Martin Eiden

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.

Epizootic emergence of Usutu virus in wild and captive birds in Germany

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.

Isolation of Usutu Virus in Germany

Usutu virus (USUV) is a mosquito-borne flavivirus that emerged 2001 in Austria and caused deaths in wild birds. In Germany, 70,378 female mosquitoes were captured in 2009 and 2010 and assayed for USUV. Virus was isolated in cell culture from one pool of Culex pipiens pipiens mosquitoes trapped exclusively in August 2010 in Weinheim, Germany. Subsequent phylogenetic analysis demonstrated a close relationship between the isolated USUV strain from Germany and a USUV strain from Austria, which was detected in a dead blackbird in 2004.

Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France

We report 2 natural scrapie cases in sheep carrying the ARR/ARR prion genotype, which is believed to confer resistance against classic scrapie and bovine spongiform encephalopathy.

BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle

The pathogenesis of bovine spongiform encephalopathy (BSE) infections in cattle has been studied in recent years by using highly sensitive transgenic-mouse bioassays. It has been shown that in this species, the BSE agent amplifies almost exclusively in the central and peripheral nervous system. Even in animals that were killed in the clinical end stage of the disease, the lymphoreticular system was shown to be free of the infectious agent. No other animal species investigated to date exhibits such a restricted BSE-infectivity distribution pattern. However, there is growing evidence for a radial spread of infection from the central nervous system (CNS) into the periphery during the late stages of the disease. In this study, we challenged transgenic mice overexpressing the bovine prion protein with homogenates prepared from a wide variety of tissue samples collected from BSE-infected cattle. As prion infections involve the conversion of the cellular prion protein into its abnormally folded isoform (PrP(Sc)), we applied various detection methods, such as the purification of scrapie-associated fibrils, immunohistochemistry, and the protein misfolding cyclic amplification technique. Despite negative results using these highly sensitive biochemical methods, we were, for the first time, able to detect BSE infectivity in the tongue and in the nasal mucosa of terminally diseased BSE field cases as well as experimentally challenged cattle by transgenic-mouse bioassay. This shows that BSE infectivity can be present in the peripheral tissues of terminally diseased cattle, including tissues used for human consumption.

BSE infectivity in jejunum, ileum and ileocaecal junction of incubating cattle

To establish bovine spongiform encephalopathy (BSE) public health protection measures it is important to precisely define the cattle tissues considered as specified risk materials (SRM). To date, in pre-clinical BSE infected cattle, no evidence of the BSE agent had been found in the gut outside of the ileal Peyers Patches. This study was undertaken to determine when and where the pathological prion protein (PrPSc) and/or BSE infectivity can be found in the small intestine of cattle 4 to 6 months of age, orally challenged with BSE. Samples of the jejunum, the ileum and the ileocaecal junction from 46 BSE infected cattle, culled from 1 up to 44 months post infection (mpi) were examined by immunohistochemistry. Samples from cattle 8 mpi to 20 mpi were additionally studied by PTA Western blot, rapid tests, and by mouse (TgbovXV) bioassay. In doing so nearly all of the cattle, from 4 up to 44 mpi, had detectable amounts of PrPSc and/or infectivity in the distal ileum. In the distal ileum clear time-dependent variations were visible concerning the amount of PrPSc, the tissue structures affected, and the cells involved. BSE infectivity was found not only in the ileum and ileocaecal junction but also in the jejunum. The systematic approach of this study provides new data for qualitative and quantitative risk assessments and allows defining bovine SRM more precisely.

Two New Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Assays with Unique Target Sites for the Specific and Sensitive Detection of Lineages 1 and 2 West Nile Virus Strains

Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5′-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2–4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5′-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.

Natural and experimental hepatitis E virus genotype 3 - infection in European wild boar is transmissible to domestic pigs

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons.

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle). Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viremia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viremia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.

St. John's Wort Reduces Beta‐Amyloid Accumulation in a Double Transgenic Alzheimer's Disease Mouse Model—Role of P‐Glycoprotein

The adenosine triphosphate‐binding cassette transport protein P‐glycoprotein (ABCB1) is involved in the export of beta‐amyloid from the brain into the blood, and there is evidence that age‐associated deficits in cerebral P‐glycoprotein content may be involved in Alzheimers disease pathogenesis. P‐glycoprotein function and expression can be pharmacologically induced by a variety of compounds including extracts of Hypericum perforatum (St. Johns Wort). To clarify the effect of St. Johns Wort on the accumulation of beta‐amyloid and P‐glycoprotein expression in the brain, St. Johns Wort extract (final hyperforin concentration 5%) was fed to 30‐day‐old male C57BL/6J‐APP/PS1+/− mice over a period of 60 or 120 days, respectively. Age‐matched male C57BL/6J‐APP/PS1+/− mice receiving a St. Johns Wort‐free diet served as controls. Mice receiving St. Johns Wort extract showed (i) significant reductions of parenchymal beta‐amyloid 1–40 and 1–42 accumulation; and (ii) moderate, but statistically significant increases in cerebrovascular P‐glycoprotein expression. Thus, the induction of cerebrovascular P‐glycoprotein may be a novel therapeutic strategy to protect the brain from beta‐amyloid accumulation, and thereby impede the progression of Alzheimers disease.