Shi Y, Andhey PS, Ising C, Wang K, Snipes LL, Boyer K, Lawson S, Yamada K, Qin W, Manis M, Serrano JR, Benitez BA, Schmidt RE, Artyomov M, Ulrich JD, Holtzman DM. Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms. Neuron. 2021 Aug 4;109(15):2413-2426.e7. Epub 2021 Jun 21 PubMed.

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Comments

  1. Twenty-eight years after the first report of an impact of polymorphisms in the apolipoprotein E gene (APOE) on risk for Alzheimer’s disease, there is still no consensus on a mechanistic explanation for this genetic association. Interactions of the ApoE proteins with Aβ peptide dominate most hypotheses, but there are well-documented, variant-specific impacts of ApoE on more complex physiological systems, including inflammation (Colton et al., 2005; Tai et al., 2015) and autophagy (Simonovitch et al., 2016; Lin et al., 2015; Schmukler et al., 2018; Li et al., 2019). Moreover, ApoE4 expression exacerbates neuropathology independently of Aβ in parkinsonian conditions (Davis et al., 2020; Zhao et al., 2020), tauopathies (Litvinchuk et al., 2021), and frontotemporal dementia (Koriath et al., 2019). The ability of ApoE to influence neurological function and pathology independently of Aβ is given further importance by very recent correlations drawn between ApoE levels and dramatic differences in gene expression on a cell-by-cell basis (Zalocusky et al., 2021). The latter is consistent with ApoE having a cell-autonomous role in gene expression.

    Here, Shi et al. have contributed further to evidence for ApoE participating in neuropathology irrespective of Aꞵ. Working in the PS19 line of Tau-transgenic mice, they previously showed that pharmacological depletion of microglia or genetic ablation of ApoE ameliorated neurodegeneration; additional evidence indicated these two manipulations overlapped in their mechanisms (Shi et al., 2019). In their present report, Shi et al. returned to the tauopathy model, this time comparing ApoE knockouts with mice overexpressing an ApoE receptor that efficiently reduces ApoE levels. Microglia were again identified as a key mediator of these effects through diverse lines of evidence, including a prominent change in lysosomal gene expression and a compatible elevation of lysosomal activity in ApoE-depleted brains. The fact that such changes were observed in relatively pure isolates of microglia suggests that the phenotype manifested through a mechanism that was cell-type sufficient and perhaps—depending on the cell density—cell-autonomous.

    Lysosomal insufficiency is connected to failures of proteostasis, which contribute to several facets of aging in cells and whole organisms. While Aꞵ plaques and neurofibrillary tangles are the most widely known aggregates in AD, there is a generalized failure of proteostasis in AD, including the accumulation of scores of proteins that do not aggregate in the normal aging brain (Nixon, 2013; Ayyadevara et al., 2016). 

    ApoE appears to contribute to reduced lysosome function via a mechanism that is surprising when first confronted. We have confirmed evidence from others that ApoE is present in the nuclei of cells that express it. Furthermore, we find that ApoE4, in particular, exhibits specific and avid binding to a DNA sequence known as the “coordinated lysosomal expression and regulation” (CLEAR) enhancer (Parcon et al., 2018). This is a cis element used by transcription factor EB (TFEB) in its role as a master regulator of lysosomal genes involved in autophagy and other responses to starvation.

    We were prompted to explore this possibility by Theendakara et al. (2016), who combined ChIP-Seq and surface plasmon resonance to find sequences that bind ApoE with high affinity. Within a 250-bp sequence they isolated, we noted a CLEAR site and used two additional biochemical techniques—supplemented with in silico molecular modeling—to document specific, high-affinity binding by ApoE4. Finally, we found that ApoE4 competed with TFEB and reduced expression of TFEB/CLEAR-driven genes in cells and human brains expressing ApoE4, as compared to those expressing ApoE3 (Parcon et al., 2018). Using two additional methods, a third laboratory subsequently confirmed that ApoE4 has greater affinity than ApoE3 for the CLEAR sequence (Lima et al., 2020). 

