Transferrin Receptor-Mediated Uptake at the BloodâBrain Barrier Is

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Transferrin receptor-mediated uptake at the blood-brain barrier is not impaired by Alzheimer’s disease neuropathology. Philippe Bourassa, Wael Alata, Cyntia Tremblay, Sarah Paris-Robidas, and Frédéric Calon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00870 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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Molecular Pharmaceutics

Transferrin receptor-mediated uptake at the blood-brain barrier is not impaired by Alzheimer’s disease neuropathology.

Philippe Bourassa1,2, Wael Alata1,2, Cyntia Tremblay1, Sarah Paris-Robidas1,2 and Frédéric Calon1,2. 1. Faculté de pharmacie, Université Laval, Québec, Québec, Canada 2. Axe Neurosciences, Centre de recherche du CHU de Québec – Université Laval, Québec, Québec, Canada

Corresponding author: Frédéric Calon, Ph.D. Centre de recherche du CHU de Québec – Université Laval 2705, Boulevard Laurier, Room T2-67 Québec, QC, G1V 4G2, Canada Tel #: +1(418) 525-4444 ext. 48697 Fax #: +1(418) 654-2761 E-mail: [email protected]

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Abstract The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB) and is therefore considered as a potential target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCEC in vivo. However, since Alzheimer's disease (AD) is accompanied by a BBB dysfunction, it raises concerns about whether TfR-mediated transport becomes inefficient during the progression of the disease. Measurements of TfR levels using Western blot analysis in whole homogenates from human post-mortem parietal cortex and hippocampus did not reveal any significant difference between individuals with or without a neuropathological diagnosis of AD (respectively n = 19 and 22 for the parietal cortex; n = 12 and 14 for hippocampus). Similarly, TfR concentrations in isolated human brain microvessels from parietal cortex were similar between controls and AD cases. TfR levels in isolated murine brain microvessels were not significantly different between groups of 12- and 18-month-old NonTg and 3xTg-AD mice, the latter modeling Aβ and tau neuropathologies. In situ brain perfusion assays were then conducted to measure the brain uptake and internalization of fluorolabeled Ri7 in BCEC upon binding. Consistently, TfR-mediated uptake in BCEC was similar between 3xTg-AD mice and non-transgenic controls (~ 0.3 µl.g-1.s-1) at 12, 18 and 22 months of age. Fluorescence microscopy analysis following intravenous administration of fluorolabeled Ri7 highlighted that the signal from the antibody was widely distributed throughout the cerebral vasculature, but not in neurons or astrocytes. Overall, our data suggest that both TfR protein levels and TfR-dependent internalization mechanisms are preserved in the presence of Aβ and tau neuropathologies, supporting the potential of TfR as a vector target for drug delivery into BCEC in AD. Keywords: transferrin receptor, 3xTg-AD, blood-brain barrier, Alzheimer's disease


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Abstract graphic



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Introduction Alzheimer's disease (AD) is currently the most prevalent neurodegenerative disorder. Despite decades of research, curative treatments for AD have yet to be identified. Crossing the blood-brain barrier (BBB) is normally considered as an essential condition for drug efficacy in brain disorders, to ensure sufficient tissue exposure and therapeutic target engagement. On the one hand, because of their unique phenotype characterized by the expression of tight junction proteins and a low pinocytic transport 1, 2, brain capillary endothelial cells (BCEC) forming the BBB restrict the passage of most bloodborne molecules, including biopharmaceuticals 3. On the other hand, BCEC also express a wide array of receptors and transporters that are involved in the selective uptake of biomolecules from the bloodstream to maintain brain homeostasis 46.

From a brain drug delivery standpoint, these receptors and transporters therefore offer unique

opportunities 7. Among them, the insulin receptor, the low density lipoprotein receptor-related protein 1 (LRP1) and the transferrin receptor (TfR) have been identified as potential BBB targets for the development of brain drug delivery approaches 8. The TfR is highly enriched in BCEC 9, 10 and is one of the most studied receptor/transporter in the context of targeted transport of therapeutics to the brain 11. Others and we have previously shown that monoclonal antibodies (mAbs) targeting this receptor, either alone or conjugated to nanoparticles, are internalized in BCEC following systemic or intracarotid administration, illustrating their potential as vectors for brain drug delivery 11-19. Several pieces of evidence lead us to suspect that BBB dysfunctions occur in AD (Table 1). Morphological changes, including thickening of the basal lamina and reduced microvessel density, have been described in both post-mortem AD brains and animal models

20-25.

It has also been shown, using various imaging

approaches, that cerebral blood flow and glucose uptake are reduced in AD patients, suggesting functional changes implicating specific transporters at the BBB

26-29.

Impaired glucose uptake is also a feature

commonly reported with mouse models of AD neuropathology, including the 3xTg-AD mouse

30-32.

Moreover, accumulation of amyloid in the cerebral vasculature, termed cerebral amyloid angiopathy (CAA), is frequently observed in AD brains

33-36,

correlating with cognitive decline

35.

Recent reports also


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highlighted a potential association between tau protein and BBB dysfunctions. Increased labeling of perivascular phosphorylated tau has been reported in AD brains 37-41, including at the vicinity of Aβ-laden brain vessels, consistent with a possible association between tau and CAA. Studies in the tetracyclineregulatable rTg4510 tau transgenic mouse, a model of tauopathy, showed that a perturbation of BBB integrity concomitantly appears when perivascular tau is detected around hippocampal vessels

42.

Doxycycline-induced reduction of tau prevented the alteration of BBB integrity, suggesting a role for tau protein in BBB dysfunction in tauopathies 42. Overall, these reports suggest that alterations in BBB integrity and/or function might be an intrinsic part of AD pathophysiology. Given the presence of such vascular dysfunctions in AD, concerns could be raised about whether specific receptor-mediated internalization mechanisms at the blood-brain interface are preserved during the progression of the disease and thus can still be considered as targets for therapeutic vectors 43. Insulin is transported across the BBB via the insulin receptor 44-46 and it has been hypothesized that insulin transport to the brain might be compromised during aging 47. Transferrin and the TfR contribute to iron homeostasis in the brain by mediating its transport at the BBB. Studies in AD patients have shown that iron accumulates in several brain regions, including the parietal cortex and hippocampus 48-51, and that this iron accumulation was correlated to Aβ and tau pathologies

52.

On the other hand, cerebral uptake of anti-TfR bispecific

antibodies has not been shown to be altered in animal models of amyloid accumulation

53.

However, it

remains unclear how TfR-mediated endocytosis into BCEC is affected by the combination of old age with Aβ and tau pathologies, the latter being the strongest neuropathological correlate of AD symptoms 54, 55. All these factors are thought to be closely interrelated during the progression of AD

54, 56, 57.

The 3xTg-AD

mouse is a widely used model that progressively develops both amyloid deposits and neurofibrillary tangles (tau pathology) 58. Therefore, in the present work, we sought to determine TfR levels in homogenates and microvessel extracts from human parietal cortex samples and to directly investigate whether TfR-mediated internalization mechanisms are impaired by Aβ and tau pathologies in the 3xTg-AD mouse model at 12, 18 and 22 months of age.



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Materials and Methods Human brain samples: cohort of the Douglas Hospital Research Center Brain Bank Parietal cortex samples from 19 AD patients and 22 controls and hippocampal samples from 12 AD patients and 14 controls, who died with no other neurological disorders, were obtained from the Douglas Hospital Research Center Brain Bank (Douglas Hospital Research Center, Montreal, Quebec, Canada)

59, 60.

The

immediate cause of death was available for 6 controls and 11 AD patients included in this study. The most reported causes of death were pulmonary (chronic obstructive pulmonary disease, edema and pneumonia) and/or cardiac (heart failure and coronary diseases) dysfunctions (4 out of 6 for controls and 10 out of 11 for AD patients). Patients included in the study were rated as having ''definite AD'' or ''probable AD'' based on the CERAD diagnostic criteria (CERAD 1-2); controls were rated either as ''possible AD'' or ''no AD'' (CERAD 3-4) 61. Informed consent forms were obtained from either the patient or a family member. Clinical and biochemical data is summarized in Table 2.

Whole parietal cortex homogenates Human parietal cortex and hippocampus samples (~ 100 mg) were sequentially centrifuged to generate a Tris-buffered saline (TBS)-soluble protein fraction containing soluble intracellular, nuclear and extracellular proteins, a detergent-soluble fraction containing membrane-bound proteins and a detergentinsoluble fraction, as previously reported 59, 60. The detergent-soluble fraction from both the parietal cortex and hippocampus was used for the TfR immunoblotting whereas the detergent-insoluble fraction from parietal cortex samples was used to probe for total tau and Aβ42, by Western blot and ELISA respectively, as previously reported. Total homogenates from the parietal cortex consisting of a pool of TBS-soluble and detergent-soluble protein fractions from the same subjects were also used to compare with and validate the microvessel enrichment procedure described below.


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Isolation of human brain microvessels Separate parietal cortex samples were used to generate microvessel-enriched fractions. The protocol used was adapted from our previous publications 15, 30, 62, 63, as well as from Yousif et al. 64 and from Boulay et al. 65,

to be used for frozen brain samples as the starting material. Parietal cortex samples (~ 400 mg) were

thawed on ice in 500 µl of a microvessel isolation buffer (MIB; 15 mM HEPES, 147 mM NaCl, 4 mM KCl, 3 mM CaCl2 and 12 mM MgCl2) containing a cocktail of protease and phosphatase inhibitors (Bimake, Houston, TX). Meninges and all visible white matter were removed with tweezers and samples were transferred in a 2-ml tissue grinder. Samples were then homogenized in a total of 1.5 ml of MIB, transferred in a 15-ml conical tube and centrifuged at 1,000 g for 10 minutes at 4°C. The supernatant was removed, the pellet was resuspended in 5 ml of MIB containing 18% dextran (from Leuconostoc mesenteroides, M.W. 60,000 – 90,000; Sigma-Aldrich, St.Louis, MO) and spun at 4,000 g for 20 minutes at 4°C. The resulting supernatant was discarded and the pellet was resuspended in 1 ml of MIB. The homogenate was then filtered through a 20-µm nylon filter (Millipore, Temecula, CA). Microvessels were retained on the filter whereas the post-vascular parenchymal fraction was contained in the filtrate. The collected vascular fraction was then homogenized in 500 µl of lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1% Triton X-100, 0.5% SDS and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Bimake) and 1 mM EDTA, sonicated (3 x 45 seconds) in a Sonic Dismembrator apparatus (Thermo Fisher Scientific, Waltham, MA) and spun at 100,000 g for 20 minutes at 4°C. The resulting supernatant was concentrated by a centrifugation at 16,000 g for 60 minutes at 4°C in a Vivaspin device (MWCO, 3 kDa; Sartorius Stedim Biotech, Aubagne, France) and preserved for Western immunoblotting analyses as the vascular fraction. In parallel, the filtrate containing the post-vascular fraction was spun at 16,000 g for 20 minutes at 4°C. The resulting pellet was homogenized in 100 µl of lysis buffer, sonicated and spun at 100,000 g for 20 minutes at 4°C. The supernatant was preserved for Western immunoblotting analyses as the post-vascular parenchymal fraction. Protein concentrations in all fractions were determined using the bicinchoninic acid assay (BCA) (Thermo



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Fisher Scientific). Bovine serum albumin standard concentrations used for the BCA ranged between 25 and 1500 µg/ml. Samples were diluted 1:4 and 1:8 to fit the standard curve.

