APOE: A major genetic risk factor for late-onset Alzheimer’s disease

• Gene Information: Human APOE is a 34 kDa glycoprotein, and there was three APOE polymorphic alleles in human, ε2, ε3 and ε4, in which ε4 increases the risk, whereas ε2 reduces the risk compared with ε3. The APOE isoforms encoded by the three corresponding gene alleles differ from one another only at position 112 and 158.
• Protein Expression: In the CNS, APOE is primarily expressed in astrocytes and microglia, and in the periphery, APOE is mainly expressed in liver and macrophages.
• Signaling Pathway: APOE is a major genetic risk factor for AD, and APOE4 is associated with an increased risk and lower age of onset through multiple pathways, such as the inhibition of amyloid-β (Aβ) clearance, promotion of Aβ aggregation, influencing on tau pathology, impairing microglial responsiveness and lipid transport.
• Therapeutic Inhibition: APOE represents a promising therapeutic target for Alzheimer’s disease. By inhibition the expression of APOE4, correcting APOE4 conformation or AAV-mediated APOE2 gene therapies/gene transition were being evaluated.

TFR1: Target for Drug Delivery Across the Blood-Brain Barrier

• Primary role: TFR1 binds transferrin (TF) to mediate cellular iron uptake via endocytosis.
• Binding: At physiological pH, TFR1 binds TF-Fe²⁺ to form the TF–TFR1 complex.
• Endocytosis: The complex undergoes clathrin-mediated endocytosis and enters endosomes.
• Iron release: Endosomal acidification induces conformational changes, releasing iron ions.
• Recycling: TFR1 is recycled to the cell surface through the Golgi, completing the iron transport cycle.

APOE3

• Part of exon 2 to exon 4 of the mouse Apoe gene, which encode the entire protein (from ATG to stop codon) and the 3’UTR, are replaced with the corresponding human sequences.
• The endogenous mouse promoter and 5’UTR are retained, allowing human APOE expression to be driven by the native mouse Apoe promoter, while endogenous mouse Apoe transcription and translation are abolished.

TFR1

• The exons 4-19 of mouse Tfr1 gene that encode extracellular domain are replaced by human counterparts in B-hTFR1 mice. The genomic region of mouse Tfr1 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric TFR1 expression is driven by endogenous mouse Tfr1 promoter, while mouse Tfr1 gene transcription and translation will be disrupted.

• Mouse Tfr1 mRNA was detectable in wild-type mice but not in homozygous B-hAPOE3/hTFR1 mice.
• Human TFR1 mRNA was only detectable in homozygous B-hAPOE3/hTFR1 mice, but not in wild-type mice.

Strain specific analysis of TFR1 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hAPOE3/hTFR1 mice by RT-PCR. Brain RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hAPOE3/hTFR1 mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TFR1 primers. Mouse Tfr1 mRNA was detectable in wild-type mice but not in homozygous B-hAPOE3/hTFR1 mice. Human TFR1 mRNA was only detectable in homozygous B-hAPOE3/hTFR1 mice, but not in wild-type mice.

• Mouse Apoe mRNA was detectable in wild-type mice but not in homozygous B-hAPOE3/hTFR1 mice.
• Human APOE mRNA was only detectable in homozygous B-hAPOE3/hTFR1 mice, and the results were confirmed via Sanger sequencing.

Strain specific analysis of APOE3 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hAPOE3/hTFR1 mice by RT-PCR. Brain RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hAPOE3/hTFR1 mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human APOE primers. Mouse Apoe mRNA was detectable in wild-type mice but not in homozygous B-hAPOE3/hTFR1 mice. Human APOE mRNA was only detectable in homozygous B-hAPOE3/hTFR1 mice, but not in wild-type mice. And the results were confirmed via Sanger sequencing.

• TFR1 was detected in spleen, lung, kidney, brain and stomach from wild-type mice and homozygous B-hAPOE3/hTFR1 mice, as the antibody used cross-reacted with both human and mouse TFR1 protein.

Western blot analysis of TFR1 protein expression in homozygous B-hAPOE3/hTFR1 mice. Various tissue lysates were collected from C57BL/6JNifdc mice (+/+) (female, 7-week-old) and homozygous B-hAPOE3/hTFR1 mice (H/H) (female, 7-week-old) , and then analyzed by western blot with anti-TFR1 antibody (Abcam, ab214039). 40 μg total proteins were loaded for western blotting analysis. TFR1 was detected in spleen, lung, kidney, brain and stomach from wild-type mice and homozygous B-hAPOE3/hTFR1 mice, as the antibody used cross-reacted with both human and mouse TFR1 protein.

• TFR1 was detected in skeletal muscle, colon, uterus, eyeballs, heart and liver from wild-type mice and homozygous B-hAPOE3/hTFR1 mice, as the antibody used cross-reacted with both human and mouse TFR1 protein.

Western blot analysis of TFR1 protein expression in homozygous B-hAPOE3/hTFR1 mice. Various tissue lysates were collected from C57BL/6JNifdc mice (+/+) (female, 7-week-old) and homozygous B-hAPOE3/hTFR1 mice (H/H) (female, 7-week-old) , and then analyzed by western blot with anti-TFR1 antibody (Abcam, ab214039). 40 μg total proteins were loaded for western blotting analysis. TFR1 was detected in skeletal muscle, colon, uterus, eyeballs, heart and liver from wild-type mice and homozygous B-hAPOE3/hTFR1 mice, as the antibody used cross-reacted with both human and mouse TFR1 protein.

• Human APOE was detected in serum from homozygous humanized APOE lines, but not from wild-type mice.

Strain specific analysis of human APOE expression in homozygous B-hAPOE3 mice, B-hAPOE3/hTFR1 mice, B-hAPOE4 mice and B-hAPOE4/hTFR1 mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+), homozygous B-hAPOE3 mice (female, n=3, 6-week-old), B-hAPOE3/hTFR1 mice (female, n=3, 6-week-old), B-hAPOE4 mice (female, n=3, 8-week-old) and B-hAPOE4/hTFR1 mice (female, n=3, 8-week-old), and serum were then analyzed by ELISA with human-specific APOE ELISA kit (Abcam, ab233623). This ELISA kit specifically recognized human APOE protein, as no signal was detected in serum from wild-type mice. Human APOE was only detectable in serum from homozygous humanized APOE lines, with comparable expression level across these groups.

• Human APOE was detected in cortex and hippocampus from homozygous humanized APOE lines.

Strain specific analysis of human APOE expression in homozygous B-hAPOE3 mice, B-hAPOE3/hTFR1 mice, B-hAPOE4 mice and B-hAPOE4/hTFR1 mice by ELISA. Cortex and hippocampus were collected from homozygous B-hAPOE3 mice (female, n=3, 6-week-old), B-hAPOE3/hTFR1 mice (female, n=3, 6-week-old), B-hAPOE4 mice (female, n=3, 8-week-old) and B-hAPOE4/hTFR1 mice (female, n=3, 8-week-old), and samples were then analyzed by ELISA with human-specific APOE ELISA kit (Abcam, ab233623). Human APOE was only detectable in cortex and hippocampus from homozygous humanized APOE lines, with comparable expression level across these groups.