Detection of cerebral tauopathy in P301L mice using high-resolution large-field multifocal illumination fluorescence microscopy

Current intravital microscopy techniques visualize tauopathy with high-resolution, but have a small field-of-view and depth-of-focus. Herein, we report a transcranial detection of tauopathy over the entire cortex of P301L tauopathy mice using large-field multifocal illumination (LMI) fluorescence microscopy technique and luminescent conjugated oligothiophenes. In vitro assays revealed that fluorescent ligand h-FTAA is optimal for in vivo tau imaging, which was confirmed by observing elevated probe retention in the cortex of P301L mice compared to non-transgenic littermates. Immunohistochemical staining further verified the specificity of h-FTAA to detect tauopathy in P301L mice. The new imaging platform can be leveraged in pre-clinical mechanistic studies of tau spreading and clearance as well as longitudinal monitoring of tau targeting therapeutics

Efficient characterization of multiple binding sites of small molecule imaging ligands on amyloid-beta, tau and alpha-synuclein

PURPOSE: There is an unmet need for compounds to detect fibrillar forms of alpha-synuclein (αSyn) and 4-repeat tau, which are critical in many neurodegenerative diseases. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based assay to facilitate the characterization of small molecules that can bind these fibrils. METHODS: SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds toward recombinant amyloid-beta (Aβ)$_{42}$, K18-tau, full-length 2N4R-tau and αSyn fibrils. In silico modeling was performed to examine the binding pockets of ligands on αSyn fibrils. Immunofluorescence staining of postmortem brain tissue slices from Parkinson's disease patients and mouse models was performed with fluorescence ligands and specific antibodies. RESULTS: We optimized the protocol for the immobilization of Aβ$_{42}$, K18-tau, full-length 2N4R-tau and αSyn fibrils in a controlled aggregation state on SPR-sensor chips and for assessing their binding to ligands. The SPR results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes, benzothiazole derivatives, nonfluorescent methylene blue and lansoprazole. In silico modeling studies for αSyn (6H6B) revealed four binding sites with a preference for one site on the surface. Immunofluorescence staining validated the detection of pS129-αSyn positivity in the brains of Parkinson's disease patients and αSyn preformed-fibril injected mice, 6E10-positive Aβ in arcAβ mice, and AT-8/AT-100-positivity in pR5 mice. CONCLUSION: SPR measurements of small molecules binding to Aβ$_{42}$, K18/full-length 2N4R-tau and αSyn fibrils suggested the existence of multiple binding sites. This approach may provide efficient characterization of compounds for neurodegenerative disease-relevant proteinopathies

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson’s disease in vivo

Abstract: Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo

BACKGROUND Abnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo . METHODS Fluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. RESULTS THK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinson's disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe $^{3+}$ form, as evinced by the STXM results. CONCLUSION We demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo

Metabolic rate of C. quadricarinatus juveniles injected with recombinant CHH

<p>Metabolic rate of crayfish injected with recombinant CHH. Oxygen consumption of crayfish was determined as an indirect measure of the metabolic rate after 30 days under stressful conditions (high salinity 10 g/L; low temperature 20 °C). Values are means ± standard errors. No significant (p>0.05) differences were detected among groups.</p

Protein structure determination in human cells by in-cell NMR and a reporter system to optimize protein delivery or transexpression

Most experimental methods for structural biology proceed in vitro and therefore the contribution of the intracellular environment on protein structure and dynamics is absent. Studying proteins at atomic resolution in living mammalian cells has been elusive due to the lack of methodologies. In-cell nuclear magnetic resonance spectroscopy (in-cell NMR) is an emerging technique with the power to do so. Here, we improved current methods of in-cell NMR by the development of a reporter system that allows monitoring the delivery of exogenous proteins into mammalian cells, a process that we called here “transexpression”. The reporter system was used to develop an efficient protocol for in-cell NMR which enables spectral acquisition with higher quality for both disordered and folded proteins. With this method, the 3D atomic resolution structure of the model protein GB1 in human cells was determined with a backbone root-mean-square deviation (RMSD) of 1.1 Å.ISSN:2399-364

At-sea distribution, movements and diving behavior of Magellanic penguins reflect small-scale changes in oceanographic conditions around the colony

Penguins are highly specialized divers that are expected to reflect environmental variation by adjusting their foraging behavior. We performed a comprehensive analysis of the at-sea distribution, diving, and foraging performance of Magellanic penguins (Spheniscus magellanicus) during the early chick-rearing period over two consecutive breeding seasons. The study was conducted at Cabo dos Bahías, (44° 54′ 50″ S; 65° 32′ 37″ W) a breeding colony located south of the latitudinal range of penguins’ main prey item Argentine anchovies (Engraulis anchoita). We also linked penguin foraging behavior to sea surface temperature (SST) to examine how birds cope with differences in oceanographic conditions. For this, we instrumented 37 adult penguins (18 in 2015 and 19 in 2016) with data loggers. In addition, we recorded chick growth in body mass during the first 12 days of life. Overall, the diving patterns of adult Magellanic penguins were similar in both breeding seasons. However, during 2015, adult breeders spent more time at the sea surface between foraging dives and performed more foraging dives per hour. The time spent foraging was higher in 2016 than in 2015. Foraging penguins also expanded their foraging range more than 100 km during 2016. Temperature records gathered by diving penguins during 2016 showed significantly higher temperatures, both at the sea surface as well as at the bottom of dives. Adults performed a higher foraging effort and chicks gained weight faster during 2016. Site-specific variability in prey distribution and abundance may be responsible for inter-seasonal discrepancies in the foraging and diving patterns. We reasoned that any environmental change could cause a shift in the distribution of anchovies, which would change the foraging behavior of penguins as they attempted to optimize their chick growth. Penguins from Cabo dos Bahías appear to face this environmental challenge with a highly sensitive at-sea foraging performance by increasing foraging effort when necessary.Fil: Blanco, Gabriela Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; ArgentinaFil: Gallo, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; ArgentinaFil: Pisoni, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Dell´Omo, G.. Ornis Italica; ItaliaFil: Gerez, N. A.. Universidad Nacional de la Patagonia. Facultad de Ciencias Naturales. Sede Puerto Madryn; ArgentinaFil: Molina, G.. Universidad del Mar; MéxicoFil: Quintana, Flavio Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentin

Transcranial detection of tauopathy in vivo in P301L mice with high‐resolution large‐field multifocal illumination fluorescence microscopy

Background Tau abnormal aggregates have been visualized in‐vivo using intravital microscopy techniques with high resolution, but are limited to a small field‐of‐view and depth. Here, we report a new method for the transcranial detection of tau deposits accross the brain with 6 μm resolution. Method In vitro Thioflavin T fluorescence assay was performed to screen fluorescence imaging probes that bind to tau fibrils. P301L (Thy1.2) mouse models of frontal temporal lobe dementia with four repeat tau and non‐transgenic littermates were imaged in vivo using a novel large‐field multifocal illumination (LMI) fluorescence microscopy technique using luminescent conjugated oligothiophenes h‐FTAA. Immunohistochemical staining were performed on P301L mouse brain tissue sections using anti‐phosphorylated tau antibodies AT8 and AT100. Result In vitro Thioflavin T fluorescence assay and LMI imaging shows that h‐FTAA bind to recombinant full‐length tau fibrils. In vivo LMI imaging using h‐FTAA showed higher retention in the cortex of P301L mice at 10 month‐of‐age compared to age‐matched non‐transgenic littermates. Immunohistochemical staining on P301L mouse brain tissue sections showed co‐localization of h‐FTAA with anti‐phosphorylated tau antibodies AT8 and AT100, thus verifying the specificity of the probe to tauopathy in P301L mice. Conclusion We demonstrate a new in vivo high‐resolution imaging platform for detection of tauopathy in murine models of frontal temporal lobe tauopathy

RSUME enhances Glucocorticoid Receptor SUMOylation and transcriptional activity

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation.We nowdemonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulatesGRtranscriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating thatGRSUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-inducedGRSUMOylation. These results show that inhibitory and stimulatorySUMOsites are present in theGRand at higher SUMOylation levels the stimulatory one becomes dominant.Fil: Druker, Jimena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Antunica Noguerol, María de Las Nieves. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Rein, Theo. Max Planck Institute of Psychiatry; AlemaniaFil: Iñiguez Lluhi, Jorge A.. University of Michigan Medical School. Department of Pharmacology; Estados UnidosFil: Holsboer, Florian. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; Argentin