Animals The 3xTg-AD mouse model used here has already been described previously

58, 66-69.

Briefly, this mouse

model of AD carries mutant presenilin-1 (PS1M146V), APPswe, and tau (P301L) transgenes, causing the progressive development of Aβ plaques and neurofibrillary tangles. Both 3xTg-AD and non-transgenic (NonTg) mouse lines are maintained in our animal facilities and backcrossed every 8 generations to ensure a uniform genetic background in both lines. Mice of 12, 18 or 22 months of age were used in this study. AD neuropathology becomes apparent at 12 months and is widespread after 18 months in the 3xTg-AD model 66-69.

All experiments in this study were approved by the Laval University animal ethics committee and were

performed in accordance with the Canadian Council on Animal Care guidelines.

Isolation of mouse brain microvessels Twelve (12)- and 18-month-old NonTg (male/female: 5/5 and 12/6, respectively) and 3xTg-AD (male/female: 5/5 and 8/7, respectively) mice were deeply anesthetized with ketamine/xylazine and sacrificed with an intracardiac perfusion of ice-cold phosphate-buffered saline (PBS) containing 0.32 M sucrose and protease (SIGMAFAST™ Protease Inhibitor tablets, Sigma-Aldrich) and phosphatase (1 mM sodium pyrophosphate and 50 mM sodium fluoride) inhibitors. The brains were immediately collected and transferred into ice-cold perfusion buffer and meninges, cerebellum and brainstem were removed. Murine brain samples were then chopped and frozen in 0.5 ml of MIB containing 0.32 M sucrose and protease and phophatase inhibitors (Bimake) until processed for microvessel enrichment, which was performed as described above for human samples. For enrichment validation of brain endothelial proteins following our microvessel enrichment protocol, the vascular and post-vascular parenchymal fractions were compared to a 8 ACS Paragon Plus Environment

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total homogenate generated from the whole hemisphere of a control mouse directly homogenized in a lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1% Triton X-100, 0.5% SDS and 0.5% sodium deoxycholate) containing the same protease and phosphatase inhibitors (Bimake). Protein concentrations in all fractions were determined using the bicinchoninic acid assay (Thermo Fisher Scientific), as described above for human brain microvessels.

Western blot Protein homogenates were added to Laemmli’s loading buffer and heated 5 minutes at 95°C (detergentsoluble homogenates) or 10 minutes at 70°C (microvessel extracts). Equal amounts of proteins per sample (12 µg for detergent-soluble homogenates and 8 µg for human or mouse microvessel extracts) were resolved on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted on PVDF membranes, which were then blocked during 1 h at room temperature (RT) with a PBS solution containing 5% non-fat dry milk, 0.5% BSA and 0.1% Tween 20. Membranes were then incubated overnight at 4°C with the primary antibodies (rabbit anti-transferrin receptor; Abcam, Toronto, ON, Canada; 1:1000; rabbit anti-cyclophilin B; Abcam, 1:2000; mouse anti-β-actin; Applied Biological Materials (ABM), Richmond, BC, Canada; 1:5000). Membranes were then washed three times with PBS containing 0.1% Tween 20 and incubated during 1 h at RT with the secondary antibody (goat anti-rabbit HRP or goat antimouse HRP; Jackson ImmunoResearch Laboratories, West Grove, PA; 1:50,000 in PBS containing 0.1% Tween 20 and 1% BSA). Membranes were probed with chemiluminescence reagent (Luminata Forte Western HRP substrate; Millipore) and imaged using the myECL imager system (Thermo Fisher Scientific). This system is designed to adjust the exposure time to achieve optimal detection and linear intensity of the bands without saturation of the signal. β-actin and cyclophilin B were respectively selected as reference proteins for measurements in protein homogenates from whole brain samples and microvessel extracts, as each was the reference protein with the lowest variation between groups in their respective set of samples. Densitometric analysis was performed using the myImage analysis software provided with the imaging 9 ACS Paragon Plus Environment


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system. Insoluble total tau in parietal cortex from the same cohort of subjects has been determined by Western immunoblotting using the tau-13 primary antibody (anti-total human tau; Covance, Princeton, NJ, USA), as previously described 59, 60 (Table 2).

ELISA Aβ42 concentrations from the parietal cortex of the same cohort of subjects have been determined previously in detergent-insoluble fractions using highly sensitive ELISA 59, 60 (Table 2).

Monoclonal Antibody Conjugation to Alexa Fluor dyes. Monoclonal antibodies (mAbs) (Ri7 and control rat IgG2a (2A3); BioXCell, West Lebanon, NH) were fluorolabeled as described previously

15, 16.

Firstly, mAbs were thiolated with a 40:1 M excess of freshly

prepared 2-iminothiolane (Traut's reagent) in 0.05 M sodium borate/0.1 mM EDTA, pH 8.5 during 1 h. Thiolated mAbs were diluted with 0.05 M HEPES/0.1 mM EDTA, pH 7.0 and concentrated using a Vivaspin filter device (MWCO, 30 kDa; Sartorius Stedim Biotech). To conjugate thiolated mAbs to Alexa Fluor (AF) dyes, AF dyes functionalized with a maleimide moiety were added and incubated overnight in 2-ml glass bottles under an inert nitrogen atmosphere. Two different dyes were used: AF750 C5-maleimide for in situ brain perfusion, and AF647 C2-maleimide for immunofluorescence (Life technologies, Burlington, ON, Canada). Vivaspin devices were then used to remove unbound AF dyes and volumes were adjusted with 0.05 M HEPES/0.1 mM EDTA, pH 7.0 to reach the desired concentrations after determination of the degree of labeling (DOL) using the manufacturer’s instructions. DOL were similar between Ri7AF647 and IgG-AF647 (9 and 9.4 molecules of dye per mAb respectively), and between Ri7-AF50 and IgG-750 (5.8 and 7.6 molecules of dye per mAb respectively).


