In JoVE (1)
Other Publications (8)
- Hepatology (Baltimore, Md.)
- Brain Research. Developmental Brain Research
- Brain Research. Developmental Brain Research
- Molecular Biology of the Cell
- International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience
- Journal of Neuroscience Research
- Journal of Alzheimer's Disease : JAD
- Journal of Neuroscience Methods
Articles by Boris Decourt in JoVE
Culturas de células neuronais de Aplysia para imagens de alta resolução de Cones de Crescimento Aih Cheun Lee1, Boris Decourt1, Daniel M. Suter1 1Department of Biological Sciences, Purdue University Neurônios Aplysia californica desenvolver cones grande crescimento na cultura que são excelentes para imagens de alta resolução da motilidade cone de crescimento e orientação. Aqui, apresentamos um protocolo para a dissecção e chapeamento de neurônios células Aplysia saco, bem como para a criação de uma câmara para imagens de células vivas.
Other articles by Boris Decourt on PubMed
Inhibition of Rat Liver Fibrogenesis Through Noradrenergic Antagonism Hepatology (Baltimore, Md.). Feb, 2002 | Pubmed ID: 11826405 The effect of adrenergic innervation and/or circulating catecholamines on the function of liver fibrogenic cells is poorly understood. Our aim was to investigate the effects of noradrenergic antagonism on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Two weeks of CCl4 induced an approximately 5-fold increase in the area of fibrosis as compared with controls. The addition of 6-hydroxydopamine (OHDA), a toxin that destroys noradrenergic fibers, decreased fibrosis by 60%. After 6 weeks of CCl4, the area of fibrosis increased about 30-fold in CCl4-treated animals and was decreased by 36% with OHDA. At 2 weeks, OHDA abrogated the CCl4-induced increase in mRNA level of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), an inhibitor of extracellular matrix degradation, and it greatly reduced it at 6 weeks. Finally, when rats treated with CCl4 for 2 weeks also received prazosin, an antagonist of alpha1-adrenergic receptors, fibrosis was decreased by 83%. In conclusion, destruction of noradrenergic fibers or antagonism of noradrenergic signaling through alpha1 receptors inhibited the development of liver fibrosis. Because adrenoreceptor antagonists have a very sound safety profile, they appear as attractive drugs to reduce liver fibrogenesis.
Expression Analysis of Neuroleukin, Calmodulin, Cortactin, and Rho7/Rnd2 in the Intact and Injured Mouse Brain Brain Research. Developmental Brain Research. Sep, 2005 | Pubmed ID: 16051374 Subtracted cDNA libraries from the mouse developing inferior colliculus were previously constructed between postnatal day (P) 6 and 10. In the P10-P6 subtracted library, neuroleukin, calmodulin I, cortactin, and Rho7 were identified. The goal of the present study was to analyze their distribution, at the mRNA and protein levels, in both the adult and the developing mouse brain. The four molecules showed a wide expression throughout the brain, with a neuronal-enriched localization in structures such as the cortex, the hippocampus, the cerebellum, and the inferior colliculus. The level of expression of their corresponding mRNAs increased during brain postnatal development. The expression of these molecules was also investigated 2 weeks after a mechanical lesion in the adult cerebral cortex. Neuroleukin and cortactin were found to be expressed by reactive astrocytes, while there were no changes in the expression of calmodulin and Rho7. The expression of neuroleukin, calmodulin, cortactin, and Rho7 is discussed in the context of their putative role in the maturation of the brain and in the axonal regeneration process.
Identification of Differentially Expressed Genes in the Developing Mouse Inferior Colliculus Brain Research. Developmental Brain Research. Sep, 2005 | Pubmed ID: 16095723 Although injured neurons of inferior colliculus (IC) display a robust axonal outgrowth through a lesion site at postnatal day six (P6) in vitro, and are capable to re-innervate their target cells, injured neurons from P10 IC are unable to regenerate their axons across the lesion site. This axonal regenerative failure has been attributed to an increase of expression of inhibitory molecules in endogenous tissue, during development. As a first step to identify such inhibitory molecules, the present study reports the isolation of molecules differentially expressed in the IC during development. A two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on IC tissue between P6 and P10 stages. One hundred cDNAs from P6 (P6-P10) and 200 cDNAs from P10 (P10-P6)-subtracted libraries were randomly sequenced. A dot-blot screening of sequenced cDNAs revealed the differential expression for the majority of these cDNAs at their respective developmental stage. Then, the analysis of sequenced clones showed that P6 library was highly enriched in molecules expressed early in the development, such as GAP43 or vimentin proteins. By contrast, the P10 library contained mostly molecules expressed at later stages of development in the central nervous system, such as myelin-related proteins. Our results show that SSH is a suitable method for identifying differentially expressed genes in the developing IC. In addition, these results provide a foundation for further studies dealing with molecules involved in the IC development before and at the onset of hearing, some of which being probably involved in the axonal outgrowth mechanism.
Microtubule-mediated Src Tyrosine Kinase Trafficking in Neuronal Growth Cones Molecular Biology of the Cell. Nov, 2008 | Pubmed ID: 18716055 Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein-positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.
Is Otospiralin Inner Ear Specific? Evidence for Its Expression in Mouse Brain International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience. Feb, 2009 | Pubmed ID: 18832023 The small protein otospiralin has initially been identified as an inner ear specific molecule. However, compelling evidence from high throughput sequencing projects suggested that otospiralin is likely expressed in the central nervous system. Here, we tested this hypothesis using a combination of molecular biology, immunological, and histological techniques, and found that otospiralin is expressed in numerous regions of the central nervous system in mouse. In situ hybridization and immunohistochemistry revealed that otospiralin is widely expressed in neuronal cell bodies and glia. Ultrastructural observations in the cerebral cortex located the small protein in close proximity to membranous organelles in perikarya, the inner face of post-synaptic neuronal membranes, and in astrocytic processes. These results are in agreement with the predicted structure of the protein which revealed a single N-terminal transmembrane helix domain followed by a C-terminus cytosolic tail. Interestingly, 2 weeks after a mechanical trauma in the cerebral cortex, otospiralin expression increased in reactive astrocytes located within the vicinity of the site of injury, but not in neurons. Collectively, our observations suggest that otospiralin is possibly involved in signaling pathways, and could play a role in repair mechanisms subsequent to an injury in the central nervous system.
Cortactin Colocalizes with Filopodial Actin and Accumulates at IgCAM Adhesion Sites in Aplysia Growth Cones Journal of Neuroscience Research. Apr, 2009 | Pubmed ID: 19021290 Both IgCAMs and the actin cytoskeleton play critical roles in neuronal growth cone motility and guidance. However, it is unclear how IgCAM receptors transduce signals from the plasma membrane to induce actin remodeling. Previous studies have shown that local clustering and immobilization of apCAM, the Aplysia homolog of NCAM, induces Src kinase activity and F-actin polymerization in the peripheral domain of cultured Aplysia bag cell growth cones. Therefore, we wanted to test whether the Src kinase substrate and actin regulator cortactin could be a molecular link between Src activity and actin assembly during apCAM-mediated growth cone guidance. Here, we cloned Aplysia cortactin and showed that it is abundant in the nervous system. Immunostaining of growth cones revealed a strong colocalization of cortactin with F-actin in filopodial bundles and at the leading edge of lamellipodia. Perturbation of the cytoskeleton indicated that cortactin distribution largely depends on actin filaments. Furthermore, active Src colocalized with cortactin in regions of actin assembly, including leading edge and filopodia tips. Finally, we observed that cortactin, like F-actin, localizes to apCAM adhesion sites mediating growth cone guidance. Altogether, these data suggest that cortactin is a mediator of IgCAM-triggered actin assembly involved in growth cone motility and guidance.
BACE1 As a Potential Biomarker for Alzheimer's Disease Journal of Alzheimer's Disease : JAD. 2011 | Pubmed ID: 21403391 The diagnosis of Alzheimer's disease (AD) relies principally on clinical criteria for probable and possible AD as defined by the NINCDS-ADRDRA. The field is desperately lacking of biological markers to assist with AD diagnosis and verification of treatment efficacy. According to the Consensus Report of the Working Group on Molecular and Biochemical Markers of Alzheimer's Disease, in order to qualify as a biomarker the sample in question must adhere to certain basic requirements, including the ability to: reflect AD pathology and differentiate it from other dementia with an 80% sensitivity; be reliable and reproducible; be easy to perform and analyze; remain relatively inexpensive. Beta secretases are crucial enzymes in the pathogenesis of AD. Given its primary role in brain amyloidogenesis and its ubiquitous expression, one may consider measuring peripheral BACE1 levels and activity as biomarkers of AD, like performed in the brain and cerebrospinal fluid. However, very little is known about the periphery and whether peripheral BACE1 is involved in AD pathogenesis or mirrors AD progression. Moreover, no investigation has focused on the possibility of monitoring peripheral BACE1 to assess the efficiency of BACE1 inhibitors during the course of clinical trials. Part of the problem may be attributed to the lack of sensitive molecular tools which are absolutely necessary to use BACE1 as a biomarker. In this review we evaluate the progress and feasibility of developing BACE1 as a biomarker for AD in different tissues.
Development of a Specific ELISA to Measure BACE1 Levels in Human Tissues Journal of Neuroscience Methods. Oct, 2011 | Pubmed ID: 21889954 The aspartyl protease BACE1 is the rate limiting enzyme in the synthesis of amyloid beta, which accumulation in the human brain is a hallmark of Alzheimer's disease (AD). BACE1 has been proposed as a surrogate marker of AD; however, very few BACE1 immunoassays have been reported. In the present study we have screened ten BACE1 antibodies by Western blot and several antibody pairs to develop a new BACE1 sandwich ELISA procedure. We identified one pair that showed little background and good reproducibility. Several dilution buffers and sample denaturation methods were tried to partially unfold BACE1 before capture. We found that dilution in PBS followed by 10 min incubation at 50Â°C critically improves the performance of the assay. Finally, we successfully measured BACE1 levels in a few human brain and platelet lysates as well as in plasma and AD CSF. We anticipate that this assay will lay the ground to accurately measure BACE1 levels in human tissues, which could facilitate the molecular diagnosis of AD in the near future.