Daniel Schwanzar

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.

Globular and Protofibrillar Aβ Aggregates Impair Neurotransmission by Different Mechanisms

In Alzheimers disease, substantial evidence indicates the causative role of soluble amyloid β (Aβ) aggregates. Although a variety of Aβ assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aβ aggregates. Here, we used different protocols for the establishment of prefibrillar Aβ assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aβ aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aβ assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aβ assemblies, based on the stability of the applied Aβ aggregates in these settings. In contrast to monomeric or fibrillar Aβ, different prefibrillar Aβ aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aβ aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aβ aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aβ assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of prefacto characterized Aβ aggregates in assigning a biological effect.

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

Humanin (HN) is a linear 24‐aa peptide recently detected in human Alzheimers disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß‐amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1–40) and characterization of the interaction structure through a molecular modeling study. Wild‐type HN and HN‐sequence mutations were synthesized by SPPS and the HPLC‐purified peptides characterized by MALDI‐MS. The interaction epitopes between HN and Aß(1–40) were identified by affinity‐MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity‐bound peptides. The affinity‐MS analyses revealed HN(5–15) as the epitope sequence of HN, whereas Aß(17–28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1–40) and by ELISA with Aß(1–40) and Aß‐partial sequences as ligands to immobilized HN. The specificity and affinity of the HN‐Aß interaction were characterized by direct ESI‐MS of the HN‐Aß(1–40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a KD of the complex of 610 n m. A molecular dynamics simulation of the HN‐Aß(1–40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1–40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN. Copyright

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.

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.

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.

Investigation of alternative binding events between the Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family and BACE1 and its influence on the processing of amyloid precursor protein (APP)

the microarray analysis, we surveyed the binding sites of transcription factors, using the database produced by UCSC Genome Bioinformatics. Results: The increase in the DNA-binding activity of NF-kappa B and AP-1 after exposure to Abeta fibrils was suppressed by PARP-1 inhibitor, 1,5-dihydroxyisoquinoline (DHIQ), and also PARP-1 siRNA. The microarray analysis demonstrated that among 31,042 probes used, 345 and 224 probe sets showed up-regulation and down-regulation, respectively, in the astrocytes after exposure to Abeta fibrils. Furthermore, 87 probe sets showed down-regulation, while only three probe sets showed up-regulation, after addition of DHIQ. Upstream and downstream of the genes detected by these probe sets, the DNA-binding sites of other transcription factors than NF-kappa B and AP-1 were identified. Conclusions: PARP-1 plays an important role in the change of gene expression profile of astrocytes after exposure to A beta fibrils through the activation of a variety of transcription factors. By regulation of these factors, PARP-1 inhibitors could be new therapeutic and/or neuroprotective agents for Alzheimer’s disease.

Interaction of Engulfment Adaptor Protein (GULP) and Amyloid Precursor Protein (APP) decreases Aβ40/42 secretion

Background: The frequent co-occurrence of Alzheimer’s disease (AD) pathology in patients with clinically diagnosed normal pressure hydrocephalus suggests a possible link between ventricular dilation and AD. If enlarging ventricles serve as a marker of potentially faulty cerebrospinal fluid (CSF) clearance mechanisms, then a relationship may be demonstrable between increasing ventricular volume and decreasing levels of A-beta in CSF in preclinical and early AD. Methods: CSF biomarker data (A-beta, tau, and phosphorylated tau) as well as direct measurements of whole brain and ventricular volume data were obtained from the Alzheimer’s Disease Neuroimaging Initiative dataset. The ratio of ventricular volume to whole brain volume was derived as a secondary independent measure. Baseline data were used for the group analyses of 819 subjects classified as being either normal (n1⁄4229), having the syndrome of mild cognitive impairment (n1⁄4397), or mild AD (n1⁄4193). Linear regression models were derived for each biomarker as the dependent variable, using the MRI volume measures and age as independent variables. Subjects in each diagnostic group were further stratified by ApoE genotype. Results: For controls, ventricular volume was negatively associated with A-beta in E4 positive subjects, independent of age. Ventricular volume as well as age was negatively associated with tau in E4 negative control subjects. A significant reversed pattern was seen in AD subjects, in whom ventricular volume was negatively associated with tau, but not A-beta, in E4 positive subjects, independent of age. Abeta and tau were not significantly related to whole brain volume in either controls or AD; however, tau was negatively related to whole brain volume in MCI subjects. Conclusions: Increased ventricular volume may be associated with decreased levels of CSF A-beta in preclinical AD. The basis for the apparent effect of ApoE4 genotype on the relationship of ventricular volume to A-beta and tau levels is unknown, but could involve altered CSF-bloodbrain barrier function early in the course of the disease. Further experimental evidence with animal models of hydrocephalus and ApoE may shed light on the exact nature of these relationships.

P1-461: The role of cholesterol level alterations upon APP and BACE proximity and APP cleavage

tion, *P 0.001 vs. Control). The MMP inhibitors did not have direct antioxidant effects. Treatment of animals with the antioxidant PBN or MMP inhibitor minocycline did not have a significant effect on CAA progression rates measured with longitudinal imaging when compared to untreated mice (untreated 0.51%, PBN 0.51, minocycline 0.54 % vessel segment affected per week). Conclusions: These data offer insights on the molecular pathways activated by vascular amyloid and suggest a close association between A -related MMP activation and ROS generation in vivo. They also raise the possibility that therapeutic treatment with MMP inhibitors may have beneficial effects in the A amyloidosis by indirectly reducing the oxidative stress associated with CAA Acknowledgements: NIH EB000768, AG020570, AG024688, AG021084, Harvard Neurodiscovery Center (R.A.B.), Leffler fellowship (CP), Bugher Foundation fellowship (M.G-A).