Régénération et croissance de l'axone

A lesion of mammalian nervous system is known to trigger a series of typical events that inevitably perturb the neural environment, a phenomenon that in turn influences the remodeling of neurons and their axons, whether they are themselves injured, or indirectly affected.

Axon degeneration is the pivotal pathological event of acute traumatic neural injury as well as many chronic neurodegenerative diseases. It is an active cellular program, and yet molecularly distinct from cell death. Much effort is devoted towards understanding the nature of axon degeneration, and promoting axon regeneration. Anterograde and retrograde axonal transport coupling the distantly located synaptic terminals with the cell soma is essential for neuronal differentiation, survival, and function. The importance of these trafficking routes is underscored by the findings that disruption of axonal transport such as by abnormal accumulation of protein aggregates and organelles along the axon is thought to be an early pathogenic event leading to the demise of neurons. In addition, retrograde axonal transport is critical to allow for peripheral neurons repairing themselves after injury, and may be absent or deficient in neurons within the central nervous system (CNS). Failure to repair, or protect CNS axons from degeneration has remained a still unsolved problem despite a century of research, and continues to be one of the biggest challenges in neuroscience (Aim 1). Our methodological expertise ranges from micro-technological approaches allowing the control of axonal outgrowth and neuronal networks reconstruction (Aim 2) to in vivo models allowing to study neuronal restoration and network reconnection (Aim 3).

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Our team has largely contributed to a better understanding of the molecular and cellular mechanisms underlying injury- or disease-related axon regeneration and degeneration, and this will continue to be one of our major aims. Our recent investigation also included the development of micro-technological approaches allowing in vitro reconstruction of human and rodent functional neuronal pathways. In addition a repair strategy that promote axon regeneration in adult CNS, particularly after traumatic lesions, constituting the second aim of our project. Thus, through interdisciplinary approaches our future investigations may be considered as linking fundamental and applied research, as both branches are interconnected. 

Aim-1 : Axon fate: Molecular mechanisms involved in axon regeneration and degeneration

In the course of traumatic and degenerative diseases of the nervous system (NS), affected neurons will degenerate their axons and disconnect from their targets. Such lesions may even spread from neurons to neurons leading to neuronal circuits weakening, even if they are principally capable of reorganization. Both cell intrinsic and non-cell autonomous mechanisms controls axonal fate under stress that will leads to either axonal die back processes or attempts to regrowth. While attempts to regenerate them are generally abortive in the CNS, lesioned axons need to be restructured by forming a growth cone at their distal end, capable of navigating in an adult neural environment, and reconnecting to its natural target. While trying to decipher the cellular and molecular mechanisms at plays during axonal degeneration/regeneration,our aim is twofold:

  1. Determine the common or divergent mechanisms that trigger either regeneration or degeneration after focal trauma or chronic degenerative condition, at the single cell level. The central element is the cytoskeleton, since it not only determines the shape of the cell, but also organizes the cellular compartments that assure proper functioning of the neuron. In particular, we address the role of intracellular trafficking, transport, and mitochondria dynamics, in relation with the state of the microtubules, by studies in vivo and in vitro.
  2. Study the consequences of axonal degeneration-induced de-afferentation on the robustness of neuronal networks. Axonal trauma and progressive neurodegenerative condition alters neuronal network connectivity and triggers the transmission of trans-synaptic deleterious signals between neurons. The impact of interneuronal exchange of protein aggregates, the role of synaptic physiology and interference with activity-dependent survival processes are analyzed at the level of neuronal networks, the topology of which is controlled by micro-technological approaches (Aim 2).

Aim-2 : CNS-on-Chip: Technological approaches for axonal degeneration and neuronal reconnections modeling

Pathological conditions progressively disrupt brain organization, and there is an urgent need to develop both integrated 2D and 3D living in vitro models of brain architectures allowing disease modelling, predictive pharmacology, and elaboration of neuro-restorative strategies for the rewiring of a damaged brain. Through established academic collaborative networks involving physicist, technologists and theoretician, we co-develop microfluidic and biomaterial derived cell culture prototypes to control cell-cell interactions. By combining microfluidic, micro-pattering and biomaterial approaches allowing to control cytoskeletal bio-mechanic and neuronal physiology, we are currently involved in designing both rodent and human (iPSC derived) 2D and 3D neuronal networks of fully controlled topology. These experimental systems bridge the gap between conventional cell culture systems and in vivo models. They allow us to model both traumatic and chronic degenerative processes in highly predictive environment and evaluate both protective and regenerative strategies.

Our seminal work showed that the extension of cell processes (axons, astrocytes extensions) can be controlled trough 1) bio-imprinted biomaterial and 2) textured micro-environment imposing mechanical constrains on neurons (3 patents). This permits the creation of microfluidic or micro-patterned cell culture chambers allowing the manipulation of neurons at the subcellular level. Based on our previous expertise we developed a unique fabrication process allowing both rapid prototyping and mass producing of simple microfluidic devices (Paul et al, 2010). These experimental systems are routinely used in our laboratory and are disseminated to our collaborative network of neuroscientist and cell biologists. Using our “axon diode” paradigm that allows the control of axonal outgrowth direction we design microfluidic chips allowing the reconstruction of oriented functional neuronal networks in microfluidic environment (Peyrin et al, 2011) which electrical activity is controllable trough pharmacological, optogenetical or neuro-electronic stimulation. More specifically we developed microfluidic chips allowing to model axonal trauma and axonal degeneration at the single cell level (Kilinc et al 2010, Magnifico et al 2013, Szelechowski et al, 2014); or at the neuronal network level (Peyrin et al, 2011, Deleglise et al 2013, 2014, Alleaume-Butaux et al 2015), together with micro-technological devices allowing the fast evaluation of therapeutic and restorative strategies. 

Aim-3 : Regenerative strategies for spinal cord after traumatic injury

Spinal cord injury (SCI) causes severe, lifelong disability, and has significant societal consequences. The only therapy approved to treat SCI is acute methylprednisolone treatment, which has very little impact on patient recovery. This is due in part to the limited ability of the adult human Central Nervous System (CNS) to self-repair after injury. Basic research has made advances in elucidating the mechanisms involved in neurodegeneration and inflammation after SCI, and the causes limiting axon regeneration. This knowledge provided the basis for developing animal models of SCI that in turn, allow for developing novel approaches for CNS repair and functional recovery. Maximal restoration following SCI will only be achieved by a combination of therapies, an approach that requires a multidisciplinary research strategy like the one we established for our studies. Our main focus is on the use novel biomaterials as a basis for creating unique, combinatorial repair strategies to treat SCI. We established a strong collaboration with experts in natural polymer research who have developed innovative glycosaminoglycan-based hydrogels capable of supporting cell-based therapies, and small molecule/drug treatment for SCI (IMP, Univ Lyon 1, headed by Laurent David). Our collaboration yielded promising results in treating SCI using chitosan biomaterial scaffolds alone. Implantation of our patented formulation in lesioned SCI tissue results in reduction of scar formation, functional revascularization, modulated inflammation, andmost remarkably, presence of high numbers of axons within the implant, and functional recovery (patent, PhD thesis, and submitted paper). These findings support the ability of our new approach to induce recovery of experimental SCI, and the feasibility of our proposed studies to advance this treatment for future clinical development.

Our project will employ novel combinatorial approaches destined to optimize the efficacy of a biomaterial by combination with neuroprotective drugs to minimize axon die-back, and to prevent secondary progressive neuron degeneration by cell therapy.