Journal Pre-proof Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS

International audienceMutations in Fused in sarcoma (FUS), an RNA-binding protein, are known to cause Amyotrophic Lateral Sclerosis (ALS). However, molecular mechanisms due to loss of FUS function remain unclear and controversial. Here, we report the characterization and phenotypic analysis of a deletion mutant of the unique FUS orthologue in zebrafish where Fus protein levels are depleted. The homozygous mutants displayed a reduced lifespan as well as impaired motor abilities associated with specific cellular deficits, including decreased motor neurons length and neuromuscular junctions (NMJ) fragmentation. Furthermore, we demonstrate that these cellular impairments are linked to the misregulation of mRNA expression of acetylcholine receptor (AChR) subunits and histone deacetylase 4, markers of denervation and reinnervation processes observed in ALS patients. In addition, fus loss of function alters tau transcripts favoring the expression of small tau isoforms. Overall, this new animal model extends our knowledge on FUS and supports the relevance of FUS loss of function in ALS physiopathology

Rescue of impaired late-phase long-term depression in a tau transgenic mouse model

Cognitive decline, the hallmark of Alzheimer's disease, and accompanying neuropsychiatric symptoms share dysfunctions of synaptic processes as a common cellular pathomechanism. Long-term potentiation has proven to be a sensitive tool for the "diagnosis" of such synaptic dysfunctions. Much less, however, is known about how long-term depression (LTD), an alternative mechanism for the storage of memory, is affected by Alzheimer's disease progression. Here, we demonstrate that impaired late LTD (>3 hours) in THY-Tau22 mice can be rescued by either inhibition of glycogen synthase kinase-3 (GSK3β) activity or by application of the protein-phosphatase 2A agonist selenate. In line with these findings, we observed increased phosphorylation of GSK3β at Y216 and reduced total phosphatase activity in biochemical assays of hippocampal tissue of THY-Tau22 mice. Interestingly, LTD induction and pharmacologic inhibition of GSK3β appeared to downregulate GSK3ß activity via a marked upregulation of phosphorylation at the inhibitory Ser9 residue. Our results point to alterations in phosphorylation and/or dephosphorylation homeostasis as key mechanisms underlying the deficits in LTD and hippocampus-dependent learning found in THY-Tau22 mice.publisher: Elsevier articletitle: Rescue of impaired late–phase long-term depression in a tau transgenic mouse model journaltitle: Neurobiology of Aging articlelink: http://dx.doi.org/10.1016/j.neurobiolaging.2014.09.015 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

The MAP kinase pathway mediates transcytosis induced by TNF-α in an in vitro blood-brain barrier model

International audienceCerebral capillary endothelial cells constitute the blood-brain barrier (BBB). In these highly specialized cells, transcellular transports rarely occur, and the presence of tight junctions between them leads to a low paracellular permeability. In order to understand the functions of this barrier, an in vitro model of the BBB has been developed and consists in a co-culture of primary cerebral capillary endothelial cells and glial cells. When these endothelial cells are subjected to an inflammatory agent, such as tumor necrosis factoralpha (TNF-α), in vitro BBB permeability is increased, as indicated by the increase in holotransferrin transcytosis. However, no significant change in the paracellular permeability is observed. In order to understand the molecular mechanisms that underlie these transcytosis processes, we investigated the implication of the mitogen-activated protein kinase (MAPK) signalling pathway, as TNF-α is known to activate this kinase family. In the present study, an increase in the activation of p42–44 MAPK is observed after TNF-α treatment. Holotransferrin transcytosis as well as p42–44 MAPK activation are inhibited after addition of a p42–44 MAPK pathway inhibitor (UO126) during TNF-α challenge. These data suggest that the MAPK pathway is involved in the transcytosis regulation in endothelial cells from an in vitro BBB model

Tau hyperphosphorylation induced by the anesthetic agent ketamine/xylazine involved the calmodulin‐dependent protein kinase II

International audienceTau hyperphosphorylation is a major neuropathological hallmark of many neurodegenerative disorders such as Alzheimer's disease. Several anesthetics have been shown previously to induced marked tau hyperphosphorylation. Although the ketamine/xylazine mixture is one of the most commonly used anesthetic agents in animal research and veterinary practice, the effect of this anesthetic agent on tau phosphorylation still remains to be determined. Here, we found that ketamine-/xylazine-induced a rapid and robust hyperphosphorylation of tau in a dose-dependent manner under normothermic and hypothermic conditions in mice. When used together, ketamine and xylazine exerted a synergistic action on tau phosphorylation most strongly not only on epitopes S396 and S262, but also on other residues (T181, and S202/T205). We observed that activation of the calmodulin-dependent protein kinase II (CaMKII) is the major upstream molecular event leading to tau hyperphosphorylation following ketamine/xylazine anesthesia in mice. Moreover, we observed that intracerebroventricular injection of the selective CaMKII inhibitor KN93 attenuated tau hyperphosphorylation. Since ketamine/xylazine also had a marked impact on other key molecular signaling pathways involving the MAP/microtubule affinity-regulating kinase (MARK), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3 (GSK3), our study calls for high caution and careful monitoring when using this anesthetic agent in laboratory animal settings across all fields of biological sciences in order to avoid artifactual results

Phosphorylation of N-methyl-D-aspartic acid receptor-associated neuronal nitric oxide synthase depends on estrogens and modulates hypothalamic nitric oxide production during the ovarian cycle.

International audienceWithin the preoptic region, nitric oxide (NO) production varies during the ovarian cycle and has the ability to impact hypothalamic reproductive function. One mechanism for the regulation of NO release mediated by estrogens during the estrous cycle includes physical association of the calcium-activated neuronal NO synthase (nNOS) enzyme with the glutamate N-methyl-d-aspartate (NMDA) receptor channels via the postsynaptic density 95 scaffolding protein. Here we demonstrate that endogenous variations in estrogens levels during the estrous cycle also coincide with corresponding changes in the state of nNOS Ser1412 phosphorylation, the level of association of this isoform with the NMDA receptor/postsynaptic density 95 complex at the plasma membrane, and the activity of NO synthase (NOS). Neuronal NOS Ser1412 phosphorylation is maximal on the afternoon of proestrus when both the levels of estrogens and the physical association of nNOS with NMDA receptors are highest. Estradiol mimicked these effects in ovariectomized (OVX) rats. In addition, the catalytic activity of NOS in membrane protein extracts from the preoptic region, i.e. independent of any functional protein-protein interactions or cell-cell signaling, was significantly increased in estradiol-treated OVX rats compared with OVX rats. Finally, lambda phosphatase-mediated nNOS dephosphorylation dramatically impaired NOS activity in preoptic region protein extracts, thus demonstrating the important role of phosphorylation in the regulation of NO production in the preoptic region. Taken together, these results yield new insights into the regulation of neuron-derived NO production by gonadal steroids within the preoptic region and raise the possibility that changes in nNOS phosphorylation during fluctuating physiological conditions may be involved in the hypothalamic control of key neuroendocrine functions, such as reproduction

Physiological pathway for low density lipoproteins across the blood-brain barrier: transcytosis through brain capillary endothelial cells in vitro

International audienceAlthough an immense knowledge has accumulated concerning regulation of cholesterol homeostasis in the body, this does not include the brain, where details are just emerging. Using an in vitro blood-brain barrier model, we have demonstrated that low density lipoprotein underwent transcytosis through the endothelial cells (ECs) by a receptor-mediated process bypassing the lysosomal compartment. Moreover, caveolae might be involved in these blood-borne molecule transports from the blood to the brain. Although several ligands are known to be internalized through cell surface caveolae, the subsequent intracellular pathways have remained elusive. By cell fractionation experiment and Western blot, we have demonstrated that the LDL receptor is located in the caveolae membrane fraction. Then, low density lipoproteins internalized were detected by electron microscopy in multivesicular bodies. We identified in brain capillary ECs a novel endosomal compartment, mildly acidic, positive for marker Lamp-1 but devoid of any degradative capability. In terms of pH point of view, cellular location and caveolae derived formation, the multivesicular organelles described here can be related to the caveosome structure. Our results could allow us to provide clues to physiological functions of caveolae-caveosome transcellular pathway in brain capillary ECs and may help in the rational design of more effective therapeutic drugs to the brain

Un cluster géographique de paralysie supranucléaire progressive dans le nord de la France

International audienceObjective: To describe a cluster of progressive supranuclear palsy (PSP) in northern France. PSP has not been reported in geographical, temporal, or occupational clusters. A unit of Neurology and Neurogeriatrics opened in 2005 at the Centre Hospitalier de Wattrelos, serving the population of Wattrelos and Leers (combined population 51,551) and parts of neighboring towns. For most of the 20th century, this area was a center for chromate and phosphate ore processing, textile dyeing, and tanning. Significant industrial waste persists close to residential areas. Methods: From 2005 to 2014, 92 patients with PSP at Centre Hospitalier de Wattrelos were identified and studied. Detailed residential data were available in the medical records. Eighty cases have had magnetic resonance head scanning and 60 have died, of whom 13 have been examined neuropathologically. Results: The ratio of observed to expected PSP incidence over the period 2005 to 2012 was 12.3 (95% confidence interval: 7.4–35.9). Mean onset age was 74.3 years. The Richardson syndrome/PSP-parkinsonism ratio was 43%/42%. Four other phenotypes each occurred in 2% to 5%. Onset was gait/balance difficulty in 52%. None of the 92 affected patients were relatives and 7 were of North African ancestry. MRI was compatible with a clinical diagnostic of PSP in all cases. Histopathologic examination confirmed neurofibrillary degeneration and tufted astrocytes in all autopsied cases. Western blots revealed a typical tau 4R doublet. The tau H1 haplotype occurred in 95.8% of cases' chromosomes. Conclusions: We have identified a cluster of PSP in a geographical area with severe environmental contamination by industrial metals

Inhibition of P-glycoprotein: rapid assessment of its implication in blood-brain barrier integrity and drug transport to the brain by an in vitro model of the blood-brain barrier

International audiencePurpose. The objective of this work was to asses, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agents. Methods. An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal or abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity