• TAU is mainly distributed in the central nervous system, most of it exists in the axons of neurons, and a small amount exists in oligodendrocytes. TAU is involved in neurodegenerative diseases, and most prominently in the pathogenesis of Alzheimer disease (AD).
• TFR1 plays a crucial role in regulating the distribution of iron in the brain, involving multiple biological processes such as cell metabolism, development, myelination, and neurotransmission. Due to the expression of TfR1 in brain endothelial cells, it serves as an ideal target for drug delivery. Various drug delivery strategies targeting TfR1 have been developed to effectively penetrate the blood-brain barrier and promote therapeutic interventions for brain diseases.
• Gene editing strategy: The full coding sequences of human TAU gene with P301S mutation that is driven by mouse Prnp promoter are inserted into mouse Hipp11 (H11) locus in B-hTFR1/hTAU*P301S mice. The exons 4-19 of mouse Tfr1 gene that encode the extracellular region were replaced by human TFR1 exons 4-19 in B-hTFR1/hTAU*P301S mice.
• Protein expression analysis: TAU and p-tau were detected in cortex and hippocampus of both wild-type C57BL/6 mice and homozygous B-hTFR1/hTAU*P301S mice. Additionally, homozygous B-hTFR1/hTAU*P301S mice showed a significant increase in TAU and p-tau levels compared to wild-type C57BL/6 mice. Mouse TFR1 was detectable in wild-type mice. Human TFR1 was exclusively detectable in homozygous hTFR1/hTAU*P301S mice but not in wild-type mice.

Gene targeting strategy for B-hTFR1/hTAU*P301S mice. The full coding sequences of human TAU gene with P301S mutation that is driven by mouse Prnp promoter are inserted into mouse Hipp11 (H11) locus in B-hTFR1/hTAU*P301S mice. The exons 4-19 of mouse Tfr1 gene that encode the extracellular region were replaced by human TFR1 exons 4-19 in B-hTFR1/hTAU*P301S mice.

Strain specific TFR1 expression analysis in homozygous B-hTFR1/hTAU*P301S by flow cytometry. Bone marrow and blood cells were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTFR1/hTAU*P301S mice (H/H), and analyzed by flow cytometry with anti-mouse TFR1 antibody (Biolegend, 113808) and anti-human TFR1 antibody (Biolegend, 334108). Mouse TFR1 was detectable in wild-type mice. Human TFR1 was exclusively detectable in homozygous hTFR1/hTAU*P301S mice but not in wild-type mice.

Western blot analysis of phosphorylation of TAU protein expression in homozygous B-hTFR1/hTAU*P301S mice. Cortex and hippocampus lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTFR1/hTAU*P301S mice (H/H, 8-week-old mice), and then analyzed by western blot (Phospho-Tau (Ser202, Thr205) antibody: Invitrogen, MN1020; anti-TAU antibody: CST, #46687). 40 μg total proteins were loaded for western blotting analysis. TAU and p-tau were detected in cortex and hippocampus of both wild-type and homozygous mice.