The APOE gene exacerbates pathology not only of the amyloid variety, but tau, as well. Previous research has tied ApoE’s effect on tau to microglial activation, but how does ApoE trigger microglia? In the June 10 Neuron online, scientists led by David Holtzman at Washington University, St. Louis, implicate the low-density lipoprotein receptor (LDLR), one of two ApoE receptors. In a tauopathy mouse model, overexpressing LDLR lowered ApoE and calmed microglia. This, in turn, slowed tau pathology and neurodegeneration. Curiously, excess LDLR was also a good thing for other glia. It dampened astrocyte reactivity and boosted the number of oligodendrocyte precursor cells (OPCs), bolstering myelination.

  • In tauopathy mice, increased LDLR dramatically lowered ApoE in the brain.
  • This shifted microglia from a DAM into protein clearance/neuron support mode ...
  • ... which, in turn, hit the brakes on tangles and neurodegeneration.

Holtzman noted that LDLR has been highly studied for cardiovascular diseases, but less so in the brain. The new findings imply that this receptor can protect neurons and might make a promising therapeutic target in AD and other tauopathies, he told Alzforum.

Others said the findings fit with existing literature on lipid dysregulation in neurodegenerative disease. “This is a great paper that highlights the important role of apolipoproteins and lipids in mediating the pathophysiology of AD,” noted Shane Liddelow at New York University.

LDLR: Guardian Angel? Synapses (red) are dense in wild-type mouse hippocampus (left), lost in P301S mouse hippocampus (center), and partially preserved by LDLR overexpression (right). [Courtesy of Shi et al., Neuron.]

The Holtzman group previously found that ApoE, especially the AD risk allele E4, drove P301S tauopathy mice to accumulate phosphorylated tau. This dialed up neuroinflammation, accelerating brain atrophy (Sep 2017 news). Later studies by this and other groups implicated microglia (Oct 2019 news; Mancuso et al., 2019).

To further dissect ApoE biology, Holtzman turned to its LDLR. He had previously found that amping up LDLR levels in amyloidosis mouse models not only squelched Aβ and amyloid plaques, but also slashed the amount of soluble ApoE by 90 percent (Dec 2009 news). This is because when ApoE binds LDLR, the complex is internalized and degraded (Heeren et al., 2006). 

What would boosting LDLR do in a tauopathy model? To answer this, first author Yang Shi crossed P301S mice with LDLR over-expressers. By 9 months of age, P301S mice normally have extensive loss of synapses and gray matter in the hippocampus. LDLR overexpression roughly halved synapse loss and atrophy (see image above). It also cut the amount of soluble p-tau in half. Knocking out APOE similarly protects these mice, strengthening the idea that LDLR acts via its effect on ApoE.

Most cell types in the brain express LDLR. To pin down its effects on microglia, the authors isolated them from P301S/LDLR mice. Microglia from 3-month-old mice made about half as much ApoE as did wild-types. In 20-month-old P301S/LDLR mice, the difference was more dramatic, with ApoE slashed by about 90 percent. Normally, ApoE expression increases with age, helping trigger the disease-associated microglial (DAM) activation state (Jun 2017 news; Sep 2017 news). LDLR overexpression seemed to prevent this age-associated microglial increase in ApoE. In keeping with this, microglia from 20-month-old mice expressed few DAM genes, suggesting this activation state was suppressed.

State Shifting. In wild-type mice (left), homeostatic microglia (red and blue) predominate, while in P301S mice (middle), the majority are DAM (green). When LDLR expression rises (right), a subtype (russet) that supports neuronal function replaces DAM. Lysosomal specialist microglia (yellow) also crop up in the presence of tau tangles. [Courtesy of Shi et al., Neuron.]

Single-nucleus RNA-Seq of hippocampal cells from 9-month-old P301S/LDLR mice and controls bolstered this idea. In P301S mice, nearly two-thirds of microglia had the DAM phenotype; in LDLR over-expressers, almost none did (see image above). Instead, P301S/LDLR hippocampi contained a microglial subtype not found in wild-type mice. This subtype, which made up about 10 percent of the microglia in their brains, ramped up lysosomal degradation while turning down cellular metabolism and protein synthesis. It seems to represent an alternate activation state specialized to clear proteins and lipids. The same subtype occurred in APOE knockouts.

Steve Barger at the University of Arkansas for Medical Sciences in Little Rock was intrigued by this lysosomal phenotype. His work has shown that ApoE binds to a DNA sequence that serves as a master regulator of lysosomal genes, suppressing their expression (Parcon et al., 2018). Thus, depleting ApoE may boost lysosomal gene expression and improve protein clearance. “A generalized effect of ApoE4 on autophagy may explain why it worsens conditions involving aggregation of Tau, α-synuclein, or TDP43, irrespective of Aꞵ,” Barger noted (full comment below).

