[Show abstract][Hide abstract] ABSTRACT: Structural microtubule associated proteins (MAPs) stabilize microtubules, a property that was thought to be essential for development, maintenance and function of neuronal circuits. However, deletion of the structural MAPs in mice does not lead to major neurodevelopment defects. Here we demonstrate a role for MAP6 in brain wiring that is independent of microtubule binding. We find that MAP6 deletion disrupts brain connectivity and is associated with a lack of post-commissural fornix fibres. MAP6 contributes to fornix development by regulating axonal elongation induced by Semaphorin 3E. We show that MAP6 acts downstream of receptor activation through a mechanism that requires a proline-rich domain distinct from its microtubule-stabilizing domains. We also show that MAP6 directly binds to SH3 domain proteins known to be involved in neurite extension and semaphorin function. We conclude that MAP6 is critical to interface guidance molecules with intracellular signalling effectors during the development of cerebral axon tracts.
[Show abstract][Hide abstract] ABSTRACT: MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.
PLoS ONE 12/2014; 9(12):e114905. DOI:10.1371/journal.pone.0114905 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for in vivo tract tracing or functional imaging of the central nervous system. However Mn2+ may be toxic at high levels. In this study, we addressed the impact of Mn2+ 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 Mn2+ 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 Mn2+ in both cell types exposed to Mn2+ 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, Mn2+ 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 Mn2+ below LC50 shortened neuritic length and decreased mitochondria velocity after 24 h of incubation. Similar concentrations of Mn2+ also facilitated the opening of the mitochondrial permeability transition pore in isolated mitochondria from rat brains. The sensitivity of primary HN to Mn2+ demonstrated here supports their use as a relevant model to study Mn2+-induced neurotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nano-pillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nano-pillars with a typical distance between adhesion points of about 500nm. These neurons produce less neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted to a differential surface topography, neurons specify an axon preferentially onto nano-pillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.
[Show abstract][Hide abstract] ABSTRACT: Seasonal or chronic vitamin D deficiency and/or insufficiency is highly prevalent in the human population. Receptors for 1,25-dihydroxyvitamin D3, the hormonal metabolite of vitamin D, are found throughout the brain. To provide further information on the role of this hormone on brain function, we analyzed the transcriptomic profiles of mixed neuron-glial cell cultures in response to 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 treatment increases the mRNA levels of 27 genes by at least 1.9 fold. Among them, 17 genes were related to neurodegenerative and psychiatric diseases, or brain morphogenesis. Notably, 10 of these genes encode proteins potentially limiting the progression of Alzheimer's disease. These data provide support for a role of 1,25-dihydroxyvitamin D3 in brain disease prevention. The possible consequences of circannual or chronic vitamin D insufficiencies on a tissue with a low regenerative potential such as the brain should be considered.
[Show abstract][Hide abstract] ABSTRACT: Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2013; 33(4):1391-9. DOI:10.1523/JNEUROSCI.2231-12.2013 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: STOP/MAP6 null (KO) mice recapitulate behavioral abnormalities related to positive and negative symptoms and cognitive deficits of schizophrenia. Here, we investigated whether decreased expression of STOP/ MAP6 proteins in heterozygous mice (only one allele expressed) would result in abnormal behavior related to those displayed by STOP null mice. Methods: Using a comprehensive test battery, we investigated the behavioral phenotype of STOP heterozygous (Het) mice compared with STOP KO and wild type (WT) mice on animals raised either in standard conditions (controls) or submit-ted to maternal deprivation. Results: Control Het mice displayed prominent deficits in social interaction and learning, resembling KO mice. In contrast, they exhib-ited short-lasting locomotor hyperreactivity to acute mild stress and no impaired locomotor response to amphet-amine, much like WT mice. Additionally, perinatal stress deteriorated Het mouse phenotype by exacerbating alter-ations related to positive symptoms such as their locomo-tor reactivity to acute mild stress and psychostimulant challenge. Conclusion: Results show that the dosage of susceptibility genes modulates their putative phenotypic contribution and that STOP expression has a high pen-etrance on cognitive abilities. Hence, STOP Het mice might be useful to investigate cognitive defects related to those observed in mental diseases and ultimately might be a valuable experimental model to evaluate preventive treatments.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 03/2012; 7(3):e33623. DOI:10.1371/journal.pone.0033623 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.
Small 03/2012; 8(5):671-5. DOI:10.1002/smll.201102325 · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2010; 5(10):e13553. DOI:10.1371/journal.pone.0013553 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies have suggested that schizophrenia is associated with alterations in the synaptic connectivity involving cytoskeletal proteins. The microtubule-associated protein stable tubule only polypeptide (STOP) plays a key role in neuronal architecture and synaptic plasticity, and it has been demonstrated that STOP gene deletion in mice leads to a phenotype mimicking aspects of positive and negative symptoms and cognitive deficits classically observed in schizophrenic patients. In STOP null mice, behavioral defects are associated with synaptic plasticity abnormalities including defects in long-term potentiation. In these mice, long-term administration of typical antipsychotics has been shown to partially alleviate behavioral defects but, as in humans, such a treatment was poorly active on deficits related to negative symptoms and cognitive impairments. Here, we assessed the effects of risperidone and clozapine, two atypical antipsychotics, on STOP null mice behavior and synaptic plasticity.
Long-term administration of either drug results in alleviation of behavioral alterations mimicking some negative symptoms and partial amelioration of some cognitive defects in STOP null mice. Interestingly, clozapine treatment also improves synaptic plasticity of the STOP null animals by restoring long-term potentiation in the hippocampus.
All together, the pharmacological reactivity of STOP null mice to antipsychotics evokes the pharmacological response of humans to such drugs. Totally, our study suggests that STOP null mice may provide a useful preclinical model to evaluate pharmacological properties of antipsychotic drugs.
[Show abstract][Hide abstract] ABSTRACT: Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.
[Show abstract][Hide abstract] ABSTRACT: In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.
The Journal of Cell Biology 07/2009; 185(7):1159-66. DOI:10.1083/jcb.200902142 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our project is based on the elaboration of in vitro neuron networks as simplified models to explore the relation between neuronal architecture and biological function. Beyond a control of soma and neurite position, our first goal was to achieve in-vitro axonal differentiation of embryonic E18 hippocampal mice neurons by the mean of geometrical growth constraints, i.e. by the use of adhesive micro-patterns on silanized glass substrates. Such a process thus excludes chemical guidance or specific adhesion mechanisms. This study explores two different types of geometrical constraints. The first one, based on the centrosome role and localization, is applied to the soma, and force a choosen neurite to differentiate into an axon with 39% of efficacy (N= 160 cells, 3 different cultures). The second one derives from the suggested relationship between neurite mechanical tension and axonal differentiation, and is based on the design of wavy neurite's shape. Its efficacy reach 0.51% (N= 300 cells, 3 different cultures). The combinaison of these two constraints into a final pattern yields an efficacy of 82% (N= 83 cells, 2 different cultures). These results not only provide an important tool for creating neural model networks but also point out an important role of intrinsic neurite tension during axon differentiation.
[Show abstract][Hide abstract] ABSTRACT: Neural circuit architecture is a fundamental characteristic of the brain, and how architecture is bound to biological functions is still an open question. Some neuronal geometries seen in the retina or the cochlea are intriguing: information is processed in parallel by several entities like in "pooling" networks which have recently drawn the attention of signal processing scientists. These systems indeed exhibit the noise-enhanced processing effect, which is also actively discussed in the neuroscience community at the neuron scale. The aim of our project is to use in-vitro ordered neuron networks as living paradigms to test ideas coming from the computational science. The different technological bolts that have to be solved are enumerated and the first results are presented. A neuron is a polarised cell, with an excitatory axon and a receiving dendritic tree. We present how soma confinement and axon differentiation can be induced by surface functionalization techniques. The recording of large neuron networks, ordered or not, is also detailed and biological signals shown. The main difficulty to access neural noise in the case of weakly connected networks grown on micro electrode arrays is explained. This open the door to a new detection technology suitable for sub-cellular analysis and stimulation, whose development will constitute the next step of this project.
Proceedings of SPIE - The International Society for Optical Engineering 06/2007; DOI:10.1117/12.724652 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STOP proteins are microtubule-associated, calmodulinregulated proteins responsible for the high degree of stabilization displayed
by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity
in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite
the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP
from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization.
We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a
key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of
STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with
actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated
STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments
where it may interact with actin, which could be important for STOP function in synaptic plasticity.
[Show abstract][Hide abstract] ABSTRACT: In this study we compare the neural activity of a population of neurons recorded with a MEA to simulations of equivalent networks obtained on a computer when bicuculline, an antagonist of inhibitory connections, is introduced into the nutritive solution. The aim of this study is to obtain a model producing extra-cellular data that match the synchronicity of two different real networks: a cell culture and an oraganotypic hippocampus slice. A one compartment model of neuron and a neuron-electrode model are used to simulate experiments. Parameters of the models are fitted to match in vivo data. It is shown that the variation of the noise level at the synaptic level induces a variation in the period of the bursting effect of bicuculline and produces variation in amplitude of the recorded signal.