Despite years of research on ApoE4, questions remain about exactly how this gene variant raises risk for Alzheimer’s disease. To boot, much of the data to date has come from mouse models and may not reflect human brain biology. Two new papers seek to rectify this by describing what the ApoE4 allele does to human neurons and glia derived from induced pluripotent stem cells. The two studies take different approaches but arrive at similar findings. They confirm some previous research on ApoE4, while also holding a few surprises.
- In iPSC-derived human neurons, ApoE4 raises Aβ production and tau phosphorylation.
- In human astrocytes and microglia, ApoE4 slows uptake and clearance of Aβ.
- ApoE4 exerts its toxic effects through multiple cell types.
In the May Nature Medicine, researchers led by Yadong Huang at the Gladstone Institute of Neurological Disease, San Francisco, reported that ApoE4 in human neurons boosted production of Aβ40 and Aβ42. It does not do that in mouse neurons. Independent of its effect on Aβ, ApoE4 triggered phosphorylation and mislocalization of tau. In mixed neuronal cultures, GABAergic neurons seemed particularly sensitive to this tau toxicity, perishing at high rates. The other study, led by Li-Huei Tsai and colleagues at Massachusetts Institute of Technology, Cambridge, took a broader approach, comparing gene-expression profiles of ApoE4 and ApoE3 neurons, astrocytes, and microglia. Published online May 31 in Neuron, these scientists likewise report a rise in Aβ42 production in ApoE4 neurons. In astrocytes and microglia, the ApoE4 allele slowed Aβ uptake and clearance. The data suggest that ApoE4 promotes AD pathology through distinct effects on different cell types, Tsai told Alzforum.
Both groups stressed that iPSC-derived cultures can answer mechanistic questions about AD and are suitable for screening potential therapies. “Human neurons may work better than mouse neurons to determine compound safety and efficacy,” Huang noted.
Others said the cell-type-specific data make an important contribution. “[The findings] suggest that despite low levels of ApoE expression by neurons, ApoE4 can still result in a neuronal phenotype … What is not clear is which phenotypes are relevant in the in vivo setting, where there are many other cell types present including glia, which produce much more ApoE than neurons,” David Holtzman at Washington University in St. Louis wrote to Alzforum (full comment below).
Earlier mouse and human imaging studies have suggested that the ApoE4 allele slows Aβ clearance, allowing amyloid to accumulate faster (Apr 2009 news; Jul 2010 conference news). It remained unclear, however, if these effects were due to loss of ApoE’s normal functions, or to a gain of toxic function. Studies conflict on this point, with some finding better brain function in mice from lowering ApoE, others from raising it (Dec 2011 news; Feb 2012 news; Feb 2012 conference news).
Huang and colleagues wanted to know what ApoE does in human cells. First author Chengzhong Wang generated iPSC lines from three people who were homozygous for ApoE4, and three who were homozygous for ApoE3. The scientists differentiated each line into mixed excitatory and inhibitory neuronal cultures and looked for differences between E3 and E4 cells.
The first surprise was that ApoE4 cultures secreted twice as much Aβ40 and Aβ42 as did E3 cultures. A direct effect on Aβ production does not happen in mouse models, Huang said. Although some papers report higher Aβ levels in ApoE4 knock-ins, this may be due to slower clearance of the peptide, he added (Pankiewicz et al., 2014; Boehm-Cagan and Michaelson, 2014; Luz et al., 2016). An in vivo microdialysis study that directly measured Aβ dynamics in knock-in mice confirmed that the E4 allele only affected clearance, not production (Jun 2011 news).
Huang and colleagues detected more soluble amyloid precursor protein as well, suggesting that enhanced processing of APP was responsible for the uptick. They found no change in APP expression, contradicting earlier findings in human neurons (Jan 2017 news).
Other findings from this study reinforced previous research. E4 neurons retained more ApoE inside their cell bodies than did E3s, and broke it down more, in agreement with reports that the E4 isoform fragments more readily (Apr 2012 news). E4 neurons developed tau pathology even when Aβ processing was inhibited, confirming a direct effect of ApoE4 on tau as first reported in mouse models (Apr 2017 conference news; Sep 2017 news). Huang noted that tau pathology was more intense in these human neurons than in mice. Antibodies against paired helical filaments, tau’s most pathological form, rarely give much signal in mice, but lit up the human neurons strongly, he told Alzforum.
