Ragna Sannerud

ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1.

Amyloid β (Aβ) peptides, the primary constituents of senile plaques and a hallmark in Alzheimers disease pathology, are generated through the sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. The early endosome is thought to represent a major compartment for APP processing; however, the mechanisms of how BACE1 encounters APP are largely unknown. In contrast to APP internalization, which is clathrin-dependent, we demonstrate that BACE1 is sorted to early endosomes via a route controlled by the small GTPase ADP ribosylation factor 6 (ARF6). Altering ARF6 levels or its activity affects endosomal sorting of BACE1, and consequently results in altered APP processing and Aβ production. Furthermore, sorting of newly internalized BACE1 from ARF6-positive towards RAB GTPase 5 (RAB5)-positive early endosomes depends on its carboxyterminal short acidic cluster-dileucine motif. This ARF6-mediated sorting of BACE1 is confined to the somatodendritic compartment of polarized neurons in agreement with Aβ peptides being primarily secreted from here. These results demonstrate a spatial separation between APP and BACE1 during surface-to-endosome transport, suggesting subcellular trafficking as a regulatory mechanism for this proteolytic processing step. It thereby provides a novel avenue to interfere with Aβ production through a selective modulation of the distinct endosomal transport routes used by BACE1 or APP.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimers disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Trafficking, a key player in regulated intramembrane proteolysis

The term regulated intramembrane proteolysis (RIP) emerged from converging mechanisms aiming to release or activate signaling fragments or transcription factors from their respective membrane-anchored precursors. To date, four families of intramembrane proteases exist each of which process their own specific target substrates. As such, RIP initiates or abrogates a multitude of signaling cascades and plays a pivotal role in many physiological processes. The spatial and temporal localization of substrates versus proteases is of major importance in the diverse regulation of intramembrane proteolysis. Here we highlight the exciting conjunction between intracellular transport and RIP through the example of the routes taken by APP and its associated proteases.

A fast growing spectrum of biological functions of γ-secretase in development and disease ☆

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already >15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimers disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1.

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC50 ∼0.76 nm), in primary neurons (EC50 ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC50 ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299–312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299–312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.

ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation

Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule‐5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP‐ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia‐to‐spine transition, and requires Rac1‐mediated dephosphorylation/release of actin‐binding ERM proteins from TLN. At the somato‐dendritic surface, TLN and EFA6A are confined to distinct, flotillin‐positive membrane subdomains. The co‐distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63‐positive late endosomes. This suggests a specific microenvironment facilitating ARF6‐mediated mobilization of TLN that contributes to promotion of dendritic spine development.

Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing

Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.

Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments

BackgroundThe mechanisms behind Aβ-peptide accumulation in non-familial Alzheimer’s disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aβ production by interacting to γ-secretase.MethodsWe searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo.ResultsTetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aβ levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF.ConclusionsTSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.

Peptides based on the presenilin-APP binding domain inhibit APP processing and Aβ production through interfering with the APP transmembrane domain

Presenilins (PSENs) form the catalytic component of the γ‐secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1‐binding domain in APP, encompassing half of the transmembrane domain following the amyloid β (Aβ) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aβ generation through interfering with enzyme‐substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aβ production (IC50: 317 nM) in cell‐free and cell‐based assays using APP‐carboxy terminal fragment as a direct γ‐secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ‐cleavage, illustrating specificity and potential therapeutic value. In cell‐based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ‐secretase, most likely through steric hindrance.—Esselens, C., Sannerud, R., Gallardo, R., Baert, V., Kaden, D., Serneels, L., De Strooper, B., Rousseau, F., Multhaup, G., Schymkowitz, J., Langedijk, J. P. M., Annaert, W. Peptides based on the presenilin‐APP binding domain inhibit APP processing and Aβ production through interfering with the APP transmembrane domain. FASEB J. 26, 3765–3778 (2012). www.fasebj.org

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P724-L725-C726, is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.