This “lysosome specialist” microglia was not the only new subtype in P301S/LDLR mice. Almost half their microglia assumed a unique phenotype not seen in wild-type or P301S mice. These microglia suppressed DAM and antigen-presenting genes, while boosting ion channels, neurotransmitter receptors such as NMDAR, and genes that regulate synaptic plasticity. Possibly, these microglia support neuronal functions. Curiously, this subtype was absent in APOE knockouts.

“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,” Susanne Krasemann at University Medical Center in Hamburg, Germany, wrote to Alzforum (full comment below).

LDLR overexpression also soothed astrocytes. Almost two-thirds of astrocytes in P301S mice are reactive, but in P301S/LDLR mice, a fifth were, barely more than in wild-type. Reactive astrocytes turn up production of lipids and extracellular matrix proteins, as well as proinflammatory cytokines (Aug 2019 news). Other work by the Holtzman group has reported benefits from lowering astrocytic ApoE in tauopathy models (Apr 2021 news). 

Better Insulated. P301S mice (middle) lose their myelin coating (black rings) with age compared to healthy young controls (top). LDLR overexpression prevents this (bottom). [Courtesy of Shi et al., Neuron.]

Perhaps most surprisingly, LDLR overexpression nearly doubled the number of OPCs. And it showed: P301S/LDLR mice had more intact myelin than did P301S controls, hinting that the larger progenitor crew repaired damage to the brain’s insulation. Knocking out APOE had similar effects. Wild-type mice accrue myelin damage with age and, intriguingly, LDLR overexpression or APOE knockout both ameliorated this. The data suggest that targeting this system could improve the health of the aging brain, Holtzman said.

Would increasing LDLR be a good strategy in Alzheimer’s, where both plaques and tangles plague the brain? This is unclear, as some evidence suggests activated microglia, with high ApoE and TREM2, help contain amyloid plaques (Jul 2019 conference news; Mar 2020 news). However, this may depend on disease stage, noted Julia TCW at Icahn School of Medicine in New York (full comment below). TREM2 expression helps early in disease, when plaques are forming, but can become harmful later, when tau pathology predominates (Oct 2017 news; Apr 2020 news). Holtzman plans to probe the issue in mouse models that develop both pathologies.

So, how about targeting LDLR in people? Because of the receptor’s central importance in cardiovascular disease, numerous medications have been developed to boost it. For example, statins increase LDLR in the liver. The challenge is to get these medications into the brain, Holtzman said.

Joachim Herz at the University of Texas Southwestern Medical Center in Dallas agreed. “This paper raises the question of whether it is practical and clinically safe … to design lipophilic statins that preferentially partition into the brain,” he wrote to Alzforum (full comment below). Meanwhile, Holtzman is exploring small molecule or gene-based approaches to boost LDLR in the brain.—Madolyn Bowman Rogers

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.

    References:

    . Sex steroids, APOE genotype and the innate immune system. Neurobiol Aging. 2005 Mar;26(3):363-72. PubMed.

    . APOE-modulated Aβ-induced neuroinflammation in Alzheimer's disease: current landscape, novel data, and future perspective. J Neurochem. 2015 May;133(4):465-88. Epub 2015 Mar 18 PubMed.

    . Impaired Autophagy in APOE4 Astrocytes. J Alzheimers Dis. 2016;51(3):915-27. PubMed.

    . Rapamycin rescues vascular, metabolic and learning deficits in apolipoprotein E4 transgenic mice with pre-symptomatic Alzheimer's disease. J Cereb Blood Flow Metab. 2015 Dec 31; PubMed.

    . The Interplay Between Apolipoprotein E4 and the Autophagic-Endocytic-Lysosomal Axis. Mol Neurobiol. 2018 Jan 20; PubMed.

    . Proteomes analysis reveals the involvement of autophagy in AD-like neuropathology induced by noise exposure and ApoE4. Environ Res. 2019 Sep;176:108537. Epub 2019 Jun 15 PubMed.

    . APOE genotype regulates pathology and disease progression in synucleinopathy. Sci Transl Med. 2020 Feb 5;12(529) PubMed.

    . APOE4 exacerbates α-synuclein pathology and related toxicity independent of amyloid. Sci Transl Med. 2020 Feb 5;12(529) PubMed.

    . 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.

    . ApoE4 lowers age at onset in patients with frontotemporal dementia and tauopathy independent of amyloid-β copathology. Alzheimers Dement (Amst). 2019 Dec;11:277-280. Epub 2019 Mar 19 PubMed.

    . Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer's disease. Nat Neurosci. 2021 Jun;24(6):786-798. Epub 2021 May 6 PubMed.

    . 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.

    . The role of autophagy in neurodegenerative disease. Nat Med. 2013 Aug;19(8):983-97. PubMed.

    . Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls. Aging Cell. 2016 Oct;15(5):924-39. Epub 2016 Jul 23 PubMed.

    . Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs. Alzheimers Dement. 2018 Feb;14(2):230-242. Epub 2017 Sep 22 PubMed.

    . Direct Transcriptional Effects of Apolipoprotein E. J Neurosci. 2016 Jan 20;36(3):685-700. PubMed.

    . Electrochemical detection of specific interactions between apolipoprotein E isoforms and DNA sequences related to Alzheimer's disease. Bioelectrochemistry. 2020 Jun;133:107447. Epub 2019 Dec 23 PubMed.

    . HMGB1, IL-1α, IL-33 and S100 proteins: dual-function alarmins. Cell Mol Immunol. 2017 Jan;14(1):43-64. Epub 2016 Aug 29 PubMed.

    . Detection of apolipoprotein A-I, B, and E immunoreactivity in the nuclei of various rat tissue cells. Biochemistry (Mosc). 2000 Dec;65(12):1419-23. PubMed.

    . Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis. Biochem J. 2004 Mar 15;378(Pt 3):753-61. PubMed.

    . Modulation of apolipoprotein D expression and translocation under specific stress conditions. Biochim Biophys Acta. 2007 Jun;1773(6):954-69. Epub 2007 Mar 24 PubMed.

    . Analysis of apolipoprotein E nuclear localization using green fluorescent protein and biotinylation approaches. Biochem J. 2008 Feb 1;409(3):701-9. PubMed.

    . Binding and Repressive Activities of Apolipoprotein E3 and E4 Isoforms on the Human ApoD Promoter. Mol Neurobiol. 2013 May 30; PubMed.

    . Effects of bariatric surgery on HDL structure and functionality: results from a prospective trial. J Clin Lipidol. 2014 Jul-Aug;8(4):408-17. Epub 2014 May 21 PubMed.

    . Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats. J Lipid Res. 1987 Apr;28(4):351-60. PubMed.

    . SIRT1 regulates macrophage self-renewal. EMBO J. 2017 Aug 15;36(16):2353-2372. Epub 2017 Jul 12 PubMed.

  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:

    . The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    . 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.

    . 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.

    . 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.

  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:

    . 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.

    . 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.

    . 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.

    . Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1. Neuron. 2007 Oct 4;56(1):66-78. PubMed.

    . Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron. 1993 Oct;11(4):575-80. PubMed.

    . 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.

    . 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.

    . 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.

    . 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.

  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:

    . 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. PubMed.

    . Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer's disease. Elife. 2018 Oct 30;7 PubMed.

    . Endosomal Acidification by NHE6-depletion Corrects ApoE4-mediated Synaptic Impairments and Reduces Amyloid Plaque Load. bioRxiv. March 23, 2021

  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:

    . Cholesterol and matrisome pathways dysregulated in human APOE ε4 glia. bioRχiv, July 25, 2019

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References

Research Models Citations

  1. Tau P301S (Line PS19)

News Citations

  1. ApoE4 Makes All Things Tau Worse, From Beginning to End
  2. In Tauopathy, ApoE Destroys Neurons Via Microglia
  3. Mind Over Heart—LDL Receptors Crimp ApoE, Aβ Accumulation
  4. Hot DAM: Specific Microglia Engulf Plaques
  5. ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
  6. ApoE4 Glia Bungle Lipid Processing, Mess with the Matrisome
  7. Squelching ApoE in Astrocytes of Tau-Ravaged Mice Dampens Degeneration
  8. TREM2, Microglia Dampen Dangerous Liaisons Between Aβ and Tau
  9. Paper Alert: Mouse TREM2 Antibody Boosts Microglial Plaque Clean-Up
  10. Changing With the Times: Disease Stage Alters TREM2 Effect on Tau
  11. With TREM2, Timing Is Everything

Paper Citations

  1. . CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice. Brain. 2019 Oct 1;142(10):3243-3264. PubMed.
  2. . Apolipoprotein E recycling: implications for dyslipidemia and atherosclerosis. Arterioscler Thromb Vasc Biol. 2006 Mar;26(3):442-8. Epub 2005 Dec 22 PubMed.
  3. . Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs. Alzheimers Dement. 2018 Feb;14(2):230-242. Epub 2017 Sep 22 PubMed.

Further Reading

Primary Papers

  1. . 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.