In the mixed neuronal cultures, GABAergic neurons appeared particularly vulnerable to the effects of tau. They developed more signs of tau toxicity, for example hyperphosphorylation and axonal degeneration, and subsequently perished at higher rates than did glutamatergic neurons in the same cultures. This, too, matches in vivo findings from mice, where degeneration of inhibitory interneurons has been blamed for learning and memory deficits (Feb 2012 conference news). It also agrees with long-standing findings on inhibitory neuron loss in human AD brain samples. However, the iPSC culture system now gives researchers tools to dissect the mechanisms behind this phenomenon, Huang believes. He is examining whether inhibitory neurons have less tau phosphatase than excitatory ones, allowing more p-tau to accumulate.
The iPSC system also answered the long-standing debate over loss-of-function versus gain-of-function mechanisms for ApoE4. When the researchers generated iPSCs from a person lacking ApoE, the resulting neuronal cultures resembled ApoE3 lines with regard to Aβ, tau, and GABAergic neuron viability. This benign knockout phenotype jibes with some human data (Aug 2014 news). The findings argue against a loss-of-function mechanism for ApoE4, hinting that E4 may be toxic directly, Huang said. Indeed, transfecting the knockout cells with ApoE4 triggered Aβ production, tau pathology, and GABAergic degeneration, whereas changing ApoE4 cells to ApoE3 using CRISPR restored their health. “Based on these data, we should lower ApoE4 to treat AD,” Huang said.
Likewise, the authors rescued ApoE4 neurons by treating them with a molecule that acts as a chaperone, refolding E4 into the E3 configuration. Huang noted that this “structural corrector” molecule is unsuitable for therapeutic development (Brodbeck et al., 2011; Chen et al., 2012). He is working on derivatives that are more potent and better enter the brain, and hopes to take them to the clinic.
Lars Ittner at the University of New South Wales in Sydney said he was particularly intrigued by the fact that the structural corrector lowered tau pathology in ApoE4 cells, whereas reducing the cells’ Aβ levels did not reduce tau pathology. “We certainly need to see if a small molecule corrector of ApoE4 will improve deficits in relevant in vivo models of Alzheimer’s disease,” Ittner wrote to Alzforum.
For their part, Tsai and colleagues focused on gene-expression changes in ApoE4 cells. Previous studies found that ApoE4 protein can act both directly on DNA and indirectly via the transcription factor AP-1 to stimulate transcription (Theendakara et al., 2016; Huang et al., 2017). Joint first authors Yuan-Ta Lin and Jinsoo Seo generated iPSCs from a healthy human donor homozygous for ApoE3, then altered both alleles to E4 in some lines. They differentiated these isogenic stem cells into pure cultures of glutamatergic neurons, astrocytes, or microglia, and analyzed gene-expression profiles for each cell type.
In the ApoE4 excitatory neuron cultures, 445 genes differed from their levels in E3 cultures. ApoE4 turned down genes involved in cell proliferation, while dialing up genes involved in cell differentiation. How did this change the cells? E4 cultures secreted 20 percent more Aβ42 and accumulated more early endosomes, suggesting more APP was being processed. In addition, ApoE4 neurons formed 25 percent more synaptic connections than E3s did, with a corresponding bump in excitatory electrical discharges. “ApoE4 neurons seem to be hyperactive. That surprised us,” Tsai said.
Astrocyte and microglial cultures showed many more gene changes than did neurons. In astrocytes, ApoE4 upregulated 418 genes and downregulated 909. Overall, genes involved in lipid metabolism were up, while genes involved in lipid transport and tissue development were down. ApoE4 astrocytes produced much more cholesterol than E3s, while making about half as much ApoE. They cleared only half as much Aβ42 from culture media over two days as did E3s, and degraded it more slowly.
In microglia, ApoE4 boosted expression of 329 genes, while dampening 1,131 more. Upregulated genes mostly pertained to inflammation, while downregulated genes were involved in cell movement and development. Overall, the transcriptome data reflects a shift from a phagocytic phenotype to a more inflammatory one, Tsai said. Supporting this, ApoE4 microglia had fewer and shorter processes than E3s, and took up less than half the Aβ as E3s. These findings dovetail with previous data that suggested ApoE promoted microglial inflammation and curbed phagocytosis (Feb 2015 conference news; Feb 2015 conference news). “[The gene changes] are consistent with several recent results suggesting effects of ApoE on microglia under certain conditions can affect the innate immune response and drive neurodegeneration,” Holtzman noted.