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In situ brain perfusion In situ brain perfusion experiments were conducted as previously described 15. Briefly, 12-, 18- and 22month-old NonTg (male/female: 3/4, 3/5 and 5/2, respectively) and 3xTg-AD (male/female: 3/3, 5/2 and 4/3, respectively) mice were perfused with 100 µg of AF750-mAbs during 1 minute at a flow rate of 2.5 ml/min, followed by a 4-minute washout at the same flow rate. Immediately after the end of perfusion, mice were decapitated and the right hemispheres were harvested, snap frozen and kept at -80°C before processing. Right hemispheres were then homogenized in 4 volumes of lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1% Triton X-100, 0.5% SDS and 0.5% sodium deoxycholate) containing inhibitors of proteases and phosphatases, as previously mentioned, and fluorescence was determined using a Kodak 4000MM digital imaging system with appropriate filters (Molecular Imaging Systems, Carestream Health, Rochester, NY). A correction factor of 0.8 was applied to IgG-AF750 values to take into account the difference in the DOL compared to Ri7-AF750. The distribution volume (VD; µl.g-1) was calculated by dividing the amount of fluorescence in the right brain hemisphere by the concentration of fluorescence in the perfusate. Specific VD values for Ri7 were obtained after substracting the VD of the control IgG. The apparent brain uptake coefficient (Clup; µl*g-1s-1) was then calculated by dividing the VD by the time of perfusion.

Immunofluorescence NonTg and 3xTg-AD mice were intravenously injected with 300 µg of AF647-mAbs at 12, 18 or 22 months of age. One hour later, mice were sacrificed by an intracardiac perfusion of phosphate-buffered saline followed by 4% paraformaldehyde (PFA) under deep anesthesia using ketamine/xylazine. Brains were collected, post-fixed in 4% PFA during 6 h and cryopreserved in a PBS solution containing 20% sucrose. Free-floating brain slices (25 µm) were blocked for 1 h in a PBS solution containing 5% normal horse serum (Invitrogen, Carlsbad, CA) and 0.2% Triton X-100. Sections were then incubated overnight at 4°C with primary antibodies in the blocking solution: goat anti-type IV collagen (1:500; Millipore), mouse anti11 ACS Paragon Plus Environment


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neuronal nuclei (NeuN, 1:1000; Millipore) and mouse anti-glial fibrillary acidic protein (GFAP, 1:1000; Sigma-Aldrich). Brain slices were then incubated with secondary antibodies (donkey anti-goat Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 555; both 1:1000; Life technologies) for 2 h at RT. Finally, brain sections were counterstained with DAPI (Thermo Fisher Scientific), mounted on SuperFrost Plus slides (Thermo Fisher Scientific) and coverslipped with Mowiol mounting medium. Images were acquired using an EVOS® FL Auto imaging system (Life technologies/Thermo Fisher Scientific).

Data and statistical analysis Data are presented as means ± SEM. Statistical analyses and the number of mice or individuals per group are specified in each Figure. Our power analysis was based on previous data and the literature, and the number of animals per group was set at a minimum of 6 to have a 95 % possibility to detect a 20% change if alpha is set at 0.05 and standard deviations are 10% of average. Equality of the variances between the groups was determined using Bartlett’s test. Normality of the data was evaluated using a Shapiro-Wilk test for groups with at least 7 values and assessed using a Kolmogorov-Smirnov test for groups with less than 5 values. When comparing two groups, an unpaired Student t test was performed, with a Welch correction included when variances were not equal. When normality could not be assumed and variances were unequal, a non-parametric Mann-Whitney test was performed. When more than two groups were compared, one-way or two-way ANOVA were used. For one-way ANOVA with comparable variances, Tukey’s post hoc analysis was performed. When variances were significantly different, we performed non-parametric Kruskal-Wallis ANOVA followed by Dunn’s multiple comparison tests. For all data, statistical significance was set at P < 0.05. All statistical analyzes were performed with Prism 6 (GraphPad, San Diego, CA, USA) or JMP (version 13; SAS Institute Inc., Cary, IL) softwares.


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Results Transferrin receptor levels are not altered in whole brain homogenates from the parietal cortex and hippocampus of individuals with a neuropathological diagnosis of AD. We probed whether TfR levels were different between individuals classified as control (CERAD 3-4) or AD (CERAD 1-2), with parietal cortex and hippocampus samples obtained from the Douglas Hospital Research Center. Neuropathological diagnosis was previously confirmed by measuring the levels of insoluble Aβ42 and insoluble tau in the parietal cortex, which were 350% and 2400% higher in AD patients compared to controls, respectively (Table 2 and references 59 and 60). As represented in Figure 1A, we did not observe any significant difference when probing for the TfR in detergent-soluble homogenates generated from whole samples of parietal cortex (Figure 1A, left graph). We also evaluated whether TfR levels were altered when differentiating subjects based on the ApoE4 carriage, as it is a critical genetic risk factor for the development of AD 70 that is also thought to be involved in the cerebrovascular dysfunctions reported in AD 71. As shown in Figure 1A (right graph), no difference was observed in ApoE4 carriers compared to non-carriers. TfR levels were also assessed in detergent-soluble protein homogenates from hippocampal samples and no significant difference was noted between AD patients and controls (Figure 1B, left graph) or between ApoE4 carriers and non-carriers (Figure 1B, right graph). Linear regression analysis revealed that TfR levels in parietal cortex were not correlated with age at death, cortical insoluble Aβ42 and tau levels, and duration of symptoms (Table 3, middle column) in the present cohort, suggesting that TfR levels are not influenced by old age or by the development of AD neuropathology.

Transferrin receptor levels are not altered in vasculature extracts from the parietal cortex of persons with a neuropathological diagnosis of AD. Since TfR expression has been reported in various brain cells, though in a lesser extent than in BCEC 72, 73, we then performed Western blots in extracts enriched in brain microvessels. We first validated that the 13 ACS Paragon Plus Environment


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material retained on the filter during our microvessel enrichment protocol was enriched in brain endothelial cells compared to the post-vascular filtrate and the total homogenate. As represented in Figure 2A, vascular fractions were enriched in CD31, also known as platelet endothelial cell adhesion molecule (PECAM), and claudin5 (a component of tight junctions), but contained a low amount of synaptophysin, a synaptic protein. Similar to CD31 and claudin5, TfR was also strongly concentrated in vascular fractions, confirming that endothelial cells are the major source of TfR in the brain. We evaluated the microvessel enrichment by calculating the vascular/post-vascular ratio for claudin5 and we did not observe any significant difference between groups (Figure S1). Our analysis in microvessel extracts from subjects of the Douglas cohort showed that TfR levels were not changed in AD patients compared to controls (Figure 2B, left graph). Moreover, no difference was observed for TfR levels in microvascular extracts from ApoE4 carriers compared to non-carriers (Figure 2B, right graph). Finally, TfR levels in microvessel extracts from the parietal cortex were not correlated with age at death, cortical insoluble Aβ42 and tau levels, and duration of symptoms (Table 3, right column).

TfR levels in microvessel extracts remain unaltered by age or genotype in 3xTg-AD mice. We assessed TfR levels in microvessel extracts from NonTg and 3xTg-AD mice aged 12 and 18 months. We also validated the mouse brain microvessel enrichment protocol by probing for the same endothelial and non-endothelial markers described above for human samples. As expected, endothelial markers as well as TfR were all enriched in the vascular fractions generated, compared with the post-vascular fraction or the total homogenate (Figure 3A). Again, the comparison of the vascular/post-vascular claudin5 ratio revealed that microvessel enrichment was similar between groups (Figure S1). As shown in Figure 3B, we did not observe any effect of older age or genotype on TfR levels in mouse microvessel extracts.


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Brain uptake of a monoclonal antibody, clone Ri7.217, targeting the murine TfR is unchanged by aging and AD-like neuropathology in the 3xTg-AD mouse After validating that TfR levels were not affected by aging or AD-like neuropathology in 3xTg-AD mice, we investigated whether its uptake capability was also preserved using in situ brain perfusion (ISBP) (Figure 4A). We previously developed an experimental paradigm to quantify the brain uptake of Ri7 using a nearinfrared dye to avoid the autofluorescence of the brain in the visible spectrum 15. We thus conjugated in this study the Ri7 antibody with AF750. The fluorescence in the brain homogenates was assessed after the perfusion of 100 µg of fluorolabeled antibodies, either Ri7 or IgG, and a subsequent 4-minute washout to flush unbound mAbs, in 12, 18 and 22-month-old NonTg and 3xTg-AD mice. As represented in Figure 4B, the fluorescence detected in the brain of mice perfused with Ri7-AF750 was higher than that in mice perfused with IgG-AF750, indicating that Ri7-AF750 is accumulating in the brain. Brain uptake values for fluorolabeled Ri7 ranged between 0.31 and 0.35 µl*g-1*s-1 for all groups, indicating that TfR-mediated uptake of Ri7-AF750 is not impaired by aging or AD-like neuropathology (Figure 4B). We previously showed that fluorolabeled Ri7 was massively internalized in BCEC of Balb/c mice 60 minutes after systemic administration 16. Similarly, immunofluorescence analysis of the distribution of fluorolabeled Ri7 in the brain of NonTg and 3xTg-AD mice sacrificed 60 minutes after intravenous injection revealed a strong colocalization with brain microvessels (Figure 4C-D; green), but not with neurons (Figure 4C; grey) or astrocytes (Figure 4D; grey), respectively labeled with collagen IV, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP). Finally, we did not observe any apparent difference in fluorolabeled Ri7 accumulation in BCEC between NonTg and 3xTg-AD mice for all ages tested.



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Discussion Given the potential of the TfR for brain drug targeting, the present work aimed to determine whether TfR levels and TfR-mediated uptake and internalization are compromised by aging and AD neuropathology. Overall, our data indicate that: (1) TfR levels are not altered by AD neuropathology in total brain homogenates and brain microvessels from human parietal cortex samples, and in brain microvessels from 3xTg-AD mice; (2) TfR levels do not significantly fluctuate with age; (3) Brain uptake of a TfR-binding mAb into brain endothelial cells remains unchanged in 12, 18 and 22 month-old 3xTg-AD mice. Such series of observations are consistent with the conclusion that TfR-related mechanisms at the BBB remain effective during the progression of AD. The observation that levels of TfR in parietal cortex are not altered in AD patients corroborates previous findings in other brain regions. For example, similar TfR levels between AD subjects and controls have been reported in samples from frontal, entorhinal and parietal cortices using Western blot measurements 53, 74, 75.

A previous macroscopic autoradiography study showed that tritiated transferrin binding was reduced

in specific subregions, namely CA1, CA2 and CA4 pyramidal layers, but not in other regions of the hippocampus and not in the temporal cortex when comparing AD patients with controls 75. This discrepancy might be explained by differences in experimental approaches. Autoradiographic analysis on hippocampal sections allowed for discrimination of the different subregions of the hippocampus, whereas immunoblotting analysis on detergent-soluble fractions containing membrane proteins did not. Nevertheless, we cannot rule out a possible region-specific effect for TfR levels that was not captured in the present analysis 50. Reports focusing on brain microvessels are more sparse. However, TfR density has been evaluated in brain microvessels from frontal cortex samples of AD patients and controls, isolated using density gradient centrifugation and separation with glass beads, and it was observed that TfR density is unchanged in AD patients compared to controls 74. Although the parietal cortex is not the earliest region affected in AD, it is still plagued by high amounts of neuropathology. For example, it has been shown that aggregated tau and 16 ACS Paragon Plus Environment

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Aβ in the parietal cortex are strongly correlated to cognitive decline

54, 55.

