Establishment of a FRET-based Aggregate Seeding Assay to study the pathogenic role of self-replicating mutant HTT fibrils in Huntington’s disease

Die Huntington-Krankheit (HK) ist eine progressive, autosomal-dominant vererbte neurodegenerative Erkrankung, die sich mit motorischen, kognitiven und psychiatrischen Symptomen manifestiert. Die HK ist bis heute unheilbar und wird durch die Expansion des CAG-Trinukleotid-Repeats im HTT-Gen verursacht. Diese hat eine Verlängerung der Polyglutamin-Sequenz im Huntingtin-Protein (HTT) zur Folge, wodurch die Konformation des Proteins instabil wird und es zur Bildung von amyloidogenen Proteinaggregaten kommt, die hauptsächlich aus N-terminalen HTT Fragmenten bestehen. Amyloidogene Aggregate des mutierten HTT (mHTT) weisen gemeinsame Merkmale mit Prionen auf, da sie in der Lage sind, ihre fehlgefaltete Struktur auf nativ gefaltetes mHTT zu übertragen und sich zwischen benachbarten Zellen auszubreiten. Die Bedeutung von mHTT-Aggregaten und deren prionenähnlichen Eigenschaften für die Pathogenese der HK ist jedoch nur wenig erforscht. Um dieser Frage nachzugehen, habe ich einen FRET-basierten HTT-Seeding-Assay (FRASE) zum Nachweis selbstreplizierender mHTT-Aggregate („seeds“) entwickelt und ihn für den sensitiven Nachweis der Replikationsaktivität (HSA) in biologischen Proben von Patienten mit der HK und Modellsystemen optimiert. mHTT „seeds“ konnten früh im Krankheitsverlauf nachgewiesen werden und nahmen in ihrer Häufigkeit zu, was darauf hindeutet, dass durch die Messung der HSA das Fortschreiten der Krankheit quantitativ verfolgt werden kann. Biochemische Untersuchungen von Maushirnhomogenaten zeigten, dass eher kleine als große mHTT-Aggregatspezies für die gemessene HSA verantwortlich sind. Mit dem FRASE-Assay untersuchte ich HSA in einem etablierten induzierbaren Drosophila-HD-Modell und beobachtete eine starke Korrelation zwischen HSA und erhöhter Mortalität in transgenen Fliegen, was nahelegt, dass mHTT „seeds“ krankheitsrelevante neurotoxische Strukturen sind. Mittels strukturgestützter Mutagenese habe ich anschließend Proteinvarianten des mHTTex1 Proteins generiert, um die Auswirkungen der Struktur von mHTTex-Aggregaten auf deren Toxizität zu untersuchen. Die generierten mHTTex1-Proteinvarianten unterschieden sich in ihren Aggregationseigenschaften und bildeten fibrilläre Aggregate mit unterschiedlicher Stabilität und Morphologie. Um die Proteotoxizität von strukturell unterschiedlichen mHTTex1 Aggregaten in vivo zu untersuchen, wurden die Proteinvarianten in neu entwickelten Drosophila-Modellen panneuronal exprimiert. Die phänotypische und biochemische Charakterisierung der transgenen Fliegen zeigte, dass die Aggregatbildung mit der Toxizität korreliert und dass diese auch von der Stabilität der Aggregate beeinflusst wird. Zusammengefasst weisen meine Studien darauf hin, dass selbstreplizierenden mHTTex1-Aggregate bei der Pathogenese der HK eine wichtige Rolle spielen. Außerdem entwickelte ich eine Vielzahl neuer Methoden und Werkzeuge, die nun in der Krankheitsforschung angewendet werden können.Huntington’s disease (HD) is progressive neurodegenerative disorder with an autosomal dominant inheritance, manifesting with a triad of motor, cognitive and behavioral symptoms. HD is an incurable disease caused by a CAG trinucleotide repeat expansion in the HTT gene, which translates into an expanded polyglutamine tract in the huntingtin (HTT) protein. This expansion renders the conformation of the HTT protein unstable and promotes the assembly of amyloidogenic protein aggregates, predominantly composed of N-terminal HTT fragments. Amyloidogenic mutant HTT (mHTT) aggregates share common features with prion proteins, being able to self-propagate their corrupted conformation and to spread between adjacent cells. However, the role of mHTT aggregates and their prion-like features in the pathogenesis of HD remains elusive. To address this question, I developed a FRET-based aggregate HTT seeding assay (FRASE) for the detection of self-replicating mHTT aggregates. I optimized the assay for the sensitive quantification of mHTT seeding activity (HSA) in biological samples from HD patients and disease models. I could show that mHTT seeds are present early in the pathogenesis and increase in abundance with progression of disease, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Using the FRASE assay, I assessed HSA in an established inducible Drosophila model of HD and found a strong correlation between HSA and increased mortality in transgenic HD flies, suggesting that self-replicating mHTT seeds are disease relevant, neurotoxic structures causing severe phenotypic consequences in vivo. Next, I used structure-guided mutagenesis to generate protein variants of the N-terminal mHTT exon 1 fragment (mHTTex1), as a tool to explore the relationship between the structural properties of mHTTex1 aggregates and their putative proteotoxicity. Recombinant mHTTex1 protein variants were distinct in their aggregation properties and revealed fibrillar aggregates with different stabilities and morphologies. In order to relate these structural features to mHTTex1-induced proteotoxicity, protein variants were pan-neuronally expressed in a newly developed Drosophila model. Behavioral and biochemical analysis of transgenic flies confirmed the concurrence of mHTTex1 aggregates and toxicity and indicated that aggregate stability influences neurotoxicity in transgenic HD flies. Taken together, my studies emphasize the importance of self-replicating mHTTex1 aggregates in HD pathogenesis and provide novel tools for basic and clinical disease research

Mutant HTT seeding activity: a marker of disease progression and neurotoxicity in models of Huntington’s disease

Self-propagating, amyloidogenic mutant huntingtin (HTT) aggregates may drive progression of Huntington’s disease (HD). Here, we report the development of a FRET-based HTT aggregate seeding (FRASE) biosensor assay that enables the quantification of mutant HTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knock-in mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that HSA is high in fractions that contain small HTT fibrils but is low in fractions with large, insoluble HTT aggregates, indicating that small rather than large mutant HTT structures possess HSA. Finally, we assessed the neurotoxicity of mutant HTT seeds in an inducible Drosophila model transgenic for HTT. We found a strong correlation between HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mutant HTT structures with severe phenotypic consequences in vivo

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.This work was supported by Wellcome Trust Grant 091157, JDRF International Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre, and the Medical Research Council Cusrow Wadia Fund. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). U.M.N. was the recipient of a Hoffmann-La Roche postdoctoral fellowship.This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S089684111400146

Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics’ ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) — brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways

Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics’ ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) — brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing

What makes a good reflective paper?

Reflective papers are increasingly recognized as potentially important contributors to clinical education and practice; however, few quality guidelines are available for potential authors or reviewers. We sought to identify key characteristics of effective reflective papers and to clarify factors that increased or reduced the probability of acceptance for publication.A 10-item survey addressing the definition, purpose, and quality characteristics of reflective papers was developed based on a literature review and analysis of the author instructions of 14 journals that regularly publish reflective papers and are likely to be read by primary care physicians. The survey was sent electronically to the editor or associate editor responsible for reflective papers at each journal.Seven completed surveys were returned. The essential element defining a reflective paper was identified as narration of a specific professional experience that resonated with readers and conveyed deeper meaning. All respondents rated emotional engagement as very important, followed by stimulating reflection in the reader, providing a lesson applicable to patient care, and stimulating discussion with colleagues and/or learners. Reasons for acceptance or rejection of reflective submissions to journals were identified in issues related to writing style, topic selection, and reader reaction.Writing and reviewing reflective papers is strongly dependent on context, personal values, experience, and emotional reaction; nevertheless, core quality features can be identified to guide both writers and editors/reviewers without destroying the unique nature of these papers

What makes a good reflective paper?

Reflective papers are increasingly recognized as potentially important contributors to clinical education and practice; however, few quality guidelines are available for potential authors or reviewers. We sought to identify key characteristics of effective reflective papers and to clarify factors that increased or reduced the probability of acceptance for publication.A 10-item survey addressing the definition, purpose, and quality characteristics of reflective papers was developed based on a literature review and analysis of the author instructions of 14 journals that regularly publish reflective papers and are likely to be read by primary care physicians. The survey was sent electronically to the editor or associate editor responsible for reflective papers at each journal.Seven completed surveys were returned. The essential element defining a reflective paper was identified as narration of a specific professional experience that resonated with readers and conveyed deeper meaning. All respondents rated emotional engagement as very important, followed by stimulating reflection in the reader, providing a lesson applicable to patient care, and stimulating discussion with colleagues and/or learners. Reasons for acceptance or rejection of reflective submissions to journals were identified in issues related to writing style, topic selection, and reader reaction.Writing and reviewing reflective papers is strongly dependent on context, personal values, experience, and emotional reaction; nevertheless, core quality features can be identified to guide both writers and editors/reviewers without destroying the unique nature of these papers