stress and calcium dysfunction. Next-generation hiPSC-based approaches should include as well in vivo analyses of human astrocytes transplanted in chimeric AD mice (12). Chimeras offer a unique opportunity to analyze human astrocyte dysfunction in the live AD brain and how it contributes to AD progression. Combination of in vitro and in vivo models with CRISPR-Cas9 editing to allow targeting specific AD risk genes or variants in human astrocytes, transcriptomics and high-resolution imaging will be critical for further exploring the role of human astrocytes in AD, and it will open new avenues enabling the development of astrocyte-specific targeting therapeutics to prevent, slow down and even cure AD. List of abbreviations: CNS: Central nervous system; AD: Alzheimer’ s disease; GWAS: Genome-wide association studies; LOAD: late-onset AD; hiPSC: human induced pluripotent stem cells; Aβ: β-amyloid; ROS: reactive oxygen species; apoE: apolipoprotein E; TAGLN3: Transgelin 3; IL-3: interleukin-3. Declarations: Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding JTCW received funding from NIH NIA K01AG062683, 1R56AG078733, Toffler Scholar Award and BrightFocus Foundation (A2022049S). AMA receives funding from the MCIN/AEI/10.13039/501100011033/FEDER (RTI2018-101850-A-I00, RYC2020029494-I, and PID2021-125443OB-100 grants), the Alzheimer’s Association (AARG21-850389 grant), the Basque Government (PIBA-2020-1-0030 grant), and IKERBASQUE; and is the recipient of an IBRO Early Career Award. 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Human astrocytes get dysfunctional at early stages of AD and exacerbate the pathology affecting major AD processes. Combination of hiPSC-based in vitro and in vivo models with gene editing, transcriptomics and high-resolution imaging will allow in depth analyses of human astrocytes in AD and enable the development of astrocyte targeting therapeutics.