Antje Willuweit

Early-Onset and Robust Amyloid Pathology in a New Homozygous Mouse Model of Alzheimer's Disease

Background Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral β-amyloidosis, one of the characteristic hallmarks of Alzheimers disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues. Methodology/Principal Findings The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APPswe) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral β-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a γ-secretase inhibitor we show a dose dependent reduction of soluble amyloid β levels in the brain. Conclusions ARTE10 mice develop a cerebral β-amyloidosis closely resembling the β-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.

Osteopontin mediates survival, proliferation and migration of neural stem cells through the chemokine receptor CXCR4

IntroductionOsteopontin (OPN) is a phosphoglycoprotein with important roles in tissue homeostasis, wound healing, immune regulation, and stress responses. It is expressed constitutively in the brain and upregulated during neuroinflammatory responses; for example, after focal cerebral ischemia. To date, its effects on neural stem cells (NSC) remain to be elucidated and are, accordingly, the subject of this study.MethodPrimary fetal rat NSC were cultured as homogenous monolayers and treated with different concentrations of OPN. Fundamental properties of NSC were assessed following OPN exposure, including proliferative activity, survival under oxidative stress, migration, and differentiation potential. To elucidate a putative action of OPN via the CXC chemokine receptor type 4 (CXCR4), the latter was blocked with AMD3100. To investigate effects of OPN on endogenous NSC in vivo, recombinant OPN was injected into the brain of healthy adult rats as well as rats subjected to focal cerebral ischemia. Effects of OPN on NSC proliferation and neurogenesis in the subventricular zone were studied immunohistochemically.ResultsOPN dose-dependently increased the number of NSC in vitro. As hypothesized, this effect was mediated through CXCR4. The increase in NSC number was due to both enhanced cell proliferation and increased survival, and was confirmed in vivo. Additionally, OPN dose-dependently stimulated the migration of NSC via CXCR4. Moreover, in the presence of OPN, differentiation of NSC led to a significant increase in neurogenesis both in vitro as well as in vivo after cerebral ischemia.ConclusionData show positive effects of OPN on survival, proliferation, migration, and neuronal differentiation of NSC. At least in part these effects were mediated via CXCR4. Results suggest that OPN is a promising substance for the targeted activation of NSC in future experimental therapies for neurological disorders such as stroke.

QIAD assay for quantitating a compound's efficacy in elimination of toxic Aβ oligomers.

Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.

Uptake of O-(2-[18F]fluoroethyl)-L-tyrosine in reactive astrocytosis in the vicinity of cerebral gliomas

UNLABELLED PET using O-(2-[(18)F]fluoroethyl)-L-tyrosine ((18)F-FET) allows improved imaging of tumor extent of cerebral gliomas in comparison to MRI. In experimental brain infarction and hematoma, an unspecific accumulation of (18)F-FET has been detected in the area of reactive astrogliosis which is a common cellular reaction in the vicinity of cerebral gliomas. The aim of this study was to investigate possible (18)F-FET uptake in the area of reactive gliosis in the vicinity of untreated and irradiated rat gliomas. METHODS F98-glioma cells were implanted into the caudate nucleus of 33 Fisher CDF rats. Sixteen animals remained untreated and in 17 animals the tumor was irradiated by Gamma Knife 5-8 days after implantation (2/50 Gy, 3/75 Gy, 6/100 Gy, 6/150 Gy). After 8-17 days of tumor growth the animals were sacrificed following injection of (18)F-FET. Brains were removed, cut in coronal sections and autoradiograms of (18)F-FET distribution were produced and compared with histology (toluidine blue) and reactive astrogliosis (GFAP staining). (18)F-FET uptake in the tumors and in areas of reactive astrocytosis was evaluated by lesion to brain ratios (L/B). RESULTS Large F98-gliomas were present in all animals showing increased (18)F-FET-uptake which was similar in irradiated and non-irradiated tumors (L/B: 3.9 ± 0.8 vs. 4.0 ± 1.3). A pronounced reactive astrogliosis was noted in the vicinity of all tumors that showed significantly lower (18)F-FET-uptake than the tumors (L/B: 1.5 ± 0.4 vs. 3.9 ± 1.1). The area of (18)F-FET-uptake in the tumor was congruent with histological tumor extent in 31/33 animals. In 2 rats irradiated with 150 Gy, however, high (18)F-FET uptake was noted in the area of astrogliosis which led to an overestimation of the tumor size. CONCLUSIONS Reactive astrogliosis in the vicinity of gliomas generally leads to only a slight (18)F-FET-enrichment that appears not to affect the correct definition of tumor extent for treatment planning.

Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease

Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.

Imaging of amino acid transport in brain tumours : Positron emission tomography with O-(2-[18F]fluoroethyl)-L-tyrosine (FET)

The assessment of cerebral gliomas using magnetic resonance imaging (MRI) provides excellent structural images but cannot solve all diagnostic problems satisfactorily. The differentiation of tumour tissue from non-neoplastic changes may be difficult especially in the post-treatment phase. In recent years, positron emission tomography (PET) using radiolabelled amino acids has gained considerable interest as an additional tool to improve the diagnosis of cerebral gliomas and brain metastases. A key step for this advancement was the development of the F-18 labelled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (FET) which has spread rapidly in the last decade and replaced carbon-11 labelled amino acid tracers such as 11C-methyl-L-methionine (MET) in many centres in Europe. FET can be produced with high efficiency and distributed in a satellite concept like 2-[18F]fluoro-2-deoxy-D-glucose (FDG). Furthermore, FET exhibits favourable properties such as high in vivo stability, high tumour to background contrast and tissue specific tracer kinetics, which provides additional information for tumour grading or differential diagnosis. The Response Assessment in Neuro-Oncology (RANO) working group - an international effort to develop new standardized response criteria for clinical trials in brain tumours - has recently recommended the additional use of amino acid PET imaging for brain tumour management. FET PET can provide important diagnostic information in crucial situations such as the definition of biopsy site, the delineation of cerebral gliomas for therapy planning, sensitive monitoring of treatment response and an improved differentiation of tumour recurrence from treatment-related changes. In this article the basic information, methodological aspects and the actual status of clinical application of FET PET are reviewed.

Epileptic Activity Increases Cerebral Amino Acid Transport Assessed by 18F-Fluoroethyl-l-Tyrosine Amino Acid PET: A Potential Brain Tumor Mimic

O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) PET is a well-established method increasingly used for diagnosis, treatment planning, and monitoring in gliomas. Epileptic activity, frequently occurring in glioma patients, can influence MRI findings. Whether seizures also affect 18F-FET PET imaging is currently unknown. The aim of this retrospective analysis was to investigate the brain amino acid metabolism during epileptic seizures by 18F-FET PET and to elucidate the pathophysiologic background. Methods: Ten patients with 11 episodes of serial seizures or status epilepticus, who underwent MRI and 18F-FET PET, were studied. The main diagnosis was glioma World Health Organization grade II–IV (n = 8); 2 patients suffered from nonneoplastic diseases. Immunohistochemical assessment of LAT1/LAT2/CD98 amino acid transporters was performed in seizure-affected cortex (n = 2) and compared with glioma tissues (n = 3). Results: All patients exhibited increased seizure-associated strict gyral 18F-FET uptake, which was reversible in follow-up studies or negative shortly before and without any histologic or clinical signs of tumor recurrence. 18F-FET uptake corresponded to structural MRI changes, compatible with cortical vasogenic and cytotoxic edema, partial contrast enhancement, and hyperperfusion. Patients with prolonged postictal symptoms lasting up to 8 wk displayed intensive and widespread (≥ 1 lobe) cortical 18F-FET uptake. LAT1/LAT2/CD98 was strongly expressed in neurons and endothelium of seizure-affected brains and less in reactive astrocytosis. Conclusion: Seizure activity, in particular status epilepticus, increases cerebral amino acid transport with a strict gyral 18F-FET uptake pattern. Such periictal pseudoprogression represents a potential pitfall of 18F-FET PET and may mimic brain tumor. Our data also indicate a seizure-induced upregulation of neuronal, endothelial, and less astroglial LAT1/LAT2/CD98 amino acid transporter expression.

Influence of bevacizumab on blood-brain barrier permeability and O-(2- 18 F-fluoroethyl)-L-tyrosine uptake in rat gliomas

Restoration of the blood–brain barrier (BBB) after antiangiogenic therapy of gliomas with bevacizumab may result in a decrease in contrast enhancement on MRI despite tumor progression. This so-called pseudoresponse is difficult to differentiate from a true tumor response with conventional MRI. Initial patient studies have indicated that PET using O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) may be helpful for solving this diagnostic problem. This study was performed to investigate the effects of bevacizumab on BBB permeability and 18F-FET uptake in a human xenograft model. Methods: Human U87 glioblastoma cells were implanted into the striatum of immunodeficient RNU rats. 18F-FET PET scans and ex vivo autoradiography were performed in animals receiving a single high dose of bevacizumab (45 mg/kg 2 d before PET; n = 9) or in animals receiving 2 lower doses (10 mg/kg 9 and 2 d before PET; n = 10) to evaluate short-term and long-term effects on the BBB, respectively, and in control animals without bevacizumab treatment (n = 8). Time–activity curves, slope, and tumor-to-brain ratios of 18F-FET uptake (18–61 min after injection) were evaluated using a volume-of-interest analysis. After PET scanning, Evans blue dye (EBD) was injected into animals, and cryosections of the brains were evaluated by autoradiography, by histology, and for EBD fluorescence to assess BBB permeability. Results: Compared with the control, short-term bevacizumab therapy resulted in a trend toward BBB restoration (P = 0.055) and long-term therapy resulted in a significant decrease (P = 0.004) in BBB permeability, as assessed by EBD fluorescence. In contrast, no significant differences in tumor-to-brain ratios or slope of 18F-FET uptake were observed in PET and autoradiography (P > 0.05). Conclusion: 8F-FET uptake in glioblastomas seems to be largely independent of BBB permeability and reflects the viability of tumor tissue during antiangiogenic therapy more reliably than contrast-enhanced MRI.

Pharmacokinetic properties of tandem d-peptides designed for treatment of Alzheimer's disease

Peptides are more and more considered for the development of drug candidates. However, they frequently exhibit severe disadvantages such as instability and unfavourable pharmacokinetic properties. Many peptides are rapidly cleared from the organism and oral bioavailabilities as well as in vivo half-lives often remain low. In contrast, some peptides consisting solely of d-enantiomeric amino acid residues were shown to combine promising therapeutic properties with high proteolytic stability and enhanced pharmacokinetic parameters. Recently, we have shown that D3 and RD2 have highly advantageous pharmacokinetic properties. Especially D3 has already proven promising properties suitable for treatment of Alzheimers disease. Here, we analyse the pharmacokinetic profiles of D3D3 and RD2D3, which are head-to-tail tandem d-peptides built of D3 and its derivative RD2. Both D3D3 and RD2D3 show proteolytic stability in mouse plasma and organ homogenates for at least 24h and in murine and human liver microsomes for 4h. Notwithstanding their high affinity to plasma proteins, both peptides are taken up into the brain following i.v. as well as i.p. administration. Although both peptides contain identical d-amino acid residues, they are arranged in a different sequence order and the peptides show differences in pharmacokinetic properties. After i.p. administration RD2D3 exhibits lower plasma clearance and higher bioavailability than D3D3. We therefore concluded that the amino acid sequence of RD2 leads to more favourable pharmacokinetic properties within the tandem peptide, which underlines the importance of particular sequence motifs, even in short peptides, for the design of further therapeutic d-peptides.

The Aβ oligomer eliminating D -enantiomeric peptide RD2 improves cognition without changing plaque pathology

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer’s disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the d-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aβ42. Our findings demonstrate that RD2 was significantly more efficient in Aβ oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aβ oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aβ oligomer elimination by RD2 treatment may be also beneficial for AD patients.