    Such evidence indicates that ApoE falls into the group of proteins—including IL-1α, IL-33, HMGB1, and S100 proteins—that have both intracellular and extracellular roles (Bertheloot and Latz, 2017). This paradigm should not have come as a surprise; ApoE was localized to the nucleus in numerous studies (Panin et al., 2000; Quinn et al., 2004; Do Carmo et al., 2007; Kim et al., 2008), and it was documented to bind the promoter of the ApoD gene and alter its transcription nearly a decade ago (Levros et al., 2013). It only seems logical that a lipoprotein, the expression of which is positively correlated with body-mass index (Zvintzou et al., 2014) and negatively correlated with fasting (Wilcox et al., 1987), would suppress lysosomal gene expression, a phenomenon associated with starvation.

    A generalized effect of ApoE4 on autophagy may explain why it worsens conditions involving aggregation of Tau, α-synuclein, or TDP43, irrespective of Aꞵ (Davis et al., 2020; Zhao et al., 2020; Litvinchuk et al., 2021; Koriath et al., 2019). A key aspect of this intersecting, interspecies work will be determination of the DNA-binding properties of murine ApoE. Shi et al. (2019, 2021) report profound results from manipulating the endogenous ApoE in mice, and this murine protein is well known to promote amyloid deposition even more aggressively than does human ApoE4.

    Incidentally, in their earlier study Shi (2019) found that the CSF1R antagonist used to deplete microglia was less effective in ApoE-knockout mice. This, too, is consistent with the ApoE DNA-binding hypothesis; SirT1 was prominently suppressed by ApoE4 in the landmark study by Theenadakara et al., and SirT1 appears to be important for macrophage renewal (Imperatore et al., 2017). ApoE-knockout microglia may have an elevated SirT1 expression that substitutes, to some extent, for CSF1R activation in preventing apoptosis and elevating steady-state levels of myeloid cells.

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    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.

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    View all comments by Steve Barger

  2. This paper is a very interesting and important continuation in a line of publications from the Holtzman lab investigating the role of ApoE in tau-associated neurodegeneration. Because ApoE4 is the strongest known risk factor to develop late-onset AD (LOAD), several studies have focused on the direct influence of the three human ApoE isoforms on Aβ deposition. During the last years, it became more and more evident that ApoE in neurodegeneration is also acting at the cellular level.

    We and others discovered that ApoE has an important role in regulating microglia dysfunction and is significantly upregulated in mouse and human microglia in neurodegeneration (Krasemann et al., 2017). We suggested Trem2, another genetic risk factor to develop LOAD, as one potential team player in this process.

    The Holtzman group focused on the role of ApoE in tau-associated neurodegeneration independent of Aβ pathology. First, they showed that ApoE4 is dramatically exacerbating tau-mediated neurodegeneration and also influencing the tau deposition pattern in the brain (Shi et al., 2017). Then, they discovered that ApoE is driving microglia activation in their model and promoting neurodegeneration (Shi et al., 2019). However, microglia are not stand-alone, and the group moved on to show that astrocytes contribute to disease as a main source for ApoE4, since its astrocyte-specific depletion markedly reduces tau pathology (Wang et al., 2021). 

    In the current paper, the authors focused on one of the known ApoE receptors, LDLR. Overexpression of the latter in transgenic mice significantly reduces brain-soluble ApoE levels. Crossing these mice with P301S-Tau mice reduced tau pathology significantly, too. Surprisingly, the authors found that the TgLDLR is also expressed in microglia and directly impacts their expression signature, especially in disease with reduction of known disease markers such as ApoE or SPP1.

    The authors speculate that the effect of LDLR overexpression might be a direct consequence of the reduced ApoE expression that is evident in these mice. However, in contrast to ApoE-KO mice, the effect of LDLR overexpression is more obvious in later disease stages.

    The authors also identified subpopulations of microglia in health and disease. The disease-associated cluster (Cluster 2) that is upregulated in the tau mice is almost unchanged in both ApoE-KO and TgLDLR. However, while the ApoE-KO microglia seem to be more stuck in homeostasis, in the TgLDLR mice one microglia sub-cluster (Cluster 3) was increased specifically in disease.

