Wang C, Xiong M, Gratuze M, Bao X, Shi Y, Andhey PS, Manis M, Schroeder C, Yin Z, Madore C, Butovsky O, Artyomov M, Ulrich JD, Holtzman DM. Selective removal of astrocytic APOE4 strongly protects against tau-mediated neurodegeneration and decreases synaptic phagocytosis by microglia. Neuron. 2021 May 19;109(10):1657-1674.e7. Epub 2021 Apr 7 PubMed.

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  1. This is a really important and definitive study demonstrating that astrocyte-derived ApoE has a major impact on tau accumulation and associated pathologies. Even though many labs are studying microglial ApoE in Alzheimer’s disease, the major source of ApoE in the brain is still astrocytes, and they have convincingly demonstrated that here, as in Fig. 1 where conditional genetic deletion in astrocytes dropped ApoE by more than 90 percent. This is not to say that microglia don’t make a lot of ApoE on a “per cell” basis, but astrocytes make the bulk of it.

    What is really interesting is that they highlighted some of the activated microglial signatures that occur during tau pathology, and these were decreased in the astrocyte ApoE4-deleted mice. Also interesting, but still requiring further study, is that many of these effects were strongest or only present in female mice. A good explanation for this is not apparent from the current data.

    Overall, this adds to the previous landmark study that the Holtzman lab published on global effects of ApoE in tau models, and suggests that astrocytes are major mediators of this, though conditional deletion of ApoE in microglia is being pursued by several labs and it effects remain to be seen.

    View all comments by John Fryer

  2. This paper by Wang et al. describes multiple effects of inducibly removing astrocytic APOE isoforms (APOE3 or APOE4) in a tau P301S mouse model. This model system is powerful in that it allows the researchers to isolate the contribution of astrocytic APOE isoforms to pathology and molecular signatures of neurodegeneration.

    Wang et al. discover that inducible knockdown of APOE4, and not APOE3, results in a number of protective effects in a tauopathy mouse model. These many effects include molecular phenotypes such as less astrocytic activation, altered oligodendrocyte transcription, changes in excitatory neuron populations, and decreased phagocytosis of synapses by microglia.

    This study re-emphasizes and expands on previous work by the Holtzman lab and others to make very clear that astrocytic APOE4 represents a toxic gain of function. This gain of toxic function can impact cell physiology in both cell autonomous and cell non-autonomous ways. It will be interesting to see if the same toxic gain-of-function effect holds true for APOE4’s influence on amyloid-centric, or other disease-centric models.

    This paper presents a number of intriguing hypotheses about interactions between cell types that are impacted by genotype and presence of APOE. These are ripe for future mechanistic investigations. Given APOE’s role in lipid metabolism, and the involvement of lipids in nearly every cellular process, it is possible that lipid state is a key mediator of astrocytic APOE’s inter- and intracellular effects.

    One interesting theme throughout the data is that knockdown of APOE4 has a stronger effect on female animals than males. This echoes the literature in both mouse models and humans—in the context of tauopathies, APOE4’s detrimental effects are far greater on females than males (Nov 2019 news). It will be interesting to see if this correlation remains true for diseases other than tauopathies for which APOE4 increases risk.

    The strong protective effect of astrocytic APOE knockdown identified in this work supports the exploration of therapeutics aimed at reducing APOE levels. A challenge does arise in designing cell type-specific therapeutics. More investigation into the cell-type-specific effects of APOE knockdown will be essential in developing targeted and nuanced therapeutics or preventative approaches.

    View all comments by Priyanka Narayan

  3. This new study fits with previous work from the Holtzman lab showing that ApoE4 knock-in exacerbates the neuroinflammation and neurodegeneration that occurs in this tauopathy model, while ApoE4 reduction by antisense oligonucleotide treatment opposes these effects (Shi et al., 2017; Litvinchuk et al., 2021). 

    One open question was whether ApoE4 derived from specific cell types was of particular importance to driving neuroinflammation and degeneration. In particular, while astrocytes are a major source of ApoE, activated microglia as found in the tauopathy model exhibit a striking induction of ApoE expression, raising the hypothesis that microglia-derived ApoE could be playing a key role during pathology.

    The new findings that the removal of ApoE4 selectively in astrocytes is sufficient to reduce neurodegeneration and associated glial activation argue against a key role for microglia ApoE during pathology in this model. Therefore, the role of activated microglia in mediating tau-dependent pathology (Shi et al., 2019; Mancuso et al., 2019) is likely not so much via to production of ApoE, but rather as a mediator of neuronal damage in response to the harmful inflammatory state of the brain that is caused by tau pathology, and exacerbated by ApoE4.

    A remaining goal for the field is to gain a better mechanistic understanding of the actions of ApoE4 on the interactions between tau pathology, glial activation, and synapse and neuronal loss.

    References:

    Shi Y, Yamada K, Liddelow SA, Smith ST, Zhao L, Luo W, Tsai RM, Spina S, Grinberg LT, Rojas JC, Gallardo G, Wang K, Roh J, Robinson G, Finn MB, Jiang H, Sullivan PM, Baufeld C, Wood MW, Sutphen C, McCue L, Xiong C, Del-Aguila JL, Morris JC, Cruchaga C, Alzheimer’s Disease Neuroimaging Initiative, Fagan AM, Miller BL, Boxer AL, Seeley WW, Butovsky O, Barres BA, Paul SM, Holtzman DM. ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy. Nature. 2017 Sep 28;549(7673):523-527. Epub 2017 Sep 20 PubMed.

    Litvinchuk A, Huynh TV, Shi Y, Jackson RJ, Finn MB, Manis M, Francis CM, Tran AC, Sullivan PM, Ulrich JD, Hyman BT, Cole T, Holtzman DM. Apolipoprotein E4 Reduction with Antisense Oligonucleotides Decreases Neurodegeneration in a Tauopathy Model. Ann Neurol. 2021 May;89(5):952-966. Epub 2021 Feb 24 PubMed.

    Shi Y, Manis M, Long J, Wang K, Sullivan PM, Remolina Serrano J, Hoyle R, Holtzman DM. Microglia drive APOE-dependent neurodegeneration in a tauopathy mouse model. J Exp Med. 2019 Nov 4;216(11):2546-2561. Epub 2019 Oct 10 PubMed.

    Mancuso R, Fryatt G, Cleal M, Obst J, Pipi E, Monzón-Sandoval J, Ribe E, Winchester L, Webber C, Nevado A, Jacobs T, Austin N, Theunis C, Grauwen K, Daniela Ruiz E, Mudher A, Vicente-Rodriguez M, Parker CA, Simmons C, Cash D, Richardson J, NIMA Consortium, Jones DN, Lovestone S, Gómez-Nicola D, Perry VH. CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice. Brain. 2019 Oct 1;142(10):3243-3264. PubMed.

    View all comments by Jesse Hanson

  4. The new study in Neuron by Wang and colleagues, led by David Holtzman, Washington University, St. Louis, strengthens the evidence that APOE4 acts not only by gain of toxic function, but that APOE4 promotes multiple features of the classical Alzheimer’s disease (AD) phenotype. The title of the paper, “Selective removal of astrocytic APOE4 strongly protects against tau-mediated neurodegeneration and decreases synaptic phagocytosis by microglia,” communicates succinctly the important conclusions for which compelling mouse model data are presented.

    Wang et al. describe use of the MAPT P301S tauopathy mouse line as the backbone for generation of an inducible cre-lox based system for APOE isotype expression and knockout (Tau/Aldh1l1-CreERT2/apoE3flox/flox or Tau/Aldh1l1-CreERT2/apoE4flox/flox). At 5.5 months of age, after the onset of tau pathology, they administered tamoxifen or vehicle and compared mice at 9.5 months of age. Removing astrocytic APOE4 markedly reduced tau-mediated neurodegeneration and decreased phospho-tau pathology. Single-nucleus RNA sequencing analysis revealed striking gene expression changes in all cell types, with astrocytic APOE4 removal decreasing disease-associated gene signatures in neurons, oligodendrocytes, astrocytes, and microglia. An exciting additional observation was that removal of astrocytic APOE4 decreased tau-induced synaptic loss and microglial phagocytosis of synaptic elements, suggesting that astrocytic ApoE plays a key role in synaptic degeneration. There is ample documentation that synaptic integrity—not severity of proteinopathy—is the best correlate of cognition during either aging or AD.

