Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx17 expression by short interfering RNA. We conclude that Snx17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.

Accumulation of extracellular amyloid ␤ peptide (A␤), generated from amyloid precursor protein (APP) processing by ␤-and ␥-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of A␤ from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in A␤ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steadystate levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to A␤. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to A␤.Mounting evidence has demonstrated that proteolytic processing of the amyloid precursor protein (APP) 4 is central to the pathogenesis of Alzheimer disease (AD) (1, 2). Many reports have shown that APP processing to A␤ is greatly affected by the subcellular localization of APP, presumably because of the specific subcellular localizations of ␤-and ␥-secretases (3). Both transmembrane receptors and cytoplasmic adaptor proteins have been shown to interact with APP and affect its trafficking. The low-density lipoprotein receptor-related protein 1 (LRP1) increases APP endocytosis and A␤ production (4), whereas SorLA decreases APP processing to A␤ by shuttling APP away from endosomes (5). Several cell surface adaptor proteins, including Fe65, X11, and Dab1, bind to the NPXY motif within the APP cytoplasmic domain and regulate its trafficking and processing to A␤ (6 -8). By overexpression or knockdown, Fe65 has been shown to affect APP processing to A␤ (9, 10). Although Dab1 has been shown to affect APP processing and A␤ production (11), the function of its homologue Dab2 in APP trafficking and processing to A␤ has not been studied (8). These studies firmly establish that APP-interacting proteins can both positively and negatively affect A␤ production by altering APP trafficking through ␤-and ␥-secretase-containing compartments. Revealing the mechanisms by which intracellular trafficking of APP is regulated may permit the development of novel therapeutic approaches for AD.Sorting nexin 17 (SNX17) is a member of the sorting nexin family characteri...

BackgroundThe glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability.MethodsSingle-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency).ResultsAll cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type–specific and injury type–specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis.ConclusionsGeneration of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.

2-tailed Student's t test or log-rank test. P < 0.05 was considered significant. Serial glucose and GTT for streptozotocin-treated WT and knockout mice ( Figure 7) were compared using multiple unpaired t tests and corrected for multiple comparisons using the Šidák-Bonferroni method, with α of 0.05.Study approval. Animal studies were approved by the Institutional Animal Care and Use Committee

Sorting nexin 17 (SNX17) is an adaptor protein present in EEA1-positive sorting endosomes that promotes the efficient recycling of low-density lipoprotein receptor-related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1-positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17-binding domain, we generated chimeric proteins in which the SNX17-binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non-polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized MDCK cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17-binding receptors and the restricted function of SNX17 in the BSE.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'--methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. However, it is not known whether snoRNA secretion is regulated or whether secreted snoRNAs are functional. Here, we show that inflammation stimulates secretion of snoRNAs U32a (SNORD32a), U33 (SNORD33), U34 (SNORD34), and U35a (SNORD35a) from cultured macrophages, in mice, and in human subjects. Secreted snoRNAs co-fractionate with extracellular vesicles and are taken up by recipient cells. In a murine parabiosis model, we demonstrate that snoRNAs travel through the circulation to function in distant tissues. These findings support a previously unappreciated link between inflammation and snoRNA secretion in mice and humans and uncover a potential role for secreted snoRNAs in cell-cell communication.

Small nucleolar RNAs (snoRNAs) guide chemical modifications of ribosomal and small nuclear RNAs, functions that are carried out in the nucleus. Although most snoRNAs reside in the nucleolus, a growing body of evidence indicates that snoRNAs are also present in the cytoplasm and that snoRNAs move between the nucleus and cytoplasm by a mechanism that is regulated by lipotoxic and oxidative stress. Here, in a genome-wide shRNA-based screen, we identified nuclear export factor 3 (NXF3) as a transporter that alters the nucleocytoplasmic distribution of box C/D snoRNAs from the ribosomal protein L13a () locus. Using RNA-sequencing analysis, we show that NXF3 associates not only with snoRNAs, but also with a broad range of box C/D and box H/ACA snoRNAs. Under homeostatic conditions, gain- or loss-of-function of NXF3, but not related family member NXF1, decreases or increases cytosolic snoRNAs, respectively. Furthermore, treatment with the adenylyl cyclase activator forskolin diminishes cytosolic localization of the snoRNAs through a mechanism that is dependent on NXF3 but not NXF1. Our results provide evidence of a new role for NXF3 in regulating the distribution of snoRNAs between the nuclear and cytoplasmic compartments.