Globular and protofibrillar aß aggregates impair neurotransmission by different mechanisms.

In Alzheimer's disease, substantial evidence indicates the causative role of soluble amyloid ß (Aß) aggregates. Although a variety of Aß 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 Aß aggregates. Here, we used different protocols for the establishment of prefibrillar Aß 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 Aß 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 Aß assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aß assemblies, based on the stability of the applied Aß aggregates in these settings. In contrast to monomeric or fibrillar Aß, different prefibrillar Aß aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aß aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aß aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aß 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 Aß aggregates in assigning a biological effect.

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

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's 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 K(D) of the complex of 610 nm. 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.

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 Alzheimer's 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.

sAPPß and sAPPα increase structural complexity and E/I input ratio in primary hippocampal neurons and alter Ca2+ homeostasis and CREB1-signaling.

One major pathophysiological hallmark of Alzheimer's disease (AD) is senile plaques composed of amyloid ß (Aß). In the amyloidogenic pathway, cleavage of the amyloid precursor protein (APP) is shifted towards Aß production and soluble APPß (sAPPß) levels. Aß is known to impair synaptic function; however, much less is known about the physiological functions of sAPPß. The neurotrophic properties of sAPPα, derived from the non-amyloidogenic pathway of APP cleavage, are well-established, whereas only a few, conflicting studies on sAPPß exist. The intracellular pathways of sAPPß are largely unknown. Since sAPPß is generated alongside Aß by ß-secretase (BACE1) cleavage, we tested the hypothesis that sAPPß effects differ from sAPPα 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 sAPPα significantly increases axon length (p = 0.0002) and that both sAPPα and sAPPß increase neurite number (p < 0.0001) of PHN at 7 days in culture (DIV7) but not at DIV4. Moreover, both sAPPα- and sAPPß-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 sAPPα 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 sAPPα 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 Aß pathophysiology.

Engulfment adapter PTB domain containing 1 interacts with and affects processing of the amyloid-ß 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 Alzheimer's disease (AD). Since AD associated proteins amyloid-ß 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 Aß40/42 and sAPPα. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.

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 Alzheimer's 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.