The Blood-Brain Barrier Is Unaffected in the Ndufs4-/- Mouse Model of Leigh Syndrome.

Mitochondrial dysfunction plays a major role in physiological aging and in many pathological conditions. Yet, no study has explored the consequence of primary mitochondrial deficiency on the blood-brain barrier (BBB) structure and function. Addressing this question has major implications for pharmacological and genetic strategies aimed at ameliorating the neurological symptoms that are often predominant in patients suffering from these conditions. In this study, we examined the permeability of the BBB in the Ndufs4-/- mouse model of Leigh syndrome (LS). Our results indicated that the structural and functional integrity of the BBB was preserved in this severe model of mitochondrial disease. Our findings suggests that pharmacological or gene therapy strategies targeting the central nervous system in this mouse model and possibly other models of mitochondrial dysfunction require the use of specific tools to bypass the BBB. In addition, they raise the need for testing the integrity of the BBB in complementary in vivo models.

Imaging Microtubules in vitro at High Resolution while Preserving their Structure.

Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can then be observed using a confocal microscope with an Airyscan module for higher resolution. This protocol allows the imaging of microtubules that have retained their original three-dimensional shape and is compatible with high-resolution immunofluorescence detection.

Studying Tau-Microtubule Interaction Using Single-Molecule TIRF Microscopy.

Microtubule architecture depends on a complex network of microtubule-associated proteins (MAPs) that act in concert to modulate microtubule assembly/disassembly and spatial arrangement. In vitro reconstitution of cytoskeleton dynamics coupled to single-molecule fluorescence assays has opened new perspectives to quantify the interaction of MAPs with microtubules. Here, we present a Total Internal Reflection Fluorescence (TIRF) microscopy-based assay enabling the characterization of Tau interaction with dynamic microtubules at the single-molecule level. We describe protein sample preparation in flow cells, single-molecule acquisitions by TIRF microscopy, and quantitative analysis of Tau oligomerization states and dwell time on microtubules.

A key function for microtubule-associated-protein 6 in activity-dependent stabilisation of actin filaments in dendritic spines.

Emerging evidence indicates that microtubule-associated proteins (MAPs) are implicated in synaptic function; in particular, mice deficient for MAP6 exhibit striking deficits in plasticity and cognition. How MAP6 connects to plasticity mechanisms is unclear. Here, we address the possible role of this protein in dendritic spines. We find that in MAP6-deficient cortical and hippocampal neurons, maintenance of mature spines is impaired, and can be restored by expressing a stretch of the MAP6 sequence called Mc modules. Mc modules directly bind actin filaments and mediate activity-dependent stabilisation of F-actin in dendritic spines, a key event of synaptic plasticity. In vitro, Mc modules enhance actin filament nucleation and promote the formation of stable, highly ordered filament bundles. Activity-induced phosphorylation of MAP6 likely controls its transfer to the spine cytoskeleton. These results provide a molecular explanation for the role of MAP6 in cognition, enlightening the connection between cytoskeletal dysfunction, synaptic impairment and neuropsychiatric illnesses.

MAP6 interacts with Tctex1 and Cav 2.2/N-type calcium channels to regulate calcium signalling in neurons.

MAP6 proteins were first described as microtubule-stabilizing agents, whose properties were thought to be essential for neuronal development and maintenance of complex neuronal networks. However, deletion of all MAP6 isoforms in MAP6 KO mice does not lead to dramatic morphological aberrations of the brain but rather to alterations in multiple neurotransmissions and severe behavioural impairments. A search for protein partners of MAP6 proteins identified Tctex1 - a dynein light chain with multiple non-microtubule-related functions. The involvement of Tctex1 in calcium signalling led to investigate it in MAP6 KO neurons. In this study, we show that functional Cav 2.2/N-type calcium channels are deficient in MAP6 KO neurons, due to improper location. We also show that MAP6 proteins interact directly with both Tctex1 and the C-terminus of Cav 2.2/N-type calcium channels. A balance of these two interactions seems to be crucial for MAP6 to modulate calcium signalling in neurons.

ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

Evidence for new C-terminally truncated variants of α- and ß-tubulins.

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α­tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3­tubulin variant corresponding to α1A/B­tubulin deleted of its last three residues (EEY). αΔ3­tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C­terminally truncated ß-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that ß2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified ßΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and ß-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.

Neuronal transport defects of the MAP6 KO mouse - a model of schizophrenia - and alleviation by Epothilone D treatment, as observed using MEMRI.