Loss of synaptic proteins like

synaptophysin and inclusions of phosphorylated TDP-43 in the parietal cortex were also reported to be correlated with cognitive deficit 55, 76. Added to the fact that the AD parietal cortex samples used here come from subjects with a heavy burden of both aggregated tau and Aβ (Table 2 and references 59 and 60), these data collectively do not support an important effect of AD neuropathology on TfR located on the human BBB. It could be argued that analysis in human post-mortem AD samples might be confounded by other variables not controlled for. To directly assess the effect of AD neuropathology, we evaluated TfR in the 3xTg-AD mouse model, which expresses an increasing amount of tau and Aβ pathologies with age 66. Of note, this model displays deficiencies in brain vasculature function, but unchanged BBB permeability 20, 30, 31, 77, 78 (see also Table 1). We previously showed, using ISBP of [14C]-sucrose and [3H]-inulin, that the integrity of the BBB is not compromised in 3xTg-AD mice at 3, 6, 8, 11, and 18 months of age 20, 30. However, a reduction of the cerebral vascular space is observed in at 8, 11 and 18 months of age in the same model, associated with a thickening of the basal membrane 20, 30. Data consistent with an intact BBB obtained with ISBP of human IgG were also obtained in 4- to 13-month-old 3xTg-AD mice 77. Decreased brain uptake clearance (Clup, µl.g-1.s-1) of [3H]-D-glucose is observed in 18-month-old 3xTg-AD mice, but not at earlier ages 20, 30, while diazepam uptake is unchanged, consistent with a similar BBB surface 30. This model was thus well suited to directly study the impact of AD neuropathology on brain TfR, along with age. However, immunoblot results from isolated brain microvessels of the 3xTg-AD mouse were in line with our observations in human samples, indicating that TfR levels are not altered by both aging and AD-like pathologies. Investigation in the 3xTg-AD mouse model allowed us to perform functional analyses of TfR-mediated transport at the BBB. We previously characterized the BBB uptake of fluorolabeled Ri7 in Balb/c mice and showed that the perfusion of this anti-TfR monoclonal antibody was not accompanied with a perturbation of the BBB integrity and that the internalization process was specific and saturable, as co-perfusion of the 17 ACS Paragon Plus Environment


Page 18 of 39

unlabeled antibody reduced its uptake by BCEC 15. ISBP data in the present work confirmed that there was no abnormal BBB uptake of anti-TfR mAbs in 12-, 18- and 22-month-old NonTg and 3xTg-AD mice, with an apparent brain uptake coefficient of the control IgG consistently lower than for Ri7. Our ISBP data revealed that brain uptake of fluorolabeled Ri7 was similar in both genotypes for all ages tested, with values (~ 0.3 µl.g-1.s-1) similar to what we previously observed in Balb/c mice 15, suggesting that TfR-mediated uptake is preserved in the 3xTg-AD mouse. These findings are in agreement with a previous report showing that brain content of TfR bispecific antibodies after systemic administration was similar in APP mice overexpressing Aβ compared to controls 53. The low Clup value and the absence of fluorescent signal in the brains of animals injected with either control IgG or anti-TfR mAbs is also consistent with a preserved BBB integrity in the 3xTg-AD model as reported previously 20, 77. These results indicate that older age as well as Aβ and tau pathologies had no frank detrimental impact on the BCEC uptake of the Ri7 anti-TfR mAb. Fluorolabeled Ri7 was only detected in microvessels 1 hour following systemic administration, in both NonTg and 3xTg-AD mice. This distribution is consistent with previous colocalization studies with CD31 and type IV collagen with Ri7 and other mAbs targeting the TfR

15, 16.

It is also in accordance with the

specific internalization of Ri7-targeted Qdots into BCEC in vivo 17. Importantly, the present results indicate that AD neuropathology had no massive influence on the brain distribution of anti-TfR mAbs. It has been proposed that anti-TfR mAbs are restricted to BCEC as a consequence of their high affinity for TfR, which hinders their dissociation from the receptor, thus leading to their entrapment into the endosomal machinery of BCEC 12, 15, 79. Thus, the present data suggest that AD neuropathology in 3xTg-AD animals had no impact on the binding affinity between Ri7 and the TfR. Altogether, our results suggest that both TfR levels and TfR-mediated internalization mechanisms at the BBB are preserved, despite the accumulation with age of Aβ and tau in the 3xTg-AD mouse. Post-mortem data in human samples indicate that targetable TfR concentrations remain unaffected by AD neuropathology, at least in the parietal cortex and hippocampus. This suggests that reported iron accumulation in the brain of AD patients 48-51 is not associated with a change in TfR content. Moreover, our 18 ACS Paragon Plus Environment

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data highlight that the development of AD is not likely to limit the access to the brain of systemically administered TfR-targeting therapeutics. Considering the potential of TfR as a gateway to the brain and the importance of target engagement for an efficient delivery to the brain, our results provide essential confirmations for future studies, especially given the growing interest for bispecific antibodies targeting both TfR and beta-amyloid for diagnostic and therapeutic applications 12, 80, 81. Because BCEC are involved in the pathogenesis of several other central nervous system disorders than AD, such as multiple sclerosis and stroke 4, 82-84, our results support the potential of TfR as a vector target for drug delivery in BCEC.