    Maybe the TgLDLR mice could respond more flexibly upon disease. It would be very interesting to study this sub-population in more detail and evaluate if it is also abundant in other neurodegenerative diseases or affected by Aβ pathology.

    Interestingly, the KO of Trem2 is beneficial in the Tau model, whereas it is worsening disease in AD models. Thus, it would be very important to see if overexpressing LDLR act exclusively in the Tau model, or also in other neurodegenerative disorders, such as prion disease, which are not at all influenced by KO of either Trem2 or ApoE.

    One surprising finding in this paper is that overexpressing LDLR, but even more KO of ApoE, is affecting the number of OPCs in health and in disease. This seems counterintuitive, since ApoE, a main lipid transporter in the brain, is missing, but investigations have shown that regulating the cellular cholesterol content is also affecting signaling complexes at the cell membrane.

    Moreover, both ApoE-KO and TgLDLR also preserved myelin integrity in disease. Proper myelin maintenance may indeed positively influence disease progression. Since receptors like LDLR are more accessible for manipulation, this new work might open up novel opportunities to therapeutically target and reduce ApoE levels in the brain. This might be especially interesting for disease-driving ApoE4. I am looking forward to seeing the results of such experiments.

    References:

    Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O'Loughlin E, Xu Y, Fanek Z, Greco DJ, Smith ST, Tweet G, Humulock Z, Zrzavy T, Conde-Sanroman P, Gacias M, Weng Z, Chen H, Tjon E, Mazaheri F, Hartmann K, Madi A, Ulrich JD, Glatzel M, Worthmann A, Heeren J, Budnik B, Lemere C, Ikezu T, Heppner FL, Litvak V, Holtzman DM, Lassmann H, Weiner HL, Ochando J, Haass C, Butovsky O. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    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.

    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.

    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.

    View all comments by Susanne Krasemann

  3. Shi et al. have shown that reducing the extracellular ApoE level by increasing LDLR in microglia potentially increases the intracellular lysosomal clearance program and suppresses tau-mediated neurodegeneration in the P301S mouse model. Here I discuss several questions raised by the Alzforum community.

    How it fits with what we know about ApoE4 and tauopathy.
    Intracellular ApoE4 has a toxic effect in a tauopathy model (Shi et al., 2017), so that the selective removal of astrocyte-derived ApoE4 but not ApoE3 reduces tau-associated neurodegeneration (Wang et al., 2021). It is interesting that in the tau model used in this study, even ApoE3 has toxic effects when ApoE is elevated in the brain. This may be associated with ApoE overexpression in microglia, as well as excessive amounts of ApoE secreted by astrocytes and taken up by microglia, leading to microglial activation during aging and tau-mediated neurodegeneration. Therefore, a removal of extracellular ApoE by overexpression of LDLR rescues tau phenotypes. That also reduces the intracellular ApoE level and the metabolic program it triggers.

    It has to be further studied how ApoE4 microglia would respond to LDLR overexpression in this tau model. I think this relates to age-related neurodegeneration and early progression of the disease in mice carrying ApoE4 and tau mutation. Presumably, similar phenotypes could be observed at earlier time points in an ApoE4 tau mouse model. The Wang et al. study measured ApoE effects in tau at 5.5 months of age, while the Shi et al. study measured it at a late stage (9 months) of the P301S tau mice.

    Could this mechanism be targeted therapeutically?
    LDLR is a receptor so that is a modifiable target for therapeutics, but this needs to be further studied. The goal for targeted therapy would be to suppress microglial activation by upregulating LDLR, which removes extracellular ApoE through increased uptake and degradation in microglia. The strategy is well-aligned with immunosuppression therapy in neurodegenerative diseases. However, several major questions remain.

    1. What are the differences between mouse and human, in APOE4 carriers, and with disease stage?

    This study shows that ApoE deficiency increases lysosomal function in mouse microglia, but it could be different in humans, as shown by reduced lysosomal function in glia from APOE4 carriers with reduced ApoE (TCW et al., 2019). Shi et al. also shows that ApoE deficiency reduced mTOR activity at 9 months but not 3 months, stressing the importance of the right treatment window and disease stage.

    2. Two different ApoE receptors: LDLR vs LRP1?

    LDLR shows a protective effect in this study, whereas LRP1 has a harmful effect in a tauopathy model by increasing tau spread through tau uptake (Rebeck et al., 1993). LRP1 should be further studied and compared with LDLR’s role in the microglia activation associated with ApoE to fully understand the role of these different ApoE receptors.

    How might that work in Alzheimer's disease, where there are both amyloid and tau pathology?
    This is a nice follow-up on ApoE and LDLR’s role in microglia. Previous work from the Holtzman group (Kim et al., 2009) showed that overexpression of LDLR in mouse brain reduces ApoE, inhibits amyloid deposition, and increases extracellular Aβ clearance. This protective effect is replicated in this tauopathy model. The authors carried out an in-depth study, focusing on a microglial role in the presence or absence of ApoE and overexpression of LDLR.

    However, there are controversial aspects when it comes to microglia-targeted therapy in different neurodegenerative diseases.

    1) There are many efforts to increase TREM2 levels in microglia to make them more active, promote a cellular clearance program, and reduce amyloid plaques (Schlepckow et al., 2020; Lee et al., 2018). However in tau models, TREM2 deficiency protects against tau-associated neurodegeneration (Leyns et al., 2017). I believe it depends on when microglial activity is promoted or suppressed to treat diseases.

    2) We need to further investigate cell-type-specific effects of different ApoE receptors in microglia (e.g. LDLR vs LRP1). When we look at AD research, for example the APP knockout study that reduces ApoE and increases cholesterol, the findings align with those from the Holtzman group, but this study increases LRP1 and not LDLR (Liu et al., 2007). This could be associated with a neuron-specific effect in which overexpression of LRP1 in an AD mouse model reduces neuronal ApoE and Aβ42 (Zerbinatti et al, 2006). 

    References:

    Kim J, Castellano JM, Jiang H, Basak JM, Parsadanian M, Pham V, Mason SM, Paul SM, Holtzman DM. Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular A beta clearance. Neuron. 2009 Dec 10;64(5):632-44. PubMed.

    Leyns CE, Ulrich JD, Finn MB, Stewart FR, Koscal LJ, Remolina Serrano J, Robinson GO, Anderson E, Colonna M, Holtzman DM. TREM2 deficiency attenuates neuroinflammation and protects against neurodegeneration in a mouse model of tauopathy. Proc Natl Acad Sci U S A. 2017 Oct 24;114(43):11524-11529. Epub 2017 Oct 9 PubMed.

    Lee CY, Daggett A, Gu X, Jiang LL, Langfelder P, Li X, Wang N, Zhao Y, Park CS, Cooper Y, Ferando I, Mody I, Coppola G, Xu H, Yang XW. Elevated TREM2 Gene Dosage Reprograms Microglia Responsivity and Ameliorates Pathological Phenotypes in Alzheimer's Disease Models. Neuron. 2018 Mar 7;97(5):1032-1048.e5. PubMed.

    Liu Q, Zerbinatti CV, Zhang J, Hoe HS, Wang B, Cole SL, Herz J, Muglia L, Bu G. Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1. Neuron. 2007 Oct 4;56(1):66-78. PubMed.

    Rebeck GW, Reiter JS, Strickland DK, Hyman BT. Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron. 1993 Oct;11(4):575-80. PubMed.

    Schlepckow K, Monroe KM, Kleinberger G, Cantuti-Castelvetri L, Parhizkar S, Xia D, Willem M, Werner G, Pettkus N, Brunner B, Sülzen A, Nuscher B, Hampel H, Xiang X, Feederle R, Tahirovic S, Park JI, Prorok R, Mahon C, Liang CC, Shi J, Kim DJ, Sabelström H, Huang F, Di Paolo G, Simons M, Lewcock JW, Haass C. Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol Med. 2020 Apr 7;12(4):e11227. Epub 2020 Mar 10 PubMed.

    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.

    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.

    Zerbinatti CV, Wahrle SE, Kim H, Cam JA, Bales K, Paul SM, Holtzman DM, Bu G. Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice. J Biol Chem. 2006 Nov 24;281(47):36180-6. PubMed.

    View all comments by Julia TCW

  4. I agree with the overall tenor of this paper, i.e., that promoting lipoprotein metabolism, and especially ApoE turnover, by increasing LDLR expression is expected to be beneficial. It also nicely fits the model we proposed in our 2010 PNAS paper (Chen et al., 2010) and 2018 eLife paper (Xian et al., 2018), where we showed how ApoE4 in particular causes an intracellular traffic jam and results in the cellular accumulation of ApoE. LDLR overexpression would be expected to partially overcome this, however without correcting the root cause. Only NHE6 inhibition would (Pohlkamp et al., 2021). 

    This new paper now raises the question of whether it is practical and clinically safe, without excessive side effects, to design lipophilic statins that preferentially partition into the brain to upregulate the LDLR there. There are some concerns about potential neurological side effects; even so, it would be worth revisiting the potential usefulness of targeting statins directly to the brain.

    References:

    Chen Y, Durakoglugil MS, Xian X, Herz J. ApoE4 reduces glutamate receptor function and synaptic plasticity by selectively impairing ApoE receptor recycling. Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):12011-6. Epub 2010 Jun 14 PubMed.

    Xian X, Pohlkamp T, Durakoglugil MS, Wong CH, Beck JK, Lane-Donovan C, Plattner F, Herz J. Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer's disease. Elife. 2018 Oct 30;7 PubMed.

    Pohlkamp T, Xian X, Wong CH, Durakoglugil M, Saido T, Evers BM, White CL, Connor J, Hammer RE, Herz J. Endosomal Acidification by NHE6-depletion Corrects ApoE4-mediated Synaptic Impairments and Reduces Amyloid Plaque Load. bioRxiv. March 23, 2021

    View all comments by Joachim Herz

  5. This great paper highlights the important role of apolipoproteins and lipids in mediating the pathophysiology of AD. The ApoE4 story fits nicely with work from Julia TCW and Alison Goate, which shows E4 astrocytes produce and secrete less cholesterol. This makes microglia more reactive, in turn activating the astrocytes, and ultimately setting up a dangerous cascade (TCW et al., 2019). Their work was completed in human organoids, while the new work from Yang Shi and David Holtzman shows similar cross-talk between microglia and astrocytes, ultimately leading to decreased reactive astrocytes.

    One point the paper does highlight, which is a continuing problem for many researchers, is how poorly astrocytes are represented in single-cell RNA-Sequencing datasets—the capture here is very low. This is a common problem that the field is collectively trying to overcome at the moment.

    These clusters will need validation using in situ or spatial transcriptomics, or functional studies in the future. As an example: Cluster 2 is only represented by less than a dozen cells in the wild-type, LDLR, and ApoE knockout mice, and maybe two to four dozen cells in the P301S-containing lines. Given that scRNA-Seq inherently has low sequencing depth, this small sample number could lead to an interpretation of artifacts of this technical limitations as novel biological insights. As a result, additional animal numbers (or improved astrocyte capture methods), and validation of these cluster-specific differentially expressed genes will be required to validate the biological importance of these clusters moving forward.

    Additionally, some integration with human AD datasets will be important to determine if this is a characteristic AD-astrocyte effect, or if this is just something that occurs in the P301S mouse. The same is true for the microglia, as well.

    As always though, the Holtzman group has carefully and methodically integrated data across modalities—scRNA-Seq, immunohistochemistry, and  several genetic mouse models—making for a compelling and exciting new avenue for AD research moving forward.

    References:

    Tcw J, Qian L, Pipalia NH, Chao MJ, Liang SA, Shi Y, Jain BR, Bertelsen SE, Kapoor M, Marcora E, Sikora E, Andrews EJ, Martini AC, Karch CM, Head E, Holtzman DM, Zhang B, Wang M, Maxfield FR, Poon WW, Goate AM. Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Cell. 2022 Jun 23;185(13):2213-2233.e25. PubMed. BioRxiv.

    View all comments by Shane Liddelow

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