Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ¿PEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.We are grateful to Stephen J. Gould (Johns Hopkins University, Baltimore), Nancy E. Braverman (McGill University, Montreal), Wolfgang Schliebs (Ruhr University, Bochum) and Daniel Passon (EMBL, Hamburg) for providing plasmids and antibodies. Work in J.E.A. lab is funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 – Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEXBCM/2311/2014), and through Norte 2020 – Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). We acknowledge support by the Open Access Publishing Fund of the University of Tübingen and the Deutsche Forschungsgemeinschaft for publishing costs

Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques

No Elevated Plasma Catecholamine Levels during Sleep in Newly Diagnosed, Untreated Hypertensives

The sympatho-adrenergic system is highly involved in regulating sleep, wake and arousal states, and abnormalities in this system are regarded as a key factor in the development and progression of arterial hypertension. While hypertension is associated with a hyperadrenergic state during wakefulness, the effect of hypertension on plasma-catecholamine levels during sleep is not yet known. Twelve young participants with newly diagnosed, untreated hypertension and twelve healthy controls slept for 7 hours in the sleep laboratory. Before and after sleep, subjects rested in a supine position for 3-h periods of wakefulness. We sampled blood at a fast rate (1/10 min) and monitored blood pressure and heart rate continuously. We show that plasma NE and E levels did not differ between hypertensives and normotensive during sleep as well as before and after sleep. Blood pressure was higher in hypertensives, reaching the largest group difference in the morning after sleep. Unlike in the normotensives, in the hypertensive participants the morning rise in blood pressure did not correlate with the rise in catecholamine levels at awakening. Our results suggest that hypertension in its early stages is not associated with a strong hyperadrenergic state during sleep. In showing a diminished control of blood pressure through sympatho-adrenergic signals in hypertensive participants, our data point towards a possible involvement of dysfunctional sleep-related blood pressure regulation in the development of hypertension

Voxel-wise comparisons of cellular microstructure and diffusion-MRI in mouse hippocampus using 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND)

A key challenge in medical imaging is determining a precise correspondence between image properties and tissue microstructure. This comparison is hindered by disparate scales and resolutions between medical imaging and histology. We present a new technique, 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND), for registering medical images with 3D histology to overcome these limitations. Ex vivo 120 × 120 × 200 μm resolution diffusion-MRI (dMRI) data was acquired at 7 T from adult C57Bl/6 mouse hippocampus. Tissue was then optically cleared using CLARITY and stained with cellular markers and confocal microscopy used to produce high-resolution images of the 3D-tissue microstructure. For each sample, a dense array of hippocampal landmarks was used to drive registration between upsampled dMRI data and the corresponding confocal images. The cell population in each MRI voxel was determined within hippocampal subregions and compared to MRI-derived metrics. 3D-BOND provided robust voxel-wise, cellular correlates of dMRI data. CA1 pyramidal and dentate gyrus granular layers had significantly different mean diffusivity (p > 0.001), which was related to microstructural features. Overall, mean and radial diffusivity correlated with cell and axon density and fractional anisotropy with astrocyte density, while apparent fibre density correlated negatively with axon density. Astrocytes, axons and blood vessels correlated to tensor orientation

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins, Is a Redox-Sensitive Protein

Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibr oblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.The authors are grateful to Dr. Ann Moser (Baltimore, USA) for the primary PEX5 null human fibroblasts. This work was supported bygrants from the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Onderzoeksproject G.0754.09)’ (to M. F. and P. P.V.V.), by the KU Leuven grants OT/09/045 (toM. F. and P. P. V. V.) and DBOF/10/059 (to P. P. V. V. and M. F.), and by FEDER funds through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Fundac¸ão para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019731 (PTDC/BIA-BCM/118577/2010) (to J. E. A.). M.N. is supported by a FLOF fellowship from the Department of Cellular and Molecular Medicine, KU Leuven. B.W. is a recipient of a DBOF fellowship (DBOF/10/059) from the KU Leuven. C. P. G. is supported by Fundac¸ão para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu

Neocortical hyperexcitability in a genetic model of absence seizures and its reduction by levetiracetam

PURPOSE: To study the effect of the antiepileptic drug levetiracetam (LEV) on the patterns of intrinsic optical signals (IOSs) generated by slices of the somatosensory cortex obtained from 3- and 6-month-old WAG/Rij and age-matched, nonepileptic control (NEC) rats. METHODS: WAG/Rij and NEC animals were anesthetized with enfluorane and decapitated. Brains were quickly removed, and neocortical slices were cut coronally with a vibratome, transferred to a submerged tissue chamber, and superfused with oxygenated artificial cerebrospinal fluid (aCSF). Slices were illuminated with a dark-field condensor and examined with a x2.5 objective; images were processed with a real time digital video image-enhancement system. Images were acquired before (background) and during electrical stimulation with a temporal resolution of 10 images/s and were displayed in pseudocolors. Extracellular stimuli (200 micros; <4 V) were delivered through bipolar stainless steel electrodes placed in the white matter. RESULTS: IOSs recorded in NEC slices bathed in control aCSF became less intense and of reduced size with age (p < 0.05); this trend was not seen in WAG/Rij slices. Age-dependent decreases in IOS intensity and area size were also seen in NEC slices superfused with aCSF containing the convulsant 4-aminopyridine (4-AP, 5 microM); in contrast, significant increases in both parameters occurred with age in 4-AP-treated WAG/Rij slices (p < 0.05). Under any of these conditions, the IOS intensity and area size slices were larger in WAG/Rij than in NEC slices. LEV (50-500 microM) application to WAG/Rij slices caused dose-dependent IOS reductions that were evident both in control and in 4-AP-containing aCSF and were more pronounced in 6-month-old tissue. CONCLUSIONS: These data demonstrate age-dependent IOS modifications in NEC and WAG/Rij rat slices and identify a clear pattern of hyperexcitability that occurs in 6-month-old WAG/Rij neocortical tissue, an age when absence seizures occur in all animals. The ability of LEV to reduce these patterns of network hyperexcitability supports the potential use of this new antiepileptic drug in primary generalized epileptic disorders

Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol

PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex.The authors thank Britta Moellers (Ruhr-Universität Bochum, Germany) for providing plasmids and recombinant protein for the generation of the anti-PEX6 antibody. This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 –Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020 – Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). A.G.P, T.F., D.B., A.F.D., A.B.B. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

Semi-Automated Reconstruction of Neural Processes from Large Numbers of Fluorescence Images

We introduce a method for large scale reconstruction of complex bundles of neural processes from fluorescent image stacks. We imaged yellow fluorescent protein labeled axons that innervated a whole muscle, as well as dendrites in cerebral cortex, in transgenic mice, at the diffraction limit with a confocal microscope. Each image stack was digitally re-sampled along an orientation such that the majority of axons appeared in cross-section. A region growing algorithm was implemented in the open-source Reconstruct software and applied to the semi-automatic tracing of individual axons in three dimensions. The progression of region growing is constrained by user-specified criteria based on pixel values and object sizes, and the user has full control over the segmentation process. A full montage of reconstructed axons was assembled from the ∼200 individually reconstructed stacks. Average reconstruction speed is ∼0.5 mm per hour. We found an error rate in the automatic tracing mode of ∼1 error per 250 um of axonal length. We demonstrated the capacity of the program by reconstructing the connectome of motor axons in a small mouse muscle