Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY3AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR3AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL3AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-β (Aβ) generation via APP internalization and thus its amyloidogenic processing. However, conditional knock-out studies in mice define LRP1 as an important mediator for the clearance of extracellular Aβ from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aβ in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer’s disease (AD). Analysis of Aβ metabolism showed that, despite reduced Aβ clearance due to LRP1 inactivation in vivo, less Aβ was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aβ-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aβ and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aβ levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1’s endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aβ generation overrules the simultaneous impaired Aβ clearance, resulting in less extracellular Aβ and reduced plaque deposition in a mouse model of AD. Electronic supplementary material The online version of this article (10.1007/s12035-019-1594-2) contains supplementary material, which is available to authorized users.

Objective-The purpose of this study was to determine the significance of the intracellular NPxYxxL motif of LRP1 for the atheroprotective role of this multifunctional receptor. Methods and Results-LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with LDLR-deficient mice, a model for atherosclerosis. In this LDLR Ϫ/Ϫ background the mutated mice showed a more atherogenic lipoprotein profile, which was associated with a decreased clearance of postprandial lipids because of a compromised endocytosis rate and reduced lipase activity. On an atherogenic diet LRP1 mutant mice revealed a 50% increased development of atherosclerosis. This aggravation was accompanied by an increase in smooth muscle cell (SMC) and collagen content and apoptotic cells in the lesions. The mutation showed, however, a limited impact on basal PDGFR-␤ expression and signaling and the antimigratory property of apoE on PDGF-BB-stimulated SMCs. Additionally, levels of LRP1 atherogenic ligands, like MMP2, t-PA, FVIII, and the inflammatory ligand TNF-␣ showed to be significantly elevated.Conclusion-These findings demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1. This motif is relevant for normal control of lipid metabolism and of atherogenic and inflammatory ligands, but has no pronounced effect on regulating PDGF-BB/PDGFR-␤ signaling in SMCs. Key Words: atherosclerosis Ⅲ growth factors Ⅲ immune system Ⅲ lipases Ⅲ lipids T he large endocytic receptor low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a multifunctional protein that binds multiple extracellular ligands including apolipoprotein E (apoE) containing lipoproteins, lipoprotein lipase, complexes of proteinases-proteinase-inhibitors, hormones, matrix proteins, and growth factors like plateletderived growth factor (PDGF; reviewed by Herz 1 ). This 600 kDa receptor is proteolytically cleaved by a furin-like endoprotease into 2 subunits of 515 kDa and 85 kDa. The very large extracellular ␣-subunit contains the ligand-binding domains and is noncovalent associated with the smaller ␤-subunit, containing an extracellular part, the membrane spanning domain and the cytoplasmatic or intracellular domain (LRP1-ICD).In several studies using conditional inactivation of LRP1 in mice, the receptor has been associated with a clear role in the pathogenesis of atherosclerosis. In the liver it has been shown that the receptor is important for the removal of atherogenic lipoproteins and other proatherogenic ligands from the circulation. 2,3 Boucher et al, moreover, showed that LRP1 has a cholesterol-independent role in atherosclerosis by modulating the activity and cellular localization of the PDGF receptor-␤ (PDGFR-␤) in vascular smooth muscle cells (SMCs). 4 Finally, 2 studies argue that LRP1 in macrophages has an effect on atherosclerosis through the modulation of the extracellular matrix and inflammatory responses. 5,6 In all these studies, the overall function of LRP1 in a specific cell type was disrupted. These app...

The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.