Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin

2008 Inter-laboratory Comparison Study of a Reference Material for Nutrients in Seawater

Autoclaved natural seawater collected in the North Pacific Ocean was used as a reference material for nutrients in seawater (RMNS) during an inter-laboratory comparison (I/C) study conducted in 2008. This study was a follow-up to previous studies conducted in 2003 and 2006. A set of six samples was distributed to each of 58 laboratories in 15 countries around the globe, and results were returned by 54 of those laboratories (15 countries). The homogeneities of samples used in the 2008 I/C study, based on analyses for three determinants, were improved compared to those of samples used in the 2003 and 2006 I/C studies. Results of these I/C studies indicate that most of the participating laboratories have an analytical technique for nutrients that is sufficient to provide data of high comparability. The differences between reported concentrations from the same laboratories in the 2006 and 2008 I/C studies for the same batch of RMNS indicate that most of the laboratories have been maintaining internal comparability for two years. Thus, with the current high level of performance in the participating laboratories, the use of a common reference material and the adaptation of an internationally accepted nutrient scale system would increase comparability among laboratories worldwide, and the use of a certified reference material would establish traceability. In the 2008 I/C study we observed a problem of non-linearity of the instruments of the participating laboratories similar to that observed among the laboratories in the 2006 I/C study. This problem of non-linearity should be investigated and discussed to improve comparability for the full range of nutrient concentrations. For silicate comparability in particular, we see relatively larger consensus standard deviations than those for nitrate and phosphate

Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides.

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides

Conductivité thermique d'une céramique affectée par un réseau de fissures localisées aux joints de granulats : simulations 2D de l'effet d'une distribution bimodale de taille de granulats

International audienc

Realistic morphological models of weakly to strongly branched pore networks for the computation of effective properties

International audienceWe provide a detailed expository report of a new methodology aiming at building a numerical model of the complex pore distribution of porous UO$_2$ ceramics, tunable to real materials, in view of computing their effective thermal behavior. First, based on 2D optical microscopy images, we characterize the material of interest, dedicating a special attention to the porous network because of its major influence on the thermal behavior. Following Meynard et al. (2022), we then propose a simple morphological model combining a Voronoi tessellation and a boolean model, involving a limited number of parameters, from which 3D virtual microstructures (and so 2D cross-sections) can be generated. These parameters are tuned in order to select within our class of models the microstructures that are the most representative of the real ones ; in practice, this optimization process minimizes a cost function based on morphological descriptors computed from the 2D cross-sections. Last, we perform 2D full-field thermal simulations on cross-sections through Representative Volume Elements of both the numerical and the experimental microstructures. We validate our approach by qualitative and quantitative comparisons relative to both global properties and local field statistics

Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL^+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL_1/2, 1∶20,000), and anti-detyrosinated tubulin (L₄, 1∶20,000).</p

Analysis of developed anti-EB1 antibodies compared to the known anti-tubulin antibodies.

<p>(A) Western-blot analysis of the indicated proteins (15 ng) separated on 10% SDS-PAGE using a commercial anti-EB1 antibody (anti-total EB1, raised against amino-acids 107–268 of mouse EB1), the presently developed antibodies (anti-Tyr EB1 and anti-deTyr EB1), and tubulin antibodies. Detyrosinated EB1 was obtained from recombinant EB1 using carboxypeptidase A. Tyrosinated and detyrosinated tubulin were obtained from purified brain tubulin, using respectively TTL and carboxypeptidase A. Both anti-Tyr EB1 and anti-deTyr EB1 are highly specific. (B) Double immunostaining with anti-total EB1 antibody and anti-Tyr EB1 on fibroblasts after transfection of plasmids allowing expression of either tyrosinated or detyrosinated EB1 with EGFP at the N-terminus. The transfected cells were detected by EGFP signal. Anti-Tyr EB1 is highly specific of tyrosinated form of EB1. (C) Immunostaining of endogenous EB1 in astrocytes with anti-Tyr EB1 and anti-total EB1.</p

Analysis of C-termini of recombinant EB1 forms overexpressed in wild type and TTL-deficient fibroblasts.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) and of fractions of immunoprecipitation experiments carried out on cells transfected with cDNA encoding different EB1 forms. EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. (A) transfection of fibroblasts (NIH3T3) with plasmids encoding tyrosinated EB1 fused with GFP at the N-terminus (GFP-EB1), followed by immunoprecipitation using anti-GFP antibody and analysis using anti-Tyr EB1 (1∶15000) and anti-deTyr EB1 (1∶200). No detyrosinated GFP-EB1 could be detected. (B) Transfection of fibroblasts with cDNA encoding GFP-EB1 ending with the C-terminus of α-tubulin GEEEGEEY (GFP-EB1-CterTub), followed by immunoprecipitation with anti-GFP antibody and analysis using anti-Tyr Tub (1∶20,000) and anti-deTyr Tub (1∶20,000). NIH3T3 were used as TTL^+/+ cells and MEFs isolated from TTL null mice were used as TTL^−/−. A very low quantity of detyrosinated protein ending with α-tubulin residues was detected (upper band in IP fractions of lower panel).</p