Bjoern von Einem

Impact of cholesterol level upon APP and BACE proximity and APP cleavage

Cleavage of APP by BACE is the first proteolytic step in the production of Amyloid beta (Abeta, which accumulates in senile plaques in Alzheimers disease. BACE-cleavage of APP is thought to happen in endosomes. However, there are controversial data whether APP and BACE can already interact on the cell surface dependent on the cholesterol level. To examine whether APP and BACE come into close proximity on the cell surface in living cells, we employed a novel technique by combining time-resolved Förster resonance energy transfer (FRET) measurements with total internal reflection microscopy (TIRET microscopy). Our data indicate that BACE and APP come into close proximity within the cell, but probably not on the cell surface. To analyze the impact of alterations in cholesterol level upon BACE-cleavage, we measured sAPP secretion. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity.

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1.

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Abeta), which accumulates in senile plaques in Alzheimers disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for beta-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPbeta, but not sAPPalpha. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that--in addition to its role in APP trafficking--LRP1 affects APP processing by competing for cleavage by BACE1.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases.

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1).

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

Engulfment adapter PTB domain containing 1 interacts with and affects processing of the amyloid-β precursor protein

Previous studies identified engulfment adapter phosphotyrosine binding (PTB) domain containing 1 (GULP1) as an NPXY-motif interactor of low-density lipoprotein receptor-related protein 1 (LRP1) and suggested a potential relevance in Alzheimers disease (AD). Since AD associated proteins amyloid-β A4 precursor protein (APP) and LRP1 were shown to interact with the PTB domain of Fe65 and several other adapters via their intracellular NPXY-motifs, we examined a possible interaction of GULP1 PTB domain with the YENPTY-motif of APP. Here we demonstrate that GULP1 is present in human hippocampal and neocortical neurons. Confocal live cell imaging revealed that coexpressed and endogenous GULP1 colocalizes with APP in the Golgi and endoplasmic reticulum. Analysis of the interacting domains by co-immunoprecipitation of point and deletion mutants revealed that the interaction depends on the PTB domain of GULP1 and the YENPTY-motif of APP. Coexpression of GULP1 affected APP cell surface localization and suppressed generation of Aβ40/42 and sAPPα. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.

The Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family alters alpha synuclein (α-syn) oligomerization and secretion

Several age-related neurodegenerative disorders are associated with protein misfolding and aggregation of toxic peptides. α-synuclein (α-syn) aggregation and the resulting cytotoxicity is a hallmark of Parkinsons disease (PD) as well as dementia with Lewy bodies. Rising evidence points to oligomeric and pre-fibrillar forms as the pathogenic species, and oligomer secretion seems to be crucial for the spreading and progression of PD pathology. Recent studies implicate that dysfunctions in endolysosomal/autophagosomal pathways increase α-syn secretion. Mutation in the retromer-complex protein VPS35, which is involved in endosome to Golgi transport, was suggested to cause familial PD. GGA proteins regulate vesicular traffic between Golgi and endosomes and might work as antagonists for retromer complex mediated transport. To investigate the role of the GGAs in the α-syn oligomerization and/or secretion process we utilized protein-fragment complementation assays (PCA). We here demonstrate that GGAs alter α-syn oligomer secretion and α-syn oligomer-mediated toxicity. Specifically, we determined that GGA3 modifies extracellular α-syn species in an exosome-independent manner. Our data suggest that GGA3 drives α-syn oligomerization in endosomal compartments and thus facilitates α-syn oligomer secretion. Preventing the early events in α-syn oligomer release may be a novel approach to halt disease spreading in PD and other synucleinopathies.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer’s disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1−/−), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1−/− cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1−/− cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1−/− cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1−/−. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Mitochondrial matrix pH as a decisive factor in neurometabolic imaging

Abstract. Alterations of cellular bioenergetics are a common feature in most neurodegenerative disorders. However, there is a selective vulnerability of different brain regions, cell types, and even mitochondrial populations to these metabolic disturbances. Thus, the aim of our study was to establish and validate an in vivo metabolic imaging technique to screen for mitochondrial function on the subcellular level. Based on nicotinamide adenine dinucleotide (phosphate) fluorescence lifetime imaging microscopy [NAD(P)H FLIM], we performed a quantitative correlation to high-resolution respirometry. Thereby, we revealed mitochondrial matrix pH as a decisive factor in imaging NAD(P)H redox state. By combining both parameters, we illustrate a quantitative, high-resolution assessment of mitochondrial function in metabolically modified cells as well as in an amyloid precursor protein-overexpressing model of Alzheimer’s disease. Our metabolic imaging technique provides the basis for dissecting mitochondrial deficits not only in a range of neurodegenerative diseases, shedding light onto bioenergetic failures of cells remaining in their metabolic microenvironment.

sAPPβ and sAPPα increase structural complexity and E/I input ratio in primary hippocampal neurons and alter Ca 2+ homeostasis and CREB1-signaling

ABSTRACT One major pathophysiological hallmark of Alzheimers disease (AD) is senile plaques composed of amyloid &bgr; (A&bgr;). In the amyloidogenic pathway, cleavage of the amyloid precursor protein (APP) is shifted towards A&bgr; production and soluble APP&bgr; (sAPP&bgr;) levels. A&bgr; is known to impair synaptic function; however, much less is known about the physiological functions of sAPP&bgr;. The neurotrophic properties of sAPP&agr;, derived from the non‐amyloidogenic pathway of APP cleavage, are well‐established, whereas only a few, conflicting studies on sAPP&bgr; exist. The intracellular pathways of sAPP&bgr; are largely unknown. Since sAPP&bgr; is generated alongside A&bgr; by &bgr;‐secretase (BACE1) cleavage, we tested the hypothesis that sAPP&bgr; effects differ from sAPP&agr; effects as a neurotrophic factor. We therefore performed a head‐to‐head comparison of both mammalian recombinant peptides in developing primary hippocampal neurons (PHN). We found that sAPP&agr; significantly increases axon length (p=0.0002) and that both sAPP&agr; and sAPP&bgr; increase neurite number (p<0.0001) of PHN at 7days in culture (DIV7) but not at DIV4. Moreover, both sAPP&agr;‐ and sAPP&bgr;‐treated neurons showed a higher neuritic complexity in Sholl analysis. The number of glutamatergic synapses (p<0.0001), as well as layer thickness of postsynaptic densities (PSDs), were significantly increased, and GABAergic synapses decreased upon sAPP overexpression in PHN. Furthermore, we showed that sAPP&agr; enhances ERK and CREB1 phosphorylation upon glutamate stimulation at DIV7, but not DIV4 or DIV14. These neurotrophic effects are further associated with increased glutamate sensitivity and CREB1‐signaling. Finally, we found that sAPP&agr; levels are significantly reduced in brain homogenates of AD patients compared to control subjects. Taken together, our data indicate critical stage‐dependent roles of sAPPs in the developing glutamatergic system in vitro, which might help to understand deleterious consequences of altered APP shedding in AD patients, beyond A&bgr; pathophysiology. HIGHLIGHTSsAPP&agr; and sAPP&bgr; have a critical stage‐dependent role in the developing glutamatergic system in vitro.sAPP&bgr; impacts in a different manner on the developing glutamatergic system compared to sAPP&agr;.Effects of sAPP&agr; are associated with CREB‐1 signaling.

NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research: NADH Autofluorescence

More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker‐free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research.