Synthesis of monomeric and dimeric acridine compounds as potential therapeutics in Alzheimer and prion diseases.

Starting from substituted 9-chloroacridines, a series of quinacrine and spacered dimeric acridine compounds was prepared. Their ability to interrupt the protein association of prion- and Alzheimer-specific proteins and Ab peptides was explored using a fast screening system based on FACS analysis. The bis-acridines displayed a higher activity than the corresponding monomers. Among these derivatives, best results were obtained with the 2,4-dimethoxy-6-nitro compound 7h for Abeta-peptides and the 2-methoxy-6-nitro compound 7f for PrP.

Inducible gene manipulations in serotonergic neurons.

An impairment of the serotonergic (5-HT) system has been implicated in the etiology of many neuropsychiatric disorders. Despite the considerable genetic evidence, the exact molecular and pathophysiological mechanisms underlying this dysfunction remain largely unknown. To address the lack of instruments for the molecular dissection of gene function in serotonergic neurons we have developed a new mouse transgenic tool that allows inducible Cre-mediated recombination of genes selectively in 5-HT neurons of all raphe nuclei. In this transgenic mouse line, the tamoxifen-inducible CreERT2 recombinase is expressed under the regulatory control of the mouse tryptophan hydroxylase 2 (Tph2) gene locus (177 kb). Tamoxifen treatment efficiently induced recombination selectively in serotonergic neurons with minimal background activity in vehicle-treated mice. These genetic manipulations can be initiated at any desired time during embryonic development, neonatal stage or adulthood. To illustrate the versatility of this new tool, we show that Brainbow-1.0L(TPH2-CreERT2) mice display highly efficient recombination in serotonergic neurons with individual 5-HT neurons labeling with multiple distinct fluorescent colors. This labeling is well suited for visualization and tracing of serotonergic neurons and their network architecture. Finally, the applicability of TPH2-CreERT2 for loxP-flanked candidate gene manipulation is evidenced by our successful knockout induction of the ubiquitously expressed glucocorticoid-receptor exclusively in 5-HT neurons of adult mice. The TPH2-CreERT2 line will allow detailed analysis of gene function in both developing and adult serotonergic neurons.

Synthesis of dimeric quinazolin-2-one, 1,4-benzodiazepin-2-one, and isoalloxazine compounds as inhibitors of amyloid peptides association.

The synthesis of dimeric compounds derived from quinazolin-2-one and 1,4-benzodiazepin-2-one possessing a piperazine or homopiperazine spacer was investigated. In addition, a piperazine spacered bis-isoalloxazine and a bis-riboflavin compound were prepared and their ability to interrupt the association of prion proteins and Alzheimer-specific Abeta peptides was investigated using a fast screening system based on flow cytometry. The bis-isoalloxazine 14 was identified as a new lead structure.

Detection of amyloid-beta oligomers in human cerebrospinal fluid by flow cytometry and fluorescence resonance energy transfer.

The neuropathology of Alzheimer's disease (AD) has been linked recently to non-fibrillar forms of neurotoxic amyloid-beta (Abeta) oligomers of which high levels are observed in the brain of AD patients. This suggests that Abeta oligomers play a key role in the early events of AD, underlining their potential for the early diagnosis of the disease. We have developed an extremely sensitive assay for the detection of oligomeric and fibrillar structures of Abeta that is based on multiparametric analysis of data obtained by flow cytometry and fluorescence resonance energy transfer (Fret). The assay readily detects Abeta oligomers in human cerebrospinal fluid (CSF) as verified by dot blot of the isolated particles. By measuring 174 CSF samples of non-demented control patients with various neurological disorders a high reliability and reproducibility of the method could be demonstrated.

On the seeding and oligomerization of pGlu-amyloid peptides (in vitro).

Oligomerization of amyloid beta (Abeta) peptides is the decisive event in the development of Alzheimer's disease (AD), the most common neurogenerative disorder in developed countries. Recent evidence links this conformation-driven process to primary- and secondary-structure modifications of Abeta. The N and C terminus of deposited Abeta has been shown to possess conspicuous heterogeneity. While the C-terminally longer form of Abeta, i.e., Abeta (42), is considered more amyloidogenic, the role of the N-terminal modifications, e.g., truncation and glutamate cyclization accounting for the majority of the deposited peptides, is less understood. In the present study, we characterized the oligomerization and seeding capacity of pGlu-amyloid peptides using two unrelated techniques based on flow cytometry or flourescence dye binding. Under different conditions and irrespective of the C terminus of Abeta, i.e., Abeta40 or 42, pGlu-modified peptides displayed an up to 250-fold accelerated initial formation of aggregates compared to unmodified Abeta. The accelerated seed formation is accompanied by a change in the oligomerization kinetics because of N-terminal pGlu formation. Furthermore, the formation of mixed aggregates consisting of either pGlu-Abeta (3-42) or ADan or ABri and Abeta (1-42) was investigated by Abeta fluorescence labeling in flow cytometry. The results suggest that pGlu-modified peptides are potential seeding species of aggregate formation in vivo. The data presented here and the abundance of pGlu peptides in amyloidoses, such as FBD and AD, suggest pGlu-amyloid peptides as a species with biophysical characteristics that might be in particular crucial for the initiation of the disease.

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy.

BACKGROUND: The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods. RESULTS: We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10(-8) nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle. CONCLUSION: We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases.

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain.

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.