Species: Mouse
Genes: APOE
Modification: APOE: Knock-In
Disease Relevance: Alzheimer's Disease, Multiple Conditions
Strain Name: B6.129P2-Apoe^{tm2(APOE*3)Mae} N8

Summary

The APOE3 Targeted Replacement model is one line in a set of three APOE knock-in lines created by Nobuyo Maeda, Patrick Sullivan, and colleagues. APOE2 and APOE4 Targeted Replacement mice are also available.

Complementary Models

Human induced pluripotent cell lines (iPSCs) are complementary models for the study of ApoE biology at the cellular level. This survey focuses on reports of the generation and use of isogenic lines—lines in which gene editing was used to change the parental APOE allele to a different APOE allele—to compare the effects of the major human ApoE isoforms on essentially identical genetic backgrounds. Isogenic human iPSC lines expressing the three major APOE alleles, as well as corresponding APOE-null lines, have been generated.

Starting with an existing iPSC line derived from an 80-year-old male Alzheimer’s patient homozygous for the E4 allele (Peitz et al., 2018), Schmid, Cabrera-Socorro and colleagues used CRISPR/Cas9 gene editing to generate APOE-knockout, E3/E4, and E3/E3 lines, and then edited the latter line to generate the isogenic E2/E2 line (Schmid et al., 2021). These lines are available through the European Bank for induced pluripotent Stem Cells (EBiSC) [UKBi011-A, parental line APOE4/4; UKBi011-A-1, APOE knockout; UKBi011-A-2, APOE2/2; UKBi011-A-3, APOE3/3; UKBi011-A-4, APOE3/4]. These cells have been used to study the responses of human microglia-like cells to amyloid pathology (Mancuso et al., 2024). IPSCs were differentiated into microglial precursors and transplanted into the brains of mice genetically engineered to lack an adaptive immune system and that have the AD-linked Swedish, Iberian, and Arctic mutations knocked into the mouse App gene. Transcriptomic analysis of microglia isolated from these brains 6 to 7 months after transplantation showed that APOE4/4 microglia resembled APOE knockout microglia, suggesting that the E4 allele has a loss-of-function effect with regards to the response of microglia to amyloid pathology. The effect of microglial APOE genotype on amyloid pathology was not reported. Microglia-like cells derived from the APOE3/3, APOE4/4 and APOE knockout lines have also been used to test the activity of anti-sense oligonucleotides directed against APOE (Vandermeulen et al., 2024).

This same group had previously attempted to create a set of isogenic lines homozygous for the three major APOE alleles starting with iPSCs derived from skin fibroblasts from a healthy 18-year-old with the E3/E4 APOE genotype (Schmid et al., 2019). However, it was found that the resulting E2, E3, and E4 lines were actually hemizygous for the APOE alleles, while the knockout line was as expected (Schmid et al., 2020). These lines are also available through the EBiSC (BIONi010-C, parental line APOE3/4; BIONi010-C-2, hemizygous for the APOE3 allele; BIONi010-C-4, hemizygous for the APOE4 allele; BIONi010-C-6, hemizygous for the APOE2 allele; BIONi010-C-3, APOE knockout). IPSCs from these lines were differentiated into astrocytes and subjected to proteomic and functional analyses (de Leeuw et al., 2022). Proteomic analysis suggested a reduction in components of cholesterol and lipid metabolic and biosynthetic pathways in E4 carriers and an enhancement in E2 carriers compared with E3. Biochemical studies confirmed decreased biosynthesis and efflux of cholesterol from APOE4 astrocytes. Conversely, proteomics showed genotype-dependent effects on immunoregulatory pathways that followed the pattern APOE4 > APOE3 > APOE2. Biochemical studies confirmed increased inflammatory signaling in E4 carriers, and decreased signaling in E2 carriers, compared with E3: When stimulated with IL-1β or TNF, astrocytes of all genotypes increased their release of cytokines, with the responses of APOE4 > APOE3 > APOE2. Uptake of glutamate and of aggregated Aβ42 were lowest in APOE4 astrocytes and highest in APOE2 and APOE knockout cells. The iPSCs have also been differentiated into neurons. In a study that measured energy metabolism in vitro, APOE4 was found to increase mitochondrial and glycolytic ATP production compared with the other genotypes, while cells expressing APOE2 or APOE3 resembled the APOE knock-out neurons (Budny et al., 2024). These results suggest that the E4 allele confers a gain-of-function with regards to neuronal energy metabolism. In another study, the E4 allele led to endolysosomal dysfunction in the iPSC-derived neurons (Somogyi et al., 2023).

The company Alstem distributes two sets of isogenic human iPSCs—E2/E2, E3/E3, E4/E4, and APOE-knockout lines. One of these sets also carries a doxycycline-inducible Ngn2 gene to facilitate differentiation into neurons. Neurons and astrocytes induced from these lines were used to study APOE genotype effects on the expression of PPP2CB—a variant in this gene having been shown to associate with AD in E2 carriers (Jun et al., 2022).

Other groups created isogenic pairs of iPSCs to compare the effects of the E2 or E4 alleles with E3. Beginning with iPSC lines carrying AD-linked mutations in APP (APP V717I or an APP duplication) or PSEN1 (PSEN1 A246E) on an E3/3 background, Brafman and colleagues generated isogenic partner lines homozygous for E2 (Brookhouser et al., 2021). Converting the E3 allele to E2 lowered the levels of Aβ42 produced in neuron-astrocyte co-cultures derived from these iPSCs, but the E3 to E2 switch decreased the Aβ42/Aβ40 ratio only in cultures carrying PSEN1 A246E. The ratio of tau phosphorylated at threonine-231 to total tau was also decreased in cells carrying the PSEN1 mutation when the APOE genotype was switched from E3 to E2.

Li-Huei Tsai and her team made two sets of isogenic pairs of iPSCs to compare the effects of the E3 and E4 alleles (Lin et al., 2018; Jun 2018 news). Beginning with cells homozygous for E3 from an individual cognitively intact at age 75, they created a matching E4/E4 line, then differentiated the iPSCs into neurons, astrocytes, and microglia. APOE-dependent differences in gene expression were seen in all three cell types: Comparing cells expressing E4 with those expressing E3, prominent differences included downregulation of genes involved in cell proliferation and upregulation of genes involved in differentiation in neurons, and upregulation of genes related to lipid metabolism and immune responses in astrocytes and microglia, respectively. Functional differences between the genotypes were also observed: E4 neurons had higher frequencies of excitatory post-synaptic currents (ePSCs), elevated levels of pre- and post-synaptic markers, generated more Aβ42 and had more early endosomes than E3 neurons. Astrocytes generated less ApoE4 than ApoE3 and took up less Aβ42 added to the culture medium. ApoE4-expressing microglia were similarly less efficient than ApoE3-expressing microglia at taking up Aβ42. In a complementary series of experiments, this group created an isogenic E3/E3 iPSC line from an E4/E4 line generated from a person with sporadic AD. Here, too, astrocytes made less ApoE4 than ApoE3, E4-expressing glia took up less Aβ than E3-expressing cells, and E4 neurons showed elevated excitatory synaptic activity and made more synapses than E3 cells. However, the two genotypes did not differ in the amount of Aβ generated by neurons or numbers of early endosomes in neurons.

Yadong Huang’s group first generated iPSCs from a 64-year-old woman with AD, homozygous for the E4 allele, then converted these cells to E3/E3 (Wang et al., 2018; Jun 2018 news). When differentiated into neurons, cells carrying the E3 allele released less Aβ and contained less hyperphosphorylated tau, compared with neurons with the E4 allele, and more E3 GABAergic neurons survived.

In another study, genotype-dependent inflammatory profiles were compared in astrocytes derived from sets of isogenic APOE knockout and E4/E4 lines generated from E3/E3 lines; elevated levels of cytokines were consistently found in astrocytes carrying the E4 allele (Arnaud et al., 2022).

An E4/E4 iPSC line was also generated from an E3/E3 parental line derived from an apparently healthy 36-year-old Caucasian woman (Watanabe et al., 2023,). When differentiated into astrocytes, cells carrying the E4 allele had larger soma sizes than E3 carriers. Dendritic spine densities were lower in mouse primary neurons co-cultured with E4/E4 IPSC-derived astrocytes, compared with E3/E3, congruent with findings of lower spine densities in the brains of APOE4 Targeted Replacement mice compared with APOE3 mice. APOE genotype did not affect dendritic complexity in co-cultures of mouse neurons with human iPSC-derived astrocytes, in contrast to findings in Targeted Replacement mice, where dendritic complexity was lower in E4 carriers—an effect that may be age- or region-dependent.

Cerebral organoids have been generated from isogenic iPSC lines homozygous for either E3 or E4 (Lin et al., 2018; Zhao et al., 2020). In both studies, lower levels of Aβ and less hyperphosphorylated tau were found in the organoids when cells carried the E3 allele.

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