Transferrin receptor-mediated transport at the blood-brain barrier is elevated during early development and maintained across aging and in an Alzheimer’s mouse model
Restricted accessResearch articleFirst published online January, 2026
Transferrin receptor-mediated transport at the blood-brain barrier is elevated during early development and maintained across aging and in an Alzheimer’s mouse model
Transferrin receptor (TfR)-targeting of biologics has emerged as a promising strategy to improve drug delivery across the blood-brain barrier (BBB). However, most preclinical studies evaluating TfR-enabled drugs have been conducted in young adult animals. It remains unclear whether age and aging-related diseases impact TfR protein levels and/or BBB transport capacity. Here, we utilized a previously described TfR-targeting antibody transport vehicle (ATVTfR) to investigate how healthy aging and disease progression in the 5xFAD mouse model of Alzheimer’s disease (AD) impact TfR protein and TfR-mediated brain delivery. ATVTfR transport capacity remained stable across 3- to 24-month-old healthy mice and 5xFAD progression did not impair ATVTfR brain transport up to 10.5 months, despite significant amyloid burden. Interestingly, neonates exhibited significantly elevated levels of vascular TfR protein and ATVTfR brain exposure compared to adult mice. Furthermore, vascular TfR in AD patient brains was similar to age-matched controls, suggesting conserved TfR transport is also likely in human AD. Overall, our data demonstrates broad functional utility for TfR-based brain delivery in both healthy aging and in an AD mouse model. Additionally, elevated TfR-mediated brain delivery during early mouse development highlights the potential of added efficacy in utilizing such platforms in disease treatment of infants and children.
AbbottNJPatabendigeAADolmanDE, et al.
Structure and function of the blood-brain barrier. Neurobiol Dis201037: 13–25.
2.
PatelMMGoyalBRBhadadaSV, et al.
Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs2009;
23: 35–58.
3.
HershDSWadajkarASRobertsN, et al.
Evolving drug delivery strategies to overcome the blood brain barrier. Curr Pharm Des2016;
22: 1177–1193.
4.
TerstappenGCMeyerAHBellRD, et al.
Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov2021;
20: 362–383.
5.
WuDChenQChenX, et al.
The blood-brain barrier: structure, regulation, and drug delivery. Signal Transduct Target Ther2023;
8: 217.
6.
RennieKYogiA, andCostainWJ.Mechanisms and methods for evaluating drug delivery via transcytosis to the brain. In: TaleviA (ed.) CNS drug development and delivery: concepts and applications.
Cham, Switzerland:
Springer Nature, 2024, pp. 31–68.
7.
YuYJZhangYKenrickM, et al.
Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med2011;
3: 84ra44.
8.
Bien-LyNYuYJBumbacaD, et al.
Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J Exp Med2014;
211: 233–244.
9.
NiewoehnerJBohrmannBCollinL, et al.
Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron2014;
81: 49–60.
10.
ZucheroYJChenXBien-LyN, et al.
Discovery of novel blood-brain barrier targets to enhance brain uptake of therapeutic antibodies. Neuron2016;
89: 70–82.
HaqqaniASThomGBurrellM, et al.
Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity. J Neurochem2018;
146: 735–752.
13.
StockiPSzaryJRasmussenCLM, et al.
Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1. FASEB J2021;
35: e21172.
14.
KariolisMSWellsRCGetzJA, et al.
Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys. Sci Transl Med2020;
12: eaay1359.
15.
YuYJAtwalJKZhangY, et al.
Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci Transl Med2014;
6: 261ra154.
16.
ZhaoLLiZVongJSL, et al.
Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain. Nat Commun2020;
11: 4413.
17.
YangACStevensMYChenMB, et al.
Physiological blood-brain transport is impaired with age by a shift in transcytosis. Nature2020;
583: 425–430.
18.
Todorov-VolgyiKGonzalez-GallegoJMullerSA, et al.
Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging. Nat Aging2024;
4: 595–612.
19.
AndjelkovicAVSituMCitalan-MadridAF, et al.
Blood-brain barrier dysfunction in normal aging and neurodegeneration: mechanisms, impact, and treatments. Stroke2023;
54: 661–672.
20.
LauSFCaoHFuAKY, et al.
Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer’s disease. Proc Natl Acad Sci U S A2020;
117: 25800–25809.
21.
YangACVestRTKernF, et al.
A human brain vascular atlas reveals diverse mediators of Alzheimer’s risk. Nature2022;
603: 885–892.
22.
SunNAkayLAMurdockMH, et al.
Single-nucleus multiregion transcriptomic analysis of brain vasculature in Alzheimer’s disease. Nat Neurosci2023;
26: 970–982.
23.
BryantALiZJayakumarR, et al.
Endothelial cells are heterogeneous in different brain regions and are dramatically altered in Alzheimer’s disease. J Neurosci2023;
43: 4541–4557.
24.
ItoSYagiROgataS, et al.
Proteomic alterations in the brain and blood-brain barrier during brain Abeta accumulation in an APP knock-in mouse model of Alzheimer’s disease. Fluids Barriers CNS2023;
20: 66.
25.
SaundersNRDreifussJJDziegielewskaKM, et al.
The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history. Front Neurosci2014;
8: 404.
26.
MontagneAZhaoZ, andZlokovicBV.Alzheimer’s disease: a matter of blood-brain barrier dysfunction?J Exp Med2017;
214: 3151–3169.
27.
MuenzerJHoCLauH, et al.
Community consensus for heparan sulfate as a biomarker to support accelerated approval in neuronopathic mucopolysaccharidoses. Mol Genet Metab2024;
142: 108535.
28.
GrimmHPSchumacherVSchaferM, et al.
Delivery of the brainshuttle amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans. MAbs2023;
15: 2261509.
29.
Bien-LyNBoswellCAJeetS, et al.
Lack of widespread BBB disruption in Alzheimer’s disease models: focus on therapeutic antibodies. Neuron2015;
88: 289–297.
30.
BourassaPAlataWTremblayC, et al.
Transferrin receptor-mediated uptake at the blood-brain barrier is not impaired by Alzheimer’s disease neuropathology. Mol Pharm2019;
16: 583–594.
31.
FaresjoRSehlinD, andSyvanenS.Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody. Fluids Barriers CNS2023;
20: 34.
32.
TaylorEMMorganEH.Developmental changes in transferrin and iron uptake by the brain in the rat. Brain Res Dev Brain Res1990;
55: 35–42.
33.
MoosTMorganEH.Evidence for low molecular weight, non-transferrin-bound iron in rat brain and cerebrospinal fluid. J Neurosci Res1998;
54: 486–494.
34.
van LengerichBZhanLXiaD, et al.
A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models. Nat Neurosci2023;
26: 416–429.
35.
Percie du SertNAhluwaliaAAlamS, et al.
Reporting animal research: explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol2020;
18: e3000411.
36.
CouncilNR.Guide for the care and use of laboratory animals. Eighth edition.
Washington, DC:
The National Academies Press, 2011, p. 246.
37.
OakleyHColeSLLoganS, et al.
Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci2006;
26: 10129–10140.
38.
PizzoMEPloweyEDKhouryN, et al.
Engineering anti-amyloid antibodies with transferrin receptor targeting improves safety and brain biodistribution. bioRxiv2024: 2024-07.
39.
LeahyDJRuleGSWhittakerMM, et al.
Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies. Proc Natl Acad Sci U S A1988;
85: 3661–3665.
40.
CalvoBRubioFFernandezM, et al.
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry. IBRO Rep2020;
8: 36–47.
41.
KisselKHammSSchulzM, et al.
Immunohistochemical localization of the murine transferrin receptor (TfR) on blood-tissue barriers using a novel anti-TfR monoclonal antibody. Histochem Cell Biol1998;
110: 63–72.
42.
LeeHJEngelhardtBLesleyJ, et al.
Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse. J Pharmacol Exp Ther2000;
292: 1048–1052.
43.
CouchJAYuYJZhangY, et al.
Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier. Sci Transl Med2013;
5: 183ra57, 1–12.
44.
SaundersNRDziegielewskaKMMollgardK, et al.
Physiology and molecular biology of barrier mechanisms in the fetal and neonatal brain. J Physiol2018;
596: 5723–5756.
45.
PizzoMEWolakDJKumarNN, et al.
Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J Physiol2018;
596: 445–475.
46.
DziegielewskaKMEvansCALaiPC, et al.
Proteins in cerebrospinal fluid and plasma of fetal rats during development. Dev Biol1981;
83: 193–200.
47.
WongMSchlaggarBLBullerRS, et al.
Cerebrospinal fluid protein concentration in pediatric patients: defining clinically relevant reference values. Arch Pediatr Adolesc Med2000;
154: 827–831.
48.
MoosTOatesPS, andMorganEH.Expression of the neuronal transferrin receptor is age dependent and susceptible to iron deficiency. J Comp Neurol1998;
398: 420–430.
ZeppenfeldDMSimonMHaswellJD, et al.
Association of perivascular localization of aquaporin-4 with cognition and Alzheimer disease in aging brains. JAMA Neurol2017;
74: 91–99.
51.
XuZXiaoNChenY, et al.
Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Abeta accumulation and memory deficits. Mol Neurodegener2015;
10: 58.
52.
SimonMWangMXIsmailO, et al.
Loss of perivascular aquaporin-4 localization impairs glymphatic exchange and promotes amyloid beta plaque formation in mice. Alzheimers Res Ther2022;
14: 59.
53.
GarciaFJSunNLeeH, et al.
Single-cell dissection of the human brain vasculature. Nature2022;
603: 893–899.
54.
WinklerEAKimCNRossJM, et al.
A single-cell atlas of the normal and malformed human brain vasculature. Science2022;
375: eabi7377.
55.
WalchliTGhobrialMSchwabM, et al.
Single-cell atlas of the human brain vasculature across development, adulthood and disease. Nature2024;
632: 603–613.
56.
VanlandewijckMHeLMaeMA, et al.
A molecular atlas of cell types and zonation in the brain vasculature. Nature2018;
554: 475–480.
57.
YangWMJungKJLeeMO, et al.
Transient expression of iron transport proteins in the capillary of the developing rat brain. Cell Mol Neurobiol2011;
31: 93–99.
58.
SiddappaAJRaoRBWobkenJD, et al.
Developmental changes in the expression of iron regulatory proteins and iron transport proteins in the perinatal rat brain. J Neurosci Res2002;
68: 761–775.
59.
BelaidiAAGunnAPWongBX, et al.
Marked age-related changes in brain iron homeostasis in amyloid protein precursor knockout mice. Neurotherapeutics2018;
15: 1055–1062.
60.
UllmanJCArguelloAGetzJA, et al.
Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice. Sci Transl Med2020;
12: eaay1163.
61.
AjiokaRSKaplanJ.Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors. Proc Natl Acad Sci U S A1986;
83: 6445–6449.
62.
CiechanoverASchwartzALDautry-VarsatA, et al.
Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem1983;
258: 9681–9689.
63.
SehlinDSyvanenS, and faculty M.
Engineered antibodies: new possibilities for brain PET?Eur J Nucl Med Mol Imaging2019;
46: 2848–2858.
64.
EdavettalSCejudo-MartinPDasguptaB, et al.
Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis. Med2022;
3: 860–882 e15.
65.
PornnoppadolGBondLGLucasMJ, et al.
Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs. Cell Chem Biol2024;
31: 361–372 e8.
66.
ChewKSWellsRCMoshkforoushA, et al.
CD98hc is a target for brain delivery of biotherapeutics. Nat Commun2023;
14: 5053.
67.
MezzanotteMAmmirataGBoidoM, et al.
Activation of the Hepcidin-Ferroportin1 pathway in the brain and astrocytic-neuronal crosstalk to counteract iron dyshomeostasis during aging. Sci Rep2022;
12: 11724.
68.
SatoTShapiroJSChangHC, et al.
Aging is associated with increased brain iron through cortex-derived hepcidin expression. Elife2022;
11: e73456.
69.
MollerHEBossoniLConnorJR, et al.
Iron, Myelin, and the brain: neuroimaging meets neurobiology. Trends Neurosci2019;
42: 384–401.
70.
TellingNDEverettJCollingwoodJF, et al.
Iron biochemistry is correlated with amyloid plaque morphology in an established mouse model of Alzheimer’s disease. Cell Chem Biol2017;
24: 1205–1215 e3.
71.
ZhangXGouYJZhangY, et al.
Hepcidin overexpression in astrocytes alters brain iron metabolism and protects against amyloid-beta induced brain damage in mice. Cell Death Discov2020;
6: 113.
72.
RogersJTBushAIChoHH, et al.
Iron and the translation of the amyloid precursor protein (APP) and ferritin mRNAs: riboregulation against neural oxidative damage in Alzheimer’s disease. Biochem Soc Trans2008;
36: 1282–1287.
73.
BaringerSLSimpsonIA, andConnorJR.Brain iron acquisition: an overview of homeostatic regulation and disease dysregulation. J Neurochem2023;
165: 625–642.
74.
AytonSWangYDioufI, et al.
Brain iron is associated with accelerated cognitive decline in people with Alzheimer pathology. Mol Psychiatry2020;
25: 2932–2941.
75.
GiannoniPArango-LievanoMNevesID, et al.
Cerebrovascular pathology during the progression of experimental Alzheimer’s disease. Neurobiol Dis2016;
88: 107–117.
76.
ArguelloAMahonCSCalvertME, et al.
Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme. J Exp Med2022;
219: e20211057.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