To learn how these different ApoE4 cell types might influence each other, the authors placed iPSCs into a blob of Matrigel in a rotating chamber, which provided a steady flow of nutrients and oxygen. Under these conditions, iPSCs differentiate and self-assemble into mini brain-like structures known as cerebral organoids (Aug 2013 news; Raja et al., 2016). Initially, the organoids contained only neurons and did not accumulate Aβ. Six months later, astrocytes appeared, and at that point ApoE4 organoids accumulated twice as much Aβ and p-tau as did E3 organoids. This demonstrates that the ApoE4 allele alone can cause AD pathology, Tsai noted.
In ongoing work, she is analyzing the gene-expression data to find signaling pathways that are altered in ApoE4 cells and might suggest potential therapeutic targets. Edwin Weeber at the University of South Florida, Tampa, agreed this holds potential, but added, “Time will tell if the utility of human cell lines to better understand fundamental AD pathogenic processes will translate into desperately needed successful human clinical trials.”—Madolyn Bowman Rogers
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- Keystone: Does ApoE Fragmentation Drive Pathology?
- ApoE and Tau: Unholy Alliance Spawns Neurodegeneration
- ApoE4 Makes All Things Tau Worse, From Beginning to End
- San Francisco: GABA Neurons Blamed for Memory Loss in ApoE Mice
- ApoE: One Man’s Brain Can Do Without It
- Microglia in Disease: Innocent Bystanders, or Agents of Destruction?
- Cytokine Takes Aβ Off the Menu for Microglia
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- Pankiewicz JE, Guridi M, Kim J, Asuni AA, Sanchez S, Sullivan PM, Holtzman DM, Sadowski MJ. Blocking the apoE/Aβ interaction ameliorates Aβ-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice. Acta Neuropathol Commun. 2014 Jun 28;2:75. PubMed.
- Boehm-Cagan A, Michaelson DM. Reversal of apoE4-driven brain pathology and behavioral deficits by bexarotene. J Neurosci. 2014 May 21;34(21):7293-301. PubMed.
- Luz I, Liraz O, Michaelson DM. An Anti-apoE4 Specific Monoclonal Antibody Counteracts the Pathological Effects of apoE4 In Vivo. Curr Alzheimer Res. 2016 Jun 2;13(8):918-29. PubMed.
- Brodbeck J, McGuire J, Liu Z, Meyer-Franke A, Balestra ME, Jeong DE, Pleiss M, McComas C, Hess F, Witter D, Peterson S, Childers M, Goulet M, Liverton N, Hargreaves R, Freedman S, Weisgraber KH, Mahley RW, Huang Y. Structure-dependent impairment of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors. J Biol Chem. 2011 May 13;286(19):17217-26. PubMed.
- Chen HK, Liu Z, Meyer-Franke A, Brodbeck J, Miranda RD, McGuire JG, Pleiss MA, Ji ZS, Balestra ME, Walker DW, Xu Q, Jeong DE, Budamagunta MS, Voss JC, Freedman SB, Weisgraber KH, Huang Y, Mahley RW. Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons. J Biol Chem. 2012 Feb 17;287(8):5253-66. PubMed.
- Theendakara V, Peters-Libeu CA, Spilman P, Poksay KS, Bredesen DE, Rao RV. Direct Transcriptional Effects of Apolipoprotein E. J Neurosci. 2016 Jan 20;36(3):685-700. PubMed.
- Huang YA, Zhou B, Wernig M, Südhof TC. ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion. Cell. 2017 Jan 26;168(3):427-441.e21. Epub 2017 Jan 19 PubMed.
- Raja WK, Mungenast AE, Lin YT, Ko T, Abdurrob F, Seo J, Tsai LH. Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes. PLoS One. 2016;11(9):e0161969. Epub 2016 Sep 13 PubMed.
- ApoE4 Promotes Amyloidosis, But Only in Plaque-Free Mice
- APOE4 Subtly Alters Brain Network Activity With Age
- Has ApoE’s Time Come as a Therapeutic Target?
- Human ApoE Antibody Nips Mouse Amyloid in the Bud
- Mind Over Heart—LDL Receptors Crimp ApoE, Aβ Accumulation
- ApoE4 Promotes Amyloidosis, But Only in Plaque-Free Mice
- Wang C, Najm R, Xu Q, Jeong DE, Walker D, Balestra ME, Yoon SY, Yuan H, Li G, Miller ZA, Miller BL, Malloy MJ, Huang Y. Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector. Nat Med. 2018 May;24(5):647-657. Epub 2018 Apr 9 PubMed.
- Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31 PubMed.