    There are also already strong independent data indicating that APOE4 apparently promotes parenchymal and cerebrovascular amyloid accumulation directly, while promoting tauopathy both via acceleration of amyloidosis as well as by direct actions of ApoE4 on tauopathy. ApoE4 disrupts the blood-brain barrier in analogous dual mechanisms, i.e., apparently both by impairing pericyte viability and by promoting cerebrovascular amyloidosis that promotes degeneration of the medial layer of the vessel wall.

    Though the new Wang et al. story involves astrocyte-derived ApoE4, we and others have observed roles played by microglial APOE in the phenotypic switching from homeostatic to disease-associated microglia (DAM). We have recently observed that APOE-related induction of the DAM phenotype can be caused by molecular pathways via TYROBP even in the absence of TREM2 (Audrain et al., 2020). This is in addition to the role, well-documented by several groups, indicating that TREM2 induces DAM via ApoE.

    Koldamova and colleagues have reported that APOE4 acts more potently in microglial phenotype switching than does APOE3 (Fitz et al., 2020). Wang et al. confirm that tauopathy can drive the switch from homeostatic microglia toward DAM, but they report the unexpected observation that knocking down astrocytic APOE4 can attenuate the microglial DAM phenotype. RNA sequencing data have implicated APOE transcripts in microglia, but, as far as we are aware, Wang et al. present the first evidence that the isotype of the ApoE protein secreted from astrocytes can modulate phenotypic switching in microglia. It is tempting to speculate that this scenario is underpinned by paracrine and autocrine signaling pathways wherein microglial cell-surface molecules are triggered by ApoE (and/or other ligands), thereby stimulating intramicroglia transcription of DAM genes.

    The potential clinical impacts of this new data are several. First, depletion of astrocytic APOE4 acts to protect neurons and synapses based on structural data. Electrophysiological characterization of the Wang et al. mice depleted of astrocytic APOE4 would be an interesting follow-up.

    Moreover, this is now a third molecule that modulates synaptophagy by microglia. Hong et al. (2016) and Shi et al. (2017) identified the Aβ soluble oligomers and CR3 as two key players in this process, one that all evidence suggests is relevant for cognitive reserve and/or functional resilience, despite the otherwise clinically important severities of the proteinopathies. Given that depletion of the most neurotoxic soluble Aβ oligomer strains may be important for clinically meaningful benefit in response to passive immunotherapy with, e.g., aducanumab, BAN2401, and/or donanemab (reviewed in Gandy and Ehrlich, 2021, J Exp Med, in press), the discovery that astrocytic APOE4 mitigates synaptophagy may be especially relevant to AD, since half or more of late-onset sporadic AD is associated with the presence of at least one APOE4 allele. Identification of APOE4 carriers as those most likely to show clinically meaningful benefit following preclinical initiation of Aβ-reducing interventions would move us closer to the identification of those in whom the risk and expense of Aβ-reducing passive immunotherapy would be potentially justifiable.

    One key piece that we are still missing is an understanding of how to intervene when genetic risk for AD is linked to those subjects in whom progression of cognitive decline is apparently underpinned by a genetic-based remission of amyloidosis over time, rather than the more typical progressive clinical decline that goes hand in hand with accumulating amyloidosis. The classical clinicopathological correlations among progressive amyloidosis, progressive tauopathy, and cognitive decline are now included as an essential troika of entry criteria for some Aβ-reducing passive immunotherapies. The outliers whose cognitive decline does not conform to this classical triad are ineligible for Aβ-reducing trials. However, Wang et al.’s Neuron data raise the possibility that the APOE4 carriers among those with noncanonical CR3-linked cognitive decline associated with “vanishing amyloidosis” might respond to depletion of astrocytic APOE4 (Thambisetty et al., 2012; Gandy et al., 2013). 

