White matter maturation of normal human fetal brain. An in vivo diffusion tensor tractography study

We demonstrate for the first time the ability to determine in vivo and in utero the transitions between the main stages of white matter (WM) maturation in normal human fetuses using magnetic resonance diffusion tensor imaging (DTI) tractography. Biophysical characteristics of water motion are used as an indirect probe to evaluate progression of the tissue matrix organization in cortico-spinal tracts (CSTs), optic radiations (OR), and corpus callosum (CC) in 17 normal human fetuses explored between 23 and 38 weeks of gestation (GW) and selected strictly on minimal motion artifacts. Nonlinear polynomial (third order) curve fittings of normalized longitudinal and radial water diffusivities (Z-scores) as a function of age identify three different phases of maturation with specific dynamics for each WM bundle type. These phases may correspond to distinct cellular events such as axonal organization, myelination gliosis, and myelination, previously reported by other groups on post-mortem fetuses using immunostaining methods. According to the DTI parameter dynamics, we suggest that myelination (phase 3) appears early in the CSTs, followed by the OR and by the CC, respectively. DTI tractography provides access to a better understanding of fetal WM maturation

Rapid neurogenesis through transcriptional activation in human stem cells

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types

Rapid, modular and reliable construction of complex mammalian gene circuits

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.Synthetic Biology Engineering Research Center (SA5284-11210)United States. Defense Advanced Research Projects Agency (HR0011-12-C-0067)United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Science Foundation (U.S.) (CBET-0939511)National Institutes of Health (U.S.). (5-R01-CA155320-02

Interictal Functional Connectivity of Human Epileptic Networks Assessed by Intracerebral EEG and BOLD Signal Fluctuations

In this study, we aimed to demonstrate whether spontaneous fluctuations in the blood oxygen level dependent (BOLD) signal derived from resting state functional magnetic resonance imaging (fMRI) reflect spontaneous neuronal activity in pathological brain regions as well as in regions spared by epileptiform discharges. This is a crucial issue as coherent fluctuations of fMRI signals between remote brain areas are now widely used to define functional connectivity in physiology and in pathophysiology. We quantified functional connectivity using non-linear measures of cross-correlation between signals obtained from intracerebral EEG (iEEG) and resting-state functional MRI (fMRI) in 5 patients suffering from intractable temporal lobe epilepsy (TLE). Functional connectivity was quantified with both modalities in areas exhibiting different electrophysiological states (epileptic and non affected regions) during the interictal period. Functional connectivity as measured from the iEEG signal was higher in regions affected by electrical epileptiform abnormalities relative to non-affected areas, whereas an opposite pattern was found for functional connectivity measured from the BOLD signal. Significant negative correlations were found between the functional connectivities of iEEG and BOLD signal when considering all pairs of signals (theta, alpha, beta and broadband) and when considering pairs of signals in regions spared by epileptiform discharges (in broadband signal). This suggests differential effects of epileptic phenomena on electrophysiological and hemodynamic signals and/or an alteration of the neurovascular coupling secondary to pathological plasticity in TLE even in regions spared by epileptiform discharges. In addition, indices of directionality calculated from both modalities were consistent showing that the epileptogenic regions exert a significant influence onto the non epileptic areas during the interictal period. This study shows that functional connectivity measured by iEEG and BOLD signals give complementary but sometimes inconsistent information in TLE

BID-F1 and BID-F2 Domains of Bartonella henselae Effector Protein BepF Trigger Together with BepC the Formation of Invasome Structures

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation

A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium

Proteins injected by the bacterial pathogen "Bartonella" subvert eukaryotic cell signaling

Conclusions What are the substrates of the VirB/VirD4 T4SS of Bartonella henselae? Starting point for the first published report (“A bipartite signal mediates the transfer of type IV secreted substrates of Bartonella henselae into human cells”, Chapter 3.1) in my Ph.D. thesis was the finding that the VirB/VirD4 T4SS of B. tribocorum was essential for the pathogen to establish an intra-erythrocytic infection in an animal model (1). Additionally, at that time, unpublished data indicated that many phenotypes which were already known for B. henselae infecting HUVECs, as anti-apoptosis (2), cytoskeletal rearrangements (3) and pro-inflammatory activation (4), were dependent on a intact VirB/VirD4 T4SS (5). These findings were suggestive of substrates being translocated through the VirB/VirD4 T4SS of Bartonella into the host cells. Sequencing 23 kb downstream of the virB locus of B. henselae revealed a coupling protein (virD4), and seven genes encoding at least one common Cterminal domain. The proteins encoded by these seven genes were later termed Bartonella exported proteins (Beps), the common C-terminal domain they contain Bartonella intracellular delivery (BID). By constructing a Hidden Markov Model from these BID domains and querying protein databases, we found similar domains in the C-terminus of relaxases from conjugative plasmids in the α-proteobacteria. We showed exemplarily that the C-terminus of the TraA relaxase from the AvhB/TraG conjugation system in A. tumefaciens – one of the top hits in the database search - could still be translocated by the VirB/VirD4 T4SS of B. henselae. Both these findings support that the BID domain evolved from conjugative relaxases, in parallel with the T4SS of B. henselae. Full-length relaxases bind covalently to plasmid DNA and mediate its transfer through T4SSs. This allows the fascinating speculation that the VirB/VirD4 T4SS of Bartonella could be used to export DNA by those means in vivo into host cells. To demonstrate exemplarily the translocation of the Beps through the VirB/VirD4 T4SS, we fused a FLAG-tag to the N-terminus of BepD and could show that BepD is translocated into infected endothelial cells in a VirB/VirD4 T4SS-dependent manner, whereupon it localizes to the cytoplasm of these cells and is tyrosine-phosphorylated by host-cell kinases. The precise experimental delineation of the domain needed for translocation was made possible by the development of the Cre-recombinase reporter assay for translocation (CRAFT), which showed the translocation domain to be bipartite. In addition to the BID domain, a short, positively charged C-terminal amino acid sequence was needed for an effective delivery of proteins. Non-polar deletions of all the ORFs encoding the Beps abolished the ability of Bh to induce a variety of host-cell phenotypes, suggesting that these proteins elicit biological effects in their eukaryotic target cells. This finding was the fundament for the two Manuscripts presented in Chapter 3.3, "A translocated protein of the vascular-tumor inducing pathogen Bartonella protects human vascular endothelial cells from Apoptosis" and “Subversion of host cell cytoskeletal function during invasome-mediated uptake of Bartonella henselae into human endothelial cells”. In each of these two manuscripts, a major phenotype of the infection of HUVECs by B. henselae is shown to depend on a single Bep. While BepA inhibits the apoptosis of endothelial cells, BepG does induce massive cytoskeletal rearrangements. Interestingly, it is the BID domain of BepA which mediates the anti-apoptotic activity in the host cell as well as the localization of this protein to the plasma membrane. This involvement of the BID in localization and function in the host cell holds also true for BepE, as we showed that the two BID domains of this protein are crucial for its localization (Chapter 3.2), and to inhibit the fragmentation of infected endothelial cells (Chapter 3.4). Many interfaces for interactions over phosphotyrosines Of the seven Bep proteins, three contain putative tyrosine-phosphorylation motifs in their N-terminus. We showed that both BepD and BepE are tyrosinephoshorylated by the c-Src kinase. Because of their similar substrate specificities and their variable expression levels depending on the cell type, any member of the SFK could potentially be the kinase of BepD and BepE in vivo. Bioinformatics and preliminary experiments suggest c-Abl as the kinase of BepF. As Crk was shown to bind BepF, and one of the functions of Crk has been described to activate c-Abl, one might speculate that a positive activation loop takes place once BepF has been phosphorylated, which would bind and activate increasing amounts of the c-Abl kinase to BepF through Crk. Whereas c-Abl has been implicated in the invasion process of other pathogens (6), the exact function of BepF in Bartonella still remains to be uncovered. BepD localizes in immunofluorescence stainings to a cytoplasmic vesicular-like compartment. Additionally, it localizes upon its tyrosine-phosphorylation to a Triton X-1oo insoluble fraction, and binds SHP2 and Csk. Lipid rafts are prominently associated to Triton X-100 insoluble fractions, and future studies might show BepD interfering with the signaling in these rafts. I subsequently focused on BepE, for which bioinformatics revealed an intriguing similarity to inhibitory immune receptors of mammals. This resulted in the second manuscript “Molecular mimicry of inhibitory immune receptors by the bacterial pathogen Bartonella” (Chapter 3.2). BepE contains two C-terminal BID domains which mediate the localization of this protein to the plasma membrane of HELA cells and to the plasma membrane and cell-cell contacts in endothelial cells, as shown by a co-localizing immunofluorescence staining with VE-Cadherin in HUVECs. This peculiar localization is very intriguing, and might modulate the cell-cell contact strength or the contact inhibition which is crucial in endothelial cells. Recently, a publication demonstrated that VE-Cadherin contains in its intracellular domain a binding site for the SH2 domain of Csk, the binding of Csk to this site being crucial for the contact inhibition of cell growth (7). We identified a very similar motif in the N-terminus of BepE which did also bind Csk. The co-localization and a similar tyrosine-phosphorylation motif binding the same protein makes this finding a good starting point for investigating the functions of BepE in the endothelium. BepE contains in addition to this Csk-binding motif two immunotyrosine inhibitory motif – immunotyrosine-based switch motif (ITIM/ITSM) tandems. These motifs are commonly found in the intracellular domain of inhibitory immune receptors. In vitro phosphorylation with the c-Src kinase and subsequent mapping by mass spectrometry analysis indicated that c-Src tyrosine-phosphorylates the Csk-binding site and both ITIMs of BepE. No phosphorylation of an ITSM could be detected, which might be due to the fact that another kinase phosphorylates these motifs in vivo. Both the inhibitory immune receptors and BepE do contain ITIMs, ITSMs, Csk-binding sites, localize to the plasma membrane, are tyrosine-phosphorylated by Src family kinases and bind SHP2 and Csk. Additionally, BepE is constitutively tyrosine-phosphorylated in HEK293T cells as well as in the primary HUVECs (Chapter 3.2), in contrast to most inhibitory immune receptors, which are thought to only become phosphorylated upon engagement of their extracellular ligands. All these lines of evidence indicate BepE mimicking inhibitory immune receptors. Csk contains one SH2 domain, SHP2 two. To elucidate the binding sites of these two proteins, and also to be able to use mutants lacking these interactions, we generated a panel of tyrosine-to-phenylalanine exchange mutants in BepE. While Csk binds to one motif (the one with the similarity to the Csk-binding site of VE-Cadherin), SHP2 interacts with motifs in the two ITIM-ITSM tandems. While this interaction study was carried out in HEK293T cells, more potential ITIM/ITSM-binding proteins such as SHP-1 and SHIP, EAT-2 and SAP might increase the complexity of the picture in myeloid and lymphoid cells. The two adapter proteins SAP and EAT-2 have been shown to bind to ITSMs. While some cells such as NK express both adapters, other cells as for example T-cell do only express SAP, and other such as DCs only EAT-2. While SAP recruits Fyn and can lead to an increase in cell activation, EAT-2 recruits phosphatases and Csk, inhibiting the activity. For BepE, this opens the possibility of switching its mode of action, depending on the cell type it is translocated into. Collaborations have been initiated to assess the immunomodulatory potential of BepE and we are currently studying the impact of this protein in our HUVEC models. To summarize, my Ph.D. thesis aimed at investigating the VirB/VirD4 T4SS of Bartonella henselae for the presence of secreted substrates and signals mediating this secretion. Additionally, to describe the functions and interaction partners of these substrates in the host cell, with an emphasis on the putative tyrosinephosphorylated effectors. The core findings of this thesis are a.) The discovery of seven modular substrates secreted by the apparatus b.) Description of the BID domain mediating the secretion of proteins and protein-DNA complexes, c.) Csk and SHP2 being interaction partners for BepD and BepE on the host cell side d.) BepE mimicking inhibitory immune receptors

Imagerie spectroscopique, imagerie de diffusion et tractographie, dans les épilepsies partielles humaines caractérisées par stéréo-électroencéphalographie

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

SPECTROMETRIE DE RMN DANS LES EPILEPSIES TEMPORALES (DES NEUROLOGIE)

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

A translocated effector required for bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner