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Increased expression of metalloprotease meprin β is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin β is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin β and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin β between Arg61 and Asn62 as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin β at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin β is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP). [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood-spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood-spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood-spinal cord barrier endothelial cells of presymptomatic hSOD1G93A mice and bEnd.3 cells stably expressing hSOD1G93A. In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1G93A cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood-spinal cord barrier. [ABSTRACT FROM AUTHOR]

The Swedish mutation within the amyloid precursor protein (APP) causes early-onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β-secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild-type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β-secretase cleaved soluble APP released from APPswe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α-secretase activity-mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α-secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild-type APP. Therefore, inhibition of α-secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells. [ABSTRACT FROM AUTHOR]

Presenilins (PSs) are components of theγ-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that γ-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of γ-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of γ-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease. [ABSTRACT FROM AUTHOR]

It is well established that the proteolytic processing of the β-amyloid precursor protein (APP) generates β-amyloid (Aβ), which plays a central role in the pathogenesis of Alzheimer's disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPsα) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APPΔCT15). Interestingly, the ΔCT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced Aβ levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPsα is sufficient to mediate the physiological functions of APP assessed by these tests. [ABSTRACT FROM AUTHOR]

Background: The most crucial area to focus on when thinking of novel pathways for drug delivery into the CNS is the blood brain barrier (BBB). A number of nanoparticulate formulations have been shown in earlier research to target receptors at the BBB and transport therapeutics into the CNS. However, no mechanism for CNS entrance and movement throughout the CNS parenchyma has been proposed yet. Here, the truncated mini low-density lipoprotein receptor-related protein 1 mLRP1_DIV* was presented as blood to brain transport carrier, exemplified by antibodies and immunoliposomes using a systematic approach to screen the receptor and its ligands' route across endothelial cells in vitro. Methods: The use of mLRP1_DIV* as liposomal carrier into the CNS was validated based on internalization and transport assays across an in vitro model of the BBB using hcMEC/D3 and bEnd.3 cells. Trafficking routes of mLRP1_DIV* and corresponding cargo across endothelial cells were analyzed using immunofluorescence. Modulation of γ-secretase activity by immunoliposomes loaded with the γ-secretase modulator BB25 was investigated in co-cultures of bEnd.3 mLRP1_DIV* cells and CHO cells overexpressing human amyloid precursor protein (APP) and presenilin 1 (PSEN1). Results: We showed that while expressed in vitro, mLRP1_DIV* transports both, antibodies and functionalized immunoliposomes from luminal to basolateral side across an in vitro model of the BBB, followed by their mLRP1_DIV* dependent release of the cargo. Importantly, functionalized liposomes loaded with the γ-secretase modulator BB25 were demonstrated to effectively reduce toxic Aß42 peptide levels after mLRP1_DIV* mediated transport across a co-cultured endothelial monolayer. Conclusion: Together, the data strongly suggest mLRP1_DIV* as a promising tool for drug delivery into the CNS, as it allows a straight transport of cargo from luminal to abluminal side across an endothelial monolayer and it's release into brain parenchyma in vitro, where it exhibits its intended therapeutic effect. [ABSTRACT FROM AUTHOR]

Neurotoxic amyloid-β (Aβ) peptides cause neurodegeneration in Alzheimer’s disease (AD) patients’ brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the β-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aβ species in the mouse brains differs from those observed in AD patients’ brains. Particularly mutations proximal to the β-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aβ1-x formation) lead to artificial Aβ production, as N-terminally truncated Aβ peptides are hardly present in these mouse brains. Meprin β is an alternative β-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aβ2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aβ2-x peptides by conditionally overexpressing meprin β in astrocytes. We chose astrocytes as meprin β was detected in this cell type in close proximity to Aβ plaques in AD patients’ brains. The meprin β-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aβ production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aβ species in future studies. [ABSTRACT FROM AUTHOR]

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer’s disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-β peptides (Aβ42 and/or Aβ43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aβ production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aβ42/Aβ40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aβ42/Aβ40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aβ production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects. [ABSTRACT FROM AUTHOR]

Despite the neurodegenerative disorder Alzheimer’s disease (AD) is the most common form of dementia in late adult life, there is currently no therapy available to prevent the onset or slow down the progression of AD. The progressive cognitive decline in AD correlates with a successive accumulation of cerebral amyloid-β (Aβ) due to impaired clearance mechanisms. A significant percentage is removed by low-density lipoprotein receptor-related protein 1 (LRP1)-mediated transport across the blood–brain barrier (BBB) into the periphery. Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to members of the low-density lipoprotein receptor protein family at the cell surface and targets them for lysosomal degradation, which reduces the number of functional receptors. However, the adverse impact of PCSK9 on LRP1-mediated brain Aβ clearance remains elusive. By using an established BBB model, we identified reduced LRP1-mediated brain-to-blood Aβ clearance due to PCSK9 across different endothelial monolayer in vitro. Consequently, the repetitive application of FDA-approved monoclonal anti-PCSK9 antibodies into 5xFAD mice decreased the cerebral Aβ burden across variants and aggregation state, which was not reproducible in brain endothelial-specific LRP1−/− 5xFAD mice. The peripheral PCSK9 inhibition reduced Aβ pathology in prefrontal cortex and hippocampus–brain areas critically involved in memory processing—and prevented disease-related impairment in hippocampus-dependent memory formation. Our data suggest that peripheral inhibition of PCSK9 by already available therapeutic antibodies may be a novel and easily applicable potential AD treatment. [ABSTRACT FROM AUTHOR]

β-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described β-secretase to generate Aβ peptides in Alzheimer’s disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aβ peptides generation is the metalloproteinase meprin β, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin β expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b−/−). We examined levels of canonical and truncated Aβ species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b−/−. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aβ1-40 and 1–42 levels are reduced in APP/lon mice when meprin β is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aβ2–x peptide deposition is decreased in APP/lon × Mep1b−/− mice. Importantly, loss of meprin β improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin β within the amyloidogenic pathway and Aβ production in vivo. [ABSTRACT FROM AUTHOR]

The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood–brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [14C]-inulin. Analysis of global Mep1b knock-out (Mep1b−/−) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b−/− mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web. [ABSTRACT FROM AUTHOR]

Brain accumulation and aggregation of amyloid-β (Aβ) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aβ peptides (mainly Aβ1-40 and Aβ1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aβ peptides are truncated at the N-terminus, with Aβ4-x peptides being particularly abundant. Aβ4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aβ peptide sequence, which facilitates Aβ4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aβ4-40 but unchanged levels of Aβ1-x peptides. In the 5xFAD mouse model of amyloidosis, Aβ4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4−/− knockout background, Aβ4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aβ species, but Aβ4-40 peptides were absent in cultures derived from ADAMTS4−/− mice indicating that the enzyme was essential for Aβ4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aβ4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aβ peptides. [ABSTRACT FROM AUTHOR]

Graphical abstract Schematic representation of Aβ trafficking across the brain capillary endothelium. PICALM directs the abluminal Aβ-LRP1 complex to Rab11-positive sorting endosomes where ABCB1/P-gp is present, leading to Aβ efflux by ABCB1/P-gp on the luminal side of the endothelium. Highlights • LRP1 and ABCB1/P-gp act in concert transporting Aβ across the blood-brain barrier. • LRP1 and ABCB1/P-gp need each other as partners for Aβ transcytosis. • Late-onset Alzheimer’s disease risk factor PICALM links LRP1 and ABCB1/P-gp. Abstract The accumulation of neurotoxic amyloid-beta (Aβ) in the brain is a characteristic hallmark of Alzheimer’s disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aβ. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aβ efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aβ through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aβ transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aβ from the brain. [ABSTRACT FROM AUTHOR]

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ (1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB. [ABSTRACT FROM AUTHOR]

The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with β1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and β1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of β1-integrin and decreased β1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57B16 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with β1-integrin mediating integrin internalization and thus correlates with downstream signaling of β1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration. [ABSTRACT FROM AUTHOR]

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aβ(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD. [ABSTRACT FROM AUTHOR]

Abstract: We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor′s glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway. [Copyright &y& Elsevier]

Polymeric micelles were prepared for the delivery of donepezil, a leading Alzheimer's disease drug, to enhance its transport across the blood-brain barrier (BBB). Poly(ethylene glycol)- b -poly( tert -butyl methacrylate) amphiphilic block copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were characterized by gel permeation chromatography and nuclear magnetic resonance spectroscopy. Empty and donepezil loaded polymer micelles were formed using the dialysis method and characterized by dynamic light scattering and transmission electron microscopy. Drug loading efficiency and release behavior were monitored using UV/Vis spectroscopy, and cytotoxicity was evaluated via colorimetric tests and impedance measurements. Additionally, the permeability of the nanocarriers across an in vitro BBB culture model was assessed. Drug-loaded micelles demonstrated similar permeability to free donepezil but offered sustained release and improved stability. This micellar delivery system holds significant potential for improving therapeutic outcomes in Alzheimer's treatment by enhancing donepezil's delivery across the BBB. Improved BBB permeability and sustained drug release could lead to more effective concentration of the drug in the brain, potentially reducing peripheral cholinergic side effects, such as nausea and vomiting, often observed with traditional donepezil administration. This could result in better patient compliance and improved cognitive outcomes, making this nanocarrier system a promising alternative for Alzheimer's therapy.

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sarah Bau and Stephen T. Buckley are employees at Novo Nordisk. Catarina Chaves and Dominique Lesuisse are employees at Sanofi. Marijke De Bock is an employee at Janssen Pharmaceutica. Axel H. Mayer is an employee at AbbVie. German Leparc and Martin Lenter are employees at Boehringer Ingelheim Pharma. Tamas Letoha is the CEO of Pharmacoidea and Robert D. Bell is the Vice President and the Head of Research at Ascidian Therapeutics. All others declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)