Acknowledgments Funding was provided by the Canadian Institutes of Health Research (CIHR) to F.C (MOP 125930). F.C is a Fonds de recherche du Québec - Santé (FRQ-S) senior research scholar. P.B held scholarships from the Réseau québécois de recherche sur le médicament (RQRM), Fondation du CHU de Québec and a joined scholarship from the FRQ-S and the Alzheimer Society of Canada (ASC) and now holds a scholarship from the CIHR. The authors are indebted to all donors who contributed to the Douglas Hospital Research Centre Brain Bank. The authors are thankful to France Couture, graphic designer of the Centre de recherche du CHU de Québec – Université Laval, for the preparation of the graphical abstract and to Dr. Vincent Émond for his proofreading of the manuscript.



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Tables Table 1: Non-exhaustive list of BBB-related observations reported in AD and in the 3xTg-AD mouse model. Among BBB-related observations made in human AD cases (upper section), few of them have also been described in the 3xTg-AD mouse model (lower section). Altogether, these observations suggest functional and morphological changes at the BBB during progression of the disease.

Alzheimer's disease

BBB-related observations

References

Deficit in glucose uptake

28, 29, 85, 86

Decreased cerebral blood flow

26, 85, 86

Thickening of basal membrane

23-25, 86

Reduced microvessel density

21, 24, 86

Cerebral amyloid angiopathy

33-36, 87

Perivascular phosphorylated tau

37-41

No alteration of TfR levels

74

and present work

Deficit in glucose uptake

30-32

Reduced DHA uptake at 11 months

88

Thickening of basal membrane

20, 22

3xTg-AD

No changes in microvessel density

20

mouse model

Weak cerebral amyloid angiopathy

89, 90

No changes in BBB permeability

20, 77

No alteration of TfR levels

present work

No alteration of TfR–mediated uptake

present work


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Table 2: Clinical and Biochemical Data of the Cohort from the Douglas Hospital Research Center Brain Bank used here

Clinical and Biochemical Data

For whole brain homogenates Hippocampus samples Parietal cortex samples AD Controls AD Controls

Statistical Analysis

n

22

19

14

12

n/a

Men, %

55

47

43

58

Contingency, Pearson test

Age at first symptoms, years

n/a

68.5 (9.0)

n/a

71.6 (10.)

n/a

Duration of symptoms, years

n/a

7.7 (3.7)

n/a

6.9 (4.0)

n/a

71.1 (9.1)

77.5 (8.8)*

70.2 (8.9)

79.4 (9.0)*

Student t-test

18 (8)

18 (9)

19 (9)

20 (10)

Student t-test

Brain mass, g

1259 (134)

1080 (141)¶

1252 (136)

1128 (133)*

Student t-test

Brain pH

6.16 (0.30)

6.22 (0.28)

6.13 (0.32)

6.25 (0.28)

Student t-test

Insoluble Aβ42 concentration

626 (967)

2190 (1521)¶

n.d.

n.d.

Student t-test

Insoluble total tau content

404 (461)

9580 (5302)&

n.d.

n.d.

Student t-test

Age at death, years Postmortem delay, hours

For isolated brain microvessels from parietal cortex samples Clinical and Biochemical Data

Controls

AD

Statistical Analysis

n

19

19

n/a

Men, %

53

47

Contingency, Pearson test

Age at first symptoms, years

n/a

68.5 (9.0)

n/a

Duration of symptoms, years

n/a

7.7 (3.7)

n/a

70.8 (9.6)

77.5 (8.8)*

Student t-test

18.6 (8)

18 (9)

Student t-test

Brain mass, g

1259 (144)

1080 (141)¶

Student t-test

Brain pH

6.13 (0.30)

6.22 (0.28)

Student t-test

Age at death, years Postmortem delay, hours


Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 22 of 39

Diagnosis was based on CERAD neuropathological criteria (controls, CERAD 3-4; AD patients, CERAD 1-2). Brain pH was measured in cerebellum extracts. Values are expressed as means (SD) unless specified otherwise. Measurements of insoluble Aβ42 and tau were made in parietal cortex samples. Concentrations of insoluble Aβ42 are expressed in picograms per milligrams of tissue. Insoluble tau content is expressed in relative optical density. Statistical comparisons between groups were performed using a Student’s t-test or a Pearson test for contingency (* p < 0.05; ¶ p < 0.001; & p < 0.0001 versus CERAD 3-4 controls). For more details on methodology and cohort characteristics, see materials and methods section and references 59 and 60. Abbreviations: Aβ, amyloid-β peptide; AD, Alzheimer’s disease; C, contingency; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; n/a, not applicable; n.d., not determined.


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Table 3: Clinical and Biochemical Correlates of TfR levels in detergent-soluble homogenates and isolated brain microvessels from parietal cortex. Clinical and Biochemical Correlates of TfR Detergent-soluble Isolated brain homogenates microvessels Markers Age at death, years All subjects Controls AD

n

r2

n

r2

41 22 19

-0.001; n.s +0.052; n.s -0.191; n.s

38 19 19

-0.031; n.s (+) < 0.001; n.s -0.085; n.s

Insoluble Aβ42, pg/mg tissue All subjects Controls AD

41 22 19

+0.019; n.s +0.009; n.s +0.012; n.s

38 19 19

-0.010; n.s -0.030; n.s (+) < 0.001; n.s

Insoluble tau, relative O.D. All subjects Controls AD

41 22 19

-0.002; n.s +0.002; n.s +0.023; n.s

38 19 19

(-) < 0.001; n.s -0.021; n.s -0.015; n.s

Duration of symptoms, years All subjects Controls AD

19 19

-0.032; n.s -0.032; n.s

19 19

-0.221; n.s -0.221; n.s

Diagnosis was based on CERAD neuropathological criteria (controls, CERAD 3-4, AD patients, CERAD 1-2). TfR protein values used for correlative analyses were normalized over actin in detergent-soluble homogenates and over cyclophilin B in isolated brain microvessels. (-), Negative correlation; (+) positive correlation; Aβ, amyloid-β peptide; AD, Alzheimer’s disease; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; n.s., not significant; O.D., optical density; TfR, transferrin receptor. .



