Uwe Bertsch

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrP(C). Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.

Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease

Abstract In neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrPSc) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood–brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.

Systematic Identification of Antiprion Drugs by High-Throughput Screening Based on Scanning for Intensely Fluorescent Targets

ABSTRACT Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimers disease, Parkinsons disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF–κB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

Conversion Efficiency of Bank Vole Prion Protein in Vitro Is Determined by Residues 155 and 170, but Does Not Correlate with the High Susceptibility of Bank Voles to Sheep Scrapie in Vivo

The misfolded infectious isoform of the prion protein (PrPSc) is thought to replicate in an autocatalytic manner by converting the cellular form (PrPC) into its pathogenic folding variant. The similarity in the amino acid sequence of PrPC and PrPSc influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrPC to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie. Different conversion efficiencies obtained with bank vole and mouse PrPC in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrPC in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrPC primary sequence is decisive for its convertibility to the strain-specific PrPSc conformation.

Toward Semisynthetic Lipoproteins by Convergent Strategies Based on Click and Ligation Chemistry

Cell-membrane proteins are anchored to the lipid bilayer by single or multiple insertion of transmembrane helices or by regioselective single or dual lipidation in coand post-translational enzymatic processes, including acylation with fatty acids, prenylation, and rather commonly C-terminal amidation with glycosylphosphatidylinositols (GPI). Procedures for native and neolipidation of peptides have been comprehensively reviewed, and, more recently, even the synthesis of complex GPIs has been reported. However, lipidation of proteins at defined sites and particularly grafting of GPIs or related mimetic structures to the C termini of proteins still represent formidable long-term goals of chemistry and molecular biology. So far, lipoproteins have been obtained by chemical ligation of synthetic lipopeptides with recombinant protein fragments by the highly selective and efficient maleinimide/thiol addition reaction, as shown for the RAS protein, for example, or by total synthesis through orthogonal protection schemes for regioselective lipidation of side-chain amino groups, for example, with 1,2-dipalmitoyl-glycero-3-succinate. In view of the recently developed efficient procedures for the semisynthesis of proteins by native-chemical [6] and expressed-protein ligation, we have performed model studies toward C-terminal lipidation of proteins by exploiting the copper(i)-catalyzed Huisgen’s 1,3-dipolar cycloaddition of terminal alkynes to azides to form a stable triazole product and the transthioesterification between peptide thioesters and N-cysteinyl-lipopeptides followed by intramolecular S!N acyl shift as the synthetic strategy set forth in Scheme 1. Aside from validating the methodology of a combined click and ligation chemistry, incubation of HeLa cells with the micellar solution of the lipopeptide confirmed its fast uptake, as visualized by confocal fluorescence microscopy. It is well established that dual vicinal lipid chains, as present in the di-fattyacyl glycerol moiety of natural GPI anchors are required for an almost irreversible capture of peptides and proteins by lipid bilayers. Correspondingly, to properly mimic the GPI anchor, phosphatidylethanolamine was converted into the corresponding azide 1 by CuSO4-catalyzed diazotransfer with triflyl azide to produce the key intermediate for subsequent application of the click chemistry. The crystalline azide 1 was then used for the 1,3-cycloaddition reaction with the Sprotected model peptide 2, which contained a C-terminal propargylglycine (Pra) residue as suitable reaction partner (Scheme 1). The azide–alkyne cycloaddition was performed with CuI as catalyst in organic solvent, and the lipopeptide derivative 3 was isolated by silica gel chromatography in yields of 70–75 %. Upon removal of the acid-labile S-trityl and NBoc groups from 3 with TFA, the subsequent native chemical ligation of the cysteinyl-lipopeptide with N-dansylor N-rhodamine B-labeled Gly-Pro-Gly-Gly-SPh ester 4 was performed in micellar solutions of 2 % octyl-b-d-glucopyranoside. Ligation was found to proceed smoothly in the presence of tris(2-carboxyethyl)phosphine (TCEP) if excess of the thioesters was carefully avoided to prevent bisacylation as a side reaction. HPLC served to isolate the fluorescence-labeled lipopeptides 5 a,b in yields of 60–70 % as analytically well-characterized compounds, as shown in Figure 1 for compound 5 b. The lipopeptide 6 was obtained in practically quantitative yield by treatment of the C-terminal propargylglycine residue with azide 1 in aqueous–organic media and in the presence of [a] H.-J. Musiol, Dr. S. Dong, Dr. M. Kaiser, Prof. Dr. L. Moroder Max-Planck-Institut f r Biochemie Am Klopferspitz 18, 82152 Martinsried (Germany) Fax. : (+ 49) 89-8578-2847 E-mail : moroder@biochem.mpg.de [b] Dipl.-Phys. R. Bausinger, Dr. A. Zumbusch Institut f r physikalische Chemie Butenandtstraße 5–13, 81377 M nchen (Germany) [c] Dr. U. Bertsch Zentrum f r Neuropathologie und Prionforschung, LMU M nchen Feodor Lynen Straße 23, 81377 M nchen (Germany)

Novel G335V mutation in the tau gene associated with early onset familial frontotemporal dementia

Mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Here we describe a novel missense mutation in exon 12 of the tau gene, G335V, in a German family with frontotemporal dementia of early age at onset, in the third decade of life. Functional analysis of recombinant tau protein with the G335V mutation showed a dramatically reduced ability to promote microtubule assembly and a more rapid and accelerated tau filament formation, suggesting that the primary effect of the mutation might be the provision of a pool of unbound tau making it available for aberrant tau aggregation.

The Second Messenger Binding Site of Inositol 1,4,5-Trisphosphate 3-Kinase Is Centered in the Catalytic Domain and Related to the Inositol Trisphosphate Receptor Site

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP3) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP3. Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP3, respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP3 binding. Part of this region is similar in sequence to an InsP3 binding segment in InsP3 receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP3 binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.

A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu 2+ complex

Since high-intensity synchrotron radiation is available, “extended X-ray absorption fine structure” spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu2+ complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

From high-throughput cell culture screening to mouse model: identification of new inhibitor classes against prion disease.

Transmissible spongiform encephalopathies (TSE) or prion diseases belong to a category of fatal and so far untreatable neurodegenerative conditions. All prion diseases are characterized by both degeneration in the central nervous system (CNS) in humans and animals and the deposition and accumulation of Proteinase K‐resistant prion protein (PrPres). Until now, no pharmaceutical product has been available to cure these diseases or to alleviate their associated symptoms. Here, a cell‐culture screening system is described that allows for the large‐scale analysis of the PrPres inhibitory potential of a library of compounds and the identification of structural motifs leading potent compounds able to cause PrPres clearance at the cellular level. Based on different scrapie‐infected cell lines, 10 000 substances were tested, out of which 530 potential inhibitors were identified. After re‐screening and validation using a series of dilutions, 14 compounds were identified as the most effective. These 14 compounds were then used for therapeutic studies in a mouse bioassay to test and verify their in vivo potency. Two compounds exhibited therapeutic potential in the mouse model by significantly extending the survival time of intracerebrally infected mice, when treated 90 days after infection with scrapie.