Ectodomain shedding of the amyloid precursor protein: Cellular control mechanisms and novel modifiers

Proteolytic cleavage in the ectodomain of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer's disease amyloid-beta (A beta) pepticle and occurs through two different protease activities termed alpha- and beta-secretase. Both proteases compete for APP cleavage, but have opposite effects on A beta generation. At present, little is known about the cellular pathways that control APP alpha- or beta-secretase cleavage and thus A beta generation. To explore the contributory pathways in more detail we have recently employed an expression cloning screen and identified several activators of APP cleavage by alpha- or beta-secretase. Among them were known activators of APP cleavage, for example protein kinase A, and novel activators, such as endophilin and the APP homolog amyloid precursor-like protein 1 (APLP1). Mechanistic analysis revealed that both endophilin and APLP1 reduce the rate of APP endocytosis and strongly increase APP cleavage by alpha-secretase. This review summarizes the results of the expression cloning screen in the context of recent developments in our understanding of the cellular regulation of APP alpha-secretase cleavage. Moreover, it highlights the particular importance of endocytic APP trafficking as a prime modulator of APP shedding. Copyright (c) 2006 S. Karger AG, Basel

The Membrane-Bound Aspartyl Protease BACE1: Molecular and Functional Properties in Alzheimer’s Disease and Beyond

The β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease involved in Alzheimer’s disease (AD) pathogenesis and in myelination. BACE1 initiates the generation of the pathogenic amyloid β-peptide, which makes BACE1 a major drug target for AD. BACE1 also cleaves and activates neuregulin 1, thereby contributing to postnatal myelination, in particular in the peripheral nervous system. Additional proteins are also cleaved by BACE1, but less is known about the physiological consequences of their cleavage. Recently, new phenotypes were described in BACE1-deficient mice. Although it remains unclear through which BACE1 substrates they are mediated, the phenotypes suggest a versatile role of this protease for diverse physiological processes. This review summarizes the enzymatic and cellular properties of BACE1 as well as its regulation by lipids, by transcriptional, and by translational mechanisms. The main focus will be on the recent progress in understanding BACE1 function and its implication for potential mechanism-based side effects upon therapeutic inhibition

Cell Type-Specific Human APP Transgene Expression by Hippocampal Interneurons in the Tg2576 Mouse Model of Alzheimer’s Disease

Amyloid precursor protein (APP) transgenic animal models of Alzheimer’s disease have become versatile tools for basic and translational research. However, there is great heterogeneity of histological, biochemical, and functional data between transgenic mouse lines, which might be due to different transgene expression patterns. Here, the expression of human APP (hAPP) by GABAergic hippocampal interneurons immunoreactive for the calcium binding proteins parvalbumin, calbindin, calretinin, and for the peptide hormone somatostatin was analyzed in Tg2576 mice by double immunofluorescent microscopy. Overall, there was no GABAergic interneuron subpopulation that did not express the transgene. On the other hand, in no case all neurons of such a subpopulation expressed hAPP. In dentate gyrus molecular layer and in stratum lacunosum moleculare less than 10% of hAPP-positive interneurons co-express any of these interneuron markers, whereas in stratum oriens hAPP-expressing neurons frequently co-express these interneuron markers to different proportions. We conclude that these neurons differentially contribute to deficits in young Tg2576 mice before the onset of Abeta plaque pathology. The detailed analysis of distinct brain region and neuron type-specific APP transgene expression patterns is indispensable to understand particular pathological features and mouse line-specific differences in neuronal and systemic functions

Niemann Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway

The link between cholesterol and Alzheimer’s disease has recently been revealed in Niemann Pick type C disease. We found that NPC1-/- cells show decreased expression of APP at the cell surface and increased processing of APP through the β-secretase pathway resulting in increased C99, sAPPβ and intracellular Aβ40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Aβ/C99 levels in NPC1-/- cells to the levels observed in wt cells. Finding that overexpression of C99, a direct gamma-secretase substrate, does not lead to increased intracellular Aβ levels in NPC1-/- cells vs. CHOwt suggests that the effect on intracellular Aβ upon cholesterol accumulation in NPC1-/- cells is not due to increased APP cleavage by gamma-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and, thus, its cleavage by β-secretase

Nonsteroidal Anti-Inflammatory Drugs and Ectodomain Shedding of the Amyloid Precursor Protein

Background: Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer's disease (AD). Several mechanisms have been proposed to explain these findings including increased shedding of the soluble ectodomain of the amyloid precursor protein (sAPP), which functions as a neurotrophic and neuroprotective factor in vitro and in vivo. Objective: To clarify whether NSAIDs consistently stimulate sAPP secretion. Methods: 293-EBNA cells with stable overexpression of an APP-alkaline phosphatase fusion protein (APP-AP), SH-SY5Y and PC12 cells or primary telencephalic chicken neurons were treated with ibuprofen or indomethacin. APP shedding was then determined by measuring AP activity in conditioned media, Western blot analysis with antibodies against total sAPP or specific for sAPP-alpha, or in a pulse-chase paradigm. Results: AP activity in conditioned media was not increased after NSAID treatment of 293-EBNA cells whereas it was elevated by phorbol ester. Surprisingly, ibuprofen or indomethacin treatment of SH-SY5Y and PC12 cells expressing endogenous APP did not cause changes in sAPP or sAPP-alpha secretion or downregulation of cellular APP. These findings were further corroborated in primary chicken neuronal cultures. Conclusions: Using various experimental settings, we were unable to confirm sAPP or sAPP-alpha stimulation with the NSAIDs ibuprofen and indomethacin in transfected and nontransfected cells of neuronal and nonneuronal origin. Importantly, these findings seem to rule out chronic sAPP stimulation as an alternative mechanism of NSAID action in AD. Copyright (C) 2008 S. Karger AG, Base

Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used “surface-spanning enrichment with click-sugars (SUSPECS)” proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding

LncRNA RUS shapes the gene expression program towards neurogenesis

The evolution of brain complexity correlates with an increased expression of long, noncoding (lnc) RNAs in neural tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during brain development. We present a first functional characterization of the lncRNA LINC01322, which we term RUS for RNA upstream of Slitrk3. The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation resulting in arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner

BACE2 distribution in major brain cell types and identification of novel substrates

β-Site APP-cleaving enzyme 1 (BACE1) inhibition is considered one of the most promising therapeutic strategies for Alzheimer's disease, but current BACE1 inhibitors also block BACE2. As the localization and function of BACE2 in the brain remain unknown, it is difficult to predict whether relevant side effects can be caused by off-target inhibition of BACE2 and whether it is important to generate BACE1-specific inhibitors. Here, we show that BACE2 is expressed in discrete subsets of neurons and glia throughout the adult mouse brain. We uncover four new substrates processed by BACE2 in cultured glia: vascular cell adhesion molecule 1, delta and notch-like epidermal growth factor-related receptor, fibroblast growth factor receptor 1, and plexin domain containing 2. Although these substrates were not prominently cleaved by BACE2 in healthy adult mice, proinflammatory TNF induced a drastic increase in BACE2-mediated shedding of vascular cell adhesion molecule 1 in CSF. Thus, although under steady-state conditions the effect of BACE2 cross-inhibition by BACE1-directed inhibitors is rather subtle, it is important to consider that side effects might become apparent under physiopathological conditions that induce TNF expression

Prion Replication in the Mammalian Cytosol: Functional Regions within a Prion Domain Driving Induction, Propagation, and Inheritance

Prions of lower eukaryotes are transmissible protein particles that propagate by converting homotypic soluble proteins into growing protein assemblies. Prion activity is conferred by so-called prion domains, regions of low complexity that are often enriched in glutamines and asparagines (Q/N). The compositional similarity of fungal prion domains with intrinsically disordered domains found in many mammalian proteins raises the question of whether similar sequence elements can drive prion-like phenomena in mammals. Here, we define sequence features of the prototype Saccharomyces cerevisiae Sup35 prion domain that govern prion activities in mammalian cells by testing the ability of deletion mutants to assemble into self-perpetuating particles. Interestingly, the amino-terminal Q/N-rich tract crucially important for prion induction in yeast was dispensable for the prion life cycle in mammalian cells. Spontaneous and template-assisted prion induction, growth, and maintenance were preferentially driven by the carboxy-terminal region of the prion domain that contains a putative soft amyloid stretch recently proposed to act as a nucleation site for prion assembly. Our data demonstrate that preferred prion nucleation domains can differ between lower and higher eukaryotes, resulting in the formation of prions with strikingly different amyloid cores

Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression

Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (alpha Syn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study alpha Syn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different alpha Syn constructs: alpha Syn, alpha Syn coupled to the fluorescence protein Venus (alpha Syn-Venus), and alpha Syn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of alpha Syn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly alpha Syn-overexpressing cell lines holding high potential for the screening for modulators of alpha Syn aggregation and alpha Syn-mediated toxicity