24 research outputs found
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
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 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 âtransexpressionâ. 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 Ă
.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 BahĂas, (44° 54âČ 50âłÂ S; 65° 32âČ 37âłÂ W) a breeding colony located south of the latitudinal range of penguinsâ 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 BahĂas 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 CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Centro Nacional PatagĂłnico. Instituto de BiologĂa de Organismos Marinos; ArgentinaFil: Gallo, Luciana. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Centro Nacional PatagĂłnico. Instituto de BiologĂa de Organismos Marinos; ArgentinaFil: Pisoni, Juan Pablo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Centro Nacional PatagĂłnico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: DellÂŽOmo, 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; MĂ©xicoFil: Quintana, Flavio Roberto. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Centro Nacional PatagĂłnico. Instituto de BiologĂa de Organismos Marinos; Argentin
Transcranial detection of tauopathy in vivo in P301L mice with highâresolution largeâfield multifocal illumination fluorescence microscopy
Background
Tau abnormal aggregates have been visualized inâvivo using intravital microscopy techniques with high resolution, but are limited to a small fieldâofâview and depth. Here, we report a new method for the transcranial detection of tau deposits accross the brain with 6 ÎŒm 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 nonâtransgenic littermates were imaged in vivo using a novel largeâfield multifocal illumination (LMI) fluorescence microscopy technique using luminescent conjugated oligothiophenes hâFTAA. Immunohistochemical staining were performed on P301L mouse brain tissue sections using antiâphosphorylated tau antibodies AT8 and AT100.
Result
In vitro Thioflavin T fluorescence assay and LMI imaging shows that hâFTAA bind to recombinant fullâlength tau fibrils. In vivo LMI imaging using hâFTAA showed higher retention in the cortex of P301L mice at 10 monthâofâage compared to ageâmatched nonâtransgenic littermates. Immunohistochemical staining on P301L mouse brain tissue sections showed coâlocalization of hâFTAA with antiâphosphorylated tau antibodies AT8 and AT100, thus verifying the specificity of the probe to tauopathy in P301L mice.
Conclusion
We demonstrate a new in vivo highâresolution 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 CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn en Biomedicina de Buenos Aires; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn en Biomedicina de Buenos Aires; ArgentinaFil: Antunica Noguerol, MarĂa de Las Nieves. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn en Biomedicina de Buenos Aires; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn 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 CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn en Biomedicina de Buenos Aires; Argentin
A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy bodyâlike pathology
Parkinsonâs disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domainâcontaining protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy bodyâlike pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies
Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography
Purpose
Abnormal tau accumulation within the brain plays an important role in tauopathies such as Alzheimerâs disease and frontotemporal dementia. High-resolution imaging of tau deposits at the whole-brain scale in animal disease models is highly desired.
Methods
We approached this challenge by non-invasively imaging the brains of P301L mice of 4-repeat tau with concurrent volumetric multi-spectral optoacoustic tomography (vMSOT) atâ~â115 ÎŒm spatial resolution using the tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 (i.v.). In vitro probe characterization, concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice, followed by ex vivo validation using AT-8 antibody for phosphorylated tau.
Results
PBB5 showed specific binding to recombinant K18 tau fibrils by fluorescence assay, to post-mortem Alzheimerâs disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brains. Dose-dependent optoacoustic and fluorescence signal intensities were observed in the mouse brains following i.v. administration of different concentrations of PBB5. In vivo vMSOT brain imaging of P301L mice showed higher retention of PBB5 in the tau-laden cortex and hippocampus compared to wild-type mice, as confirmed by ex vivo vMSOT, epi-fluorescence, multiphoton microscopy, and immunofluorescence staining.
Conclusions
We demonstrated non-invasive whole-brain imaging of tau in P301L mice with vMSOT system using PBB5 at a previously unachievedâ~â115 ÎŒm spatial resolution. This platform provides a new tool to study tau spreading and clearance in a tauopathy mouse model, foreseeable in monitoring tau targeting putative therapeutics.ISSN:1619-7070ISSN:1619-708