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1.
J Neural Transm (Vienna) ; 122(7): 975-91, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25976431

RESUMEN

Brain banks manage and store fully clinically and pathologically characterised brains. The diversity of techniques used in research projects increases. These biological resource centres are made to adapt brain tissue processing. Furthermore, the development of more sensitive techniques to analyse nucleic acids and proteins offers new fields of exploration when combined with laser capture microdissection in order to decipher the physiopathology of diseases at the cell level. In this study, our goal was to evaluate procedures and set a workflow compatible with the constraints of brain banks, from brain sampling to laser capture microdissection and pre-analytical quality assessment. We compared various methods of freezing brain tissue, focused on morphological quality preservation of brain microscopical structures and on the quality of nucleic acid or protein yields. Staining protocols combined with strategies to lower neurones autofluorescence were adapted for the same purpose. Finally, we found that laser capture microdissection is possible in the setting of brain banks. However, the entire process has to be envisioned from the autopsy to the analysis. The impact on protein or nucleic acid quality is a limitation that restricts the amount of samples available for this purpose.


Asunto(s)
Encéfalo/patología , Microdisección , Neuronas/patología , Bancos de Tejidos , Flujo de Trabajo , Adulto , Anciano , Anciano de 80 o más Años , Encefalopatías/patología , Europa (Continente) , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Cambios Post Mortem , Proteínas/genética , Proteínas/metabolismo , Manejo de Especímenes , Coloración y Etiquetado
2.
Proc Natl Acad Sci U S A ; 108(33): 13782-7, 2011 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-21804034

RESUMEN

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).


Asunto(s)
Cerebelo/citología , Neuronas/ultraestructura , Receptores de N-Metil-D-Aspartato/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Familia-src Quinasas/metabolismo , Inductores de la Angiogénesis , Animales , Calcio/metabolismo , Ratones , Complejos Multiproteicos , Fosforilación , Receptores de Neurotransmisores , Sinapsis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
3.
J Neurosci ; 30(45): 15052-66, 2010 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-21068311

RESUMEN

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.


Asunto(s)
Movimiento Celular/fisiología , Cerebelo/fisiología , Neuronas/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Células Cultivadas , Cerebelo/citología , Ensayo de Inmunoadsorción Enzimática , Conos de Crecimiento/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuronas/citología , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
4.
J Neurosci ; 27(46): 12546-54, 2007 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-18003833

RESUMEN

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1+/- mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1-/- mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)-/- neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1-/- neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1-/- layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.


Asunto(s)
Diferenciación Celular/fisiología , Corteza Cerebral/crecimiento & desarrollo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Espinas Dendríticas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Semaforina-3A/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Cricetinae , Quinasa 5 Dependiente de la Ciclina/genética , Espinas Dendríticas/ultraestructura , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Fosforilación , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Semaforina-3A/genética , Sinapsis/genética , Sinapsis/metabolismo , Sinapsis/ultraestructura , Sinapsinas/genética , Sinapsinas/metabolismo , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura
5.
J Neurosci ; 26(51): 13357-62, 2006 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-17182786

RESUMEN

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1-/- mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A-/- and crmp1+/-;sema3A+/- cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/fisiología , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Fosfoproteínas/fisiología , Serina Endopeptidasas/fisiología , Transducción de Señal/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular , Movimiento Celular/genética , Corteza Cerebral/citología , Proteínas de la Matriz Extracelular/genética , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Fosfoproteínas/genética , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal/genética
6.
Cancer Res ; 75(17): 3519-28, 2015 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-26122847

RESUMEN

Collapsin response mediator protein 5 (CRMP5) belongs to a family of five cytosolic proteins that play a major role in nervous system development. This protein was first described in cancer-induced autoimmune processes, causing neurodegenerative disorders (paraneoplastic neurologic syndromes). CRMP5 expression has been reported to serve as a biomarker for high-grade lung neuroendocrine carcinomas; however, its functional roles have not been examined in any setting of cancer pathophysiology. In this study, we report two different CRMP5 expression patterns observed in human glioblastoma (GBM) biopsies that establish connections between CRMP5 expression, Notch receptor signaling, and GBM cell proliferation. We demonstrated that elevated CRMP5 promotes Notch receptor expression and Akt activation in human tumor cell lines, GBM stem cells, and primary tumor biopsies. We have shown that the high CRMP5 and Notch expression in GBM xenograft is related to stem cells. This suggests that high CRMP5 expression pattern in GBM biopsies encompasses a subset of stem cells. Mechanistically, CRMP5 functioned by hijacking Notch receptors from Itch-dependent lysosomal degradation. Our findings suggest that CRMP5 serves as a major mediator of Notch signaling and Akt activation by controlling the degradation of the Notch receptor, with implications for defining a biomarker signature in GBM that correlates with and may predict patient survival.


Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Proteínas del Tejido Nervioso/biosíntesis , Receptores Notch/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Humanos , Hidrolasas , Masculino , Ratones , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Receptores Notch/metabolismo , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Mol Neurobiol ; 28(1): 51-64, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-14514985

RESUMEN

The members of the collapsin response mediator protein (CRMP) family-five cytosolic phosphoproteins -are highly expressed throughout brain development. The first member to be cloned, CRMP2, was identified as an intracellular messenger required for the growth cone-collapse induced by semaphorin 3A (Sema3A). A rapidly expanding body of study indicates that the functions of CRMPs are not solely limited to the signaling transduction of the Sema3A guidance cue. They are probably involved in multiple cellular and molecular events involved in apoptosis/proliferation, cell migration, and differentiation. In the adult brain, the expression of CRMPs is dramatically downregulated. However, they remain expressed in structures that retain their capacity for differentiation and plasticity and also in a subpopulation of oligodendrocytes (CRMP2 and CRMP5). Moreover, the expression of CRMPs is altered in neurodegenerative diseases, and these proteins may be of key importance in the physiopathology of the adult nervous system.


Asunto(s)
Envejecimiento/fisiología , Proteínas Aviares , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Semaforinas , Envejecimiento/metabolismo , Animales , Proteínas Portadoras/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Sistema Nervioso/citología , Enfermedades Neurodegenerativas/patología , Fosfoproteínas/fisiología
8.
Mol Cell Neurosci ; 37(2): 222-35, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17997325

RESUMEN

We examined whether Sema3A, which is upregulated at the site of spinal cord injury, exerts a direct effect on axons. We used ASNKL peptide that prevents specifically the inhibitory effect of Sema3A on L1/Neuropilin1 (Nrp1)-expressing axons. In the naïve mouse spinal cord, L1 is located on a subset of corticospinal axons, whereas Nrp1 is barely detectable. After contusion injury, Nrp1 is found on L1-negative immune cells, whereas its expression does not increase on severed axons. L1-expressing axons sprout extensively into the lesion site but no difference in axon density could be detected in the lesion area of mice treated with ASNKL. In agreement, these mice did not recover a better motor function than controls. Similarly, culture of neurons sensitive to ASNKL on cryosections of lesioned spinal cords revealed no effect of Sema3A. Our data indicate a limited direct effect of Sema3A on axonal growth at the site of a contusion injury, and suggest that alternative mechanisms underlie positive effects of Sema3A inhibition on motor recovery.


Asunto(s)
Regeneración Nerviosa/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Recuperación de la Función/fisiología , Semaforina-3A/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/ultraestructura , Línea Celular , Células Cultivadas , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Femenino , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Regeneración Nerviosa/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/agonistas , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/lesiones , Vías Nerviosas/metabolismo , Neuropilina-1/metabolismo , Fragmentos de Péptidos/farmacología , Recuperación de la Función/efectos de los fármacos , Semaforina-3A/antagonistas & inhibidores , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/fisiopatología , Insuficiencia del Tratamiento
9.
Am J Surg Pathol ; 32(11): 1699-708, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18769332

RESUMEN

The diagnosis of high-grade neuroendocrine tumors has strong clinical relevance because it identifies patients at higher risk of an unfavorable outcome who should receive multimodal treatment. However, these tumors can be mistaken for poorly differentiated nonsmall cell carcinoma or carcinoid lung tumors. In fact, no immunohistochemical marker can currently distinguish between histologic lung subtypes. Because the collapsin response mediator protein (CRMP) family is involved in an autoimmune disease associated with small cell lung carcinoma, we explored the relationship between CRMP5 expression and lung tumor behavior. Using World Health Organization morphologic criteria, 123 lung neuroendocrine tumors and 41 randomly selected non-neuroendocrine tumors were classified. CRMP5 protein expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining method. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade neuroendocrine lung tumors, including small cell lung carcinoma and large cell lung neuroendocrine carcinoma, but not in any of the squamous cell carcinomas or lung adenocarcinomas in our series. In contrast, the majority of low-grade neuroendocrine lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with 2 different staining patterns of either scattered positive cells or small foci of positive cells. Our findings point at CRMP5 as a novel marker for routine pathologic evaluation of lung tumors surgical samples in distinguishing between highly aggressive neuroendocrine carcinoma and the other lung cancers.


Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Neuroendocrino/metabolismo , Carcinoma Neuroendocrino/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas del Tejido Nervioso/biosíntesis , Adulto , Western Blotting , Carcinoma Neuroendocrino/mortalidad , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Humanos , Hidrolasas , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Masculino , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
10.
Genes Cells ; 11(12): 1337-52, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17121542

RESUMEN

Collapsin response mediator proteins (CRMPs) consist of five homologous cytosolic proteins that participate in signal transduction involved in a variety of physiological events. CRMP1 is highly expressed during brain development; however, its functions remains unclear. To gain insight into its function, we generated CRMP1(-/-) mice with a knock-in LacZ gene. No gross anatomical changes or behavioral alterations were observed. Expression of CRMP1 was examined by the expression of the knocked-in LacZ gene, in situ hybridization with riboprobes and by imunohistochemistry. CRMP1 was found to be highly expressed in the developing the cerebellum, olfactory bulbs, hypothalamus and retina. In adults, expression level was high in the olfactory bulbs and hippocampus but very low in the retina and cerebellum and undetectable in hypothalamus. To study potential roles of CRMP1, we focused on cerebellum development. CRMP1(-/-) mice showed a decrease in the number of granule cells migrating out of explants of developing cerebellum, as did treatment of the explants from normal mice with anti-CRMP1 specific antibodies. CRMP1(-/-) mice showed a decrease in granule cell proliferation and apoptosis in external granule cell layers in vivo. Adult cerebellum of CRMP1(-/-) did not show any abnormalities.


Asunto(s)
Apoptosis , Movimiento Celular/fisiología , Proliferación Celular , Cerebelo/crecimiento & desarrollo , Gránulos Citoplasmáticos/fisiología , Proteínas del Tejido Nervioso/genética , Animales , Bromodesoxiuridina/metabolismo , Células Cultivadas , Cerebelo/citología , Técnica del Anticuerpo Fluorescente Indirecta , Marcación de Gen , Genes Reporteros , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Cultivo de Órganos , Sondas ARN
11.
Int Immunol ; 17(4): 439-47, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15746246

RESUMEN

CD100 represents the first semaphorin described in the immune system. It is expressed as a 300-kDa homodimer at the surface of most hematopoietic cells, but is also found in a soluble form following a proteolytic cleavage upon cell activation. We herein established that soluble CD100 (sCD100) impaired the migration of human monocytes and immature dendritic cells (DCs), but not of mature DCs. Performing competition assays, we identified plexin C1 (VESPR/CD232) as being involved in sCD100-mediated effects on human monocytes. Interestingly, we observed a complete down-regulation of plexin C1 expression during the in vitro differentiation process of monocytes to immature DCs, while concomitantly the surface expression of plexin B1 was induced. The latter receptor then binds sCD100 on immature DCs, mediating its inhibitory effect on cell migration. Finally, we showed that sCD100 modulated the cytokine production from monocytes and immature DCs. Together these results suggest that sCD100 plays a critical role in the regulation of antigen-presenting cell migration and functions via a tightly regulated process of receptor expression.


Asunto(s)
Antígenos CD/inmunología , Células Dendríticas/inmunología , Monocitos/inmunología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Virales/metabolismo , Semaforinas/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Movimiento Celular/inmunología , Células Dendríticas/metabolismo , Humanos , Monocitos/metabolismo
12.
Mol Cell Neurosci ; 24(2): 395-408, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14572461

RESUMEN

Matrix metalloproteinases (MMPs) are responsible for the extensive extracellular proteolysis that plays a central role in regulating the pericellular environment, contributing to morphogenesis and developmental remodeling. In the CNS, there is increasing in vitro evidence for the involvement of MMPs in neurite elongation and axonal guidance. Here, we show that expression of MMP-9 is spatiotemporally related to cerebellar granule cell migration during postnatal development. Using cerebellar explant cultures, we demonstrated that a specific MMP-9-blocking antibody affects granular cell axonal outgrowth and migration in a dose-dependent manner. In addition, the in vivo analysis of MMP-9-deficient mice revealed abnormal accumulation of granular precursors (GPs) in the external granular layer (EGL) at a time when migration is normally extensive. Furthermore, GP migration was delayed and their programmed cell death was reduced in MMP-9-deficient mice, suggesting that MMP-9 is involved in the control of granule cell migration and apoptosis. These results provide direct evidence for a physiological role of MMP-9 in neuronal precursor migration and apoptosis in the developing cerebellum, and emphasize the importance of MMP-9 in the temporal regulation of the cerebellar microenvironment.


Asunto(s)
Apoptosis/fisiología , Axones/fisiología , Movimiento Celular/fisiología , Cerebelo/enzimología , Metaloproteinasa 9 de la Matriz/deficiencia , Metaloproteinasa 9 de la Matriz/genética , Animales , Animales Recién Nacidos , Axones/enzimología , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
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