The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with synaptic plasticity anomalies. These defects are alleviated not only by neuroleptics, which are the gold standard molecules for the treatment of schizophrenia, but also by Epothilone D (Epo D), which is a microtubule-stabilizing molecule. To compare the neuronal transport between MAP6 KO and wild-type mice and to measure the effect of Epo D treatment on neuronal transport in KO mice, MnCl2 was injected in the primary somatosensory cortex. Then, using manganese-enhanced magnetic resonance imaging (MEMRI), we followed the propagation of Mn(2+) through axonal tracts and brain regions that are connected to the somatosensory cortex. In MAP6 KO mice, the measure of the MRI relative signal intensity over 24h revealed that the Mn(2+) transport rate was affected with a stronger effect on long-range and polysynaptic connections than in short-range and monosynaptic tracts. The chronic treatment of MAP6 KO mice with Epo D strongly increased Mn(2+) propagation within both mono- and polysynaptic connections. Our results clearly indicate an in vivo deficit in neuronal Mn(2+) transport in KO MAP6 mice, which might be due to both axonal transport defects and synaptic transmission impairments. Epo D treatment alleviated the axonal transport defects, and this improvement most likely contributes to the positive effect of Epo D on behavioral defects in KO MAP6 mice.

Impact of manganese on primary hippocampal neurons from rodents.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for in vivo tract tracing or functional imaging of the central nervous system. However Mn(2+) may be toxic at high levels. In this study, we addressed the impact of Mn(2+) on mouse hippocampal neurons (HN) and neuron-like N2a cells in culture, using several approaches. Both HN and N2a cells not exposed to exogenous MnCl2 were shown by synchrotron X-ray fluorescence to contain 5 mg/g Mn. Concentrations of Mn(2+) leading to 50% lethality (LC50) after 24 h of incubation were much higher for N2a cells (863 mM) than for HN (90 mM). The distribution of Mn(2+) in both cell types exposed to Mn(2+) concentrations below LC50 was perinuclear whereas that in cells exposed to concentrations above LC50 was more diffuse, suggesting an overloading of cell storage/detoxification capacity. In addition, Mn(2+) had a cell-type and dose-dependent impact on the total amount of intracellular P, Ca, Fe and Zn measured by synchrotron X-ray fluorescence. For HN neurons, immunofluorescence studies revealed that concentrations of Mn(2+) below LC50 shortened neuritic length and decreased mitochondria velocity after 24 h of incubation. Similar concentrations of Mn(2+) also facilitated the opening of the mitochondrial permeability transition pore in isolated mitochondria from rat brains. The sensitivity of primary HN to Mn(2+) demonstrated here supports their use as a relevant model to study Mn(2+) -induced neurotoxicity.

Prion protein expression and functional importance in developmental angiogenesis: role in oxidative stress and copper homeostasis.

AIM: It has been convincingly shown that oxidative stress and toxicity by deregulated metals, such as copper (Cu), are tightly linked to the development of pre-eclampsia and intrauterine growth retardation (IUGR), the most threatening pathologies of human pregnancy. However, mechanisms implemented to control these effects are far from being understood. Among proteins that bind Cu and insure cellular protection against oxidative stress is the cellular prion protein (PrP(C)), a glycosyl phosphatidyl inositol-anchored glycoprotein, which we reported to be highly expressed in human placenta. Herein, we investigated the pathophysiological role of PrP(C) in Cu and oxidative stress homeostasis in vitro using human placenta and trophoblast cells, and in vivo using three strains of mice (C57Bl6, PrP(C) knockout mice [PrP(-/-)], and PrP(C) overexpressing mice [Tga20]). RESULTS: At the cellular level, PrP(C) protection against oxidative stress was established in multiple angiogenic processes: proliferation, migration, and tube-like organization. For the animal models, lack (PrP(-/-)) or overexpression (Tga20) of PrP(C) in gravid mice caused severe IUGR that was correlated with a decrease in litter size, changes in Cu homeostasis, increase in oxidative stress response, development of hypoxic environment, failure in placental function, and maintenance of growth defects of the offspring even 7.5 months after delivery. INNOVATION: PrP(C) could serve as a marker for the idiopathic IUGR disease. CONCLUSION: These findings demonstrate the stress-protective role of PrP(C) during development, and propose PrP(C) dysregulation as a novel causative element of IUGR.

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.

How morphological constraints affect axonal polarity in mouse neurons.

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

Role of EG-VEGF in human placentation: Physiological and pathological implications.

Pre-eclampsia (PE), the major cause of maternal morbidity and mortality, is thought to be caused by shallow invasion of the maternal decidua by extravillous trophoblasts (EVT). Data suggest that a fine balance between the expressions of pro- and anti-invasive factors might regulate EVT invasiveness. Recently, we showed that the expression of the new growth factor endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is high in early pregnancy but falls after 11 weeks, suggesting an essential role for this factor in early pregnancy. Using human villous explants and HTR-8/SVneo, a first trimester extravillous trophoblast cell line, we showed differential expression of EG-VEGF receptors, PKR1 and PKR2, in the placenta and demonstrated that EG-VEGF inhibits EVT migration, invasion and tube-like organisation. EG-VEGF inhibitory effect on invasion was supported by a decrease in matrix metalloproteinase (MMP)-2 and MMP-9 production. Interference with PKR2 expression, using specific siRNAs, reversed the EG-VEGF-induced inhibitory effects. Furthermore, we determined EG-VEGF circulating levels in normal and PE patients. Our results showed that EG-VEGF levels were highest during the first trimester of pregnancy and decreased thereafter to non-pregnant levels. More important, EG-VEGF levels were significantly elevated in PE patients compared with age-matched controls. These findings identify EG-VEGF as a novel paracrine regulator of trophoblast invasion. We speculate that a failure to correctly down-regulate placental expression of EG-VEGF at the end of the first trimester of pregnancy might lead to PE.

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5.

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.

IQGAP1 regulates adult neural progenitors in vivo and vascular endothelial growth factor-triggered neural progenitor migration in vitro.

In the germinative zone of the adult rodent brain, neural progenitors migrate into niches delimited by astrocyte processes and differentiate into neuronal precursors. In the present study, we report a modulating role for the scaffolding protein IQGAP1 in neural progenitor migration. We have identified IQGAP1 as a new marker of amplifying neural progenitor and neuronal precursor cells of the subventricular zone (SVZ) and the rostral migratory stream (RMS) in the adult mouse brain. To determine functions for IQGAP1 in neural progenitors, we compared the properties of neural progenitor cells from wild-type and Iqgap1-null mutant mice in vivo and in vitro. The in vivo studies reveal a delay in the transition of de novo neural progenitors into neuronal precursor cells in Iqgap1-null mice. In vitro, we demonstrated that IQGAP1 acts as a downstream effector in the vascular endothelial growth factor (VEGF)-dependent migratory response of neural progenitors that also impacts on their neuronal differentiation. The Rho-family GTPases cdc42/Rac1 and Lis1 are major partners of IQGAP1 in this migratory process. Finally, astrocytes of the neurogenic SVZ and RMS are shown to express VEGF. We propose that VEGF synthesized by astrocytes could be involved in the guidance of neural progenitors to neurogenic niches and that IQGAP1 is an effector of the VEGF-dependent migratory signal.

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends.

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe.

N'-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye.

S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression.

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app) <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-trityl-L-cysteine binds more tightly than monastrol because it has both an approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-1) s(-1) and 15 s(-1) for monastrol). S-trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC(50) of 500 nm. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.

Cellular disorders induced by high magnetic fields.

PURPOSE: To evaluate whether static high magnetic fields (HMFs), in the range of 10-17 T, affect the cytoskeleton and cell organization in different types of mammalian cells, including fibroblasts, epithelial cells, and differentiating neurons. MATERIALS AND METHODS: Cells were exposed to HMF for 30 or 60 minutes and subsequently assessed for viability. Cytoskeleton arrays and focal adhesions were visualized using immunofluorescence microscopy. RESULTS: Cell exposure to HMF over 10 T in the case of cycling cells, and over 15 T in the case of neurons, affected cell viability, apparently because of cell detachment from culture dishes. In the remaining adherent cells, the organization of actin assemblies was perturbed, and both cell adhesion and spreading were impaired. Moreover, in the case of neurons, exposure to HMF induced growth cone retraction and delayed cell differentiation. CONCLUSION: Cell exposure to HMF (over 10T and 15 T in the case of cycling cells and neurons, respectively) affects the cell cytoskeleton, with deleterious effects on cell viability, organization, and differentiation. Further studies are needed to determine whether such perturbations, as observed here in cultured cells, have consequences in whole animals.

A vital role of tubulin-tyrosine-ligase for neuronal organization.

Tubulin is subject to a special cycle of detyrosination/tyrosination in which the C-terminal tyrosine of alpha-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persists in TTL null tissues, being present mainly in dividing TTL null cells where it originates from tubulin synthesis, but it is lacking in postmitotic TTL null cells such as neurons, which is apparently deleterious because early death in TTL null mice is, at least in part, accounted for by a disorganization of neuronal networks, including a disruption of the cortico-thalamic loop. Correlatively, cultured TTL null neurons display morphogenetic anomalies including an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. These anomalies may involve a mislocalization of CLIP170, which we find lacking in neurite extensions and growth cones of TTL null neurons. Our results demonstrate a vital role of TTL for neuronal organization and suggest a requirement of Tyr-tubulin for proper control of neurite extensions.