Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

Background

STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis.

Methodology/Principal Findings

Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice.

Conclusions/Significance

In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.     

Mutation of Ser172 in yeast β tubulin induces defects in microtubule dynamics and cell division

Ser172 of β tubulin is an important residue that is mutated in a human brain disease and phosphorylated by the cyclin-dependent kinase Cdk1 in mammalian cells. To examine the role of this residue, we used the yeast S. cerevisiae as a model and produced two different mutations (S172A and S172E) of the conserved Ser172 in the yeast β tubulin Tub2p. The two mutants showed impaired cell growth on benomyl-containing medium and at cold temperatures, altered microtubule (MT) dynamics, and altered nucleus positioning and segregation. When cytoplasmic MT effectors Dyn1p or Kar9p were deleted in S172A and S172E mutants, cells were viable but presented increased ploidy. Furthermore, the two β tubulin mutations exhibited synthetic lethal interactions with Bik1p, Bim1p or Kar3p, which are effectors of cytoplasmic and spindle MTs. In the absence of Mad2p-dependent spindle checkpoint, both mutations are deleterious. These findings show the importance of Ser172 for the correct function of both cytoplasmic and spindle MTs and for normal cell division.     

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6^+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Synthesis of β-Substituted Naphth-1-yl Ethylamido Derivatives as New Melatoninergic agonists

Naphthalene melatoninergic ligands with alkyl groups (Me, Et, Pr, Bz) in the β position of the ethylamido chain were synthesised. The affinity of the compounds for chicken brain melatonin receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as the radioligand. An increase in the affinity was observed with the β-methyl derivatives and the greatest increase was seen with the (−) enantiomers. The introduction of a 2- or 7-MeO group on the naphthalene ring and the lengthening (Et, Pr) of the alkylamido chain gave potent compounds such as (−)1h (K_i=24 pM). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles. The potency to produce lightening of the skin of Xenopus laevis was related to the affinities values of the molecules at melatonin chicken brain receptors. The most potent ligands were found to be full agonists and compound 1h was 25 fold more potent than melatonin in this bioassay.     

MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.     

Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.

We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.     

Degradation and fermentation of α-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats

The ability of several human gut bacteria to break down α-1,2 and α-1,6 glycosidic linkages in α-gluco-oligosaccharides (GOS) was investigated in vitro in substrate utilization tests. Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostridium butyricum , which are usually found in the infant gut and have been associated with both beneficial and deleterious effects on health, were studied. α-Gluco-oligosaccharide degradation was compared in vitro and in vivo in gnotobiotic rats associated with these organisms, inoculated alone or in combination. Oligomer breakdown and short chain fatty acid and gas production indicated hydrolysis and fermentation of the substrate. In vitro and in vivo, Cl. butyricum was the least efficient in utilizing GOS, whereas Bact. thetaiotaomicron was the most efficient. Kinetic studies on GOS hydrolysis in pH-regulated fermenters showed that α-1,2 glucosidic bonds, which characterize the substrate, were more resistant than α-1,6 linkages. Adaptation of gnotobiotic rats to a diet containing 2% (w/w) GOS significantly increased the hydrolysis of α 1,2 glucosidic bonds. Combination of bacteria in trixenic rats improved GOS degradation and inhibited Cl. butyricum metabolism. This inhibition was confirmed in pH-regulated fermenters containing GOS as the principal carbon source. The association of beneficial bacteria and GOS may therefore have a potential health-promoting effect in human necnates.     

Gauging scale effects and biogeographical signals in similarity distance decay analyses: an Early Jurassic ammonite case study

In biogeography, the similarity distance decay (SDD) relationship refers to the decrease in compositional similarity between communities with geographical distance. Although representing one of the most widely used relationships in biogeography, a review of the literature reveals that: (1) SDD is influenced by both spatial extent and sample size; (2) the potential effect of the phylogenetic level has yet to be tested; (3) the effect of a marked biogeographical structuring upon SDD patterns is largely unknown; and (4) the SDD relationship is usually explored with modern, mainly terrestrial organisms, whereas fossil taxa are seldom used in that perspective. Using this relationship, we explore the long‐distance dispersal of the Early Jurassic (early Pliensbachian, c. 190.8 Ma to 187.6 Ma) ammonites of the western Tethys and adjacent areas, in a context of marked provincialism. We show that the long‐distance dispersal of these ammonites is not related to shell size and shape, but rather to the environmental characteristics of the province to which they belong. This suggests that their long‐distance dispersal may have been essentially driven by passive planktonic drift during early juvenile, post‐hatching stages. Furthermore, it seems that the SDD relationship is not always an appropriate method to characterize the existence of a biogeographical structuring. We conducted SDD analyses at various spatial, sampling and phylogenetic scales in order to evaluate their sensitivity to scale effects. This multi‐scale approach indicates that the sampling scale may influence SDD rates in an unpredictable way and that the phylogenetic level has a major impact on SDD patterns.