RESUMO
Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.
Assuntos
Axônios/metabolismo , Retículo Endoplasmático/metabolismo , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Substituição de Aminoácidos , Animais , Axônios/patologia , Linhagem Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Éxons/genética , Feminino , Ligação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteínas do Tecido Nervoso/genética , Linhagem , Proteínas/genética , Ratos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia , Xenopus , Peixe-ZebraRESUMO
The left and right sides of the nervous system communicate via commissural axons that cross the midline during development using evolutionarily conserved molecules. These guidance cues have been particularly well studied in the mammalian spinal cord, but it remains unclear whether these guidance mechanisms for commissural axons are similar in the developing forebrain, in particular for the corpus callosum, the largest and most important commissure for cortical function. Here, we show that Netrin1 initially attracts callosal pioneering axons derived from the cingulate cortex, but surprisingly is not attractive for the neocortical callosal axons that make up the bulk of the projection. Instead, we show that Netrin-deleted in colorectal cancer signaling acts in a fundamentally different manner, to prevent the Slit2-mediated repulsion of precrossing axons thereby allowing them to approach and cross the midline. These results provide the first evidence for how callosal axons integrate multiple guidance cues to navigate the midline.
Assuntos
Axônios/fisiologia , Corpo Caloso/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Receptor DCC , Embrião de Mamíferos , Feminino , Lateralidade Funcional/genética , Lateralidade Funcional/fisiologia , Humanos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Netrina-1 , Gravidez , Ratos Wistar , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Proteínas RoundaboutRESUMO
Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Dendritos/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Plasticidade Neuronal/fisiologia , Neurônios/metabolismoRESUMO
The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1(-/-) mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2(-/-) and Robo1(-/-) mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2(-/-) mice and 30% of Slit3(-/-) mice, whereas 100% of Slit1(-/-); Slit2(-/-) mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1(-/-) tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.
Assuntos
Corpo Caloso/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Diferenciação Celular , Técnicas de Cocultura , Corpo Caloso/citologia , Corpo Caloso/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Imageamento por Ressonância Magnética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/fisiologia , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Transdução de Sinais , Proteínas RoundaboutRESUMO
The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.
Assuntos
Astrócitos/metabolismo , Corpo Caloso/metabolismo , Receptor DCC/metabolismo , Telencéfalo/metabolismo , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Células COS , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Chlorocebus aethiops , Corpo Caloso/embriologia , Receptor DCC/genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Mutação , Netrina-1/genética , Netrina-1/metabolismo , Fenótipo , Transdução de Sinais , Telencéfalo/embriologiaRESUMO
Wiring up the nervous system depends on the precise guidance of axonal growth cones to their targets. A key mechanism underlying this guidance is chemotaxis, whereby growth cones detect and follow molecular gradients. Although recent work has uncovered many of the molecules involved in this process, the mechanisms underlying chemotactic axon guidance are still unclear. Here we compare growth cones with neutrophils and Dictyostelium discoideum, systems for which a clear conceptual framework for chemotaxis has recently emerged. This analogy suggests particular ways in which the three key steps of directional sensing, polarisation and motility might be implemented in chemotaxing growth cones.
Assuntos
Comunicação Celular/fisiologia , Quimiotaxia/fisiologia , Cones de Crescimento/fisiologia , Animais , Movimento Celular/fisiologia , Corrente Citoplasmática/fisiologia , Humanos , Sistema Nervoso/crescimento & desenvolvimento , Transdução de Sinais/fisiologiaRESUMO
Pioneer axons from the cingulate cortex initiate corpus callosum (CC) development, yet nothing is known about the molecules that regulate their guidance. We demonstrate that neuropilin 1 (Npn1) plays an integral role in the development of the CC. Npn1 is localized to axons of cingulate neurons as they cross the midline, and multiple class 3 semaphorins (Semas) are expressed around the developing CC, implicating these guidance molecules in the regulation of Npn1-expressing axons emanating from the cingulate cortex. Furthermore, axons from the cingulate cortex display guidance errors in Npn1(Sema-) mice, a knockin mouse line in which Npn1 is unable to bind Semas. Analysis of mice deficient in the transcription factor Emx2 demonstrated that the cingulate cortex of these mice was significantly reduced in comparison to wild-type controls at E17 and that the CC was absent in rostral sections. Expression of Npn1 was absent in rostral sections of Emx2 mutants, suggesting that Npn1-expressing cingulate pioneers are required for CC formation. These data highlight a central role for Npn1 in the development of projections from the cingulate cortex and further illustrate the importance of these pioneer axons in the formation of the CC.
Assuntos
Axônios/fisiologia , Corpo Caloso/embriologia , Corpo Caloso/metabolismo , Giro do Cíngulo/fisiologia , Neuropilina-1/metabolismo , Transdução de Sinais/fisiologia , Animais , Corpo Caloso/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Vias NeuraisRESUMO
Commissural formation in the mammalian brain is highly organised and regulated both by the cell-autonomous expression of transcription factors, and by non-cell-autonomous mechanisms including the formation of midline glial structures and their expression of specific axon guidance molecules. These mechanisms channel axons into the correct path and enable the subsequent connection of specific brain areas to their appropriate targets. Several key findings have been made over the past two years, including the discovery of novel mechanisms of action that 'classical' guidance factors such as the Slits, Netrins, and their receptors have in axon guidance. Moreover, novel guidance factors such as members of the Wnt family, and extracellular matrix components such as heparan sulphate proteoglycans, have been shown to be important for mammalian brain commissure formation. Additionally, there have been significant discoveries regarding the role of FGF signalling in the formation of midline glial structures. In this review, we discuss the most recent advances in the field that have contributed to our current understanding of commissural development in the telencephalon.
Assuntos
Corpo Caloso/embriologia , Cones de Crescimento/metabolismo , Vias Neurais/embriologia , Neuroglia/metabolismo , Prosencéfalo/embriologia , Animais , Diferenciação Celular/fisiologia , Corpo Caloso/fisiologia , Cones de Crescimento/ultraestrutura , Humanos , Fatores de Crescimento Neural/metabolismo , Vias Neurais/fisiologia , Neuroglia/citologia , Prosencéfalo/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Wnt5a guides cortical axons in vivo by repulsion and in vitro evokes cortical axon outgrowth and repulsion by calcium signaling pathways. Here we examined the role of microtubule (MT) reorganization and dynamics in mediating effects of Wnt5a. Inhibiting MT dynamics with nocodazole and taxol abolished Wnt5a evoked axon outgrowth and repulsion of cultured hamster cortical neurons. EGFP-EB3 labeled dynamic MTs visualized in live cell imaging revealed that growth cone MTs align with the nascent axon. Wnt5a increased axon outgrowth by reorganization of dynamic MTs from a splayed to a bundled array oriented in the direction of axon extension, and Wnt5a gradients induced asymmetric redistribution of dynamic MTs toward the far side of the growth cone. Wnt5a gradients also evoked calcium transients that were highest on the far side of the growth cone. Calcium signaling and the reorganization of dynamic MTs could be linked by tau, a MT associated protein that stabilizes MTs. Tau is phosphorylated at the Ser 262 MT binding site by CaMKII, and is required for Wnt5a induced axon outgrowth and repulsive turning. Phosphorylation of tau at Ser262 is known to detach tau from MTs to increase their dynamics. Using transfection with tau constructs mutated at Ser262, we found that this site is required for the growth and guidance effects of Wnt5a by mediating reorganization of dynamic MTs in cortical growth cones. Moreover, CaMKII inhibition also prevents MT reorganization required for Wnt5a induced axon outgrowth, thus linking Wnt/calcium signaling to tau mediated MT reorganization during growth cone behaviors.
Assuntos
Axônios/metabolismo , Cones de Crescimento/fisiologia , Microtúbulos/metabolismo , Neurônios/fisiologia , Proteínas Wnt/fisiologia , Proteínas tau/metabolismo , Animais , Células Cultivadas , Cricetinae , Feminino , Masculino , Transdução de Sinais/fisiologiaRESUMO
Adhesive micro-lines of various sub-cellular geometries were created using a non-traditional micro stamping technique. This technique employed the use of commercially available diffraction gratings as the molds for the micro stamps, a method which is quick and inexpensive, and which could easily be adopted as a patterning tool in a variety of research efforts. The atypical saw-tooth profile of the micro stamps enabled a unique degree of control and flexibility over patterned line and gap widths. Cortical neurons cultured on patterned poly-lysine micro-lines on PDMS exhibit a startling transition in axonal guidance: From the expected parallel guidance to an unexpected perpendicular guidance that becomes dominant as patterned lines and gaps become sufficiently narrow. This transition is most obvious when the lines are narrow relative to gaps, while the periodicity of the pattern is reduced. Axons growing perpendicular to micro-lines exhibited 'vinculated' growth, a unique morphological phenotype consisting of periodic orthogonal extensions along the axon.
Assuntos
Axônios/fisiologia , Técnicas de Cultura de Células , Adesividade , Animais , Células Cultivadas , Dimetilpolisiloxanos/química , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Polilisina/química , Silício/química , Propriedades de SuperfícieRESUMO
We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 31, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3beta. We also present evidence that PV signaling is activated via the alpha6beta4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection.