Susanne Aileen Funke

Pyroglutamate Formation Influences Solubility and Amyloidogenicity of Amyloid Peptides

N-Terminally truncated and pyroglutamate (pGlu) modified amyloid beta (Abeta) peptides are major constituents of amyloid deposits in sporadic and inherited Alzheimers disease (AD). Formation of pGlu at the N-terminus confers resistance against cleavage by most aminopeptidases, increases toxicity of the peptides, and may seed Abeta aggregate formation. Similarly, the deposited amyloid peptides ABri and ADan, which cause a very similar histopathology in familial British dementia (FBD) and familial Danish dementia (FDD), are N-terminally blocked by pGlu. Triggered by the coincidence of pGlu-modified amyloid peptides and similar pathology in AD, FBD, and FDD, we investigated the impact of N-terminal pGlu on biochemical and biophysical properties of Abeta, ABri, and ADan. N-Terminal pGlu increases the hydrophobicity and changes the pH-dependent solubility profile, rendering the pGlu-modified peptides less soluble in the basic pH range. The pGlu residue increases the aggregation propensity of all amyloid peptides as evidenced by ThT fluorescence assays and dynamic light scattering. The far-UV CD spectroscopic analysis points toward an enhanced beta-sheet structure of the pGlu-Abeta. Importantly, changes in fibril morphology are clearly caused by the N-terminal pGlu, resulting in the formation of short fibers, which are frequently arranged in bundles. The effect of pGlu on the morphology is virtually indistinguishable between ABri, ADan, and Abeta. The data provide evidence for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD. The main driving force for the aggregation is apparently an increase in the hydrophobicity and thus an accelerated seed formation.

Reduction of Alzheimer's disease amyloid plaque load in transgenic mice by D3, A D-enantiomeric peptide identified by mirror image phage display.

Alzheimer’s disease (AD) is a multifactorial disorder, which is characterized by progressive memory deficits, cognitive impairments and personality changes. More than 20 million people are affected worldwide. The histopathological hallmarks of AD are aggregated protein deposits (i.e. , senile plaques and neurofibrillary tangles) in the brain. Senile plaques consist mainly of extracellular amyloid-b peptide (Ab) deposits. While there is still debated over whether Ab is the causative agent in AD, the inhibition of Ab production and aggregation is often targeted for therapy development. Recently, we used mirror image phage display to identify a novel d-amino acid peptide binding specifically to Ab ACHTUNGTRENNUNG(1–42) with a binding affinity in the submicromolar dissociation constant range and called it “d-pep” or “D1”. 3] d-peptides are known to be extremely protease resistant and less immunogenic than their respective l-enantiomers, thus being more suitable for in vivo use. d-Peptides have previously been used as inhibitors of amyloid formation to prevent the associated Ab cytotoxicity. Recently, another strategy to obtain d-peptides specifically interacting with amyloid stretches, inhibiting amyloid formation and cell toxicity, was presented. In the present work, we identified a novel d-enantiomeric amino acid peptide “D3” which might provide a novel basis for therapeutic and preventive approaches to AD. D3 might also be useful as a tool to study the role of Ab plaques in AD progression. We performed phage display selections of a peptide library encoding more than 1 10 randomly different 12amino acid sequences with d-enantiomeric Ab ACHTUNGTRENNUNG(1–42) (d-Ab) as the target. d-Ab was dissolved to obtain a low final concentration of 2 nm. Under those conditions, we expected monomeric Ab or small Ab oligomers to be the dominant target species during the phage display screening. After 6 rounds of biopanning, we determined the peptide sequences of the enriched phage displayed peptides by DNA sequence analysis of the respective genome region. The dominant peptide sequence obtained from the selection was RPRTRLHTHRNR, referred to as D3. This sequence was found in 9 out of 23 randomly chosen phage clones. Additionally, 9 of the selected peptides were related to the dominating peptide by at least 9 amino acids (table S1, Supporting Information). We investigated the influence of D3 on Ab aggregation. The content of amyloid fibrils was determined by Thioflavin T (ThT) fluorescence upon incubation of various Ab/D3 mixtures. ThT is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils, and is commonly used to detect amyloid fibrils. ThT fluorescence of Ab mixtures with D3 was significantly lower than those without D3 (Figure 1 a). These results suggest that D3 significantly decreased the formation of ThT-positive Ab aggregates. These results were confirmed using a fluorescence correlation spectroscopy (FCS) based assay. FCS allows the detection of Ab aggregates in highly dilute samples, with concentrations in the nanomolar range. Fluorescence fluctuations generated by single molecules passing the confocal volume of the focused laser beam are measured, and can be evaluated by autocorrelation to obtain the diffusion time of the studied molecules. When Ab aggregates, containing at least one molecule of Oregon Green (OG) labeled Ab, are present within a given solution, they can be directly detected as spikes in the fluorescence fluctuation recordings. Due to their size, they have an increased duration of stay in the confocal volume and increased fluorescence intensities because most or all of them contain more than one fluorescence label. FCS measurements were carried out with 5 nm OG-labeled Ab in the absence or presence of D3 in various concentrations. Figure 1 b shows how the number of peaks decreases with increasing amounts of added D3. A 50 % inhibition of aggregate formation by D3 is obtained at ~1 mm. This result suggests that D3 prevents aggregation of Ab in the nanomolar concentration range. To assay the ability of D3 to dissolve pre-existing ThT positive Ab aggregates, Ab was preincubated without D3 for seven days to allow aggregation. Then, D3 at various concentrations was added and ThT fluorescence was followed (Figure 1 c). The results clearly show a dose-dependent aggregate disassembly activity of D3 for preformed ThT positive Ab aggregates without stirring, ultrasonic treatment, or any other mechanical support. We studied the effect of D3 on Ab-induced cytotoxicity in rat pheochromocytoma (PC12) cells. Ab (10 mm) was incubated without or with varying concentrations of D3 for 6 days at 37 8C. Cells were then treated with various Ab/D3 mixtures and their viability was measured by MTT reduction (Figure 1 d). In the presence of 2 mm of Ab cell viability dropped to 40 %, this effect was reversed in a dose-dependant manner by the addition of D3, and cell viability could be completely rescued in [a] Dr. K. Wiesehan, Dr. S. A. Funke, Prof. Dr. D. Willbold INB-2/Molekulare Biophysik II Forschungszentrum J lich, 52425 J lich (Germany) Fax: (+ 49) 2461612233 E-mail : d.willbold@fz-juelich.de [b] Dr. L. Nagel-Steger, Prof. Dr. D. Willbold Institut f r Physikalische Biologie, BMFZ, Heinrich-Heine-Universit t 40225 D sseldorf (Germany) [c] Dr. T. van Groen, Dr. I. Kadish Dept. Cell Biology, University of Alabama at Birmingham, AL 35294 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.200800273.

Oral treatment with the d-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer's Disease transgenic mice.

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimers disease (AD). Not only Aβ fibrils but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aβ targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

Peptides for therapy and diagnosis of Alzheimer's disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. The greatest risk factor to develop AD is age. Today, only symptomatic therapies are available. Additionally, AD can be diagnosed with certainty only post mortem, whereas the diagnosis “probable AD” can be established earliest when severe clinical symptoms appear. Specific neuropathological changes like neurofibrillary tangles and amyloid plaques define AD. Amyloid plaques are mainly composed of the amyloid-β peptide (Aβ). Several lines of evidence suggest that the progressive concentration and subsequent aggregation and accumulation of Aβ play a fundamental role in the disease progress. Therefore, substances which bind to Aβ and influence aggregation thereof are of great interest. An enormous number of organic substances for therapeutic purposes are described. This review focuses on peptides developed for diagnosis and therapy of AD and discusses the pre- and disadvantages of peptide drugs.

Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex‐PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full‐length genes by a recombination‐PCR step. The process of multiplex‐PCR‐based recombination (MUPREC) does not require DNase I digestion for gene‐fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild‐type background. Furthermore, the low error rate results in high‐quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso‐compound. Thereby, the method was proved to be useful in producing a reliable second‐generation library of true recombinants from which better performing variants were identified by using a high‐throughput electrospray ionization mass spectrometry (ESI‐MS) screening system.

Single-Particle Detection System for Aβ Aggregates: Adaptation of Surface-Fluorescence Intensity Distribution Analysis to Laser Scanning Microscopy

Today, Alzheimer disease (AD) can be diagnosed with certainty only post mortem. A biomarker method for early diagnosis of AD is urgently needed. Abeta aggregates are directly involved in AD progression and therefore might be useful as biomarker in body fluids. We have developed an ultrasensitive assay system for the detection of Abeta aggregates in body fluids, called surface-fluorescence intensity distribution analysis (FIDA). Surface-FIDA in its first version was based on fluorescence correlation spectroscopy. Here we show that surface-FIDA can also be performed using a laser scanning microscope setup and is then nicely suitable for the characterization of Abeta aggregates.

Structural analysis of the pyroglutamate‐modified isoform of the Alzheimer's disease‐related amyloid‐β using NMR spectroscopy

The aggregation of the Aβ plays a fundamental role in the pathology of AD. Recently, N‐terminally modified Aβ species, pE‐Aβ, have been described as major constituents of Aβ deposits in the brains of AD patients. pE‐Aβ has an increased aggregation propensity and shows increased toxicity compared with Aβ1‐40 and Aβ1‐42. In the present work, high‐resolution NMR spectroscopy was performed to study pE‐Aβ3‐40 in aqueous TFE‐containing solution. Two‐dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE‐Aβ3‐40 was shown to contain two helical regions formed by residues 14–22 and 30–36. This is similar as previously described for Aβ1‐40. However, the secondary chemical shift data indicate decreased helical propensity in pE‐Aβ3‐40 when compared with Aβ1‐40 under exactly the same conditions. This is in agreement with the observation that pE‐Aβ3‐40 shows a drastically increased tendency to form β‐sheet‐rich structures under more physiologic conditions. Structural studies of pE‐Aβ are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE‐Aβ for the pathogenesis of AD. Copyright

Transport of Alzheimer Disease Amyloid-β-Binding d-Amino Acid Peptides across an In Vitro Blood–Brain Barrier Model

Previously, two D-enantiomeric amino acid peptides, D1 and D3, which specifically bind to the amyloid-beta peptide Abeta(1-42), were identified by phage display selection. To assess the diagnostic and therapeutic potentials of D1 and D3 for the diagnosis and treatment of Alzheimer disease, the blood-brain barrier transport of these D-peptides was quantitatively evaluated in vitro. Our results showed that the apical-to-basolateral transport of D3 was more efficient than that of D1. An active efflux transport mechanism seems to oppose the transport of D1, whereas D3 is likely to be transported through the blood-brain barrier via an adsorptive-mediated transcytosis mechanism.

Differently Selected d-Enantiomeric Peptides Act on Different Aβ Species

Aging is the most significant risk factor for Alzheimer disease (AD). The pathological hallmark of AD is the accumulation of aggregated amyloid-beta (Abeta) forms and insoluble plaques, mainly composed of Abeta, in the brain of the patient. Recently, we reported on the selection of D-enantiomeric, Abeta-binding peptides D1 and D3. D1 was selected against aggregated Abeta species to address diagnosis by in vivo imaging of amyloid plaques, whereas D3 was selected using low-molecular-weight Abeta species, therefore addressing therapeutical studies. Here, we use a surface plasmon resonance method to confirm that both peptides show the desired binding specificities.

Treatment with D3 removes amyloid deposits, reduces inflammation, and improves cognition in aged AβPP/PS1 double transgenic mice.

One of the characteristic pathological hallmarks of Alzheimers disease (AD) is neuritic plaques. The sequence of events leading to deposition of amyloid-β (Aβ) peptides in plaques is not clear. Here we investigate the effects of D3, an Aβ oligomer directed D-enantiomeric peptide that was obtained from a mirror image phage display selection against monomeric or small oligomeric forms of Aβ42, on Aβ deposition in aged AβPP/PS1 double transgenic AD-model mice. Using Alzet minipumps, we infused the brains of these AD model mice for 8 weeks with FITC-labeled D3, and examined the subsequent changes in pathology and cognitive deficits. Initial cognitive deficits are similar comparing control and D3-FITC-treated mice, but the treated mice show a significant improvement on the last day of testing. Further, we show that there is a substantial reduction in the amount of amyloid deposits in the animals treated with D3-FITC, compared to the control mice. Finally, the amount of activated microglia and astrocytes surrounding Aβ deposits is dramatically reduced in the D3-FITC-treated mice. Our findings demonstrate that treatments with the high affinity Aβ42 oligomer binding D-enantiomeric peptide D3 significantly decrease Aβ deposits and the associated inflammatory response, and improve cognition even when applied only at late stages and high age. Together, this suggests that the treatment reduces the level of Aβ peptide in the brains of AβPP/PS1 mice, possibly by increasing Aβ outflow from the brain. In conclusion, treatments with this D-peptide have great potential to be successful in AD patients.