Mutant Huntingtin Fragments Form Oligomers in a Polyglutamine Length-Dependent Manner \u3cem\u3ein Vitro\u3c/em\u3e and \u3cem\u3ein Vivo\u3c/em\u3e

Huntington disease (HD) is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis

Detection of Mutant-Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having ab-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that can be modulated by small molecules with protective potential in HD models

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-Dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD

Loss of Hsp70 Exacerbates Pathogenesis But Not Levels of Fibrillar Aggregates in a Mouse Model of Huntington's Disease

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates

Methylene Blue Modulates Huntingtin Aggregation Intermediates and is Protective in Huntington\u27s Disease Models

Huntington\u27s disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD

Modification of the ω\omega-Meson Lifetime in Nuclear Matter

The photo production of $\omega$ mesons on the nuclei C, Ca, Nb and Pb has been measured using the Crystal Barrel/TAPS detector at the ELSA tagged photon facility in Bonn. The dependence of the $\omega$ meson cross section on the nuclear mass number has been compared with three different types of models, a Glauber analysis, a BUU analysis of the Giessen theory group and a calculation by the Valencia theory group. In all three cases, the inelastic $\omega$ width is found to be $130-150 \rm{MeV/c^2}$ at normal nuclear matter density for an average 3-momentum of 1.1 GeV/c. In the restframe of the $\omega$ meson, this inelastic $\omega$ width corresponds to a reduction of the $\omega$ lifetime by a factor $\approx 30$. For the first time, the momentum dependent $\omega$N cross section has been extracted from the experiment and is in the range of 70 mb.Comment: 5 pages, 4 figure

In-medium ω\omega mass from the γ+Nb→π0γ+X\gamma + Nb \to \pi^{0}\gamma + X reaction

Data on the photoproduction of $\omega$ mesons on nuclei have been re-analyzed in a search for in-medium modifications. The data were taken with the Crystal Barrel(CB)/TAPS detector system at the ELSA accelerator facility in Bonn. First results from the analysis of the data set were published by D. Trnka et al. in Phys. Rev. Lett 94 (2005) 192303 \cite{david}, claiming a lowering of the $\omega$ mass in the nuclear medium by 14$$ at normal nuclear matter density. The extracted $\omega$ line shape was found to be sensitive to the background subtraction. For this reason a re-analysis of the same data set has been initiated and a new method has been developed to reduce the background and to determine the shape and absolute magnitude of the background directly from the data. Details of the re-analysis and of the background determination are described. The $\omega$ signal on the $Nb$ target, extracted in the re-analysis, does not show a deviation from the corresponding line shape on a $LH_2$ target, measured as reference. The earlier claim of an in-medium mass shift is thus not confirmed. The sensitivity of the $\omega$ line shape to different in-medium modification scenarios is discussed.Comment: 13 pages and 11 figures, submitted for publicatio

Quasi-free photoproduction of eta-mesons of the neutron

Quasi-free photoproduction of eta-mesons off nucleons bound in the deuteron has been measured with the CBELSA/TAPS detector for incident photon energies up to 2.5 GeV at the Bonn ELSA accelerator. The eta-mesons have been detected in coincidence with recoil protons and recoil neutrons, which allows a detailed comparison of the quasi-free n(gamma,eta)n and p(gamma,eta)p reactions. The excitation function for eta-production off the neutron shows a pronounced bump-like structure at W=1.68 GeV (E_g ~ 1 GeV), which is absent for the proton.Comment: accepted for publication in Phys. Rev. Let

K^0 pi^0 Sigma^+ and K^*0 Sigma^+ photoproduction off the proton

The exclusive reactions $\gamma p \to K^{*0} \Sigma^+(1189)$ and $\gamma p \to K^{0} \pi^{0}\Sigma^+(1189)$, leading to the p 4$\pi^{0}$ final state, have been measured with a tagged photon beam for incident energies from threshold up to 2.5 GeV. The experiment has been performed at the tagged photon facility of the ELSA accelerator (Bonn). The Crystal Barrel and TAPS detectors were combined to a photon detector system of almost 4$\pi$ geometrical acceptance. Differential and total cross sections are reported. At energies close to the threshold, a flat angular distribution has been observed for the reaction $\gamma p\to K^{0} \pi^{0}\Sigma^+$ suggesting dominant s-channel production. $\Sigma^*(1385)$ and higher lying hyperon states have been observed. An enhancement in the forward direction in the angular distributions of the reaction $\gamma p \to K^{*0}\Sigma^+$ indicates a $t$-channel exchange contribution to the reaction mechanism. The experimental data are in reasonable agreement with recent theoretical predictions.Comment: 11 pages, 13 figures, submitted to EPJ

Photoproduction of η -mesons off nuclei for Eγ ⩽ 2.2 GeV

The photoproduction of η -mesons off 12C , 40Ca , 93Nb , and nat Pb nuclei has been measured with a tagged photon beam with energies between 0.6 and 2.2GeV. The experiment was performed at the Bonn ELSA accelerator with the combined setup of the Crystal Barrel and TAPS calorimeters. It aimed at the in-medium properties of the S 11(1535) nucleon resonance and the study of the absorption properties of nuclear matter for η -mesons. Careful consideration was given to contributions from ηπ final states and secondary production mechanisms of η -mesons, e.g. from inelastic πN reactions of intermediate pions. The analysis of the mass number scaling shows that the nuclear absorption cross-section $\sigma_{{N\eta}}^{}$ for η -mesons is constant over a wide range of the η momentum. The comparison of the excitation functions to data off the deuteron and to calculations in the framework of a BUU model show no unexplained in-medium modifications of the S 11(1535