    When contemplating how APOE might be therapeutically reduced, Holtzman has championed the use of passive immunotherapy to target ApoE rather than Aβ (Xiong et al., 2021). The new data on the benefit of APOE4 depletion should add momentum to anti-ApoE passive immunotherapies. Yet another way of targeting APOE is already in human clinical trials: Crystal and colleagues at Weill Cornell Medical College have reported rodent and primate data suggesting that infusion and seeding of AAV-APOE2 into the subarachnoid space of APOE4 carriers might neutralize the untoward consequences of APOE4 for neurons, synapses, and pericytes (Zhao et al., 2016; Rosenberg et al., 2018). No formal reports from this Phase 1 trial have yet emerged, and recruitment of symptomatic APOE4 homozygotes continues. At the very least, linking common and readily identifiable genetic risks (such as APOE genotype) to clinically relevant molecular pathogenesis and patient selection might well move us closer to the eventual goal of prevention or arrest of progressive cognitive decline associated with AD.

    References:

    Audrain M, Haure-Mirande JV, Mleczko J, Wang M, Griffin JK, St George-Hyslop PH, Fraser P, Zhang B, Gandy S, Ehrlich ME. Reactive or transgenic increase in microglial TYROBP reveals a TREM2-independent TYROBP-APOE link in wild-type and Alzheimer's-related mice. Alzheimers Dement. 2021 Feb;17(2):149-163. Epub 2020 Dec 12 PubMed.

    Fitz NF, Wolfe CM, Playso BE, Biedrzycki RJ, Lu Y, Nam KN, Lefterov I, Koldamova R. Trem2 deficiency differentially affects phenotype and transcriptome of human APOE3 and APOE4 mice. Mol Neurodegener. 2020 Jul 23;15(1):41. PubMed.

    Gandy S, Haroutunian V, Dekosky ST, Sano M, Schadt EE. CR1 and the "vanishing amyloid" hypothesis of Alzheimer's disease. Biol Psychiatry. 2013 Mar 1;73(5):393-5. PubMed.

    Hong S, Beja-Glasser VF, Nfonoyim BM, Frouin A, Li S, Ramakrishnan S, Merry KM, Shi Q, Rosenthal A, Barres BA, Lemere CA, Selkoe DJ, Stevens B. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science. 2016 May 6;352(6286):712-6. Epub 2016 Mar 31 PubMed.

    Rosenberg JB, Kaplitt MG, De BP, Chen A, Flagiello T, Salami C, Pey E, Zhao L, Ricart Arbona RJ, Monette S, Dyke JP, Ballon DJ, Kaminsky SM, Sondhi D, Petsko GA, Paul SM, Crystal RG. AAVrh.10-Mediated APOE2 Central Nervous System Gene Therapy for APOE4-Associated Alzheimer's Disease. Hum Gene Ther Clin Dev. 2018 Mar;29(1):24-47. Epub 2018 Mar 13 PubMed.

    Shi Q, Chowdhury S, Ma R, Le KX, Hong S, Caldarone BJ, Stevens B, Lemere CA. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci Transl Med. 2017 May 31;9(392) PubMed.

    Thambisetty M, An Y, Nalls M, Sojkova J, Swaminathan S, Zhou Y, Singleton AB, Wong DF, Ferrucci L, Saykin AJ, Resnick SM, . Effect of Complement CR1 on Brain Amyloid Burden During Aging and Its Modification byAPOEGenotype. Biol Psychiatry. 2012 Sep 27; PubMed.

    Xiong M, Jiang H, Serrano JR, Gonzales ER, Wang C, Gratuze M, Hoyle R, Bien-Ly N, Silverman AP, Sullivan PM, Watts RJ, Ulrich JD, Zipfel GJ, Holtzman DM. APOE immunotherapy reduces cerebral amyloid angiopathy and amyloid plaques while improving cerebrovascular function. Sci Transl Med. 2021 Feb 17;13(581) PubMed.

    Zhao L, Gottesdiener AJ, Parmar M, Li M, Kaminsky SM, Chiuchiolo MJ, Sondhi D, Sullivan PM, Holtzman DM, Crystal RG, Paul SM. Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models. Neurobiol Aging. 2016 Aug;44:159-72. Epub 2016 Apr 30 PubMed.

    View all comments by Michelle Ehrlich

  5. Two comments:

    1) This study, however impressive, has a limitation: There is no Aβ pathology. Aβ uptake by astrocytes damages their endolysosomal system (Sanchez-Mico et al., 2021). Thus, the question is: How would astrocytes damaged by Aβ respond to Tau pathology in the context of different APOE alleles? It is essential to consider the interplay between Tau and Aβ in order to establish the contribution of astrocytes to Alzheimer's disease.

    2) As recommended in a recent consensus article about reactive astrocytes (Escartin et al., 2021), the terminology "homeostatic" and "senescent" or even "reactive" should be avoided for any transcriptional cluster until functions are clarified. Plausibly, the so-called "reactive" and "senescent" clusters encompass a mixed set of adaptive and maladaptive responses that respectively maintain and disrupt astrocytic homeostasis.

    References:

    Sanchez-Mico MV, Jimenez S, Gomez-Arboledas A, Muñoz-Castro C, Romero-Molina C, Navarro V, Sanchez-Mejias E, Nuñez-Diaz C, Sanchez-Varo R, Galea E, Davila JC, Vizuete M, Gutierrez A, Vitorica J. Amyloid-β impairs the phagocytosis of dystrophic synapses by astrocytes in Alzheimer's disease. Glia. 2021 Apr;69(4):997-1011. Epub 2020 Dec 7 PubMed.

    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.

    View all comments by Elena Galea

  6. This is an exciting set of data from the Holtzman group that adds another piece to the puzzle of APOE’s role as a risk factor for Alzheimer’s Disease (AD). In this paper, Wang et al. use the P301S mouse model of AD, expressing either ApoE3 or ApoE4, and genetically remove the APOE isoforms using a Cre/flox system with the pan-astrocytic, astrocyte-specific Aldh1l1 promoter as the Cre driver.

    They find that removing APOE4 specifically in astrocytes reduces brain atrophy, slows accumulation of phosphorylated tau, and improves behavioral deficits. Using single-nucleus RNA-sequencing, they find that APOE4 expression not only has drastic consequences for astrocytes, but also affects gene expression in non-astrocytic cells. Indeed, they find a shift in the cluster composition of neurons, including a surprising APOE4-dependent increase in Rorb-positive neurons, and among microglia they find an increase in major histocompatibility complex (MHC)-expressing cells, which are in turn repressed by removing APOE from astrocytes, which themselves show a shift in their relative subtype abundance. Indeed, ApoE4-carrying mice show an increase in numbers of astrocytes expressing genes classically defined as “reactive,” including Gfap and Vim, which are repressed by the genetic removal of APOE4 from astrocytes. Given the abundance of studies now highlighting the single cell heterogeneity of astrocyte (and microglia) responses to both acute insults and chronic neurodegenerative diseases, the authors also investigated these discrete astrocyte responses across different APOEe isoform-expressing mouse models.

    The use of single cell/nucleus RNA-sequencing is a powerful approach to study acute and chronic diseases in a cell-type-wide and unbiased manner. Particularly the bioinformatic integration of samples across different conditions, as applied here, allows scientists to pinpoint the disease progression of once-healthy cells. This paper and others have shown how astrocytes can be quite heterogenous and how an insult can add an additional layer of complexity. While the numbers of cells collected is quite low, they add to the growing repertoire of astrocyte single-cell datasets that can be used for ongoing meta-analysis and integration to further understand the subtle and heterogeneous transcriptomic changes that may depend on time, sex, or disease-associated mutations.

    Indeed, adding additional time points, pseudotime, and pseudobulk analyses, as well as increasing the number of sequenced astrocytes will help us understand the reactive transition astrocytes can undergo. Similarly, adding additional modalities, such as spatial transcriptomics, and performing multimodal data integration are powerful tools to unravel how both the subtype and anatomical location of an astrocyte determines its response to an inflammatory insult.

    All in all, this is another beautiful example of how a cell-type-specific intervention can have non-cell autonomous consequences, highlighting the need to consider the multicell type composition of the brain when studying disease. The use and integration of multimodal sequencing tools, such as single cell/nucleus RNA-sequencing and spatial transcriptomics, will help us uncover what and where things go awry in brain cells in disease, and aid us in identifying novel therapeutic strategies.

    View all comments by Philip Hasel

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This paper appears in the following:

News

  1. Squelching ApoE in Astrocytes of Tau-Ravaged Mice Dampens Degeneration
  2. Single Synapse Mass Spec Snags CD47 as Alzheimer’s Resilience Factor
  3. Secreted by Neurons, ApoE4 Makes Tangles and Degeneration Worse

Mutations

  1. APOE C130R (ApoE4)