Expression of an alternatively spliced variant of SORL1 in neuronal dendrites is decreased in patients with Alzheimer's disease.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Reelin-Dab1 signaling system in human colorectal cancer.

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-ß1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

Sorting receptor SORLA: cellular mechanisms and implications for disease.

Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor that directs cargo proteins, such as kinases, phosphatases, and signaling receptors, to their correct location within the cell. The activity of SORLA assures proper function of cells and tissues, and receptor dysfunction is the underlying cause of common human malignancies, including Alzheimer's disease, atherosclerosis, and obesity. Here, we discuss the molecular mechanisms that govern sorting of SORLA and its cargo in multiple cell types, and why genetic defects in this receptor results in devastating diseases.

Immunohistochemical analysis of transporters related to clearance of amyloid-ß peptides through blood-cerebrospinal fluid barrier in human brain.

A large number of previous reports have focused on the transport of amyloid-ß peptides through cerebral endothelial cells via the blood-brain barrier, while fewer reports have mentioned the transport through the choroid plexus epithelium via the blood-cerebrospinal fluid barrier. Concrete roles of these two pathways remain to be clarified. In this study, we immunohistochemically examined the expression of transporters/receptors that are supposed to be related to the clearance of amyloid-ß peptides in the choroid plexus epithelium, the ventricular ependymal cells and the brain microvessels, using seven autopsied human brains. In the choroid plexus epithelium, immunoreactivity for low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), LRP2, formylpeptide receptor-like 1 (FPRL1), ATP-binding cassette (ABC) transporter-A1 (ABCA1), ABCC1 and ABCG4 was seen in 7 of 7 brains, while that for ABCB1, ABCG2, RAGE and CD36 was seen in 0-2 brains. In the ventricular ependymal cells, immunoreactivity for CD36, LDLR, LRP1, LRP2, FPRL1, ABCA1, ABCC1 and ABCG4 was seen in 6-7 brains, while that for ABCB1, ABCG2 and RAGE was seen in 0-1 brain. Immunoreactivity for insulin-degrading enzyme (IDE) was seen in three and four brains in the choroid plexus epithelium and the ventricular ependymal cells, respectively. In addition, immunoreactivity for LDLR, ABCB1 and ABCG2 was seen in over 40 % of the microvessels (all seven brains), and that for FPRL1, ABCA1, ABCC1 and RAGE was seen in over 5 % of the microvessels (4-6 brains), while that for CD36, IDE, LRP1, LRP2 and ABCG4 was seen in less than 5 % of the microvessels (0-2 brains). These findings may suggest that these multiple transporters/receptors and IDE expressed on the choroid plexus epithelium, ventricular ependymal cells and brain microvessels complementarily or cooperatively contribute to the clearance of amyloid-ß peptides from the brain.

In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR.

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimer's disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure.

The Vps10p-domain receptor family.

The family of mammalian type-I transmembrane receptors containing a Vps10p domain contains five members, Sortilin, SorCS1, SorCS2, SorCS3, and SorLA. The common characteristic of these receptors is an N-terminal Vps10p domain, which either represents the only module of the luminal/extracellular moiety or is combined with additional domains. Family members play roles in protein transport and signal transduction. The individual receptors bind and internalize a variety of ligands, such as neuropeptides and trophic factors, and Sortilin and SorLA mediate trans-Golgi network-to-endosome sorting. Their prominent neuronal expression, several of the identified ligands, and recent results support the notion that members of this receptor family have important functions in neurogenesis, plasticity-related processes, and functional maintenance of the nervous system. For instance, it has been demonstrated that Sortilin partakes in the transduction of proapoptotic effects, and there is converging biochemical and genetic evidence that implies that SorLA is an Alzheimer's disease risk factor.

Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.

Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration.

Migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. Here, we show that efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. Our results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP).

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.