Understanding the role of the hematopoietic niche in Huntington's disease's phenotypic expression: in vivo evidence using a parabiosis model.

In a previous study, we have shown that parabiotic coupling of a knock-in mouse model (zQ175) of Huntington's disease (HD) to wild-type (WT) littermates resulted in a worsening of the normal phenotype as seen by detection of mutant huntingtin protein (mHTT) aggregates within peripheral organs and the cerebral cortex as well as vascular abnormalities in WT mice. In contrast, parabiosis improved disease features in the zQ175 mice such as reduction of mHTT aggregate number in the liver and cortex, decrease in blood-brain barrier (BBB) permeability and attenuation of mitochondrial impairments. While the shared circulation mediated these effects, no specific factor was identified. To better understand which blood elements were involved in the aforementioned changes, WT and zQ175 mice underwent parabiotic surgery prior to exposing one of the paired animals to irradiation. The irradiation procedure successfully eliminated the hematopoietic niche followed by repopulation with cells originating from the non-irradiated parabiont, as measured by the quantification of mHTT levels in peripheral blood mononuclear cells. Although irradiation of the WT parabiont, causing the loss of healthy hematopoietic cells, did lead to a few alterations in mitochondrial function in the muscle (TOM40 levels), and increased neuroinflammation in the striatum (GFAP levels), most of the changes observed were likely attributable to the irradiation procedure itself (e.g. mHTT aggregates in cortex and liver; cellular stress in peripheral organs). However, factors such as mHTT aggregation in the brain and periphery, and BBB leakage, which were improved in zQ175 mice when paired to WT littermates in the previous parabiosis experiment, were unaffected by perturbation of the hematopoietic niche. It would therefore appear that cells of the hematopoietic stem cell niche are largely uninvolved in the beneficial effects of parabiosis.

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

A standardised frankincense extract reduces disease activity in relapsing-remitting multiple sclerosis (the SABA phase IIa trial).

OBJECTIVE: To investigate whether oral administration of a standardised frankincense extract (SFE) is safe and reduces disease activity in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: We performed an investigator-initiated, bicentric phase IIa, open-label, baseline-to-treatment pilot study with an oral SFE in patients with RRMS (NCT01450124). After a 4-month baseline observation phase, patients were treated for 8 months with an option to extend treatment for up to 36 months. The primary outcome measures were the number and volume of contrast-enhancing lesions (CEL) measured in MRI during the 4-month treatment period compared with the 4-month baseline period. Eighty patients were screened at two centres, 38 patients were included in the trial, 28 completed the 8-month treatment period and 18 of these participated in the extension period. RESULTS: The SFE significantly reduced the median number of monthly CELs from 1.00 (IQR 0.75-3.38) to 0.50 (IQR 0.00-1.13; difference -0.625, 95% CI -1.25 to -0.50; P<0.0001) at months 5-8. We observed significantly less brain atrophy as assessed by parenchymal brain volume change (P=0.0081). Adverse events were generally mild (57.7%) or moderate (38.6%) and comprised mainly gastrointestinal symptoms and minor infections. Mechanistic studies showed a significant increase in regulatory CD4+ T cell markers and a significant decrease in interleukin-17A-producing CD8+ T cells indicating a distinct mechanism of action of the study drug. INTERPRETATION: The oral SFE was safe, tolerated well and exhibited beneficial effects on RRMS disease activity warranting further investigation in a controlled phase IIb or III trial. CLINICAL TRIAL REGISTRATION: NCT01450124; Results.

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

Comparison of high-resolution X-ray and micro-CT for experimental evaluation of intracranial stent prototypes: quality evaluation beyond CE mark.

INTRODUCTION: As digital mammography and micro-computed tomography (CT) have been used for evaluation of stents deployed in experimental animal models, we compared the two methods regarding their sensitivity to detect abnormalities in three prototypes of intracranial stents. METHODS: Three different prototypes of intracranial stents (n = 84) were implanted in various animal models. Explanted stents were examined using digital mammography and micro-CT. The images were compared with respect to maintenance of material and form and the stents were compared to one another. Histological analysis was performed as well. RESULTS: In the open-cell stents, expansion of the stent cells was detected in the majority of cases (57.1 %) using micro-CT and less frequently using mammography (42.3 %). The closed-cell stent revealed kink stenoses in mammography as well as in micro-CT (3/7, 42.9 %). Detailed reconstructions of micro-CT images showed high-grade kink stenoses of the flow-diverter stent in two extremely curved vessels. Strut breaks were observed more frequently using micro-CT (6/84, 7.1 %) than by mammography (4/84, 4.8 %). Histology confirmed all changes of stent architecture. CONCLUSION: Significant changes of stent architecture can be observed and assessed even in the two-dimensional mammographic images. The use of micro-CT is recommended to detect subtle changes like single strut breaks and for three-dimensional information.

Onset and phoneme awareness and its relationship to letter knowledge in German-speaking preschool children.

OBJECTIVES: The aim was to explore whether word-initial onset awareness is acquired before phoneme awareness and whether onset complexity influences performance on identification tasks. In addition, the relationship between onset and phoneme awareness and letter knowledge was investigated. METHOD: In this study, 22 monolingual German-speaking preschool children aged 5;00-5;11 were tested. Onset, phoneme identification, and letter knowledge tasks were administered. The children were presented with pictures of word pairs. Both words in each pair shared a single-consonant onset, a two-consonant onset cluster or the first consonant of a consonant cluster. The children were asked to pronounce the shared sound(s). Additionally, they were asked to name all 26 upper-case letters. RESULTS: Onset awareness tasks were significantly easier to complete than phoneme awareness tasks. However, no influence of onset complexity on onset awareness performance was found. Moreover, letter knowledge correlated with all phonological awareness tasks. CONCLUSIONS: The results corroborate that phoneme awareness develops already at preschool age irrespective of explicit literacy tuition. Nevertheless, letter knowledge is closely related and should be linked to onset/phoneme awareness tasks.

Seasonal variations of microplastic pollution in the German River Weser.

Rivers play a major role in the distribution of microplastics (MPs) in the environment, however, research on temporal variations in these highly dynamic systems is still in its infancy. To date, most studies dealing with the seasonality of MP contamination in rivers focus on bi-yearly analysis, while temporal fluctuations over the course of the year are rarely studied. To shed more light on seasonal variability of MP abundance and potential driving factors, we have thus sampled the water surface of one location in the Weser River in Germany monthly over one entire year. In our study, we targeted MP in the size range 10-5000 µm, using two different state-of-the-art sampling methods (manta net for large MP (l-MP; 500-5000 µm) and a filtration system for small MP (s-MP; 10-500 µm)) and analysis techniques (ATR-FTIR and FPA-µFTIR) for chemical identification. Our findings show a strong size-dependent positive correlation of the MP concentration with discharge rates (specifically direct runoff) and suspended particulate matter (SPM) for s-MPs, specifically in the size range 10-149 µm. L-MPs, however, show a different environmental behaviour and do not follow these patterns. With our study, we were able to deliver a much higher temporal resolution, covering a broader size range of MPs compared to most studies. Our findings point towards an interplay of two possible mechanisms: a) the riverbeds play an important role in large-scale MP and SPM release via resuspension during high discharge events, and b) precipitation-driven soil erosion and runoff from urban surfaces (e.g. rain sewers) introduce MP and SPM. Hence, our study serves as a basis for more detailed investigations of MP transport in and between ecosystems.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Development of a ligand for in vivo imaging of mutant huntingtin in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (polyQ) tract. Whereas several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, methods to visualize mHTT protein species in the living brain are lacking. Here, we demonstrate the development and characterization of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) allowed noninvasive monitoring of mHTT pathology in the brain and could track region- and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression in a rodent model. We further showed that in these animals, therapeutic agents that lowered mHTT in the striatum had a functional restorative effect that could be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

Reduced basis catalogs for gravitational wave templates.

We introduce a reduced basis approach as a new paradigm for modeling, representing and searching for gravitational waves. We construct waveform catalogs for nonspinning compact binary coalescences, and we find that for accuracies of 99% and 99.999% the method generates a factor of about 10-10(5) fewer templates than standard placement methods. The continuum of gravitational waves can be represented by a finite and comparatively compact basis. The method is robust under variations in the noise of detectors, implying that only a single catalog needs to be generated.

Ethical issues in autologous stem cell transplantation (ASCT) in advanced breast cancer: a systematic literature review.

BACKGROUND: An effectiveness assessment on ASCT in locally advanced and metastatic breast cancer identified serious ethical issues associated with this intervention. Our objective was to systematically review these aspects by means of a literature analysis. METHODS: We chose the reflexive Socratic approach as the review method using Hofmann's question list, conducted a comprehensive literature search in biomedical, psychological and ethics bibliographic databases and screened the resulting hits in a 2-step selection process. Relevant arguments were assembled from the included articles, and were assessed and assigned to the question list. Hofmann's questions were addressed by synthesizing these arguments. RESULTS: Of the identified 879 documents 102 included arguments related to one or more questions from Hofmann's question list. The most important ethical issues were the implementation of ASCT in clinical practice on the basis of phase-II trials in the 1990s and the publication of falsified data in the first randomized controlled trials (Bezwoda fraud), which caused significant negative effects on recruiting patients for further clinical trials and the doctor-patient relationship. Recent meta-analyses report a marginal effect in prolonging disease-free survival, accompanied by severe harms, including death. ASCT in breast cancer remains a stigmatized technology. Reported health-related-quality-of-life data are often at high risk of bias in favor of the survivors. Furthermore little attention has been paid to those patients who were dying. CONCLUSIONS: The questions were addressed in different degrees of completeness. All arguments were assignable to the questions. The central ethical dimensions of ASCT could be discussed by reviewing the published literature.

Assessment of patient-reported outcomes after polytrauma - instruments and methods: a systematic review.

OBJECTIVES: We (1) collected instruments that assess health-related quality of life (HRQoL), activities of daily living (ADL) and social participation during follow-up after polytrauma, (2) described their use and (3) investigated other relevant patient-reported outcomes (PROs) assessed in the studies. DESIGN: Systematic Review using the Preferred Reporting Items for Systematic Review and Meta-Analysis guideline. DATA SOURCES: MEDLINE, Embase, CINAHL, PsycINFO, CENTRAL, as well as the trials registers ClinicalTrials.gov and WHO ICTRP were searched from January 2005 to April 2018. ELIGIBILITY CRITERIA: All original empirical research published in English or German including PROs of patients aged 18-75 years with an Injury Severity Score≥16 and/or an Abbreviated Injury Scale≥3. Studies with defined injuries or diseases (e.g. low-energy injuries) and some text types (e.g. grey literature and books) were excluded. Systematic reviews and meta-analyses were excluded, but references screened for appropriate studies. DATA EXTRACTION AND SYNTHESIS: Data extraction, narrative content analysis and a critical appraisal (e.g. UK National Institute for Health and Care Excellence) were performed by two reviewers independently. RESULTS: The search yielded 3496 hits; 54 publications were included. Predominantly, HRQoL was assessed, with Short Form-36 Health Survey applied most frequently. ADL and (social) participation were rarely assessed. The methods most used were postal surveys and single assessments of PROs, with a follow-up period of one to one and a half years. Other relevant PRO areas reported were function, mental disorders and pain. CONCLUSIONS: There is a large variation in the assessment of PROs after polytrauma, impairing comparability of outcomes. First efforts to standardise the collection of PROs have been initiated, but require further harmonisation between central players. Additional knowledge on rarely reported PRO areas (e.g. (social) participation, social networks) may lead to their consideration in health services provision. PROSPERO REGISTRATION NUMBER: CRD42017060825.

Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Nucleo-cytoplasmic shuttling of protein arginine methyltransferase 1 (PRMT1) requires enzymatic activity.

Methylation of arginine residues is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMT), of which PRMT1 is the predominant member in human cells. We have previously described the localization and mobility of PRMT1 in live cells, and found that it shuttles between the nucleus and the cytoplasm depending on the methylation status of substrate proteins. Recently, amino-terminal splicing isoforms of PRMT1 were shown to differ significantly in intracellular localization, the most interesting being splice variant 2 that carries a nuclear export signal in its amino terminus, and is expressed in increased levels in breast cancer cells. We show here that enzymatic activity is required for nucleo-cytoplasmic shuttling of PRMT1v2, as a catalytically inactive mutant highly accumulates in the nucleus and displays altered intranuclear mobility as determined by fluorescence recovery after photobleaching experiments. Our results indicate that nuclear export of PRMT1v2 is dominant over activity-independent nuclear import, but can only occur after activity-dependent release of the enzyme from substrates, suggesting that shuttling of the enzyme provides a dynamic mechanism for the regulation of substrate methylation.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Huntingtin Aggregates and Mitochondrial Pathology in Skeletal Muscle but not Heart of Late-Stage R6/2 Mice.

BACKGROUND: Cell or tissue specific background may influence the consequences of expressing the Huntington's disease (HD) mutation. Aggregate formation is known to occur in skeletal muscle, but not heart of the R6/2 fragment HD model. OBJECTIVE: We asked whether aggregate formation and the expression and subcellular localization of huntingtin species was associated with mitochondrial dysfunction. METHODS: We analyzed levels of soluble HTT and HTT aggregates, as well as important fission and fusion proteins and mitochondrial respiratory chain activities, in quadriceps and heart of the R6/2 N-terminal fragment mouse model (12 weeks, 160±10 CAG repeats). RESULTS: Soluble mutant HTT was present in both tissues with expression higher in cytoplasmic/mitochondrial than nuclear fractions. HTT aggregates were only detectable in R6/2 quadriceps, in association with increased levels of the pro-fission factor DRP1 and its phosphorylated active form, and decreased levels of the pro-fusion factor MFN2. In addition, respiratory chain complex activities were decreased. In heart that was without detectable HTT aggregates, we found no evidence for mitochondrial dysfunction. CONCLUSION: Tissue specific factors may exist that protect the R6/2 heart from HTT aggregate formation and mitochondrial pathology.