Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors

Bronchoscopic assessment of airway retention time of aerosolized xylitol

BACKGROUND: Human airway surface liquid (ASL) has abundant antimicrobial peptides whose potency increases as the salt concentration decreases. Xylitol is a 5-carbon sugar that has the ability to lower ASL salt concentration, potentially enhancing innate immunity. Xylitol was detected for 8 hours in the ASL after application in airway epithelium in vitro. We tested the airway retention time of aerosolized iso-osmotic xylitol in healthy volunteers. METHODS: After a screening spirometry, volunteers received 10 ml of nebulized 5% xylitol. Bronchoscopy was done at 20 minutes (n = 6), 90 minutes (n = 6), and 3 hours (n = 5) after nebulization and ASL was collected using microsampling probes, followed by bronchoalveolar lavage (BAL). Xylitol concentration was measured by nuclear magnetic resonance spectroscopy and corrected for dilution using urea concentration. RESULTS: All subjects tolerated nebulization and bronchoscopy well. Mean ASL volume recovered from the probes was 49 ± 23 μl. The mean ASL xylitol concentration at 20, 90, and 180 minutes was 1.6 ± 1.9 μg/μl, 0.6 ± 0.6 μg/μl, and 0.1 ± 0.1 μg/μl, respectively. Corresponding BAL concentration corrected for dilution was consistently lower at all time points. The terminal half-life of aerosolized xylitol obtained by the probes was 45 minutes with a mean residence time of 65 minutes in ASL. Corresponding BAL values were 36 and 50 minutes, respectively. CONCLUSION: After a single dose nebulization, xylitol was detected in ASL for 3 hours, which was shorter than our in vitro measurement. The microsampling probe performed superior to BAL when sampling bronchial ASL

Immunological characterization of chromogranins A and B and secretogranin II in the bovine pancreatic islet

Antisera against chromogranin A and B and secretogranin II were used for analysing the bovine pancreas by immunoblotting and immunohistochemistry. All three antigens were found in extracts of fetal pancreas by one dimensional immunoblotting. A comparison with the soluble proteins of chromaffin granules revealed that in adrenal medulla and in pancreas antigens which migrated identically in electrophoresis were present. In immunohistochemistry, chromogranin A was found in all pancreatic endocrine cell types with the exception of most pancreatic polypeptide-(PP-) producing cells. For chromogranin B, only a faint immunostaining was obtained. For secretorgranin II, A-and B-cells were faintly positive, whereas the majority of PP-cells exhibited a strong immunostaining for this antigen. These results establish that chromogranins A and B and secretogranin II are present in the endocrine pancreas, but that they exhibit a distinct cellular localization

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD

Absence of Ca2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory

Ca2+-stimulated adenylyl cyclase (AC) 1 and 8 are two genes that have been shown to play critical roles in fear memory. AC1 and AC8 couple neuronal activity and intracellular Ca2+ increases to the production of cyclic adenosine monophosphate and are localized synaptically, suggesting that Ca2+-stimulated ACs may modulate synaptic plasticity. Here, we first established that Ca2+-stimulated ACs modulate protein markers of synaptic activity at baseline and after learning. Primary hippocampal cell cultures showed that AC1/AC8 double-knockout (DKO) mice have reduced SV2, a synaptic vesicle protein, abundance along their dendritic processes, and this reduction can be rescued through lentivirus delivery of AC8 to the DKO cells. Additionally, phospho-synapsin, a protein implicated in the regulation of neurotransmitter release at the synapse, is decreased in vivo 1 h after conditioned fear (CF) training in DKO mice. Importantly, additional experiments showed that long-term potentiation deficits present in DKO mice are rescued by acutely replacing AC8 in the forebrain, further supporting the idea that Ca2+-stimulated AC activity is a crucial modulator of synaptic plasticity. Previous studies have demonstrated that memory is continually modulated by gene–environment interactions. The last set of experiments evaluated the effects of knocking out AC1 and AC8 genes on experience-dependent changes in CF memory. We showed that the strength of CF memory in wild-type mice is determined by previous environment, minimal or enriched, whereas memory in DKO mice is unaffected. Thus, overall these results show that AC1 and AC8 modulate markers of synaptic activity and help integrate environmental information to modulate fear memory

The Probable Cell of Origin of NF1- and PDGF-Driven Glioblastomas

Primary glioblastomas are subdivided into several molecular subtypes. There is an ongoing debate over the cell of origin for these tumor types where some suggest a progenitor while others argue for a stem cell origin. Even within the same molecular subgroup, and using lineage tracing in mouse models, different groups have reached different conclusions. We addressed this problem from a combined mathematical modeling and experimental standpoint. We designed a novel mathematical framework to identify the most likely cells of origin of two glioma subtypes. Our mathematical model of the unperturbed in vivo system predicts that if a genetic event contributing to tumor initiation imparts symmetric self-renewing cell division (such as PDGF overexpression), then the cell of origin is a transit amplifier. Otherwise, the initiating mutations arise in stem cells. The mathematical framework was validated with the RCAS/tv-a system of somatic gene transfer in mice. We demonstrated that PDGF-induced gliomas can be derived from GFAP-expressing cells of the subventricular zone or the cortex (reactive astrocytes), thus validating the predictions of our mathematical model. This interdisciplinary approach allowed us to determine the likelihood that individual cell types serve as the cells of origin of gliomas in an unperturbed system

NESH Regulates Dendritic Spine Morphology and Synapse Formation

Background: Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown. Methodology/Principal Findings: NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines. Conclusions/Significance: NESH is a novel F-actin binding protein that likely plays important roles in the regulation o

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs