María Paz Marzolo

Adaptor Protein Sorting Nexin 17 Regulates Amyloid Precursor Protein Trafficking and Processing in the Early Endosomes

Accumulation of extracellular amyloid β peptide (Aβ), generated from amyloid precursor protein (APP) processing by β- and γ-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Aβ 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 Aβ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state 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 Aβ. 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 Aβ.

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains.

Megalin and the low‐density lipoprotein (LDL) receptor‐related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin‐Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand‐binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC‐PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand‐binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts.

Expression of α2‐macroglobulin receptor/low density lipoprotein receptor‐related protein (LRP) in rat microglial cells

Low density lipoprotein receptor‐related protein (LRP) participates in the uptake and degradation of several ligands implicated in neuronal pathophysiology including apolipoprotein E (apoE), activated α2 ‐macroglobulin (α2M*) and β‐amyloid precursor protein (APP). The receptor is expressed in a variety of tissues. In the brain LRP is present in pyramidal‐type neurons in cortical and hippocampal regions and in astrocytes that are activated as a result of injury or neoplasmic transformation. As LRP is expressed in the monocyte/macrophage cell system, we were interested in examining whether LRP is expressed in microglia. We isolated glial cells from the brain of neonatal rats and LRP was immunodetected both in microglial cells and in astrocytes expressing glial fibrillar acidic protein (GFAP). Microglial cells were able to bind and internalize LRP‐specific ligand, α2M*. The internalization was inhibitable by RAP, with a Kd of 1.7 nM. The expression of LRP was up‐regulated by dexamethasone, and down‐regulated by lipopolysaccharide (LPS), gamma interferon (IFN‐γ) or a combination of both. LRP was less sensitive to dexamethasone in activated astrocytes than in microglia. We provided the first analysis of LRP expression and regulation in microglia. Our results open the possibility that microglial cells could be related to the participation of LRP and its ligands in different pathophysiological states in brain. J. Neurosci. Res. 60:401–411, 2000

ApoER2 expression increases Aβ production while decreasing Amyloid Precursor Protein (APP) endocytosis: Possible role in the partitioning of APP into lipid rafts and in the regulation of γ-secretase activity

BackgroundThe generation of the amyloid-β peptide (Aβ) through the proteolytic processing of the amyloid precursor protein (APP) is a central event in the pathogenesis of Alzheimers disease (AD). Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R) family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing.ResultsHere, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Aβ production and reduced levels of APP-CTFs. The increased Aβ production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased γ-secretase activity, both of which might contribute to increased Aβ production.ConclusionThese findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Aβ production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting β-secretase and γ-secretase mediated amyloidogenic processing and also by incrementing the activity of γ-secretase.

ApoER2 is Endocytosed by a Clathrin-Mediated Process Involving the Adaptor Protein Dab2 Independent of its Rafts' Association

The apolipoprotein E receptor 2 (apoER2) is a member of the low‐density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin‐1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin‐mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin‐mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptors endocytosis. Neither the transmembrane nor the proline‐rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin‐mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin‐mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis.

Mannose receptor is present in a functional state in rat microglial cells.

We studied the expression of the mannose receptor (ManR) in rat microglial cells. Microglial cells are the central nervous system resident macrophages, key participants of the innate immune response. ManR is a differentiation marker and a relevant glycoprotein for the phagocytic and endocytic function of macrophages. Because there is evidence suggesting that ManR could mediate some of the nonenzymatic effects of acetilcholinesterase (AchE) and the enzyme seems to be involved in Alzheimers disease (AD), we looked for ManR in microglia, evaluating the functionality of the receptor. We isolated microglial cells from the brain of 2‐day‐old neonatal rats. Microglial cells, identified by their specific staining with the lectin Griffonia simplicifolia, expressed ManR, being detected by immunocytochemistry, Western blot, and immunoprecipitation. Microglial ManR was downregulated by lipopolysaccharide (LPS) and upregulated by dexamethasone, as described for peripheral macrophages. Microglial ManR was functional and able to internalize horseradish peroxidase (HRP), a known ManR ligand, in a mannan‐inhibitable manner. The presence of a functional ManR in microglia opens the possibility that ManR could participate in multiple physiologic and pathologic conditions in the central nervous system (CNS), including inflammation, ischaemia, and neurodegerative diseases such as AD. J. Neurosci. Res. 58:387–395, 1999.

α-Secretase-derived Fragment of Cellular Prion, N1, Protects against Monomeric and Oligomeric Amyloid β (Aβ)-associated Cell Death

Background: Cellular prion undergoes α-secretase cleavage, yielding N1. We examined whether N1 protects against Aβ monomers and oligomers. Results: N1 protects against Aβ monomers and oligomers prepared from APP-London-expressing human cells and Alzheimer disease-affected brains. Conclusion: N1 could protect from Aβ-associated toxicity at the early asymptomatic phase of Alzheimer disease. Significance: These data emphasize the cross-talk between PrPc and βAPP catabolites. In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrPc) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APPLDN) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.

Protein Kinase D Regulates Trafficking of Dendritic Membrane Proteins in Developing Neurons

In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.

The low density lipoprotein receptor-related protein functions as an endocytic receptor for decorin

Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type β and other growth factors and thereby influences the processes of proliferation and differentiation in a wide array of physiological and pathological reactions. Hence, understanding the regulatory mechanisms of decorin activity has broad implications. Here we report that the extracellular levels of decorin are controlled by receptor-mediated catabolism, involving the low density lipoprotein receptor family member, low density lipoprotein receptor-related protein (LRP). We show that decorin is endocytosed and degraded by C2C12 myoblast cells and that both processes are blocked by suppressing LRP expression using short interfering RNA. The same occurs with CHO cells, but not with CHO cells genetically deficient in LRP. Finally, we show that LRP-null CHO cells, transfected to express mini-LRP polypeptides containing either the second or fourth LRP ligand-binding domains, carry out decorin endocytosis and lysosomal degradation. These findings point to LRP-mediated catabolism as a new control pathway for the biological activities of decorin, specifically for its ability to influence extracellular matrix signaling.

Polarized traffic of LRP1 involves AP1B and SNX17 operating on Y-dependent sorting motifs in different pathways.

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y(29) but not N(26) from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N(26)A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptors exit from BSE. An endocytic Y(63)ATL(66) motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL(86,87)), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes.