Estudio de las vías de señalización intracelular asociadas a las proteínas inhibitorias de la mielina

[eng] Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3beta) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections. On the other hand, and focused in the OMgp, by using recording electrophysiological nano-devices we found that, OMgp has a role in synaptic transmission, since it can induce excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons

A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. Traditional approaches to model axonal lesions, such as high intensity power laser ablation or sharp metal scratching, are complex to implement, have low throughputs, and generate cuts that are difficult to modulate. We present here a novel reproducible microfluidic approach to model in vitro mechanical lesion of tens to hundreds of axons simultaneously in a controlled manner. The dimensions of the induced axonal injury and its distance from the neuronal cell body are precisely controlled while preserving both the proximal and distal portions of axons. We have observed that distal axons undergo Wallerianlike anterograde degeneration after axotomy; in contrast, proximal portions of the axons remain un-degenerated, possessing the potential to re-grow. More importantly, surpassing the previous axotomy methods performed in petridish in which local microenvironments cannot be tailored, our platform holds the capability to implement fine-tuned treatments to lesioned axon stumps in a local, controlled manner. Specifically, molecules such as chondroitin sulphate proteoglycans and its degrading enzyme Chondroitinase ABC, hydrogels, and supporting cells have been shown to be deliverable to the lesioned site of injured axons. In addition, this system also permits double interventions at the level of the lesioned axons and the perikaryon. This proves the potentiality of our model by demonstrating how axonal regrowth can be evaluated under circumstances that are better mimicking biological problems. We believe that this novel mechanical microfluidic axotomy approach is easy to perform, yields high throughput axon lesions, is physiologically relevant, and offers a simplified platform for screening of potential new neurological drugs

A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. Traditional approaches to model axonal lesions, such as high intensity power laser ablation or sharp metal scratching, are complex to implement, have low throughputs, and generate cuts that are difficult to modulate. We present here a novel reproducible microfluidic approach to model in vitro mechanical lesion of tens to hundreds of axons simultaneously in a controlled manner. The dimensions of the induced axonal injury and its distance from the neuronal cell body are precisely controlled while preserving both the proximal and distal portions of axons. We have observed that distal axons undergo Wallerianlike anterograde degeneration after axotomy; in contrast, proximal portions of the axons remain un-degenerated, possessing the potential to re-grow. More importantly, surpassing the previous axotomy methods performed in petridish in which local microenvironments cannot be tailored, our platform holds the capability to implement fine-tuned treatments to lesioned axon stumps in a local, controlled manner. Specifically, molecules such as chondroitin sulphate proteoglycans and its degrading enzyme Chondroitinase ABC, hydrogels, and supporting cells have been shown to be deliverable to the lesioned site of injured axons. In addition, this system also permits double interventions at the level of the lesioned axons and the perikaryon. This proves the potentiality of our model by demonstrating how axonal regrowth can be evaluated under circumstances that are better mimicking biological problems. We believe that this novel mechanical microfluidic axotomy approach is easy to perform, yields high throughput axon lesions, is physiologically relevant, and offers a simplified platform for screening of potential new neurological drugs

Neurites regrowth of cortical neurons by GSK3b inhibition independently of Nogo Receptor 1

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3 and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3 inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections

Developmental Expression of the Oligodendrocyte Myelin Glycoprotein in the Mouse Telencephalon

The oligodendrocyte myelin glycoprotein is a glycosylphosphatidylinositol-anchored protein expressed by neurons and oligodendrocytes in the central nervous system. Attempts have been made to identify the functions of the myelin-associated inhibitory proteins (MAIPs) after axonal lesion or in neurodegeneration. However, the developmental roles of some of these proteins and their receptors remain elusive. Recent studies indicate that NgR1 and the recently discovered receptor PirB restrict cortical synaptic plasticity. However, the putative factors that trigger these effects are unknown. Because Nogo-A is mostly associated with the endoplasmic reticulum and myelin associated glycoprotein appears late during development, the putative participation of OMgp should be considered. Here, we examine the pattern of development of OMgp immunoreactive elements during mouse telencephalic development. OMgp immunoreactivity in the developing cortex follows the establishment of the thalamo-cortical barrel field. At the cellular level, we located OMgp neuronal membranes in dendrites and axons as well as in brain synaptosome fractions and axon varicosities. Lastly, the analysis of the barrel field in OMgp-deficient mice revealed that although thalamo-cortical connections were formed, their targeting in layer IV was altered, and numerous axons ectopically invaded layers II–III. Our data support the idea that early expressed MAIPs play an active role during development and point to OMgp participating in thalamo-cortical connections