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Figures Figure 1

Figure 1: TfR levels are not altered in detergent-soluble homogenates from parietal cortex and hippocampus samples of AD patients. A-B) Determination of TfR levels by Western immunoblotting in detergent-soluble homogenates from parietal cortex (A) and hippocampus (B) samples. Subjects were differentiated based on the CERAD neuropathological diagnosis (controls, CERAD 3-4, n = 22; AD, CERAD 12, n =19 for parietal cortex; controls, CERAD 3-4, n = 14; AD, CERAD 1-2, n =12 for hippocampus) (left graphs) or the ApoE4 allele carriage (non carriers, n = 22; carriers, n = 19 for parietal cortex; non carriers, n = 15; carriers, n = 11 for hippocampus) (right graphs). Data were normalized over actin and are represented as a scatterplot. Horizontal lines indicate mean ± S.E.M. No difference based on CERAD neuropathological diagnosis or ApoE4 24 ACS Paragon Plus Environment

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carriage was observed for TfR levels in detergent-soluble homogenates from both regions. Statistical analysis: unpaired Student's t-test. Examples were taken from the same immunoblot experiment and consecutive bands loaded in random order are shown. Abbreviations: C, ApoE4 carriers; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; NC, ApoE4 non carriers; TfR, transferrin receptor.



Page 26 of 39

Figure 2

Figure 2: The TfR is not altered in isolated brain microvessels from parietal cortex samples of AD patients. A) Validation that known endothelial markers, like CD31, claudin5, and TfR are enriched in the brain vascular fraction (Va), enriched in brain endothelial cells, compared to post-vascular parenchyma (P), depleted in endothelial cells, and total homogenate (T) samples. For comparison purposes, synaptophysin, a synaptic/neuron marker, is shown. Eight (8) µg of proteins per sample. B) Determination of TfR levels by Western immunoblotting in isolated brain microvessels from parietal cortex samples. Subjects were differentiated based on the CERAD neuropathological diagnosis (controls, CERAD 3-4, n = 19; AD, CERAD 1-2, n =19) (left graph) or the ApoE4 allele carriage (non carriers, n = 21; carriers, n = 17) (right graph). Data were normalized over cyclophilin B and are represented as a scatterplot. Horizontal lines indicate mean ± S.E.M. No difference based on CERAD neuropathological diagnosis or ApoE4 carriage was observed for TfR levels in microvessel-enriched extracts from parietal cortex samples. Statistical analysis: unpaired Student's t-test. Examples were taken from the same immunoblot experiment and consecutive bands loaded in random order are shown. Abbreviations: C, ApoE4 carriers; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; CypB, cyclophilin B; NC, ApoE4 non carriers; TfR, transferrin receptor.


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Figure 3

Figure 3: TfR is not altered by aging or AD neuropathology in the 3xTg-AD mouse model. A) Validation that known endothelial markers, such as CD31 and claudin5, as well as TfR are also enriched in mouse brain microvessels following our microvessel enrichment protocol (Va, vascular fraction; P, post-vascular fraction; T, total homogenate). On the opposite, synaptophysin is found in higher proportion in the post-vascular fraction. Examples were taken from the same immunoblot experiment, but consecutive bands were not taken for all representative photo examples (8 µg of proteins per sample). A black vertical line was inserted to indicate nonconsecutive bands. B) Determination of TfR levels by Western immunoblotting in brain microvessel extracts from NonTg and 3xTg-AD mice aged 12 and 18 months. Data were normalized with cyclophilin B and are represented as mean ± S.E.M. Sample size is indicated in the graph bars. No difference was observed according to age or genotype in TfR levels. Statistical analysis: one-way ANOVA and two-way ANOVA. Examples were taken from



Page 28 of 39

the same immunoblot experiment and consecutive bands loaded in random order are shown. Abbreviations: 3x3xTg-AD, triple transgenic mice; CypB, cyclophilin B; NT-NonTg, non transgenic mice; TfR, transferrin receptor.


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Figure 4



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Figure 4: TfR-mediated internalization at the BBB is not compromised by aging and AD-like neuropathology in the 3xTg-AD model. A) Schematic representation of the in situ brain perfusion. B) NonTg and 3xTg-AD mice aged of 12, 18 and 22 months were perfused with 100 µg of Ri7-AF750 or IgG-AF750. Fluorescence quantification revealed that the brain uptake of Ri7 was significantly higher than control IgG and that there was no difference between genotypes (NonTg: open bars; 3xTg-AD: blue bars) for all ages tested. Data are represented as mean ± S.E.M. Sample size for each group is indicated in the graph bars. * p < 0.05 versus all Ri7-perfused groups. Statistical analysis: non-parametric one-way ANOVA followed by a Dunn's post hoc test. C-D) NonTg and 3xTg-AD aged of 12, 18 and 22 months were intravenously injected with 300 µg of Ri7-AF647 or IgG-AF647 and sacrificed 1 h after the injection. Immunofluorescence analysis showed that the signal from fluorolabeled antibodies (red) was only detectable in mice that were injected with Ri7-AF647. Ri7-AF647 (red) only colocalized with immunostaining of type IV collagen on the basal lamina of BCEC (panels C-D; green), whereas no colocalization, which would have been visible in pink, was observed with the neuronal marker NeuN (panel C; grey) or with the astrocyte marker GFAP (panel D; grey). Scale bar: 10 µm. N = 3 per group. Abbreviations: 3xTg-AD, triple transgenic mice; AF647, Alexa Fluor 647; AF750, Alexa Fluor 750; Clup, brain uptake coefficient; IgG, control rat IgG2a (2A3); NonTg, non-transgenic mice.

Supporting Information: Microvessel enrichment comparisons between groups for human and murine brain microvessel extracts.


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